The mechanisms of resistance have developed in response to the mechanisms of action of antimicrobial drugs. Antimicrobial agents are generally categorized according to their targeted sites of action. To date, three classes of antibiotic resistance have been described intrinsic resistance, acquired resistance, and genetic resistance (Hancock, 1998). 2.4.1 Intrinsic resistance: This includes outer membrane impermeability, efflux pumps, and enzymes. 2.4.1.1 Outer membrane impermeability: The outer membrane constitutes a semi-permeable barrier to the uptake of antibiotics and substrate molecules. Because uptake of small hydrophilic molecules such as β-lactams is restricted to a small portion of the outer membrane (namely the water filled channels of porin proteins), the outer membrane limits the movement of such molecules into the cell (Bellido et al., 1992). Compared with Gram-positive cells, Gram-negative bacteria are
covered with one additional membrane layer, the outer membrane (OM). It was thus hypothesized that the OM would serve as a general permeability barrier that the slows down the diffusion of various types of solutes, including drugs, and that this would contribute to the intrinsic drug resistance that find in Gram-negative bacteria (Nikaido and Vaara, 1985). It was recognized early in the history of antibiotic development that penicillin is effective against Gram-positive bacteria but not against Gram-negative ones. The difference in susceptibility to pencillin is due in large part to the outer membrane, a lipid bilayer that acts as a barrier to the penetration of antibiotics into the cell (Nikaido, 1985). 2.4.1.2 Efflux pumps: By a change in membrane permeability that makes the drug unable to penetrate through the membrane into the cell. This may be due to a change in structural protein, a decrease in pore size or an alteration in the transport system. Alternatively the rate of efflux of the drug from the cell may be increased making the drug unable to attain a sufficiently high concentration inside the cell to cause inhibition. This is the basic of resistance to tetracyclines (Delgadillo et al., 1993). An efflux system, involving three proteins (Mex A, Mex B, and Opr M) is critical for the intrinsic resistance of P. aeruginosa. Mutation in any of the genes encoding these proteins led to a fourfold to tenfold increase in susceptibility to quinolones, β-lactams (except imipenem), tetracycline, and chloramphanicol (Poole et al., 1993). Promotion of antibiotic efflux: In some strains of S. pneumoniae, S. aureus, Strept. pyogenes and S. epidermidis, an active efflux mechanism causes resistance to macrolides, streptogramins and azalides (Sutcliffe et al., 1996). Active efflux mechanisms may also contribute to the full expression of β-lactam resistance in Pseudomonas aeruginosa (Stikumar and Poolek, 1997). 2.4.1.3 Enzymes: Resistance due to Enzymic Inactivation: Two types of enzymes inactivate penicillins and cephalosporins. These are the acylases (amidases), which split the peptide bond linking the side chains to the 6-aminopenicillanic acid or 7-aminocephalosporanic acid nucleus, and the β-lactamases, which hydrolyse the CO__N bond of the β-lactam ring (Figure 1). Acylases are produced by a wide range of bacteria and moulds Hamilton-Miller, (1966). Since 6-amino penicillanic acid, the product of the reaction, has a low antibacterial activity against most micro-organisms, it has been suggested that the ability to produce acylases may contribute to the resistance of an organism to penicillin. However, the amount of enzyme produced is small and the conditions required for maximal activity are unlikely to obtain in vivo Cole and Sutherland, (1966). It seems, therefore, that these enzymes are unimportant factors in penicillin resistance. The acylases are of great value in breaking down natural penicillins to yield the penicillin nucleus from which the semi-synthetic penicillins are derived (Batchelor et al. 1961). The β-Lactamases: β-lactam antibiotics are antibacterial agents that share the structure feature of a β-lactam ring are known to be very diverse (Greenwood, 1995). Resistance to β-lactam antibiotics is due mainly to the production of β-lactamases, enzymes that inactivates these antibiotics by splitting the amide bond of the β-lactam ring. Numerous β-lactamases exist, encoded either by chromosomal genes or by transferable genes located on plasmids or transposons (Medeiros, 1984). These enzymes new first detected by Abraham and Chain, (1940) in extracts of penicillin-resistant strains of E. Coli and other Gram-negative bacteria, and have since been demonstrated in penicillin- or cephalosporin-resistant strains of most bacterial species. One of the first-recognized ways for bacteria to resist the actions of antimicrobial agents was the production of enzymes that inactivate a drug. Aminoglycosides and chloramphenicol have been found to be affected by inactivating enzymes, but the classic example of this phenomenon is β-lactamase production. Since their introduction into clinical practice, the effectiveness of β-lactam antibiotics has been reduced by the occurrence of bacteria that are resistant to their mode of action. Resistant Staphylococcus aureus strains were reported very soon after the introduction of benzyl penicillin into clinical practice (Abraham and Chain, 1940). Other than the intrinsic resistance resulting from insusceptible targets or inadequate penetration of the drug through the Gram-negative outer membrane, resistance to this class of antibiotic is most frequently due to the production of β-lactamase enzymes that hydrolyze the β-lactam bond in these antibiotics, thus destroying their functionality (Livermore, 1995). need to know more read www.amazon.com/Manual-Antibiotics-Mechanisms-Resistance
Wednesday, August 31, 2011
Methods for Detection of Antimycobacterial Drug Resistance
Early detection of drug resistance constitutes one of the priorities of TB control programs. It allows initiation of the appropriate treatment in patients and also surveillance of drug resistance. Detection of drug resistance has been performed in the past by so-called ‘conventional methods’ based on detection of growth of M. tuberculosis in the presence of the antibiotics. However, due to the laboriousness of some of these methods, and most of all, the long period of time necessary to obtain results, in recent years new technologies and approaches have been proposed.
These include both phenotypic and genotypic methods. In many cases, the genotypic methods in particular have been directed towards detection of RIF resistance, since it is considered a good surrogate marker for MDR-TB, especially in settings with a high prevalence of MDR-TB. Genotypic methods have the advantage of a shorter turnaround time, no need for growth of the organism, the possibility of direct application in clinical samples, lower biohazard risks, and the feasibility of automation; however, not all molecular mechanisms of drug resistance are known.
Phenotypic methods, on the other hand, are in general simpler to perform and might be closer to implementation on a routine basis in clinical mycobacteriology laboratories. The following section describes the conventional phenotypic methodologies proposed for drug resistance detection in TB.
These include both phenotypic and genotypic methods. In many cases, the genotypic methods in particular have been directed towards detection of RIF resistance, since it is considered a good surrogate marker for MDR-TB, especially in settings with a high prevalence of MDR-TB. Genotypic methods have the advantage of a shorter turnaround time, no need for growth of the organism, the possibility of direct application in clinical samples, lower biohazard risks, and the feasibility of automation; however, not all molecular mechanisms of drug resistance are known.
Phenotypic methods, on the other hand, are in general simpler to perform and might be closer to implementation on a routine basis in clinical mycobacteriology laboratories. The following section describes the conventional phenotypic methodologies proposed for drug resistance detection in TB.
Second line drugs used in TB treatment
According to the WHO the following drugs can be classified as second line drugs: aminoglycosides (kanamycin and amikacin) polypeptides (capreomycin, viomycin and enviomycin), fluoroquinolones (ofloxacin, ciprofloxacin, and gatifloxacin), D-cycloserine andthionamides (ethionamide and prothionamide) (WorldHealth Organization, 2001). Unfortunately, second-linedrugs are inherently more toxic and less effective thanfirst-line drugs (World Health Organization, 2001). Secondline drugs are mostly used in the treatment of MDR-TBand as a result prolong the total treatment time from 6 to9 months (Cheng et al., 2004).
The phenotypic methods to detect resistance to second line drugs are less well established and the molecular mechanisms of resistance are also less defined. Need to read more, read Mycobacterium Tuberculosis, Current status in Rapid Laborato…
The phenotypic methods to detect resistance to second line drugs are less well established and the molecular mechanisms of resistance are also less defined. Need to read more, read Mycobacterium Tuberculosis, Current status in Rapid Laborato…
Antibiotics Resistance of Mycobacterium tuberculosis
Shortly after the first anti-tuberculosis (TB) drugs were introduced, streptomycin (STR), para-aminosalicylic acid (PAS), isoniazid (INH) resistance to these drugs was observed in clinical isolates of Mycobacterium tuberculosis (Crofton and Mitchison, 1948). This led to the need to measure resistance accurately and easily. The Pasteur Institute introduced the critical proportion method in 1961 for drug susceptibility testing in TBand this method became the standard method of use (Espinal, 2003). Studies on drug resistance in various countries in the 1960s showed that developing countries had a much higher incidence of drug resistance than developed countries (Espinal, 2003). By the end of the 1960s rifampicin (RIF) was introduced and with the use of combination therapy, there was a decline in drug resistant and drug susceptible TB in developed countries. This led to a decline in funding and interest in TB control programs.
As a result, no concrete monitoring of drug resistance was carried out for the following 20 years (Espinal, 2003). The arrival of HIV/AIDS in the 1980s resulted in an increase in transmission of TB associated with outbreaks of multidrug-resistant TB (MDR-TB) (Edlin et al., 1992; Fischl et al., 1992) i.e. resistant to INH and RIF. In the early 1990s drug resistance surveillance was resumed in developed countries, but the true incidence remained unclear in the developing world (Cohn et al., 1997).
Any drug used in the anti-TB regiment is supposed to have an effective sterilizing activity that is capable of shortening the duration of treatment. Currently, a four-drug regiment is used consisting of INH, RIF, pyrazinamide (PZA) and ethambutol (EMB). Resistance to first line anti-TB drugs has been linked to mutations in at least 10 genes; katG inhA, ahpC, kasA and ndh for INH resistance; rpoB for RIF resistance, embB for EMB resistance, pncA for PZA resistance and rpsL and rrs for STR resistance.
As a result, no concrete monitoring of drug resistance was carried out for the following 20 years (Espinal, 2003). The arrival of HIV/AIDS in the 1980s resulted in an increase in transmission of TB associated with outbreaks of multidrug-resistant TB (MDR-TB) (Edlin et al., 1992; Fischl et al., 1992) i.e. resistant to INH and RIF. In the early 1990s drug resistance surveillance was resumed in developed countries, but the true incidence remained unclear in the developing world (Cohn et al., 1997).
Any drug used in the anti-TB regiment is supposed to have an effective sterilizing activity that is capable of shortening the duration of treatment. Currently, a four-drug regiment is used consisting of INH, RIF, pyrazinamide (PZA) and ethambutol (EMB). Resistance to first line anti-TB drugs has been linked to mutations in at least 10 genes; katG inhA, ahpC, kasA and ndh for INH resistance; rpoB for RIF resistance, embB for EMB resistance, pncA for PZA resistance and rpsL and rrs for STR resistance.
Tuesday, August 30, 2011
Hospital Acquired Infections and Hematological Malignancies
Patients with hematological malignancies are high risk group for nosocomial infections because of many factors as impaired immunity, high parenteral exposure, and administration of cytotoxic chemotherapy or receiving bone marrow transplantation. They are vulnerable to infections including viruses, mainly herpes viruses and respiratory viruses (Pizzo, 2007).
Viral infections are important causes of morbidity and mortality for patients with hematological malignancies. However the true incidence and consequences of viral Infections for these patients who undergo conventional nontransplant therapy are poorly defined. Differences in incidence and outcome of viral infections among these patient groups are based on intensity and duration of T-cell-mediated immune suppression (Wade, 2006).
Respiratory Viruses:
Respiratory viruses, including RSV, PIV and influenza virus, are wide spread in the community and easily transmitted to patients with hematological malignancy (England, 2001).
Infection control measures are critical and should consist of hand washing, annual influenza vaccination, early detection for infection and both respiratory and contact isolation of infection health care workers and patients. Patients, who develop respiratory virus infection prior to the initiation of treatment or transplantation, should if possible, have their therapy delayed. Respiratory virus infection among patients with hematological malignancy are associated with a more prolonged infection, higher frequency of nosocomial infection (55-83 % of exposed immunocompromized patients will become infected), a higher rate of pneumonia, co-pathogens and death. The risk of death from pneumonia has ranged from 9% to 82%, and appears to vary little between the different groups of patients with active hematological malignancy (Peck et al., 2004).
Other Viral Infections
• Adenovirus:
Primary infection is acquired from either a respiratory droplet or oral-fecal route. Most infection among compromised hosts is postulated to be viral reactivation (Kojaoghlanian et al., 2003). Clinical manifestations vary with serotype and include viremia, pneumonia, hepatitis, gastrointestinal disease, cystitis, nephritis and conjunctivitis (Bruno et al., 2004). Control of adenovirus appears T-cell mediated, and allogenic SCT recipients appear to be at greatest risk of infection and disease. However fatal adenovirus infections have been reported in patients with B-cell lymphoma, multiple myeloma and AML (Fianchi et al., 2003).
Risk factors for infection and disease include unrelated donor transplantation, GVHD, T-cell depletion, younger patient age, total body irradiation, and viremia. The incidence of infection in SCT recipients has been reported to range from 5-29% with disease occurring in 5-8% of patients. Death secondary to adenovirus disease range from 30-50% (Lion et al., 2003).
Severe disseminated disease can develop following BMT. Typical presentations in immunocompromised include pneumonitis, enterocolitis, hemorrhagic cystitis, hepatitis, encephalitis, and disseminated disease. In one study, 46% of BMT patients with adenovirus infections died; seven patients with pneumonia and six with disseminated disease (Slatter et al., 2005).
• Human Metapneumovirus ( hMPV ):
hMPV is newly discovered RNA Paramyxovirus. Most children by age 5 years are seropositive. Infection occurs primarily during winter and manifests as both upper and lower respiratory tract disease (Williams et al., 2004).
More serious disease has been reported among immunosuppressed patients. It could be an important cause of idiopathic pneumonia syndrome after SCT; Five out of 200 tested patients had hMPV detected in archived bronchial alveolar lavage specimens. All 5 positive patients had upper respiratory tract prodromes that preceded their pneumonia, and 4 of 5 patients died. Lung tissue of the died patients had histologic picture of idiopathic pneumonia syndrome at autopsy. Prospective studies of the role of hMPV as a cause of infection for patients with hematological malignancy are needed. There is no established treatment for hMPV infections although Ribavirin appears to have antiviral activity (Englund, 2001).
Herpes simplex virus:
HSV infections in patients with hematological malignancy are almost exclusively reactivation Infections (Lungman, 2004). They are common, ranging from 15% among chronic leukemia patients treated with Fludarabine, to 90% of patients with acute leukemia or stem cell transplant recipients. HSV infections and disease occur early after therapy, and frequently recur with future treatment. Mucocutaneous HSV disease will frequently present with an atypical appearance and can mimic other pathogens (i.e., Candida) or treatment induced mucositis. HSV infections among immunocompromised patients are characteristically more invasive, heal more slowly, are associated with prolonged viral shedding and may disseminate (Sandherr et al., 2006).
Varicella-Zoster virus:
The majority of VZV infections in adult patients with hematological malignancy are reactivation infections and 80% present with localized disease. The incidence of VZV infection ranges from 2% among patients with CML receiving Imatinib; to 10-15% in patients with CLL receiving Fludarabine; to 25% of patients with Hodgkin lymphoma or autologous stem cell transplantation (SCT) recipients; and to 45-60% among allogenic SCT recipients (Mattiuzzi et al., 2003).
Infection risk is greatest within the first 12 months following treatment or transplant, but late onset disease occurs because of persistent immunosuppression. Patients who are VZV naive are at risk for primary infection with either wild type or vaccine strains and should be counseled about the risk of developing such infection. Primary VZV infection can be very severe, and measures to prevent exposure and intervene early are recommended (Weinstock et al., 2004).
Unusual VZV syndromes of importance include trigeminal zoster with keratitis and yeast infections, and post–zoster pain. Hepatic or gastro-intestinal VZV disease is an important entity, and may present with few or no skin lesions. This presentation may result in delayed diagnosis, and has been associated with significant mortality (David et al., 1998).
Human herpes virus – 6 (HHV-6):
HHV-6 is ubiquitous Herpes virus that infects most persons early in life. Two major viral variants have been identified (A and B), but the B variant is most frequently associated with disease among immunocompromised patients. Longitudinal studies in SCT recipients found that viral reactivation occurred a median of 20 days after transplantation, and that viral shedding for some patients was prolonged, and correlated poorly with clinical improvement. HHV-6 viremia among allogenic transplant recipients is associated with an increased mortality, and increased when patients are transplanted for disease other than first remission, when donor and recipient are sex mismatched and among younger patients (Zerr et al., 2005).
Wade (2006) treated 4 patients for HHV-6 viremia who developed CNS dysfunction and delayed platelet recovery; three of them had autologous transplant for myeloma following Melphalan, and the fourth was treated with Imatinib for relapsed ALL. All 4 responded to prolonged antiviral therapy.
Quantitative real-time PCR analysis on blood and CSF is the method of choice for diagnosis. Foscarnet and Ganciclovir, alone or in combination, have been used as treatment for HHV-6 infection. Prospective studies are needed to better understand the importance of HHV -6 infections among patients with hematological malignancy, and to define disease spectrum, and appropriate therapy (Zerr et al., 2005).
BK Virus:
BK virus is DNA polygonal virus that is believed to cause nephropathy and graft loss among renal transplant recipients and also may cause pneumonia (Boeckh et al., 2005). There is increasing evidence that BK virus plays an important role in renal impairment in patients with hematological malignancy, but viral tissue invasion has only recently been demonstrated (Erard et al., 2005). BK virus was reported up to 95% of SCT recipients, with the onset of viral shedding occurring a median of 41 days after transplant, BK viruria may be prolonged, can be severe and some patients remain symptomatic for more than one month. Highly sensitive PCR assay for BK virus detection in blood and urine is now available (Bridges et al., 2006).
Cytomegalovirus:
T-cell function is paramount in the control of CMV and T-cell depleting agents as Alemtuzamab and aggressive chemotherapy e.g. CVAD and acute leukemia induction appear to increase the risk of CMV infection and disease. In the absence of effective antiviral prophylaxis, the incidence of CMV infection in patients with hematological malignancy ranges from 5-75% (Boeckh and Nichols, 2004). Patients undergoing an autologous stem cell transplant have a low risk of CMV infection but CD 34 selection of the autologous stem cell product increases the risk of CMV disease and death (Holmberg et al., 1999).
CMV attributable mortality for patients with hematological malignancies who receive conventional therapy ranged from 30% -57%. CMV disease in patients with acute leukemia was associated with the use of high dose Fludarabine, Cyclophosphamide, or Alemtuzumab. CMV viremia occurred in 15% of these patients with a median 28 days after starting therapy, and a similar incidence was reported for patients with lymphoid malignancies who were treated with Alemtuzumab or Rituximab (Faderl et al., 2003).
Late CMV infection after stem cell transplant is common (3-17%) of allogeneic transplant recipients and is associated with 13-fold increase in post transplant mortality. The primary risk factor for late CMV infection is specific T-cell dysfunction (Hakki et al., 2003). Late CMV disease have a varied presentation, with retinitis, sinusitis, encephalitis and marrow failure being more common than in early CMV disease (Ayala et al., 2006).
Hepatitis B Virus:
HBV infection in diffuse large B-cell lymphoma (DLBCL) patients is a common complication in China. However, the clinical relevance of HBV infection with respect to DLBCL disease stages and patient survival remains unclear. Compared with HBsAg negative patients, the HBsAg positive DLBCL patients had earlier onset and more advanced stage. The disease stage and hepatic dysfunction during chemotherapy were two significant prognostic factors in the HBsAg positive DLBCL patients. This study suggests that the prophylactic treatment of HBV may be of great importance in the cases of HBsAg-positive patients (Wang et al., 2008).
Hepatitis C Virus:
HCV infection is frequent among patients with hematological malignancies, especially those with lymphoproliferative disorders. The molecular data suggest patient-to patient nosocomial HCV transmission. A series of preventive measures should be adopted as screening for Anti-HCV search for HCV-RNA in newly admitted patients and at fixed intervals during follow up and isolation of patients during neutropenic phases and avoidance of multidose vials (Silini et al., 2002).
Viral infections are important causes of morbidity and mortality for patients with hematological malignancies. However the true incidence and consequences of viral Infections for these patients who undergo conventional nontransplant therapy are poorly defined. Differences in incidence and outcome of viral infections among these patient groups are based on intensity and duration of T-cell-mediated immune suppression (Wade, 2006).
Respiratory Viruses:
Respiratory viruses, including RSV, PIV and influenza virus, are wide spread in the community and easily transmitted to patients with hematological malignancy (England, 2001).
Infection control measures are critical and should consist of hand washing, annual influenza vaccination, early detection for infection and both respiratory and contact isolation of infection health care workers and patients. Patients, who develop respiratory virus infection prior to the initiation of treatment or transplantation, should if possible, have their therapy delayed. Respiratory virus infection among patients with hematological malignancy are associated with a more prolonged infection, higher frequency of nosocomial infection (55-83 % of exposed immunocompromized patients will become infected), a higher rate of pneumonia, co-pathogens and death. The risk of death from pneumonia has ranged from 9% to 82%, and appears to vary little between the different groups of patients with active hematological malignancy (Peck et al., 2004).
Other Viral Infections
• Adenovirus:
Primary infection is acquired from either a respiratory droplet or oral-fecal route. Most infection among compromised hosts is postulated to be viral reactivation (Kojaoghlanian et al., 2003). Clinical manifestations vary with serotype and include viremia, pneumonia, hepatitis, gastrointestinal disease, cystitis, nephritis and conjunctivitis (Bruno et al., 2004). Control of adenovirus appears T-cell mediated, and allogenic SCT recipients appear to be at greatest risk of infection and disease. However fatal adenovirus infections have been reported in patients with B-cell lymphoma, multiple myeloma and AML (Fianchi et al., 2003).
Risk factors for infection and disease include unrelated donor transplantation, GVHD, T-cell depletion, younger patient age, total body irradiation, and viremia. The incidence of infection in SCT recipients has been reported to range from 5-29% with disease occurring in 5-8% of patients. Death secondary to adenovirus disease range from 30-50% (Lion et al., 2003).
Severe disseminated disease can develop following BMT. Typical presentations in immunocompromised include pneumonitis, enterocolitis, hemorrhagic cystitis, hepatitis, encephalitis, and disseminated disease. In one study, 46% of BMT patients with adenovirus infections died; seven patients with pneumonia and six with disseminated disease (Slatter et al., 2005).
• Human Metapneumovirus ( hMPV ):
hMPV is newly discovered RNA Paramyxovirus. Most children by age 5 years are seropositive. Infection occurs primarily during winter and manifests as both upper and lower respiratory tract disease (Williams et al., 2004).
More serious disease has been reported among immunosuppressed patients. It could be an important cause of idiopathic pneumonia syndrome after SCT; Five out of 200 tested patients had hMPV detected in archived bronchial alveolar lavage specimens. All 5 positive patients had upper respiratory tract prodromes that preceded their pneumonia, and 4 of 5 patients died. Lung tissue of the died patients had histologic picture of idiopathic pneumonia syndrome at autopsy. Prospective studies of the role of hMPV as a cause of infection for patients with hematological malignancy are needed. There is no established treatment for hMPV infections although Ribavirin appears to have antiviral activity (Englund, 2001).
Herpes simplex virus:
HSV infections in patients with hematological malignancy are almost exclusively reactivation Infections (Lungman, 2004). They are common, ranging from 15% among chronic leukemia patients treated with Fludarabine, to 90% of patients with acute leukemia or stem cell transplant recipients. HSV infections and disease occur early after therapy, and frequently recur with future treatment. Mucocutaneous HSV disease will frequently present with an atypical appearance and can mimic other pathogens (i.e., Candida) or treatment induced mucositis. HSV infections among immunocompromised patients are characteristically more invasive, heal more slowly, are associated with prolonged viral shedding and may disseminate (Sandherr et al., 2006).
Varicella-Zoster virus:
The majority of VZV infections in adult patients with hematological malignancy are reactivation infections and 80% present with localized disease. The incidence of VZV infection ranges from 2% among patients with CML receiving Imatinib; to 10-15% in patients with CLL receiving Fludarabine; to 25% of patients with Hodgkin lymphoma or autologous stem cell transplantation (SCT) recipients; and to 45-60% among allogenic SCT recipients (Mattiuzzi et al., 2003).
Infection risk is greatest within the first 12 months following treatment or transplant, but late onset disease occurs because of persistent immunosuppression. Patients who are VZV naive are at risk for primary infection with either wild type or vaccine strains and should be counseled about the risk of developing such infection. Primary VZV infection can be very severe, and measures to prevent exposure and intervene early are recommended (Weinstock et al., 2004).
Unusual VZV syndromes of importance include trigeminal zoster with keratitis and yeast infections, and post–zoster pain. Hepatic or gastro-intestinal VZV disease is an important entity, and may present with few or no skin lesions. This presentation may result in delayed diagnosis, and has been associated with significant mortality (David et al., 1998).
Human herpes virus – 6 (HHV-6):
HHV-6 is ubiquitous Herpes virus that infects most persons early in life. Two major viral variants have been identified (A and B), but the B variant is most frequently associated with disease among immunocompromised patients. Longitudinal studies in SCT recipients found that viral reactivation occurred a median of 20 days after transplantation, and that viral shedding for some patients was prolonged, and correlated poorly with clinical improvement. HHV-6 viremia among allogenic transplant recipients is associated with an increased mortality, and increased when patients are transplanted for disease other than first remission, when donor and recipient are sex mismatched and among younger patients (Zerr et al., 2005).
Wade (2006) treated 4 patients for HHV-6 viremia who developed CNS dysfunction and delayed platelet recovery; three of them had autologous transplant for myeloma following Melphalan, and the fourth was treated with Imatinib for relapsed ALL. All 4 responded to prolonged antiviral therapy.
Quantitative real-time PCR analysis on blood and CSF is the method of choice for diagnosis. Foscarnet and Ganciclovir, alone or in combination, have been used as treatment for HHV-6 infection. Prospective studies are needed to better understand the importance of HHV -6 infections among patients with hematological malignancy, and to define disease spectrum, and appropriate therapy (Zerr et al., 2005).
BK Virus:
BK virus is DNA polygonal virus that is believed to cause nephropathy and graft loss among renal transplant recipients and also may cause pneumonia (Boeckh et al., 2005). There is increasing evidence that BK virus plays an important role in renal impairment in patients with hematological malignancy, but viral tissue invasion has only recently been demonstrated (Erard et al., 2005). BK virus was reported up to 95% of SCT recipients, with the onset of viral shedding occurring a median of 41 days after transplant, BK viruria may be prolonged, can be severe and some patients remain symptomatic for more than one month. Highly sensitive PCR assay for BK virus detection in blood and urine is now available (Bridges et al., 2006).
Cytomegalovirus:
T-cell function is paramount in the control of CMV and T-cell depleting agents as Alemtuzamab and aggressive chemotherapy e.g. CVAD and acute leukemia induction appear to increase the risk of CMV infection and disease. In the absence of effective antiviral prophylaxis, the incidence of CMV infection in patients with hematological malignancy ranges from 5-75% (Boeckh and Nichols, 2004). Patients undergoing an autologous stem cell transplant have a low risk of CMV infection but CD 34 selection of the autologous stem cell product increases the risk of CMV disease and death (Holmberg et al., 1999).
CMV attributable mortality for patients with hematological malignancies who receive conventional therapy ranged from 30% -57%. CMV disease in patients with acute leukemia was associated with the use of high dose Fludarabine, Cyclophosphamide, or Alemtuzumab. CMV viremia occurred in 15% of these patients with a median 28 days after starting therapy, and a similar incidence was reported for patients with lymphoid malignancies who were treated with Alemtuzumab or Rituximab (Faderl et al., 2003).
Late CMV infection after stem cell transplant is common (3-17%) of allogeneic transplant recipients and is associated with 13-fold increase in post transplant mortality. The primary risk factor for late CMV infection is specific T-cell dysfunction (Hakki et al., 2003). Late CMV disease have a varied presentation, with retinitis, sinusitis, encephalitis and marrow failure being more common than in early CMV disease (Ayala et al., 2006).
Hepatitis B Virus:
HBV infection in diffuse large B-cell lymphoma (DLBCL) patients is a common complication in China. However, the clinical relevance of HBV infection with respect to DLBCL disease stages and patient survival remains unclear. Compared with HBsAg negative patients, the HBsAg positive DLBCL patients had earlier onset and more advanced stage. The disease stage and hepatic dysfunction during chemotherapy were two significant prognostic factors in the HBsAg positive DLBCL patients. This study suggests that the prophylactic treatment of HBV may be of great importance in the cases of HBsAg-positive patients (Wang et al., 2008).
Hepatitis C Virus:
HCV infection is frequent among patients with hematological malignancies, especially those with lymphoproliferative disorders. The molecular data suggest patient-to patient nosocomial HCV transmission. A series of preventive measures should be adopted as screening for Anti-HCV search for HCV-RNA in newly admitted patients and at fixed intervals during follow up and isolation of patients during neutropenic phases and avoidance of multidose vials (Silini et al., 2002).
Monday, August 29, 2011
Culture Methods for Mycobacterium tuberculosis
Cultivation of M. tuberculosis from clinical samples is the gold standard for the diagnosis of active TB. It can detect 100 bacilli/mL-1 of sputum in comparison with 5,000–10,000 bacilli/mL-1 needed for microscopy [18]. It also provides material for further identification and drug susceptibility testing. Conventional methods of culture have relied on egg based and agar-based media, such as the Lowenstein-Jensen (LJ) medium and Middlebrook agar [19, 20]. Following decontamination and liquefaction procedures, sputum samples are inoculated and incubated for morphological growth, which usually occurs after several weeks of incubation. Identification of M. tuberculosis is done by performing several further biochemical tests [19, 21]. However, it is laborious and time consuming requiring from 3–8 weeks to obtain the results.
The introduction of the BACTEC radiometric system (BACTECTB-460; Becton Dickinson, Sparks, MD, USA) in the 1980s wasa breakthrough since it allowed the detection of M. tuberculosisin a few days compared with weeks in the conventional culture media [22]. However, the use of radioisotopes and the cost of the equipment precluded its use on a routine basis, except in reference laboratories predominantly in developed countries. A few years ago Becton Dickinson proposed another system based on fluorescence detection of mycobacterial growth [23].
The Mycobacteria growth indicator tube (MGIT) system is based on a glass tube containing a modified Middle brook 7H9 broth together with a fluorescence quenching-based oxygen sensor embedded at the bottom of the tube. When inoculated with M. tuberculosis, consumption of the dissolved oxygen produces fluorescence when illuminated by a UV lamp. The MGIT system has been thoroughly evaluated in clinical settings for the detection and recovery of mycobacteria. BADAK et al. [24] compared the MGIT system with the BACTEC TB-460 and LJ culture medium in 1,441 clinical specimens. Out of 178 isolates recovered, 30 (17%) were M. tuberculosis with the MGIT system recovering 28 (93%) compared with 25 (83%) recovered with the LJ medium. In another multicentre study, PFYFFER et al. [25] analysed 1,500clinical specimens detecting a total of 180 mycobacterial species comprising 113 M. tuberculosis complex isolates. The combination of MGIT and BACTEC detected 171 (95%) of all isolates with a time to detection of M. tuberculosis of 9.9 days compared with 9.7 days with BACTEC and 20.2 days with solid medium proving that MGIT was a valuable alternative to the radiometric system [25]. More recently the MGIT system has been fully automated and turned into the BACTEC MGIT 960 system, which is a non radiometric, non invasive system with the tubes incubated in a compact system that reads them automatically. In a multicentre study the BACTEC MGIT 960 system was compared with the radiometric BACTEC TB-460 system and LJ medium. Analysing 2,576 specimens, the best yield was obtained with BACTEC TB-460 (201 isolates), compared with190 isolates with BACTEC MGIT 960 and 168 isolates with LJ medium [26]. In another study IDIGORAS et al. [27] compared the BACTEC MGIT 960 system for sensitivity and time to detection of mycobacteria with solid medium, and microscopy on solid media. Sensitivity of each media compared with all mediacombined for growth of M. tuberculosis was 93%, 76%, 79% and75% for MGIT 960, Middlebrook 7H11, LJ and Coletsos.The mean time to detection was 12.9 days by BACTEC MGIT960, and 15.0 days with BACTEC 460, compared with 27.0 days with Lowenstein Jensen solid medium.148 Thus, WHO recently endorsed the use of liquid tuberculosis culture and drug susceptibility testing for M. tuberculosis in low-resource settings.149 The newly developed rapid liquid culture systems have unique sensing systems to detect a small amount of bacterial growth, such as by radioactivity or oxygen concentration changes, as quickly as possible. These systems can also be used for drug susceptibility testing as well as detecting M. tuberculosis.150,151
Novel diagnostic test using mycobacteriophages to identify M. tuberculosis from biological specimen require only 2 days of turnaround time read more at
The introduction of the BACTEC radiometric system (BACTECTB-460; Becton Dickinson, Sparks, MD, USA) in the 1980s wasa breakthrough since it allowed the detection of M. tuberculosisin a few days compared with weeks in the conventional culture media [22]. However, the use of radioisotopes and the cost of the equipment precluded its use on a routine basis, except in reference laboratories predominantly in developed countries. A few years ago Becton Dickinson proposed another system based on fluorescence detection of mycobacterial growth [23].
The Mycobacteria growth indicator tube (MGIT) system is based on a glass tube containing a modified Middle brook 7H9 broth together with a fluorescence quenching-based oxygen sensor embedded at the bottom of the tube. When inoculated with M. tuberculosis, consumption of the dissolved oxygen produces fluorescence when illuminated by a UV lamp. The MGIT system has been thoroughly evaluated in clinical settings for the detection and recovery of mycobacteria. BADAK et al. [24] compared the MGIT system with the BACTEC TB-460 and LJ culture medium in 1,441 clinical specimens. Out of 178 isolates recovered, 30 (17%) were M. tuberculosis with the MGIT system recovering 28 (93%) compared with 25 (83%) recovered with the LJ medium. In another multicentre study, PFYFFER et al. [25] analysed 1,500clinical specimens detecting a total of 180 mycobacterial species comprising 113 M. tuberculosis complex isolates. The combination of MGIT and BACTEC detected 171 (95%) of all isolates with a time to detection of M. tuberculosis of 9.9 days compared with 9.7 days with BACTEC and 20.2 days with solid medium proving that MGIT was a valuable alternative to the radiometric system [25]. More recently the MGIT system has been fully automated and turned into the BACTEC MGIT 960 system, which is a non radiometric, non invasive system with the tubes incubated in a compact system that reads them automatically. In a multicentre study the BACTEC MGIT 960 system was compared with the radiometric BACTEC TB-460 system and LJ medium. Analysing 2,576 specimens, the best yield was obtained with BACTEC TB-460 (201 isolates), compared with190 isolates with BACTEC MGIT 960 and 168 isolates with LJ medium [26]. In another study IDIGORAS et al. [27] compared the BACTEC MGIT 960 system for sensitivity and time to detection of mycobacteria with solid medium, and microscopy on solid media. Sensitivity of each media compared with all mediacombined for growth of M. tuberculosis was 93%, 76%, 79% and75% for MGIT 960, Middlebrook 7H11, LJ and Coletsos.The mean time to detection was 12.9 days by BACTEC MGIT960, and 15.0 days with BACTEC 460, compared with 27.0 days with Lowenstein Jensen solid medium.148 Thus, WHO recently endorsed the use of liquid tuberculosis culture and drug susceptibility testing for M. tuberculosis in low-resource settings.149 The newly developed rapid liquid culture systems have unique sensing systems to detect a small amount of bacterial growth, such as by radioactivity or oxygen concentration changes, as quickly as possible. These systems can also be used for drug susceptibility testing as well as detecting M. tuberculosis.150,151
Novel diagnostic test using mycobacteriophages to identify M. tuberculosis from biological specimen require only 2 days of turnaround time read more at
Immune Response to Pathogens
Innate Immune Function
The innate subdivision of the immune system is the most primitive. An important component consists of anatomic barriers such as intact skin and mucous membranes.
The most important cells of the innate immune system are the neutropbils (polymorphonuclear cells), macrophages (transformed monocyte cells located in tissues), and lymphocytes known as natural killer cells.
These cells roam in all body tissues and are able to identify microbes via recognition signals of patterns of surface molecules indigenous to microorganisms. Invading microbes are identified and attacked by the release of toxic molecules or by phagocytosis and destruction following ingestion.
Innate cells initiate the process of inflammation following infection or tissue damage. Inflammatory mediator molecules such as histamine are released that produce local vasodilatation and increased blood flow, accompanied by increased capillary permeability.
Circulating complement protein molecules are also activated and turn into complexes that enhance inflammation and, along with the mediator molecules released by the innate cells, act as chemoattractants, drawing more immune cells into the area. The outcome of inflammation is the destruction of invading microbes, clearance of damaged tissues, and activation of the adaptive immune system.
Adaptive Immune Function
The response of the adaptive division of lymphocyte immune cells is characterized by delay. Lymphocytes must be activated in a days-long process before they can respond; specificity—cells and antibodies are generated targeted to unique proteins found on an invading microbe; and memory dormant cells are generated that can quickly mount a defense against the invader on re exposure.
Each CD4 "helper" lymphocyte (T-helper or Th) carries T-cell receptors that respond to a single antigen microbial protein component; on exposure, the CD4 cells begin secreting activator "helper" molecules that can further enhance macrophage function and activate the CD8 (cellular immunity) and B-lymphocyte cells (humoral immunity). CD8 lymphocytes ("cytotoxic"
cells, cellular adaptive response) seek out and destroy body ceils infected with the microbe specific to its T-cell receptor. These cells are the body's primary defense against intracellular invaders such as viruses and some bacteria .
B lymphocytes, when activated by the antigen specific to the B-cell receptor and molecules from Th cells, begin the process of secreting their receptors into the plasma as soluble antibodies (humoral adaptive response) that attack invading microbes.
To "turn on" the adaptive response, specialized antigen-presenting and innate macrophage cells present in tissues migrate to regional lymph nodes, therein displaying antigens bonded to major histocompatibility complex class 1 or class II surface molecules for recognition by antigenically specific lymphocyte T-cell receptors.
In the specialized environment of the lymph node, the interactions of these cells result in the clonal expansion of lymphocyte populations and antibodies, generated to attack and destroy invading microbes.
In summary, the physiologic objective of the integrated innate and adaptive immune response is to detect and prevent infection and clear damaged cells. There is a complex and partially reciprocal relationship between
these arms of the immune system: the first-line innate immune system response is necessary to activate the adaptive immune cells, yet produces inflammatory mediator molecules that restrain lymphocyte function.
Activated Th lymphocytes produce cytokines that enhance macrophage and neutrophil function.
The innate subdivision of the immune system is the most primitive. An important component consists of anatomic barriers such as intact skin and mucous membranes.
The most important cells of the innate immune system are the neutropbils (polymorphonuclear cells), macrophages (transformed monocyte cells located in tissues), and lymphocytes known as natural killer cells.
These cells roam in all body tissues and are able to identify microbes via recognition signals of patterns of surface molecules indigenous to microorganisms. Invading microbes are identified and attacked by the release of toxic molecules or by phagocytosis and destruction following ingestion.
Innate cells initiate the process of inflammation following infection or tissue damage. Inflammatory mediator molecules such as histamine are released that produce local vasodilatation and increased blood flow, accompanied by increased capillary permeability.
Circulating complement protein molecules are also activated and turn into complexes that enhance inflammation and, along with the mediator molecules released by the innate cells, act as chemoattractants, drawing more immune cells into the area. The outcome of inflammation is the destruction of invading microbes, clearance of damaged tissues, and activation of the adaptive immune system.
Adaptive Immune Function
The response of the adaptive division of lymphocyte immune cells is characterized by delay. Lymphocytes must be activated in a days-long process before they can respond; specificity—cells and antibodies are generated targeted to unique proteins found on an invading microbe; and memory dormant cells are generated that can quickly mount a defense against the invader on re exposure.
Each CD4 "helper" lymphocyte (T-helper or Th) carries T-cell receptors that respond to a single antigen microbial protein component; on exposure, the CD4 cells begin secreting activator "helper" molecules that can further enhance macrophage function and activate the CD8 (cellular immunity) and B-lymphocyte cells (humoral immunity). CD8 lymphocytes ("cytotoxic"
cells, cellular adaptive response) seek out and destroy body ceils infected with the microbe specific to its T-cell receptor. These cells are the body's primary defense against intracellular invaders such as viruses and some bacteria .
B lymphocytes, when activated by the antigen specific to the B-cell receptor and molecules from Th cells, begin the process of secreting their receptors into the plasma as soluble antibodies (humoral adaptive response) that attack invading microbes.
To "turn on" the adaptive response, specialized antigen-presenting and innate macrophage cells present in tissues migrate to regional lymph nodes, therein displaying antigens bonded to major histocompatibility complex class 1 or class II surface molecules for recognition by antigenically specific lymphocyte T-cell receptors.
In the specialized environment of the lymph node, the interactions of these cells result in the clonal expansion of lymphocyte populations and antibodies, generated to attack and destroy invading microbes.
In summary, the physiologic objective of the integrated innate and adaptive immune response is to detect and prevent infection and clear damaged cells. There is a complex and partially reciprocal relationship between
these arms of the immune system: the first-line innate immune system response is necessary to activate the adaptive immune cells, yet produces inflammatory mediator molecules that restrain lymphocyte function.
Activated Th lymphocytes produce cytokines that enhance macrophage and neutrophil function.
Sunday, August 28, 2011
Identification of bacteria within blood culture by Molecular Methods
Detection of bacteria with blood culture starts with testing if there are actually bacteria in blood or not.Only growth-positive cultures are used in subsequent analyses. Thereafter, pathogenic organisms are isolated using agar plates.Methods for identification of bacteria can be divided into two groups: hybridization based and amplification based.
Fluorescent in situ hybridization (FISH)
It is one of extensively used method for identification, visualization and localization of microorganisms in many fields. In brief FISH involves next steps. At first samples are fixed with aqueous solutions (paraformaldehyde for Gram-negative bacteria and 50% ethanol for Gram-positive bacteria) which are specific for Gram-negative and Gram-positive bacteria. Then bacterial samples are hybridized using fluorescently labeled probes that are complementary to the 16S rRNA in the cell.
Next steps include incubation and washing to remove unspecific probes. And finally samples can be visualizated with fluorescent or laser scanning microscopy (Barken et al., 2007)
At the present this technique allows identification within 2 hours of more than 95% of the bacteria usually found in blood. As long as this method needs species-specific probes, some bacteria can be identified only at the genus level. For example, detection of coagulase-negative staphylococci has problems with specific probe design (Oliveira at al., 2002)
Another methods are built upon identification using PCR.
Amplification based diagnostics are particularly helpful when the bacterial load is low or subculturing takes time. Moreover fast identification of bacteria associated with severe morbility and mortality is very helpful, because allows to rapidly detect most common bacterial infections (Peters at al., 2004)
PCR technique is important in molecular diagnostics of such bacterial infections in blood as Streptococcus pneumoniae and methicillin-resistance Staphylococcus aureus infections.
Using blood cultures is limited, since they are fastidious organisms, and PCR detection is more suitable. For example, there is a assay, which permits to idetify seven Streptococcus pneumoniae serotypes using multiplex PCR and has reliable results in dignostics (O’Halloran and Cafferkey, 2005)
Molecular methods based on polymerase chain reaction (PCR) technology have been developed for infection diagnosis and pathogen identification. These methods offer a new approach based on detection and recognition of pathogen DNA in the blood, or indeed other clinical samples, with the potential to obtain results in a much shorter time frame (hours) than is possible with conventional culture (Dark et al., 2009)
PCR based pathogen detection depends on the ability of the reaction to selectively amplify specific regions of DNA, allowing even minute amounts of pathogen DNA in clinical samples to be detected and analysed (Jordan JA et al., 2005)
The DNA sequence that is amplified is determined by the design of oligonucleotide primers, short pieces of synthesised DNA that bind to either end of the sequence and form the starting point for DNA replication by DNA polymerase.
For bacterial pathogens, two basic approaches have been taken in assay design, using either specific primers that detect a particular organism or, more commonly, universal primers that bind to conserved sequences in bacterial but not human DNA (Dark et al., 2009)
The latter approach has the potential to detect a large number of bacterial species in a given sample.
Assays that are limited to the detection of a specific organism have often been developed to address a specific clinical need (for example, rapid confirmation of the presence of meningococcoci in patients with meningitis) (Seward RJ et al., 2000)
In most cases, however, the detection of bloodstream infection requires assays capable of detecting a broad range of pathogens given that several microbial species may be involved, including infections with multiple organisms (Munson El et al., 2003)
Furthermore terminal restriction fragment length polymorphism (T-RFLP) and single strand conformation polymorphism (SSCP) analyses are a choice. They are generally used for typing of microorganisms from blood cultures.
SSCP method is based on species-unique SSCP patterns which now are determined only for 25 of usually meeting bacteria in blood culture (Turenne et al., 2000)
Whereas T-RFLP profiles for more bacteria were found by sizing fragments from restriction digests of PCR products deduced from two sets of 16S rDNA-specific fluorescent dye-labeled primers (Christensen et al., 2003)
Fluorescent in situ hybridization (FISH)
It is one of extensively used method for identification, visualization and localization of microorganisms in many fields. In brief FISH involves next steps. At first samples are fixed with aqueous solutions (paraformaldehyde for Gram-negative bacteria and 50% ethanol for Gram-positive bacteria) which are specific for Gram-negative and Gram-positive bacteria. Then bacterial samples are hybridized using fluorescently labeled probes that are complementary to the 16S rRNA in the cell.
Next steps include incubation and washing to remove unspecific probes. And finally samples can be visualizated with fluorescent or laser scanning microscopy (Barken et al., 2007)
At the present this technique allows identification within 2 hours of more than 95% of the bacteria usually found in blood. As long as this method needs species-specific probes, some bacteria can be identified only at the genus level. For example, detection of coagulase-negative staphylococci has problems with specific probe design (Oliveira at al., 2002)
Another methods are built upon identification using PCR.
Amplification based diagnostics are particularly helpful when the bacterial load is low or subculturing takes time. Moreover fast identification of bacteria associated with severe morbility and mortality is very helpful, because allows to rapidly detect most common bacterial infections (Peters at al., 2004)
PCR technique is important in molecular diagnostics of such bacterial infections in blood as Streptococcus pneumoniae and methicillin-resistance Staphylococcus aureus infections.
Using blood cultures is limited, since they are fastidious organisms, and PCR detection is more suitable. For example, there is a assay, which permits to idetify seven Streptococcus pneumoniae serotypes using multiplex PCR and has reliable results in dignostics (O’Halloran and Cafferkey, 2005)
Molecular methods based on polymerase chain reaction (PCR) technology have been developed for infection diagnosis and pathogen identification. These methods offer a new approach based on detection and recognition of pathogen DNA in the blood, or indeed other clinical samples, with the potential to obtain results in a much shorter time frame (hours) than is possible with conventional culture (Dark et al., 2009)
PCR based pathogen detection depends on the ability of the reaction to selectively amplify specific regions of DNA, allowing even minute amounts of pathogen DNA in clinical samples to be detected and analysed (Jordan JA et al., 2005)
The DNA sequence that is amplified is determined by the design of oligonucleotide primers, short pieces of synthesised DNA that bind to either end of the sequence and form the starting point for DNA replication by DNA polymerase.
For bacterial pathogens, two basic approaches have been taken in assay design, using either specific primers that detect a particular organism or, more commonly, universal primers that bind to conserved sequences in bacterial but not human DNA (Dark et al., 2009)
The latter approach has the potential to detect a large number of bacterial species in a given sample.
Assays that are limited to the detection of a specific organism have often been developed to address a specific clinical need (for example, rapid confirmation of the presence of meningococcoci in patients with meningitis) (Seward RJ et al., 2000)
In most cases, however, the detection of bloodstream infection requires assays capable of detecting a broad range of pathogens given that several microbial species may be involved, including infections with multiple organisms (Munson El et al., 2003)
Furthermore terminal restriction fragment length polymorphism (T-RFLP) and single strand conformation polymorphism (SSCP) analyses are a choice. They are generally used for typing of microorganisms from blood cultures.
SSCP method is based on species-unique SSCP patterns which now are determined only for 25 of usually meeting bacteria in blood culture (Turenne et al., 2000)
Whereas T-RFLP profiles for more bacteria were found by sizing fragments from restriction digests of PCR products deduced from two sets of 16S rDNA-specific fluorescent dye-labeled primers (Christensen et al., 2003)
Molecular Methods in Diagnosis of Fastidious organisms
I- Fastidious Bacteria
Together with virology, the diagnosis of infections due to fastidious bacteria has benefited greatly from molecular detection. Many of these fastidious bacteria have public health implications such as Mycobacterium tuberculosis, Chlamydia trachomatis, Neisseria gonorrheae and Bordetella pertussis (David J Speers., 2006)
Non-culture-based molecular testing has the advantage of avoiding the delays of days to weeks for conventional culture to allow early recognition and treatment as a public health imperative. Commercial assays are available for M. tuberculosis and Mycobacterium avium complex, C. trachomatosis, and N.gonorrhoeae.
Several nucleic acid detection technologies are in use including PCR, transcription based amplification, ligase chain reaction, strand displacement amplification and the Qβ replicase system (Yang S, Rothman RE 2004)
In conclusion, molecular diagnostic techniques have a significant role to play in clinical bacteriology, although their adoption will never replace conventional methodologies, which continue to be the cornerstone of modern bacteriological methods. Indeed, such molecular diagnostic assays may only be implemented in specialized laboratories to enhance laboratory diagnostic efficiency (Millar et al., 2003) where the use of such assays will be mainly confined to diagnosis, identification and genotyping, where current conventional approaches are grossly inadequate
1-Fastidious sexually transmitted bacteria
The introduction of molecular detection for the fastidious sexually transmitted bacteria has led to a large increase in the proportion of laboratory confirmed cases due to its increased sensitivity allowing more effective contact tracing.
In the management of sexual health traditional screening methods require speculum examination in women and urethral swabs in men. These require special equipment and cause embarrassment and discomfort, thus reducing compliance.
Molecular detection is useful since noninvasive specimens unsuitable for traditional culture, such as initial stream urine and self-collected vaginal swabs can be used. These are more convenient and acceptable increasing the compliance with testing (David J Speers., 2006)
Although molecular testing for C. trachomatis and N.gonorrhoeae does not allow monitoring of antibiotic resistance or detect other sexually transmitted diseases, urine testing has shown equivalent sensitivity and specificity to invasive specimens for detection of C. trachomatis in men and women, and for detection of N.gonorrhoeae in men when compared to urethral swabs.
In women the sensitivity and specificity of the PCR assay for N. gonorrhoeae was lower for urine compared to cervical samples, however self-collected vaginal swabs may help in this regard.
The PCR assay for C. trachomatis has equal sensitivity for vaginal and cervical swabs and a transcription mediated amplification assay has been approved by the U.S. FDA for testing C. trachomatis and N. gonorrhoeae from vaginal specimens (Cook RL, Hutchison SL et al., 2005)
In remote areas, molecular methods have the advantage of being performed on dry swabs with little degradation of the DNA during transit compared to the difficulties of transporting samples in specialised transport medium to preserve viability.
In addition, molecular methods can test for multiple genital pathogens such as C. trachomatis, N. gonorrhoeae, the Donovanosis agent and the genital mycoplasmata from the same swab.
2- Mycobacteriology
Mycobacteriology has been aided by the introduction of molecular methods. However, it is important to note that molecular detection of M. tuberculosis is one of the few examples where conventional culture remains more sensitive.
This is possibly due to the difficulty in releasing the DNA from the bacterial cells during the extraction process. Despite this limitation, molecular detection of M. tuberculosis has a definite role as it allows confirmation of acid-fast bacilli seen on microscopy with up to 98% sensitivity in pulmonary tuberculosis within a day compared to two weeks or more by culture.
Specimens that are smear-negative have a much lower chance of molecular confirmation, with reported sensitivities as low as 40% (Bergmann JS, Woods GL.1996).
In addition to direct detection from clinical specimens, molecular methods can confirm a positive culture within a day compared to approximately four weeks using phenotypic methods.
This has shortened the time for laboratory confirmation of suspected tuberculosis even for smear-negative but culture-positive cases Mycobacteriology has also advanced through the use of molecular methods for the speciation of the many non tuberculous mycobacterial species (Kapur Vet al., 1995).
Detection of Mycobacterium tuberculosis nucleic acid in respiratory specimens has high specificity but relatively poor sensitivity, particularly for smear negative disease. The recent development of an integrated specimen processing and real-time PCR testing platform for M. tuberculosis and rifampicin resistance is an important advance that requires evaluation in childhood TB.( Nicol MP, Zar HJ., 2011)
Together with virology, the diagnosis of infections due to fastidious bacteria has benefited greatly from molecular detection. Many of these fastidious bacteria have public health implications such as Mycobacterium tuberculosis, Chlamydia trachomatis, Neisseria gonorrheae and Bordetella pertussis (David J Speers., 2006)
Non-culture-based molecular testing has the advantage of avoiding the delays of days to weeks for conventional culture to allow early recognition and treatment as a public health imperative. Commercial assays are available for M. tuberculosis and Mycobacterium avium complex, C. trachomatosis, and N.gonorrhoeae.
Several nucleic acid detection technologies are in use including PCR, transcription based amplification, ligase chain reaction, strand displacement amplification and the Qβ replicase system (Yang S, Rothman RE 2004)
In conclusion, molecular diagnostic techniques have a significant role to play in clinical bacteriology, although their adoption will never replace conventional methodologies, which continue to be the cornerstone of modern bacteriological methods. Indeed, such molecular diagnostic assays may only be implemented in specialized laboratories to enhance laboratory diagnostic efficiency (Millar et al., 2003) where the use of such assays will be mainly confined to diagnosis, identification and genotyping, where current conventional approaches are grossly inadequate
1-Fastidious sexually transmitted bacteria
The introduction of molecular detection for the fastidious sexually transmitted bacteria has led to a large increase in the proportion of laboratory confirmed cases due to its increased sensitivity allowing more effective contact tracing.
In the management of sexual health traditional screening methods require speculum examination in women and urethral swabs in men. These require special equipment and cause embarrassment and discomfort, thus reducing compliance.
Molecular detection is useful since noninvasive specimens unsuitable for traditional culture, such as initial stream urine and self-collected vaginal swabs can be used. These are more convenient and acceptable increasing the compliance with testing (David J Speers., 2006)
Although molecular testing for C. trachomatis and N.gonorrhoeae does not allow monitoring of antibiotic resistance or detect other sexually transmitted diseases, urine testing has shown equivalent sensitivity and specificity to invasive specimens for detection of C. trachomatis in men and women, and for detection of N.gonorrhoeae in men when compared to urethral swabs.
In women the sensitivity and specificity of the PCR assay for N. gonorrhoeae was lower for urine compared to cervical samples, however self-collected vaginal swabs may help in this regard.
The PCR assay for C. trachomatis has equal sensitivity for vaginal and cervical swabs and a transcription mediated amplification assay has been approved by the U.S. FDA for testing C. trachomatis and N. gonorrhoeae from vaginal specimens (Cook RL, Hutchison SL et al., 2005)
In remote areas, molecular methods have the advantage of being performed on dry swabs with little degradation of the DNA during transit compared to the difficulties of transporting samples in specialised transport medium to preserve viability.
In addition, molecular methods can test for multiple genital pathogens such as C. trachomatis, N. gonorrhoeae, the Donovanosis agent and the genital mycoplasmata from the same swab.
2- Mycobacteriology
Mycobacteriology has been aided by the introduction of molecular methods. However, it is important to note that molecular detection of M. tuberculosis is one of the few examples where conventional culture remains more sensitive.
This is possibly due to the difficulty in releasing the DNA from the bacterial cells during the extraction process. Despite this limitation, molecular detection of M. tuberculosis has a definite role as it allows confirmation of acid-fast bacilli seen on microscopy with up to 98% sensitivity in pulmonary tuberculosis within a day compared to two weeks or more by culture.
Specimens that are smear-negative have a much lower chance of molecular confirmation, with reported sensitivities as low as 40% (Bergmann JS, Woods GL.1996).
In addition to direct detection from clinical specimens, molecular methods can confirm a positive culture within a day compared to approximately four weeks using phenotypic methods.
This has shortened the time for laboratory confirmation of suspected tuberculosis even for smear-negative but culture-positive cases Mycobacteriology has also advanced through the use of molecular methods for the speciation of the many non tuberculous mycobacterial species (Kapur Vet al., 1995).
Detection of Mycobacterium tuberculosis nucleic acid in respiratory specimens has high specificity but relatively poor sensitivity, particularly for smear negative disease. The recent development of an integrated specimen processing and real-time PCR testing platform for M. tuberculosis and rifampicin resistance is an important advance that requires evaluation in childhood TB.( Nicol MP, Zar HJ., 2011)
Saturday, August 27, 2011
Mechanism of resistance to carbapenems in Ps. aeruginosa (Part 1)
1- Loss of OprD
The outer membrane of gram-negative bacteria constitutes an asymmetric bilayer, in which the inner monolayer is composed of phospholipid, whereas the outer monolayer contains the unique lipid species lipopolysaccharide (LPS) (fig. 6). Porins are group of proteins forming trans-outer-membrane, water filled channels. In general porins have monomer molecular weights in the range of 28 KD to 48 KD, are present in membrane as oligomers (usually trimers), are often strongly but non-covalentely associated with the underlying peptidoglycan and with LPS, and have a high content of β-sheet structure. In Ps. aeruginosa, OprB, OprC, OprD, OprE, OprF, OprP and OprO have been identified as porins (Hancock et al., 1990).
Porins are generally divided into two classes: non-specific (general) porins and specific porins (Nikaido and Vaara, 1985). General porins form water-filled channels that permit the passive diffusion of hydrophilic molecules below a certain size. Specific porins also produce water-filled channels, which contain stereospecific substrate-binding sites (Hancock, 1987). OprF is a major non-specific porin in Ps. aeruginosa, it allows the passage of saccharides with molecular weights of approximately 3,000. however, only 400 out of 200,000 OprF molecules per cell are proposed to form such large channels; the rest appear to form small channels that are predicted to be antibiotic impermeable. OprC and OprE are also general porins with small channel size. The above cited data explains the low outer membrane permeability of Ps. aeruginosa compared to E. coli. This, in turn, was proposed to be the major basis for the high intrinsic reistance of Ps. aeruginosa to hydrophilic antibiotics (Nikaido and Hancock, 1986).
Fig 6: Gram negative bacteria is surrounded by the outer membrane, which functions as an efficient permeability barrier because it contains lipopolysaccharide (LPS) and porins with narrow, restrictive channels (Nikaido, 1994).
To overcome the low permeability and to permit the effective uptake of essential nutrients available at low concentrations in the medium, several specific porins are present in Ps. aeruginosa outer membrane. OprB, which is induced by the presence of glucose (Hancock and Carey, 1980), form a channel that prefers D-glucose and D-xylose. OprP is induced by growth under phosphate starvation conditions (Hancock et al., 1982). Porin OprO, which is highly homologus to OprP, forms pyrophosphate-specific channels (Hancock et al., 1992). OprD was discovered due to its role in the facilitated uptake of imipenem. However the natural substrate for OprD is not imipenem, but its structural analogues, presumably basic amino acids and small peptides containing those amino acids. Lack of an outer membrane protein D2 (now called OprD) with molecular weight range 46 KD lead to mainly resistance to imipenem but only a low degree of resistance to meropenem (Naenna et al., 2010).
Genetic analysis shows that the elimination of OprD results from gene rearrangements in the OprD coding region or the upstream promoter region (Trias and Nikaido, 1990a). The loss of OprD expression occur at the levels of transcription and translation (Köhler et al., 1997); (Kolayli et al., 2004). Mutations (base transitions or deletions) in oprD structural gene generate a premature stop codon and early termination of translation. Deletions have also been shown to interfere with expression of oprD at the transcriptional level. (Yoneyama and Nakae, 1993) observed a large deletion encompassing the promoter, initiation codon and putative Shine-Dalgarno sequence of oprD preventing transcription initiation. In addition to the deletion of oprD gene, (Wolter et al., 2004) have shown the first report of carbapenem resistance occurring through insertional inactivation of the oprD gene by insertion sequence (IS) elements. It was suggested that elimination of OprD porin from most imipenem resistant Ps. aeruginosa isolates is due to efficient selection of oprD gene mutation. Thus, imipenem resistance mechanism of Ps. aeruginosa results from a loss-of function mutation, and detection of the mutated structural gene alone is not always possible to determine this type of resistance (Naenna et al., 2010).
The outer membrane of gram-negative bacteria constitutes an asymmetric bilayer, in which the inner monolayer is composed of phospholipid, whereas the outer monolayer contains the unique lipid species lipopolysaccharide (LPS) (fig. 6). Porins are group of proteins forming trans-outer-membrane, water filled channels. In general porins have monomer molecular weights in the range of 28 KD to 48 KD, are present in membrane as oligomers (usually trimers), are often strongly but non-covalentely associated with the underlying peptidoglycan and with LPS, and have a high content of β-sheet structure. In Ps. aeruginosa, OprB, OprC, OprD, OprE, OprF, OprP and OprO have been identified as porins (Hancock et al., 1990).
Porins are generally divided into two classes: non-specific (general) porins and specific porins (Nikaido and Vaara, 1985). General porins form water-filled channels that permit the passive diffusion of hydrophilic molecules below a certain size. Specific porins also produce water-filled channels, which contain stereospecific substrate-binding sites (Hancock, 1987). OprF is a major non-specific porin in Ps. aeruginosa, it allows the passage of saccharides with molecular weights of approximately 3,000. however, only 400 out of 200,000 OprF molecules per cell are proposed to form such large channels; the rest appear to form small channels that are predicted to be antibiotic impermeable. OprC and OprE are also general porins with small channel size. The above cited data explains the low outer membrane permeability of Ps. aeruginosa compared to E. coli. This, in turn, was proposed to be the major basis for the high intrinsic reistance of Ps. aeruginosa to hydrophilic antibiotics (Nikaido and Hancock, 1986).
Fig 6: Gram negative bacteria is surrounded by the outer membrane, which functions as an efficient permeability barrier because it contains lipopolysaccharide (LPS) and porins with narrow, restrictive channels (Nikaido, 1994).
To overcome the low permeability and to permit the effective uptake of essential nutrients available at low concentrations in the medium, several specific porins are present in Ps. aeruginosa outer membrane. OprB, which is induced by the presence of glucose (Hancock and Carey, 1980), form a channel that prefers D-glucose and D-xylose. OprP is induced by growth under phosphate starvation conditions (Hancock et al., 1982). Porin OprO, which is highly homologus to OprP, forms pyrophosphate-specific channels (Hancock et al., 1992). OprD was discovered due to its role in the facilitated uptake of imipenem. However the natural substrate for OprD is not imipenem, but its structural analogues, presumably basic amino acids and small peptides containing those amino acids. Lack of an outer membrane protein D2 (now called OprD) with molecular weight range 46 KD lead to mainly resistance to imipenem but only a low degree of resistance to meropenem (Naenna et al., 2010).
Genetic analysis shows that the elimination of OprD results from gene rearrangements in the OprD coding region or the upstream promoter region (Trias and Nikaido, 1990a). The loss of OprD expression occur at the levels of transcription and translation (Köhler et al., 1997); (Kolayli et al., 2004). Mutations (base transitions or deletions) in oprD structural gene generate a premature stop codon and early termination of translation. Deletions have also been shown to interfere with expression of oprD at the transcriptional level. (Yoneyama and Nakae, 1993) observed a large deletion encompassing the promoter, initiation codon and putative Shine-Dalgarno sequence of oprD preventing transcription initiation. In addition to the deletion of oprD gene, (Wolter et al., 2004) have shown the first report of carbapenem resistance occurring through insertional inactivation of the oprD gene by insertion sequence (IS) elements. It was suggested that elimination of OprD porin from most imipenem resistant Ps. aeruginosa isolates is due to efficient selection of oprD gene mutation. Thus, imipenem resistance mechanism of Ps. aeruginosa results from a loss-of function mutation, and detection of the mutated structural gene alone is not always possible to determine this type of resistance (Naenna et al., 2010).
Resistance to carbapenems
Carbapenems are commonly used as last resort drugs for treatment of infections caused by multiresistant Ps. aeruginosa isolates. Carbapenem resistance was driven mainly by mutation mediated resistance leading to the repression or inactivation of the porin OprD, conferring resistance to imipenem and reduced susceptibility to meropenem (Quale et al., 2006), and those leading to the hyperexpression of the chromosomally encoded cephalosporinase AmpC. Also remarkable, mutations leading to the up-regulation of one of the several efflux pumps encoded in the Ps. aeruginosa genome may confer resistance or reduced susceptibility to multiple agents, including all β-lactams (except imipenem), fluoroquinolones, and aminoglycosides. Furthermore, the accumulation of various combinations of these chromosomal mutations can certainly lead to the emergence of multidrug resistant (Cavallo et al., 2007).
In addition to the mutation-mediated resistance, the presence of horizontally acquired resistance determinants in Ps. aeruginosa has been increasingly reported over the last decade. Among the certainly noteworthy determinants are those encoding metalloβ-lactamases, particularly IMP and VIM enzymes, which are able to hydrolyze efficiently all β-lactams with the exception of aztreonam (Gutierrez et al., 2007).
In addition to the mutation-mediated resistance, the presence of horizontally acquired resistance determinants in Ps. aeruginosa has been increasingly reported over the last decade. Among the certainly noteworthy determinants are those encoding metalloβ-lactamases, particularly IMP and VIM enzymes, which are able to hydrolyze efficiently all β-lactams with the exception of aztreonam (Gutierrez et al., 2007).
Carbapenem Use for Pseudomonas treatment
Carbapenems consist of a series of β-lactam antibiotics with a unique nuclear structure that differs from the penam nucleus of the penicillins in having a carbon atom replacing sulphur at position 1 and in having an unsaturated bond between carbon atoms 2 and 3 in the 5-membered ring (fig. 1 and 2). The first two types of carbapenems (the olivanic acids and thienamycin) were discovered almost at the same time in the mid-1970s. The olivanic acids produced by Streptomyces olivaceus were discovered as part of the systematic screening program that also led to the discovery of the oxapenam, clavulanic acid (Brown et al., 1976). Thienamycin (fig. 3) was found in the course of a soil-screening program looking for antibiotics that inhibit cell wall synthesis (Kahan et al., 1983). It was produced by a previously unknown Streptomyces species which was named Str. cattleya because the pigment in its aerial mycelium resembled the colour of the cattleya orchid. Its structure was determined by Albers-Schonberg (Albers-Schonberg et al., 1978).
Not only is its nuclear structure remarkable but it also contains a unique hydroxyethyl side chain in the trans (α) configuration at position 6. The classic
penicillins (penams) and cephalosporins (cephems) have side chains in the cis (β) configuration at positions 6 and 7 relative to one another. Subsequent studies confirmed that it is trans configuration accounts for the unique resistance of thienamycin to a wide variety of β-lactamases. The side chain at position 2 (a basic alkylthio group) has been shown to be responsible for the striking activity of thienamycin against Ps. aeruginosa (Christensen, 1981). In addition to the olivanic acids and thienamycins, a number of naturally occurring carbapenems have been discovered in the past decade and a variety of semi-synthetic derivatives have been synthesized (Wise, 1986).
Need more Information Read Manual of Antibiotics: Method of Actions, Mechanisms of Resistan…
Not only is its nuclear structure remarkable but it also contains a unique hydroxyethyl side chain in the trans (α) configuration at position 6. The classic
penicillins (penams) and cephalosporins (cephems) have side chains in the cis (β) configuration at positions 6 and 7 relative to one another. Subsequent studies confirmed that it is trans configuration accounts for the unique resistance of thienamycin to a wide variety of β-lactamases. The side chain at position 2 (a basic alkylthio group) has been shown to be responsible for the striking activity of thienamycin against Ps. aeruginosa (Christensen, 1981). In addition to the olivanic acids and thienamycins, a number of naturally occurring carbapenems have been discovered in the past decade and a variety of semi-synthetic derivatives have been synthesized (Wise, 1986).
Need more Information Read Manual of Antibiotics: Method of Actions, Mechanisms of Resistan…
Friday, August 26, 2011
Biocides: Definition, Mode of action
Biocides are inorganic or synthetic organic molecules used to disinfect, sanitize, or sterilize objects and surfaces, and to preserve materials or processes from microbiological degradation(Chapman, 2003). Because biocides range in antimicrobial activity, other terms maybe more specific, including “-static,” referring to agents which inhibit growth (e.g., bacteriostatic, fungistatic, and sporistatic) and “-cidal,” referring to agents which kill the target organism (e.g., sporicidal, virucidal, and bactericidal) (McDonnell and Russell, 1999).
Biocides are used extensively in hospitals for a variety of topical and hard-surface applications. In particular, they are an essential part of infection control practices and aid in the prevention of nosocomial infections (McDonnell and Russell, 1999).
Table (1) shows the chemical structure and examples of the common groups of disinfectants and antiseptics:
The mechanisms of the antibacterial action of biocides are still not perfectly understood. Biocides are likely to have multiple target sites within a bacterial cell. Biocides are known to interact with bacterial cell walls or envelopes (e.g. glutaraldehyde), produce changes in cytoplasmic membrane integrity (cationic agents), dissipate the proton-motive force
(organic acids and esters), inhibit membrane enzymes (thiol interactors), act as alkylating agents (ethylene oxide), cross-linking agents (aldehydes) and intercalating agents (acridines), or otherwise interact with identifiable chemical groups in the cell (Russell, 2002b).
A summary of the mechanisms of antibacterial action of antiseptics and disinfectants is shown in table (2).
Because the mechanisms of action of biocides are often poorly understood, detailed evaluation of bacterial resistance mechanisms remains disappointing. Nevertheless, it is known that at least some (efflux, impermeability, modification of target sites) of the general mechanisms responsible for antibiotic resistance are also applicable to biocides. The possibility exists of ‘cross-resistance’ arising between antibiotics and biocides(Russell, 2002b).
Research on antibiotics and biocides has traditionally proceeded along separate lines. The reasons for this is probably because antibiotics rely on selective toxicity for their activity, although this does not imply that they are without side effects on human and animal cells. Selective toxicity, on the other hand, is not a prerequisite for the use of biocides, although their actual and potential toxicity should never be ignored (Russell, 2002a).
By the very nature of the usage of the two types of antimicrobial agents, tests for evaluating their antibacterial potency differ considerably. With antibiotics, bacterial susceptibility is determined mainly by disc sensitivity and minimum inhibitory concentration (MIC) procedures. MICs can be linked to blood or serum levels and peak drug concentration and mutant prevention concentration in vivo. By contrast, such methodology has limited applications for most types of biocide evaluations. Many biocides diffuse poorly in agar, some biocides interact with agar constituents, and MICs often provide little more than a starting point for the information needed about the lethal effects of ‘in-use’ concentrations. Therefore, Standard European tests are increasingly being made available to measure such lethal effects for a variety of purposes(Russell, 2002a).
Biocides are used extensively in hospitals for a variety of topical and hard-surface applications. In particular, they are an essential part of infection control practices and aid in the prevention of nosocomial infections (McDonnell and Russell, 1999).
Table (1) shows the chemical structure and examples of the common groups of disinfectants and antiseptics:
(organic acids and esters), inhibit membrane enzymes (thiol interactors), act as alkylating agents (ethylene oxide), cross-linking agents (aldehydes) and intercalating agents (acridines), or otherwise interact with identifiable chemical groups in the cell (Russell, 2002b).
A summary of the mechanisms of antibacterial action of antiseptics and disinfectants is shown in table (2).
Because the mechanisms of action of biocides are often poorly understood, detailed evaluation of bacterial resistance mechanisms remains disappointing. Nevertheless, it is known that at least some (efflux, impermeability, modification of target sites) of the general mechanisms responsible for antibiotic resistance are also applicable to biocides. The possibility exists of ‘cross-resistance’ arising between antibiotics and biocides(Russell, 2002b).
Research on antibiotics and biocides has traditionally proceeded along separate lines. The reasons for this is probably because antibiotics rely on selective toxicity for their activity, although this does not imply that they are without side effects on human and animal cells. Selective toxicity, on the other hand, is not a prerequisite for the use of biocides, although their actual and potential toxicity should never be ignored (Russell, 2002a).
By the very nature of the usage of the two types of antimicrobial agents, tests for evaluating their antibacterial potency differ considerably. With antibiotics, bacterial susceptibility is determined mainly by disc sensitivity and minimum inhibitory concentration (MIC) procedures. MICs can be linked to blood or serum levels and peak drug concentration and mutant prevention concentration in vivo. By contrast, such methodology has limited applications for most types of biocide evaluations. Many biocides diffuse poorly in agar, some biocides interact with agar constituents, and MICs often provide little more than a starting point for the information needed about the lethal effects of ‘in-use’ concentrations. Therefore, Standard European tests are increasingly being made available to measure such lethal effects for a variety of purposes(Russell, 2002a).
Sepsis, bacteremia and septicemia
There have been several attempts to define sepsis. The various definitions which exist to describe sepsis demonstrate the complexities of the septic process. In general, sepsis can be defined as the systemic host response to infection. It is usually associated with several signs, which include: fever, increased C-reactive protein (CRP), increased heart rate and cardiac output, tachypnea, and increased oxygen consumption among others. However, these may not occur in all patients. Bacteremia can be defined as the presence of viable bacteria in the blood and septicemia is the association of bacteremia with sepsis. Severe sepsis is usually associated with organ dysnfuction, alterations in mental status, coagulation dysfunction and hypoxemia (Vincent, 2002).
Regardless of the definitions used, there is no doubt of the magnitude of the problem, with sepsis affecting some 2-14% of intensive care unit (ICU) admissions, with mortality rates of 35-50%. Aside from the high levels of morbidity and mortality, sepsis is also associated with increased hospital and ICU stays, expensive antimicrobial therapies, and prolonged duration of mechanical ventilation, which all represent a serious economic burden (Vincent, 2002).
The most common isolates in infections causing sepsis are in descending order S.aureus, coagulase-negative Staphylococci, Pseudomonas aeruginosa, Enterobacter, Klebsiella, Acinetobacter, Serratia and Candida (Vincent, 2002). It is thus apparent that Gram-positive organisms can be considered the most important etiological factors for septicemia.
The prognosis of septic patients is influenced not only by the severity of infection, but also by the previous health status and the host response (degree of immunocompetence)(Vincent, 2002). In general, logistic regression or Cox modeling techniques are used to identify those clinical factors that are independently associated with the probability of death (Wax et al., 2002).
Sepsis is a also major health problem in children with high mortality rates. Neonates in particular are at highest risk, especially those with low birth weights. Staphylococci are the most common causes of sepsis in children as well, and are associated with considerable mortality rates (Watson et al., 2003).
Regardless of the definitions used, there is no doubt of the magnitude of the problem, with sepsis affecting some 2-14% of intensive care unit (ICU) admissions, with mortality rates of 35-50%. Aside from the high levels of morbidity and mortality, sepsis is also associated with increased hospital and ICU stays, expensive antimicrobial therapies, and prolonged duration of mechanical ventilation, which all represent a serious economic burden (Vincent, 2002).
The most common isolates in infections causing sepsis are in descending order S.aureus, coagulase-negative Staphylococci, Pseudomonas aeruginosa, Enterobacter, Klebsiella, Acinetobacter, Serratia and Candida (Vincent, 2002). It is thus apparent that Gram-positive organisms can be considered the most important etiological factors for septicemia.
The prognosis of septic patients is influenced not only by the severity of infection, but also by the previous health status and the host response (degree of immunocompetence)(Vincent, 2002). In general, logistic regression or Cox modeling techniques are used to identify those clinical factors that are independently associated with the probability of death (Wax et al., 2002).
Sepsis is a also major health problem in children with high mortality rates. Neonates in particular are at highest risk, especially those with low birth weights. Staphylococci are the most common causes of sepsis in children as well, and are associated with considerable mortality rates (Watson et al., 2003).
Sepsis, bacteremia and septicemia
There have been several attempts to define sepsis. The various definitions which exist to describe sepsis demonstrate the complexities of the septic process. In general, sepsis can be defined as the systemic host response to infection. It is usually associated with several signs, which include: fever, increased C-reactive protein (CRP), increased heart rate and cardiac output, tachypnea, and increased oxygen consumption among others. However, these may not occur in all patients. Bacteremia can be defined as the presence of viable bacteria in the blood and septicemia is the association of bacteremia with sepsis. Severe sepsis is usually associated with organ dysnfuction, alterations in mental status, coagulation dysfunction and hypoxemia (Vincent, 2002).
Regardless of the definitions used, there is no doubt of the magnitude of the problem, with sepsis affecting some 2-14% of intensive care unit (ICU) admissions, with mortality rates of 35-50%. Aside from the high levels of morbidity and mortality, sepsis is also associated with increased hospital and ICU stays, expensive antimicrobial therapies, and prolonged duration of mechanical ventilation, which all represent a serious economic burden (Vincent, 2002).
The most common isolates in infections causing sepsis are in descending order S.aureus, coagulase-negative Staphylococci, Pseudomonas aeruginosa, Enterobacter, Klebsiella, Acinetobacter, Serratia and Candida (Vincent, 2002). It is thus apparent that Gram-positive organisms can be considered the most important etiological factors for septicemia.
The prognosis of septic patients is influenced not only by the severity of infection, but also by the previous health status and the host response (degree of immunocompetence)(Vincent, 2002). In general, logistic regression or Cox modeling techniques are used to identify those clinical factors that are independently associated with the probability of death (Wax et al., 2002).
Sepsis is a also major health problem in children with high mortality rates. Neonates in particular are at highest risk, especially those with low birth weights. Staphylococci are the most common causes of sepsis in children as well, and are associated with considerable mortality rates (Watson et al., 2003).
Regardless of the definitions used, there is no doubt of the magnitude of the problem, with sepsis affecting some 2-14% of intensive care unit (ICU) admissions, with mortality rates of 35-50%. Aside from the high levels of morbidity and mortality, sepsis is also associated with increased hospital and ICU stays, expensive antimicrobial therapies, and prolonged duration of mechanical ventilation, which all represent a serious economic burden (Vincent, 2002).
The most common isolates in infections causing sepsis are in descending order S.aureus, coagulase-negative Staphylococci, Pseudomonas aeruginosa, Enterobacter, Klebsiella, Acinetobacter, Serratia and Candida (Vincent, 2002). It is thus apparent that Gram-positive organisms can be considered the most important etiological factors for septicemia.
The prognosis of septic patients is influenced not only by the severity of infection, but also by the previous health status and the host response (degree of immunocompetence)(Vincent, 2002). In general, logistic regression or Cox modeling techniques are used to identify those clinical factors that are independently associated with the probability of death (Wax et al., 2002).
Sepsis is a also major health problem in children with high mortality rates. Neonates in particular are at highest risk, especially those with low birth weights. Staphylococci are the most common causes of sepsis in children as well, and are associated with considerable mortality rates (Watson et al., 2003).
Thursday, August 25, 2011
Automated Blood Culture Systems
These systems employ equipment that automatically detects an early sign of bacterial growth in a special blood culture bottle. The system most used is Bactec (Becton Dickinson). It depends on the release of carbon dioxide (C02) into the atmosphere in the culture bottle by the bacterial degradation nutrients in a special culture medium and PH of the bottle becomes acidic leading to release of fluorescent signal detected by specific sensor in Bactec system. Other system like Bact/alert use chromogenic substrate that change in color due to PH change by bacterial growth.
Separate bottles of special medium are supplied for aerobic and anaerobic culture, and bottles of medium containing resins to inactivate any antibiotics that may be present in the specimen.
Normally 10 ml blood would be collected in a syringe and up to 5 ml injected through their rubber caps into aerobic and anaerobic culture. Bottles containing 30 ml medium. Up to 60 bottles of the one kind seeded from different patients are loaded into an instrument in which they are incubated, agitated and periodically flushed with an appropriate gas (air + CO2, or N2 + H2 + C02) through two heat-sterilized needles inserted automatically through the rubber cap). It can indicate early bacterial growth, which is often detectable on the same day as the specimen is received in the laboratory.
When growth is thus first detected, the bottle is removed and examined in the usual way by filming and subculture. Negatively reacting bottles do not need to be filmed or subcultured at any stage.
Automated Culture for Mycobacterium tuberculosis
- Radiometrie BACTEC 460 TB system: BACTEC 460 TB system and BACTEC 12B vials (Becton Dickinson Microbiology system, Cockeysville, Md.) are used for radiometrie culture of MTB. BACTEC 12B vials are prepared by addition of antimicrobial agents "PANTA" which contains polymyxin, amphotericin B, nalidixic acid, trimethoprim, and azlocillin. One half ml of concentrated sputum sediments are inoculated into each BACTEC 12B vial and kept at 37°C and then tested 3 times a week. Positive cultures as indicated by BACTEC460 are subjected to further identification by NAP test (P-Nitro-acetyl amino hydroxyl-Proionophenone) for the presence of typical Myucobacterium tuberculosis.
The BACTEC MGIT 960 System is an in vitro diagnostic instrument for rapid detection of Mycobacteria in clinical specimens other than blood.
The system is designed to meet the needs of medium and high volume labs,capable of processing about 8,000 cultures per year.
This system is simple, efficient , safe to use and occupies small laboratory space. The MGIT 7ml tube contains modified middle brook 7H9 broth. Culture tubes contain a fluorescent sensor at the bottom which responds to the concentration of oxygen.
Initial concentration of dissolved oxygen quenches the emission from the compound, and little fluorescence can be detected.
Actively respiring micro organisms consume the oxygen which allows the compound to fluorescence.
MGIT can be used in laboratory without the system and read by UV lamps of the electrophoresis
-Antimicrobial Drug susceptibility testing (AST): Isolated TB colonies from BACTEC 12B media and from LJ media will be subjected to AST using both Agar proportion method and BACTEC 460 TB system.
Recently BACTEC system is replaced by non radiometric system MGIT
Separate bottles of special medium are supplied for aerobic and anaerobic culture, and bottles of medium containing resins to inactivate any antibiotics that may be present in the specimen.
Normally 10 ml blood would be collected in a syringe and up to 5 ml injected through their rubber caps into aerobic and anaerobic culture. Bottles containing 30 ml medium. Up to 60 bottles of the one kind seeded from different patients are loaded into an instrument in which they are incubated, agitated and periodically flushed with an appropriate gas (air + CO2, or N2 + H2 + C02) through two heat-sterilized needles inserted automatically through the rubber cap). It can indicate early bacterial growth, which is often detectable on the same day as the specimen is received in the laboratory.
When growth is thus first detected, the bottle is removed and examined in the usual way by filming and subculture. Negatively reacting bottles do not need to be filmed or subcultured at any stage.
Automated Culture for Mycobacterium tuberculosis
- Radiometrie BACTEC 460 TB system: BACTEC 460 TB system and BACTEC 12B vials (Becton Dickinson Microbiology system, Cockeysville, Md.) are used for radiometrie culture of MTB. BACTEC 12B vials are prepared by addition of antimicrobial agents "PANTA" which contains polymyxin, amphotericin B, nalidixic acid, trimethoprim, and azlocillin. One half ml of concentrated sputum sediments are inoculated into each BACTEC 12B vial and kept at 37°C and then tested 3 times a week. Positive cultures as indicated by BACTEC460 are subjected to further identification by NAP test (P-Nitro-acetyl amino hydroxyl-Proionophenone) for the presence of typical Myucobacterium tuberculosis.
The BACTEC MGIT 960 System is an in vitro diagnostic instrument for rapid detection of Mycobacteria in clinical specimens other than blood.
The system is designed to meet the needs of medium and high volume labs,capable of processing about 8,000 cultures per year.
This system is simple, efficient , safe to use and occupies small laboratory space. The MGIT 7ml tube contains modified middle brook 7H9 broth. Culture tubes contain a fluorescent sensor at the bottom which responds to the concentration of oxygen.
Initial concentration of dissolved oxygen quenches the emission from the compound, and little fluorescence can be detected.
Actively respiring micro organisms consume the oxygen which allows the compound to fluorescence.
MGIT can be used in laboratory without the system and read by UV lamps of the electrophoresis
-Antimicrobial Drug susceptibility testing (AST): Isolated TB colonies from BACTEC 12B media and from LJ media will be subjected to AST using both Agar proportion method and BACTEC 460 TB system.
Recently BACTEC system is replaced by non radiometric system MGIT
Automated Blood Culture Systems
These systems employ equipment that automatically detects an early sign of bacterial growth in a special blood culture bottle. The system most used is Bactec (Becton Dickinson). It depends on the release of carbon dioxide (C02) into the atmosphere in the culture bottle by the bacterial degradation nutrients in a special culture medium and PH of the bottle becomes acidic leading to release of fluorescent signal detected by specific sensor in Bactec system. Other system like Bact/alert use chromogenic substrate that change in color due to PH change by bacterial growth.
Separate bottles of special medium are supplied for aerobic and anaerobic culture, and bottles of medium containing resins to inactivate any antibiotics that may be present in the specimen.
Normally 10 ml blood would be collected in a syringe and up to 5 ml injected through their rubber caps into aerobic and anaerobic culture. Bottles containing 30 ml medium. Up to 60 bottles of the one kind seeded from different patients are loaded into an instrument in which they are incubated, agitated and periodically flushed with an appropriate gas (air + CO2, or N2 + H2 + C02) through two heat-sterilized needles inserted automatically through the rubber cap). It can indicate early bacterial growth, which is often detectable on the same day as the specimen is received in the laboratory.
When growth is thus first detected, the bottle is removed and examined in the usual way by filming and subculture. Negatively reacting bottles do not need to be filmed or subcultured at any stage.
Automated Culture for Mycobacterium tuberculosis
- Radiometrie BACTEC 460 TB system: BACTEC 460 TB system and BACTEC 12B vials (Becton Dickinson Microbiology system, Cockeysville, Md.) are used for radiometrie culture of MTB. BACTEC 12B vials are prepared by addition of antimicrobial agents "PANTA" which contains polymyxin, amphotericin B, nalidixic acid, trimethoprim, and azlocillin. One half ml of concentrated sputum sediments are inoculated into each BACTEC 12B vial and kept at 37°C and then tested 3 times a week. Positive cultures as indicated by BACTEC460 are subjected to further identification by NAP test (P-Nitro-acetyl amino hydroxyl-Proionophenone) for the presence of typical Myucobacterium tuberculosis.
The BACTEC MGIT 960 System is an in vitro diagnostic instrument for rapid detection of Mycobacteria in clinical specimens other than blood.
The system is designed to meet the needs of medium and high volume labs,capable of processing about 8,000 cultures per year.
This system is simple, efficient , safe to use and occupies small laboratory space. The MGIT 7ml tube contains modified middle brook 7H9 broth. Culture tubes contain a fluorescent sensor at the bottom which responds to the concentration of oxygen.
Initial concentration of dissolved oxygen quenches the emission from the compound, and little fluorescence can be detected.
Actively respiring micro organisms consume the oxygen which allows the compound to fluorescence.
MGIT can be used in laboratory without the system and read by UV lamps of the electrophoresis
-Antimicrobial Drug susceptibility testing (AST): Isolated TB colonies from BACTEC 12B media and from LJ media will be subjected to AST using both Agar proportion method and BACTEC 460 TB system.
Recently BACTEC system is replaced by non radiometric system MGIT
Separate bottles of special medium are supplied for aerobic and anaerobic culture, and bottles of medium containing resins to inactivate any antibiotics that may be present in the specimen.
Normally 10 ml blood would be collected in a syringe and up to 5 ml injected through their rubber caps into aerobic and anaerobic culture. Bottles containing 30 ml medium. Up to 60 bottles of the one kind seeded from different patients are loaded into an instrument in which they are incubated, agitated and periodically flushed with an appropriate gas (air + CO2, or N2 + H2 + C02) through two heat-sterilized needles inserted automatically through the rubber cap). It can indicate early bacterial growth, which is often detectable on the same day as the specimen is received in the laboratory.
When growth is thus first detected, the bottle is removed and examined in the usual way by filming and subculture. Negatively reacting bottles do not need to be filmed or subcultured at any stage.
Automated Culture for Mycobacterium tuberculosis
- Radiometrie BACTEC 460 TB system: BACTEC 460 TB system and BACTEC 12B vials (Becton Dickinson Microbiology system, Cockeysville, Md.) are used for radiometrie culture of MTB. BACTEC 12B vials are prepared by addition of antimicrobial agents "PANTA" which contains polymyxin, amphotericin B, nalidixic acid, trimethoprim, and azlocillin. One half ml of concentrated sputum sediments are inoculated into each BACTEC 12B vial and kept at 37°C and then tested 3 times a week. Positive cultures as indicated by BACTEC460 are subjected to further identification by NAP test (P-Nitro-acetyl amino hydroxyl-Proionophenone) for the presence of typical Myucobacterium tuberculosis.
The BACTEC MGIT 960 System is an in vitro diagnostic instrument for rapid detection of Mycobacteria in clinical specimens other than blood.
The system is designed to meet the needs of medium and high volume labs,capable of processing about 8,000 cultures per year.
This system is simple, efficient , safe to use and occupies small laboratory space. The MGIT 7ml tube contains modified middle brook 7H9 broth. Culture tubes contain a fluorescent sensor at the bottom which responds to the concentration of oxygen.
Initial concentration of dissolved oxygen quenches the emission from the compound, and little fluorescence can be detected.
Actively respiring micro organisms consume the oxygen which allows the compound to fluorescence.
MGIT can be used in laboratory without the system and read by UV lamps of the electrophoresis
-Antimicrobial Drug susceptibility testing (AST): Isolated TB colonies from BACTEC 12B media and from LJ media will be subjected to AST using both Agar proportion method and BACTEC 460 TB system.
Recently BACTEC system is replaced by non radiometric system MGIT
Automated Microbiology Identification Systems
The development of the first generation of automated equipment for clinical microbiology involved essentially two approaches. One can be described as the mechanization of existing techniques. The second combined mechanization with other changes, such as miniaturization and/or incorporation of innovative substrates, inhibitors, or indicators. The primary goal was to enhance data acquisition and processing, particularly with regard to decreasing turnaround time.
The systems reviewed here vary considerably in their approach to the identification of microorganisms. The MIS, for example, analyzes cellular material, whereas others use more conventional end points such as increase in cell density or color changes due to shifts in pH. There is also some variability in the degree of automation, spectrum of organisms identified, and turnaround time. The constant is that most of these instruments have proven their applicability in clinical microbiology laboratories throughout the country.
The devices described here are, with the exception of the Vitek urine card, based on pure culture techniques. In other words, the organism must be isolated before the identification process is performed.
There are, however, procedures, both manual and automated, that can identify organisms directly in specimens by using antibodies or nucleic acid probes.
The Vitek system, based on bacterial growth in micro wells of thin plastic cards These 30 microwell cards contained antibiotics or biochemical substrates. Susceptibility test cards are available for both gram-negative bacilli and gram-positive bacteria with 11 antimicrobial agents per card. Results are available in 4 to 8 h. Vitek has a variety of standard test kits, and
custom-defined test kits can be purchased. Over 40 antimicrobial agents are currently available on the cards, and results from each test include an interpolated MIC, as well as the National Committee for Clinical Laboratory Standards categories of susceptible, moderately susceptible, intermediate, and resistant.
Identification cards automatically interpreted by the Vitek system are the Gram-Negative Identification Test Kit (GNI), the Gram-Positive Identification Test Kit (GPI), and the Yeast Biochemical Test Kit (YBC).
Identification cards that require off-line incubation and manual entry of the results into the Vitek computer are the Anaerobe Identification Test Kit, Neisseria/Haemophilus Identification Test Kit, and Enteric Pathogen Screen Test Kit.
The GNI and GPI each contain 29 substrates and a growth control medium. The GNI substrates include 25 conventional biochemical substrates, 3 proprietary substrates, and 1 antibiotic. The GNI must be marked if the organism is oxidase positive. The GNI data base includes information for identification of 46 species of members of the family Enterobactenaceae and 39 species of other gram-negative organisms. The GPI substrates include 26 based on conventional biochemical tests, two antibiotics, and one dye. The
GPI must be marked for catalase-negative, beta-hemolytic organisms or for coagulase-positive organisms that are catalase positive.
The GPI data base includes information for identification of 23 Streptococcus species, 4 Enterococcus species, 16 Staphylococcus species, and 4 Corynebacterium, Aerococcus, Listenia monocytogenes, and Erysipelothrix rhusiopathiae species or groups.
The YBC contains 26 substrates which are based on conventional methods. The YBC data base includes information for identification of 16 Candida species, 6 Cryptococcus species, 3 Rhodotorula species, 2 Tnichosporon species, 3 Geotnichum species, 2 Prototheca species, and single species of four additional genera.
The Vitek system is an integrated modular system consisting of a filling-sealer unit, reader-incubator, computer control module, data terminal, and multicopy printer. The Vitek system can be purchased with a capacity of 30, 60, 120, or 240 cards and can be interfaced with other computers. A
data management center can be added. Inocula for the identification cards are prepared from selective (GNI or GPI) or nonselective Inocula for the GNI, GPI, and YBC are prepared by suspending several colonies in 1.8 ml of 0.45 to 0.5% saline and adjusting the suspension to the equivalent of
a no. 1 (GNI and GPI) or a no. 2 (YBC) McFarland standard.
The inoculum is automatically transferred to the card via a transfer tube during the vacuum cycle of the filling module.
The GNI and GPI are placed in plastic trays, each tray holding up to 30 cards. The tray is placed in the reader incubator at 35°C, and at hourly intervals a digitized analog optical reading, proportional to the light attenuation for each test well, is obtained for each card. The first reading usually establishes a baseline value, and the amount of light reduction
caused by growth or a biochemical reaction in the microwell is determined on subsequent readings. A predetermined minimum change is required to differentiate between positive and negative reactions. Final identification by
the GNI is reported between 4 and 18 h. Most of the non-glucose-fermenting gram-negative bacilli are reported at 18 h. Organisms are identified by the GPI between 4 and 15 h. The YBC is incubated off-line at 30°C for 24 h and then placed in the reader-incubator for a single reading. A message "reincubate for 24 h at 30°C" indicates that a definitive identification requires more incubation time. At 48 h, one must fill in the 48H mark on the card and obtain a second reading. The biochemical test results for all cards are compared with the data base, and the first and second choices, as well as their absolute likelihoods and normalized percent probabilities, are reported. The biochemical test results, as well as supplemental tests if required, are printed.
SENSITITRE
The Sensititre fluorogenic system (Radiometer America, Inc., Westlake, Ohio) is a modular system composed of a computer and an automated reader. This system identifies gram-negative bacilli and performs susceptibility tests on both gram-positive and gram-negative bacteria in either 5 or 18 h. Breakpoint or MIC capability for gram-negative and gram-positive bacteria is available for 54 antimicrobial agents. The Sensititre AP80 panel data base includes information for the identification of 84 members of the family Enterobacteriaceae, 24 oxidase-positive fermenters, 16 pseudomonads, and 16 other nonfermenters.
The WalkAway-96 (formerly called the autoSCAN-W/A) and WalkAway-40 (Baxter Diagnostics, Inc., MicroScan Division, West Sacramento, Calif.) are computer-controlled systems that will incubate microtiter panels and automatically interpret biochemical or susceptibility results with either a photometric or a fluorogenic reader. MicroScan also manufactures the autoSCAN-4, which requires off-line incubation and, with the exception of fluorogenic panels, will test the same panels as the other two automated systems. All three systems perform susceptibility tests on aerobic gram negative bacilli, gram-positive bacteria, and anaerobes.
Fluorogenic panels provides a 3.5- to 7-h susceptibility result for aerobic gram-negative bacilli and a 3.5- to 15-h susceptibility result for aerobic gram-positive bacteria. Conventional panels provides a 15- to 24-h susceptibility result for aerobic bacteria and a 24- to 48-h susceptibility result for anaerobes.
MicroScan has numerous panel types, including both MIC and breakpoint susceptibility panels. Custom panels are also available. The three systems automatically identify gram-
negative bacilli, gram-positive bacteria, fastidious aerobic bacteria, anaerobes, and yeasts.
The rapid fluorogenic panels for identification of gram negative bacilli (R-GNB) and gram-positive bacteria (RGPB) use fluorogenic substrates (4-methylumbelliferone or 7-amino-4-methylcoumarin attached to a phosphate, sugarmoiety, or amino acid) or fluorometric indicators. Identification is based on hydrolysis of fluorogenic substrates, pH changes following substrate utilization, production of specific metabolic by-products, or the rate of production of specific metabolic by-products after 2 h of incubation. The modified conventional panel for identification of gram-negative bacilli (GNB) has 29 modified conventional or chromogenic tests and six antibiotics, and results are available in 15 to 42 h. Both the R-GNB and GNB data bases include information for identification of 59 groups, genera, or species of members of the Enterobacteriaceae and 57 groups, genera,
or species of nonfermentative and oxidase-positive gram-negative bacilli. The modified conventional panel for identification of gram-positive bacteria (GPB) has 25 modified conventional or chromogenic substrates, one dye, and three antibiotics and yields an identification in 15 to 42 h.
Both the R-GPB and GPB data bases include information for identification of 24 genera or species of the family Micrococcaceae, 18 members of the family Streptococcaceae, 4Enterococcus spp., Aerococcus viridans, and L. monocytogenes.
The MicroScan Rapid Haemophilus and Neisseria Identification Panel (HNID) has 17 modified conventional or chromogenic tests and one antibiotic, and the data base includes information for identification of Haemophilus influenzae (seven biotypes), Haemophilus parainfluenzae (four biotypes), Haemophilus aphrophilus-Haemophilus paraphrophilus,
Haemophilus haemolyticus, four Neisseria species, Moraxella catarrhalis, and Gardnerella vaginalis in 4h. The MicroScan Rapid Anaerobe Identification Panel (AIP) has 24 modified conventional or chromogenic substrates, and the data base includes information for identification of 21 anaerobic gram-negative bacilli, 13 anaerobic non-spore-forming gram-positive bacilli, 8 anaerobic gram positive cocci, and 16 clostridia in 4 h without anaerobic incubation. The MicroScan Rapid Yeast Identification Panel (YIP) has 27 modified conventional or chromogenic substrates, and the data base includes information for identification of 16 Candida spp. or biotypes, 8 Cryptococcus spp., and 11 genera (representing 16 species) of yeasts or yeast like organisms in 4 h.
The systems reviewed here vary considerably in their approach to the identification of microorganisms. The MIS, for example, analyzes cellular material, whereas others use more conventional end points such as increase in cell density or color changes due to shifts in pH. There is also some variability in the degree of automation, spectrum of organisms identified, and turnaround time. The constant is that most of these instruments have proven their applicability in clinical microbiology laboratories throughout the country.
The devices described here are, with the exception of the Vitek urine card, based on pure culture techniques. In other words, the organism must be isolated before the identification process is performed.
There are, however, procedures, both manual and automated, that can identify organisms directly in specimens by using antibodies or nucleic acid probes.
The Vitek system, based on bacterial growth in micro wells of thin plastic cards These 30 microwell cards contained antibiotics or biochemical substrates. Susceptibility test cards are available for both gram-negative bacilli and gram-positive bacteria with 11 antimicrobial agents per card. Results are available in 4 to 8 h. Vitek has a variety of standard test kits, and
custom-defined test kits can be purchased. Over 40 antimicrobial agents are currently available on the cards, and results from each test include an interpolated MIC, as well as the National Committee for Clinical Laboratory Standards categories of susceptible, moderately susceptible, intermediate, and resistant.
Identification cards automatically interpreted by the Vitek system are the Gram-Negative Identification Test Kit (GNI), the Gram-Positive Identification Test Kit (GPI), and the Yeast Biochemical Test Kit (YBC).
Identification cards that require off-line incubation and manual entry of the results into the Vitek computer are the Anaerobe Identification Test Kit, Neisseria/Haemophilus Identification Test Kit, and Enteric Pathogen Screen Test Kit.
The GNI and GPI each contain 29 substrates and a growth control medium. The GNI substrates include 25 conventional biochemical substrates, 3 proprietary substrates, and 1 antibiotic. The GNI must be marked if the organism is oxidase positive. The GNI data base includes information for identification of 46 species of members of the family Enterobactenaceae and 39 species of other gram-negative organisms. The GPI substrates include 26 based on conventional biochemical tests, two antibiotics, and one dye. The
GPI must be marked for catalase-negative, beta-hemolytic organisms or for coagulase-positive organisms that are catalase positive.
The GPI data base includes information for identification of 23 Streptococcus species, 4 Enterococcus species, 16 Staphylococcus species, and 4 Corynebacterium, Aerococcus, Listenia monocytogenes, and Erysipelothrix rhusiopathiae species or groups.
The YBC contains 26 substrates which are based on conventional methods. The YBC data base includes information for identification of 16 Candida species, 6 Cryptococcus species, 3 Rhodotorula species, 2 Tnichosporon species, 3 Geotnichum species, 2 Prototheca species, and single species of four additional genera.
The Vitek system is an integrated modular system consisting of a filling-sealer unit, reader-incubator, computer control module, data terminal, and multicopy printer. The Vitek system can be purchased with a capacity of 30, 60, 120, or 240 cards and can be interfaced with other computers. A
data management center can be added. Inocula for the identification cards are prepared from selective (GNI or GPI) or nonselective Inocula for the GNI, GPI, and YBC are prepared by suspending several colonies in 1.8 ml of 0.45 to 0.5% saline and adjusting the suspension to the equivalent of
a no. 1 (GNI and GPI) or a no. 2 (YBC) McFarland standard.
The inoculum is automatically transferred to the card via a transfer tube during the vacuum cycle of the filling module.
The GNI and GPI are placed in plastic trays, each tray holding up to 30 cards. The tray is placed in the reader incubator at 35°C, and at hourly intervals a digitized analog optical reading, proportional to the light attenuation for each test well, is obtained for each card. The first reading usually establishes a baseline value, and the amount of light reduction
caused by growth or a biochemical reaction in the microwell is determined on subsequent readings. A predetermined minimum change is required to differentiate between positive and negative reactions. Final identification by
the GNI is reported between 4 and 18 h. Most of the non-glucose-fermenting gram-negative bacilli are reported at 18 h. Organisms are identified by the GPI between 4 and 15 h. The YBC is incubated off-line at 30°C for 24 h and then placed in the reader-incubator for a single reading. A message "reincubate for 24 h at 30°C" indicates that a definitive identification requires more incubation time. At 48 h, one must fill in the 48H mark on the card and obtain a second reading. The biochemical test results for all cards are compared with the data base, and the first and second choices, as well as their absolute likelihoods and normalized percent probabilities, are reported. The biochemical test results, as well as supplemental tests if required, are printed.
SENSITITRE
The Sensititre fluorogenic system (Radiometer America, Inc., Westlake, Ohio) is a modular system composed of a computer and an automated reader. This system identifies gram-negative bacilli and performs susceptibility tests on both gram-positive and gram-negative bacteria in either 5 or 18 h. Breakpoint or MIC capability for gram-negative and gram-positive bacteria is available for 54 antimicrobial agents. The Sensititre AP80 panel data base includes information for the identification of 84 members of the family Enterobacteriaceae, 24 oxidase-positive fermenters, 16 pseudomonads, and 16 other nonfermenters.
The WalkAway-96 (formerly called the autoSCAN-W/A) and WalkAway-40 (Baxter Diagnostics, Inc., MicroScan Division, West Sacramento, Calif.) are computer-controlled systems that will incubate microtiter panels and automatically interpret biochemical or susceptibility results with either a photometric or a fluorogenic reader. MicroScan also manufactures the autoSCAN-4, which requires off-line incubation and, with the exception of fluorogenic panels, will test the same panels as the other two automated systems. All three systems perform susceptibility tests on aerobic gram negative bacilli, gram-positive bacteria, and anaerobes.
Fluorogenic panels provides a 3.5- to 7-h susceptibility result for aerobic gram-negative bacilli and a 3.5- to 15-h susceptibility result for aerobic gram-positive bacteria. Conventional panels provides a 15- to 24-h susceptibility result for aerobic bacteria and a 24- to 48-h susceptibility result for anaerobes.
MicroScan has numerous panel types, including both MIC and breakpoint susceptibility panels. Custom panels are also available. The three systems automatically identify gram-
negative bacilli, gram-positive bacteria, fastidious aerobic bacteria, anaerobes, and yeasts.
The rapid fluorogenic panels for identification of gram negative bacilli (R-GNB) and gram-positive bacteria (RGPB) use fluorogenic substrates (4-methylumbelliferone or 7-amino-4-methylcoumarin attached to a phosphate, sugarmoiety, or amino acid) or fluorometric indicators. Identification is based on hydrolysis of fluorogenic substrates, pH changes following substrate utilization, production of specific metabolic by-products, or the rate of production of specific metabolic by-products after 2 h of incubation. The modified conventional panel for identification of gram-negative bacilli (GNB) has 29 modified conventional or chromogenic tests and six antibiotics, and results are available in 15 to 42 h. Both the R-GNB and GNB data bases include information for identification of 59 groups, genera, or species of members of the Enterobacteriaceae and 57 groups, genera,
or species of nonfermentative and oxidase-positive gram-negative bacilli. The modified conventional panel for identification of gram-positive bacteria (GPB) has 25 modified conventional or chromogenic substrates, one dye, and three antibiotics and yields an identification in 15 to 42 h.
Both the R-GPB and GPB data bases include information for identification of 24 genera or species of the family Micrococcaceae, 18 members of the family Streptococcaceae, 4Enterococcus spp., Aerococcus viridans, and L. monocytogenes.
The MicroScan Rapid Haemophilus and Neisseria Identification Panel (HNID) has 17 modified conventional or chromogenic tests and one antibiotic, and the data base includes information for identification of Haemophilus influenzae (seven biotypes), Haemophilus parainfluenzae (four biotypes), Haemophilus aphrophilus-Haemophilus paraphrophilus,
Haemophilus haemolyticus, four Neisseria species, Moraxella catarrhalis, and Gardnerella vaginalis in 4h. The MicroScan Rapid Anaerobe Identification Panel (AIP) has 24 modified conventional or chromogenic substrates, and the data base includes information for identification of 21 anaerobic gram-negative bacilli, 13 anaerobic non-spore-forming gram-positive bacilli, 8 anaerobic gram positive cocci, and 16 clostridia in 4 h without anaerobic incubation. The MicroScan Rapid Yeast Identification Panel (YIP) has 27 modified conventional or chromogenic substrates, and the data base includes information for identification of 16 Candida spp. or biotypes, 8 Cryptococcus spp., and 11 genera (representing 16 species) of yeasts or yeast like organisms in 4 h.
Antimycobacterial tuberculosis Drug Susceptibility
The wide spread emergence of isolates of Mycobacterium tuberculosis(M. tuberculosis) resistant to one or more antituberculous drugs represents one of the most alarming corollaries of AIDS related tuberculosis during recent years. Delayed detection, identification, and susceptibility testing of drug resistant isolates and failure to appropriately isolate contagious patients and to begin adequate chemotherapy have all been identified as predisposing factors of transmission of drug resistant M. tuberculosis (1). Molecular bases of drug resistance have been identified for all of the main antituberculous drugs, and drug resistance results from changes in several target genes, some of which are still undefined (2,3). These factors highlights the need to implement the rapid detection of drug resistance, for better management of patients as well as for control of the outbreaks and prevention of future nosocomial drug- resistant tuberculosis transmission (4).
Drug resistance in M. tuberculosis is due to the acquisition of mutations in chromosomally encoded genes and the generation of multidrug resistance is a consequence of serial accumulation of mutations primarily due to inadequate therapy (5, 6).
Several studies have shown that resistance to isoniazid (INTI) is due to mutaions in kat G gene. The rpo B gene, which encodes te subunit of RNA polymerase, harbors a mutation in an 81 bp region in about 95% of rifampicin (RIF) reistant M. tuberculosis strains recovered globally (6,7). Streptomycin (STR) resistance is due to mutations in rrs and rpsl genes which encodes 16S SrRNA and ribosomal protein S12 respectively (6, 8). Approximately 65% of clinical isolates resistant to ethambutol have a mutation in the embB gene (9, 10)
Drug resistance in M. tuberculosis is due to the acquisition of mutations in chromosomally encoded genes and the generation of multidrug resistance is a consequence of serial accumulation of mutations primarily due to inadequate therapy (5, 6).
Several studies have shown that resistance to isoniazid (INTI) is due to mutaions in kat G gene. The rpo B gene, which encodes te subunit of RNA polymerase, harbors a mutation in an 81 bp region in about 95% of rifampicin (RIF) reistant M. tuberculosis strains recovered globally (6,7). Streptomycin (STR) resistance is due to mutations in rrs and rpsl genes which encodes 16S SrRNA and ribosomal protein S12 respectively (6, 8). Approximately 65% of clinical isolates resistant to ethambutol have a mutation in the embB gene (9, 10)
Obstetric, Perinatal and neonatal infections
Infections in pregnancy may cause
spontaneous abortion.
premature labour, still birth,
perinatal
congenital infection
Infections in early pregnancy
TORCH
Toxoplasma, Rubella,Cytomegalovirus, Herpes Simplex
Other include Influenza, Mumps, Measles, Coxsakie A16 virus
These infections either lead to abortions or congenital anomalies
Laboratory Diagnosis of Pregnancy Infections
For diagnosis of TORCH
Samples Can be
-Maternal Blood
Fetal blood samples if available or tissues (post morteum)
-Serological diagnosis by IgM for recent infections or raised IgG four folds within 10 days
PCR
Perinatal and neonatal bacterial infections
PERINATAL INFECTIONS
The organisms that can infect the infant during birth from the genital tract or perineum are similar to those already mentioned for later intra-uterine infection via the ascending route.
Neisseria gonorrhoeae, Chlamydia trachomatis (TR1C) agent and herpes simplex type II
Diagnosis of Perinatal Infections
Complete Microbiological Diagnosis according to site of infections in the infant
NEONATAL AND CONGENITAL INFECTIONS
Early onset infections due to Lancefield group B streptococci and Gram-negative bacilli
After 4 – 7 days of life, ‘late onset’ neonatal infections due to these organisms may appear. The source of the organisms in late onset infections need not necessarily be the maternal perineal or genital tract flora. The source may he the already infected neonates in a baby unit or the hands of hospital staff which transmit these infections from one neonate to the next.
Occasionally moist contaminated equipment, such as baby incubators which are humidified or baby resuscitation equipment, is the source of infection in a common source outbreak due to Pseudomonas aeruginosa or other Gram-negative bacilli. Late onset serious Lancefield group B streptococcal or Gram-negative infection is often characterized by the development of meningitis.
Congenital infections by TORCH can be diagnosed by specific IgM
Diagnosis of Syphilis can be performed by speciic IgM
Laboratory Diagnosis of Perinatal Infections
Investigations should include the taking of blood cultures, swabs of umbilicus, skin, eye or any septic site, faeces for culture.
Microscopy and culture of urine and cerebrospinal fluid.
When pus is present, a Gram—stain of this can frequently give a rapid indication of the likely causative group of organisms; predominant numerous Gram-positive cocci could suggest the possibility of a Lancefield group B haemolvtic streptococcus or Staphylococcus aureus infection.
However, the results of microscopy and culture of skin sites are often difficult to interpret in practice since colonization of the neonatal skin by streptococci, staphylococci and Gram-negative bacilli is also common and difficult to distinguish in the laboratory from infection due to these same organisms.
Laboratory Diagnosis of Perinatal Infections
Investigations should include the taking of blood cultures, swabs of umbilicus, skin, eye or any septic site, faeces for culture.
Microscopy and culture of urine and cerebrospinal fluid.
When pus is present, a Gram—stain of this can frequently give a rapid indication of the likely causative group of organisms; predominant numerous Gram-positive cocci could suggest the possibility of a Lancefield group B haemolvtic streptococcus or Staphylococcus aureus infection.
However, the results of microscopy and culture of skin sites are often difficult to interpret in practice since colonization of the neonatal skin by streptococci, staphylococci and Gram-negative bacilli is also common and difficult to distinguish in the laboratory from infection due to these same organisms.
You need more details read
Contents
1-The use of the Clinical Microbiology laboratory
(General principles )
2-Basic Laboratory procedures for microbiological
Diagnosis
3- Classification & pathogenicity of Microbes
4-Basic Bacterial Culture and Identification
5-Identification of Gram-Positive Bacteria
6-Culture and Identification of Fastidious Bacteria
7-Identification of the Enterobacteriacae
8-TESTS FOR SUSCEPTIBILITY TO
9-ANTIMICROBIAL AGENTS
10-Bacterial Staining
11-Sampling for FUNGAL Infections and Culture
12-Laboratory Diagnosis of Viral Diseases
13-BLOOD CULTURE
14-UPPER RESPIRATORY INFECTIONS
15-Lower respiratory tract infections
16-Wound, skin and deep sepsis
17-Genital tract infections
18-Meningitis
18-Gastrointestinal infections
20-Urinary tract infections
21-Pyrexia of unknown origin
22-Children Specific Infections
23-Clinical Groupings for Fungal Infections
24-Obesteric, Perineatal and neonatal Infections
25-Hospital Acquired Infections
26-Opportunistic Infections
27-Sterilization
28-Hepatitis
29-AIDs 138-141
30-Anaerobic Infections
31-Zoonosis
32-Antimicrobials
33-Monitoring antimicrobials therapy
34-Biosafety and Biohazard
35-Microbiology Quality control
36- Skeletal Infections
37-Bacterial Skin Infections
References
spontaneous abortion.
premature labour, still birth,
perinatal
congenital infection
Infections in early pregnancy
TORCH
Toxoplasma, Rubella,Cytomegalovirus, Herpes Simplex
Other include Influenza, Mumps, Measles, Coxsakie A16 virus
These infections either lead to abortions or congenital anomalies
Laboratory Diagnosis of Pregnancy Infections
For diagnosis of TORCH
Samples Can be
-Maternal Blood
Fetal blood samples if available or tissues (post morteum)
-Serological diagnosis by IgM for recent infections or raised IgG four folds within 10 days
PCR
Perinatal and neonatal bacterial infections
PERINATAL INFECTIONS
The organisms that can infect the infant during birth from the genital tract or perineum are similar to those already mentioned for later intra-uterine infection via the ascending route.
Neisseria gonorrhoeae, Chlamydia trachomatis (TR1C) agent and herpes simplex type II
Diagnosis of Perinatal Infections
Complete Microbiological Diagnosis according to site of infections in the infant
NEONATAL AND CONGENITAL INFECTIONS
Early onset infections due to Lancefield group B streptococci and Gram-negative bacilli
After 4 – 7 days of life, ‘late onset’ neonatal infections due to these organisms may appear. The source of the organisms in late onset infections need not necessarily be the maternal perineal or genital tract flora. The source may he the already infected neonates in a baby unit or the hands of hospital staff which transmit these infections from one neonate to the next.
Occasionally moist contaminated equipment, such as baby incubators which are humidified or baby resuscitation equipment, is the source of infection in a common source outbreak due to Pseudomonas aeruginosa or other Gram-negative bacilli. Late onset serious Lancefield group B streptococcal or Gram-negative infection is often characterized by the development of meningitis.
Congenital infections by TORCH can be diagnosed by specific IgM
Diagnosis of Syphilis can be performed by speciic IgM
Laboratory Diagnosis of Perinatal Infections
Investigations should include the taking of blood cultures, swabs of umbilicus, skin, eye or any septic site, faeces for culture.
Microscopy and culture of urine and cerebrospinal fluid.
When pus is present, a Gram—stain of this can frequently give a rapid indication of the likely causative group of organisms; predominant numerous Gram-positive cocci could suggest the possibility of a Lancefield group B haemolvtic streptococcus or Staphylococcus aureus infection.
However, the results of microscopy and culture of skin sites are often difficult to interpret in practice since colonization of the neonatal skin by streptococci, staphylococci and Gram-negative bacilli is also common and difficult to distinguish in the laboratory from infection due to these same organisms.
Laboratory Diagnosis of Perinatal Infections
Investigations should include the taking of blood cultures, swabs of umbilicus, skin, eye or any septic site, faeces for culture.
Microscopy and culture of urine and cerebrospinal fluid.
When pus is present, a Gram—stain of this can frequently give a rapid indication of the likely causative group of organisms; predominant numerous Gram-positive cocci could suggest the possibility of a Lancefield group B haemolvtic streptococcus or Staphylococcus aureus infection.
However, the results of microscopy and culture of skin sites are often difficult to interpret in practice since colonization of the neonatal skin by streptococci, staphylococci and Gram-negative bacilli is also common and difficult to distinguish in the laboratory from infection due to these same organisms.
You need more details read
Contents
1-The use of the Clinical Microbiology laboratory
(General principles )
2-Basic Laboratory procedures for microbiological
Diagnosis
3- Classification & pathogenicity of Microbes
4-Basic Bacterial Culture and Identification
5-Identification of Gram-Positive Bacteria
6-Culture and Identification of Fastidious Bacteria
7-Identification of the Enterobacteriacae
8-TESTS FOR SUSCEPTIBILITY TO
9-ANTIMICROBIAL AGENTS
10-Bacterial Staining
11-Sampling for FUNGAL Infections and Culture
12-Laboratory Diagnosis of Viral Diseases
13-BLOOD CULTURE
14-UPPER RESPIRATORY INFECTIONS
15-Lower respiratory tract infections
16-Wound, skin and deep sepsis
17-Genital tract infections
18-Meningitis
18-Gastrointestinal infections
20-Urinary tract infections
21-Pyrexia of unknown origin
22-Children Specific Infections
23-Clinical Groupings for Fungal Infections
24-Obesteric, Perineatal and neonatal Infections
25-Hospital Acquired Infections
26-Opportunistic Infections
27-Sterilization
28-Hepatitis
29-AIDs 138-141
30-Anaerobic Infections
31-Zoonosis
32-Antimicrobials
33-Monitoring antimicrobials therapy
34-Biosafety and Biohazard
35-Microbiology Quality control
36- Skeletal Infections
37-Bacterial Skin Infections
References
Wednesday, August 24, 2011
Congenital Viral Infections
Intrauterine Viral Infections
Rubella
Cytomegalovirus (CMV)
Parvovirus B19
Varicella-Zoster (VZV)
Enteroviruses
HIV
HTLV-1
Hepatitis C
Hepatitis B
Lassa Fever
Japanese Encephalitis
Perinatal and Neonatal Infections
Human Herpes Simplex
VZV
Enteroviruses
HIV
Hepatitis B
Hepatitis C
HTLV-1
Rubella
History
1881 Rubella accepted as a distinct disease
1941 Associated with congenital disease (Gregg)
1961 Rubella virus first isolated
1967 Serological tests available
1969 Rubella vaccines available
Characteristics of Rubella
RNA enveloped virus, member of the togavirus family
Spread by respiratory droplets.
In the prevaccination era, 80% of women were already infected by childbearing age.
Clinical Features
maculopapular rash
lymphadenopathy
fever
arthropathy (up to 60% of cases)
Rash of Rubella
Risks of rubella infection during pregnancy
Preconception minimal risk
0-12 weeks 100% risk of fetus being congenitally infected
resulting in major congenital abnormalities.
Spontaneous abortion occurs in 20% of cases.
13-16 weeks deafness and retinopathy 15%
after 16 weeks normal development, slight risk of deafness and retinopathy
Congenital Rubella Syndrome
Classical triad consists of cataracts, heart defects, and sensorineural deafness. Many other abnormalities had been described and these are divided into transient, permanent and developmental.
Transient low birth weight, hepatosplenomegaly, thrombocytopenic purpura bone lesions, meningoencephalitis, hepatitis, haemolytic anemia pneumonitis, lymphadenopathy
Permanent Sensorineural deafness, Heart Defects (peripheral pulmonary stenosis,
pulmonary valvular stenosis, patent ductus arteriosus, ventricular septal defect) Eye Defects (retinopathy, cataract, microopthalmia, glaucoma, severe myopia) Other Defects (microcephaly, diabetes mellitis, thyroid disorders, dermatoglyptic abnormalities
Developmental Sensorineural deafness, Mental retardation, Diabetes Mellitus, thyroid disorder
Outcome
1/3 rd will lead normal independent lives
1/3 rd will live with parents
1/3rd will be institutionalised
The only effective way to prevent CRS is to terminate the pregnancy
Prevention (1)
Antenatal screening
All pregnant women attending antenatal clinics are tested for immune status against rubella.
Non-immune women are offered rubella vaccination in the immediate post partum period.
Prevention (2)
Since 1968, a highly effective live attenuated vaccine has been available with 95% efficacy
Universal vaccination is now offered to all infants as part of the MMR regimen in the USA, UK and a number of other countries.
Some countries such as the Czech Republic continue to selectively vaccinate schoolgirls before they reach childbearing age.
Both universal and selective vaccination policies will work provided that the coverage is high enough.
Laboratory Diagnosis
Diagnosis of acute infection
Rising titres of antibody (mainly IgG) - HAI, EIA
Presence of rubella-specific IgM - EIA
Immune Status Screen
HAI is too insensitive for immune status screening
SRH, EIA and latex agglutination are routinely used
15 IU/ml is regarded as the cut-off for immunity
Typical Serological Events following acute rubella infection
Note that in reinfection, IgM is usually absent or only present transiently at a low level
Cytomegalovirus
member of the herpesvirus
primary infection usually asymptomatic. Virus then becomes latent and is reactivated from time to time.
transmitted by infected saliva, breast milk, sexually and through infected blood
60% of the population eventually become infected. In some developing countries, the figure is up to 95%.
Congenital Infection
Defined as the isolation of CMV from the saliva or urine within 3 weeks of birth.
Commonest congenital viral infection, affects 0.3 - 1% of all live births. The second most common cause of mental handicap after Down's syndrome and is responsible for more cases of congenital damage than rubella.
Transmission to the fetus may occur following primary or recurrent CMV infection. 40% chance of transmission to the fetus following a primary infection.
May be transmitted to the fetus during all stages of pregnancy.
No evidence of teratogenecity, damage to the fetus results from destruction of target cells once they are formed.
Cytomegalic Inclusion Disease
CNS abnormalities - microcephaly, mental retardation, spasticity, epilepsy, periventricular calcification.
Eye - choroidoretinitis and optic atrophy
Ear - sensorineural deafness
Liver - hepatosplenomegaly and jaundice which is due to hepatitis.
Lung - pneumonitis
Heart - myocarditis
Thrombocytopenic purpura, Haemolytic anaemia
Late sequelae in individuals asymptomatic at birth - hearing defects and reduced intelligence.
Incidence of Cytomegalic Disease
Diagnosis
Isolation of CMV from the urine or saliva of the neonate.
Presence of CMV IgM from the blood of the neonate.
Detection of Cytomegalic Inclusion Bodies from affected tissue (rarely used)
Management
Primary Infection - consider termination of pregnancy.
40% chance of the fetus being infected.
10% chance that congenitally infected baby will be symptomatic at birth or develop sequelae later in life.
Therefore in case of primary infection, there is a 4% chance (1 in 25) of giving birth to an infant with CMV problems.
Recurrent Infection - termination not recommended as risk of transmission to the fetus is much lower.
Antenatal Screening – impractical.
Vaccination - may become available in the near future.
Neonatal Herpes Simplex (1)
Incidence of neonatal HSV infection varies inexplicably from country to country e.g. from 1 in 4000 live births in the U.S. to 1 in 10000 live births in the UK.
The baby is usually infected perinatally during passage through the birth canal.
Premature rupturing of the membranes is a well recognized risk factor.
The risk of perinatal transmission is greatest when there is a florid primary infection in the mother.
There is an appreciably smaller risk from recurrent lesions in the mother, probably because of the lower viral load and the presence of specific antibody.
The baby may also be infected from other sources such as oral lesions from the mother or a herpetic whitlow in a nurse.
Neonatal Herpes Simplex (2)
The spectrum of neonatal HSV infection varies from a mild disease localized to the skin to a fatal disseminated infection.
Infection is particularly dangerous in premature infants.
Where dissemination occurs, the organs most commonly involved are the liver, adrenals and the brain.
Where the brain is involved, the prognosis is particularly severe. The encephalitis is global and of such severity that the brain may be liquefied.
A large proportion of survivors of neonatal HSV infection have residual disabilities.
Acyclovir should be promptly given in all suspected cases of neonatal HSV infection.
The only means of prevention is to offer caesarean section to mothers with florid genital HSV lesions.
Parvovirus
Causative agent of Fifth disease (erythema infectiosum), clinically difficult to distinguish from rubella.
Also causes aplastic crisis in individuals with haemolytic anaemias as erythrocyte progenitors are targeted.
Spread by the respiratory route, 60-70% of the population is eventually infected.
50% of women of childbearing age are susceptible to infection.
Congenital Parvovirus Infection
Known to cause fetal loss through hydrops fetalis; severe anaemia, congestive heart failure, generalized oedema and fetal death
No evidence of teratogenecity.
Risk of fetal death highest when infection occurs during the second trimester of pregnancy (12%).
Minimal risk to the fetus if infection occurred during the first or third trimesters of pregnancy.
Maternal infection during pregnancy does not warrant termination of pregnancy.
Cases of diagnosed hydrops fetalis had been successfully treated in utero by intrauterine transfusions and administration of digoxin to the fetus.
Varicella-Zoster Virus
90% of pregnant women already immune, therefore primary infection is rare during pregnancy
Primary infection during pregnancy carries a greater risk of severe disease, in particular pneumonia
First 20 weeks of Pregnancy
up to 3% chance of transmission to the fetus,
recognised congenital varicella syndrome;
Scarring of skin
Hypoplasia of limbs
CNS and eye defects
Death in infancy normal
Neonatal Varicella
VZV can cross the placenta in the late stages of pregnancy to infect the fetus congenitally.
Neonatal varicella may vary from a mild disease to a fatal disseminated infection.
If rash in mother occurs more than 1 week before delivery, then sufficient immunity would have been transferred to the fetus.
Zoster immunoglobulin should be given to susceptible pregnant women who had contact with suspected cases of varicella.
Zoster immunoglobulin should also be given to infants whose mothers develop varicella during the last 7 days of pregnancy or the first 14 days after delivery.
Rubella
Cytomegalovirus (CMV)
Parvovirus B19
Varicella-Zoster (VZV)
Enteroviruses
HIV
HTLV-1
Hepatitis C
Hepatitis B
Lassa Fever
Japanese Encephalitis
Perinatal and Neonatal Infections
Human Herpes Simplex
VZV
Enteroviruses
HIV
Hepatitis B
Hepatitis C
HTLV-1
Rubella
History
1881 Rubella accepted as a distinct disease
1941 Associated with congenital disease (Gregg)
1961 Rubella virus first isolated
1967 Serological tests available
1969 Rubella vaccines available
Characteristics of Rubella
RNA enveloped virus, member of the togavirus family
Spread by respiratory droplets.
In the prevaccination era, 80% of women were already infected by childbearing age.
Clinical Features
maculopapular rash
lymphadenopathy
fever
arthropathy (up to 60% of cases)
Rash of Rubella
Risks of rubella infection during pregnancy
Preconception minimal risk
0-12 weeks 100% risk of fetus being congenitally infected
resulting in major congenital abnormalities.
Spontaneous abortion occurs in 20% of cases.
13-16 weeks deafness and retinopathy 15%
after 16 weeks normal development, slight risk of deafness and retinopathy
Congenital Rubella Syndrome
Classical triad consists of cataracts, heart defects, and sensorineural deafness. Many other abnormalities had been described and these are divided into transient, permanent and developmental.
Transient low birth weight, hepatosplenomegaly, thrombocytopenic purpura bone lesions, meningoencephalitis, hepatitis, haemolytic anemia pneumonitis, lymphadenopathy
Permanent Sensorineural deafness, Heart Defects (peripheral pulmonary stenosis,
pulmonary valvular stenosis, patent ductus arteriosus, ventricular septal defect) Eye Defects (retinopathy, cataract, microopthalmia, glaucoma, severe myopia) Other Defects (microcephaly, diabetes mellitis, thyroid disorders, dermatoglyptic abnormalities
Developmental Sensorineural deafness, Mental retardation, Diabetes Mellitus, thyroid disorder
Outcome
1/3 rd will lead normal independent lives
1/3 rd will live with parents
1/3rd will be institutionalised
The only effective way to prevent CRS is to terminate the pregnancy
Prevention (1)
Antenatal screening
All pregnant women attending antenatal clinics are tested for immune status against rubella.
Non-immune women are offered rubella vaccination in the immediate post partum period.
Prevention (2)
Since 1968, a highly effective live attenuated vaccine has been available with 95% efficacy
Universal vaccination is now offered to all infants as part of the MMR regimen in the USA, UK and a number of other countries.
Some countries such as the Czech Republic continue to selectively vaccinate schoolgirls before they reach childbearing age.
Both universal and selective vaccination policies will work provided that the coverage is high enough.
Laboratory Diagnosis
Diagnosis of acute infection
Rising titres of antibody (mainly IgG) - HAI, EIA
Presence of rubella-specific IgM - EIA
Immune Status Screen
HAI is too insensitive for immune status screening
SRH, EIA and latex agglutination are routinely used
15 IU/ml is regarded as the cut-off for immunity
Typical Serological Events following acute rubella infection
Note that in reinfection, IgM is usually absent or only present transiently at a low level
Cytomegalovirus
member of the herpesvirus
primary infection usually asymptomatic. Virus then becomes latent and is reactivated from time to time.
transmitted by infected saliva, breast milk, sexually and through infected blood
60% of the population eventually become infected. In some developing countries, the figure is up to 95%.
Congenital Infection
Defined as the isolation of CMV from the saliva or urine within 3 weeks of birth.
Commonest congenital viral infection, affects 0.3 - 1% of all live births. The second most common cause of mental handicap after Down's syndrome and is responsible for more cases of congenital damage than rubella.
Transmission to the fetus may occur following primary or recurrent CMV infection. 40% chance of transmission to the fetus following a primary infection.
May be transmitted to the fetus during all stages of pregnancy.
No evidence of teratogenecity, damage to the fetus results from destruction of target cells once they are formed.
Cytomegalic Inclusion Disease
CNS abnormalities - microcephaly, mental retardation, spasticity, epilepsy, periventricular calcification.
Eye - choroidoretinitis and optic atrophy
Ear - sensorineural deafness
Liver - hepatosplenomegaly and jaundice which is due to hepatitis.
Lung - pneumonitis
Heart - myocarditis
Thrombocytopenic purpura, Haemolytic anaemia
Late sequelae in individuals asymptomatic at birth - hearing defects and reduced intelligence.
Incidence of Cytomegalic Disease
Diagnosis
Isolation of CMV from the urine or saliva of the neonate.
Presence of CMV IgM from the blood of the neonate.
Detection of Cytomegalic Inclusion Bodies from affected tissue (rarely used)
Management
Primary Infection - consider termination of pregnancy.
40% chance of the fetus being infected.
10% chance that congenitally infected baby will be symptomatic at birth or develop sequelae later in life.
Therefore in case of primary infection, there is a 4% chance (1 in 25) of giving birth to an infant with CMV problems.
Recurrent Infection - termination not recommended as risk of transmission to the fetus is much lower.
Antenatal Screening – impractical.
Vaccination - may become available in the near future.
Neonatal Herpes Simplex (1)
Incidence of neonatal HSV infection varies inexplicably from country to country e.g. from 1 in 4000 live births in the U.S. to 1 in 10000 live births in the UK.
The baby is usually infected perinatally during passage through the birth canal.
Premature rupturing of the membranes is a well recognized risk factor.
The risk of perinatal transmission is greatest when there is a florid primary infection in the mother.
There is an appreciably smaller risk from recurrent lesions in the mother, probably because of the lower viral load and the presence of specific antibody.
The baby may also be infected from other sources such as oral lesions from the mother or a herpetic whitlow in a nurse.
Neonatal Herpes Simplex (2)
The spectrum of neonatal HSV infection varies from a mild disease localized to the skin to a fatal disseminated infection.
Infection is particularly dangerous in premature infants.
Where dissemination occurs, the organs most commonly involved are the liver, adrenals and the brain.
Where the brain is involved, the prognosis is particularly severe. The encephalitis is global and of such severity that the brain may be liquefied.
A large proportion of survivors of neonatal HSV infection have residual disabilities.
Acyclovir should be promptly given in all suspected cases of neonatal HSV infection.
The only means of prevention is to offer caesarean section to mothers with florid genital HSV lesions.
Parvovirus
Causative agent of Fifth disease (erythema infectiosum), clinically difficult to distinguish from rubella.
Also causes aplastic crisis in individuals with haemolytic anaemias as erythrocyte progenitors are targeted.
Spread by the respiratory route, 60-70% of the population is eventually infected.
50% of women of childbearing age are susceptible to infection.
Congenital Parvovirus Infection
Known to cause fetal loss through hydrops fetalis; severe anaemia, congestive heart failure, generalized oedema and fetal death
No evidence of teratogenecity.
Risk of fetal death highest when infection occurs during the second trimester of pregnancy (12%).
Minimal risk to the fetus if infection occurred during the first or third trimesters of pregnancy.
Maternal infection during pregnancy does not warrant termination of pregnancy.
Cases of diagnosed hydrops fetalis had been successfully treated in utero by intrauterine transfusions and administration of digoxin to the fetus.
Varicella-Zoster Virus
90% of pregnant women already immune, therefore primary infection is rare during pregnancy
Primary infection during pregnancy carries a greater risk of severe disease, in particular pneumonia
First 20 weeks of Pregnancy
up to 3% chance of transmission to the fetus,
recognised congenital varicella syndrome;
Scarring of skin
Hypoplasia of limbs
CNS and eye defects
Death in infancy normal
Neonatal Varicella
VZV can cross the placenta in the late stages of pregnancy to infect the fetus congenitally.
Neonatal varicella may vary from a mild disease to a fatal disseminated infection.
If rash in mother occurs more than 1 week before delivery, then sufficient immunity would have been transferred to the fetus.
Zoster immunoglobulin should be given to susceptible pregnant women who had contact with suspected cases of varicella.
Zoster immunoglobulin should also be given to infants whose mothers develop varicella during the last 7 days of pregnancy or the first 14 days after delivery.
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