Lectures in Clinical Microbiology: Commercial Kits for Identification of Gram negativ...

Lectures in Clinical Microbiology: Commercial Kits for Identification of Gram negativ...: All commercial identification systems are based on one of five different technologies or a combination thereof. These include pH-based react...

Commercial Kits for Identification of Gram negative bacilli

All commercial identification systems are based on one of five different technologies or a combination thereof. These include pH-based reactions that require from 15 to 24 h of incubation, enzyme-based reactions that require 2 to 4 h, utilization of carbon sources, visual detection of bacterial growth, or detection of volatile or nonvolatile fatty acids via gas chromatography (O’Hara,et al, 2003).

In pH-based reactions, a positive test is indicated by a change in the color of one or more dyes. When a carbohydrate is utilized, the pH becomes acidic; when protein is utilized or there is release of a nitrogen-containing compound, the pH becomes alkaline. These reactions are influenced by the inoculum size, incubation time, and temperature of the reaction. In enzyme-based system, color changes were due to the hydrolysis of a colorless complex by an appropriate enzyme with the resulting release of a chromogen or fluorogen. Because the incubation times needed for assay of enzymatic activities were shorter than those required for pH-based media, chance of contamination was not a critical factor (Caroline M and O’Hara., 2005).


In the third type of reaction, utilization of carbon sources, there is a transfer of electrons from an organic product to the dye tetrazolium violet, which is incorporated within each test well. That transfer causes a colorimetric change in the dye, signaling the increased cellular respiration that occurs during the oxidation process. These reactions may occur in as little as 4 h. The fourth method is a simple visual detection of growth of the test organism (increased turbidity) in the presence of a substrate. Results are determined by comparing a control well to the test well and may utilize a Wickerham card to read turbidity. This type of reaction may be difficult to read and always involves a minimum of overnight incubation. The last technology involves detecting the end products of cellular fatty acid metabolism. The end products are displayed on chromatographic tracings that are compared to a library of known patterns. This technology is not commonly used as it is more complex (Caroline M and O’Hara., 2005).

Manual Identification System

The studies cited are those which compared product identifications to identifications obtained by using conventional biochemicals. (Caroline M and O’Hara., 2005)

API 20E
In 1971, Washington et al. published the first evaluation of the API 20E, originally owned by the Analytab Products Division of American Home Products, which has been owned since 1986 by bioMérieux, Inc. (Durham, N.C.). In the evaluation by Washington et al., approximately 93.0% of the 129


Enterobacteriaceae and five Aeromonas strains were correctly identified to species level (Washington, et al ., 1971).

An impermeable plastic backing support 20 cupules that contain pH-based substrates that have not changed since the product was originally designed in 1970.The database has expanded from 87 taxa in 1977 to 102 taxa in 2003 and includes Y. pestis. The current database is version 4.0 (Caroline M and O’Hara., 2005).

API 20NE
Constructed along the same lines as the API 20E, the API 20NE (bioMe´rieux) has 20 cupules that contain 8 conventional substrates and 12 assimilation tests. Suspensions are prepared in 0.85% NaCl for inoculation into the 8 conventional substrates and in AUX medium for inoculation into the 12 assimilation cupules. The database contains 32 genera and 64 species of nonfastidious gram-negative rods not belonging to the Enterobacteriaceae. The seven-digit profile number is converted to an identification by using the APILAB software, version 3.3.3 (Caroline M and O’Hara., 2005).

API RapiD 20E
Originally marketed in 1982 as the Rapid E system (DMS Laboratories, Flemington, N.J.) but owned since 1986 by API and subsequently by bioMe´rieux Inc., the API RapiD 20E system is designed to identify Enterobacteriaceae in 4 h. Similar to the API 20E in its test configuration, this system has 20 microtubes that contain substrates for the demonstration of enzymatic activity or fermentation of carbohydrates. The seven-digit profile number that is compiled from the test reactions is entered into the APILAB software. The current version of the RapiD 20E software is 3.0. The database

contains 26 genera and 65 species. Identifications are also available by using the Analytical Profile Index (Caroline M and O’Hara., 2005).

Crystal E/NF
Introduced in 1993 by Becton Dickinson (Sparks, Md.), the BBL Crystal Enteric/Nonfermenter (E/NF) ID kit is for the identification of Enterobacteriaceae and more commonly isolated glucose-fermenting and nonfermenting gram-negative bacilli. The plastic panels include 30 tests for the fermentation, oxidation, degradation, or hydrolysis of various substrates. Once the panel is inoculated and snapped together with its lid, it becomes a sealed system posing little risk of exposure to the technologist. The current software version is 4.0 and contains 38 genera and 104 species (Caroline M and O’Hara., 2005).

Automated Identification Systems

With the advancement of automated testing in chemistry and hematology laboratories in the mid-1970s, it was only logical that some degree of automation in microbiology would eventually follow. (Caroline M, et al., 2005). From that, in 1973, the automicrobic system (AMS) (McDonnell Douglas Corp., St. Louis, Mo.) was born. It incorporated a disposable miniaturized plastic specimen-handling system, solid state optics for microbial detection, and a minicomputer for control and processing. Now it is recognized as the first generation of the Vitek instruments. (Aldridge, et al., 1977). Within 10 years, Vitek's competitors included the MS-2 (Abbott Diagnostics, Inc., Chicago, Ill.), the Autobac IDX (Pfizer Inc., Groton, Conn. And General Diagnostics, Morris Plains. N.J.), and the Autoscan-3 (Microscan Corp., Hillsdale, N.J.). Technology
had enabled valid results to be obtained in as little as 4 h. Microbiology was definitely on the fast track to rapid testing and shorter turnaround times (Caroline M and O’Hara., 2005).

Dade Behring MicroScan
In 1981, American MicroScan (then located in Hillsdale, N.J.) introduced the autoSCAN-3, a semiautomated instrument that utilized microdilution trays containing frozen conventional substrates for identification of bacterial isolates. An early evaluation, incorporating both Enterobacteriaceae and nonfermenters, by Ellner and Myers in 1981 reported an agreement of 95.0% between visually read and automated identification thus ensuring that machines were capable of accurate interpretations of the reactions in each well (Ellner, P. D., and D. A. Myers 1981). The company then introduced the autoSCAN-4 in 1983, which brought with it improved dry panels that did not require refrigeration and included an updated database. Baxter Healthcare Corporation and subsequently Dade Behring have owned the company since its move to West Sacramento, Calif. In 1986, the auto-SCAN-WalkAway came into the marketplace.

This instrument is a combination incubator-reader that monitors the growth in bacterial identification panels in a completely “hands-off” method. This instrument has become known as the WalkAway. One of MicroScan’s goals was to shorten the turnaround times for test results by using fluorogenic substrates in the panels.These “rapid” panels were first marketed in 1989. The data management system, called LabPro, runs on an adjacent computer (Caroline M and O’Hara., 2005).



Neg ID type 2. The Neg ID type 2 panel was introduced in 1988 for the identification to species level of aerobic and facultatively anaerobic gram-negative bacilli and was designed to be read either manually or on the WalkAway instrument. The clear plastic 96-well tray contains 26 conventional substrates and 6 antimicrobials for inhibition of growth, all in dried form, and requires overnight incubation (Caroline M and O’Hara., 2005).

Rapid Neg ID type 3. The Rapid Neg ID type 3 panel was introduced in approximately 1998 as an update of the Neg ID type 2 panel. The Rapid Neg ID type 3 replaced 10 of the substrates on the Neg ID type 2 panel with newer ones and eliminated the need for the mineral oil overlay on the decarboxylase test. It also increased the shelf life from 6 months to 1 year when stored at 2 to 8°C. The rapid panel utilizes 36 that work by one of the following mechanisms: hydrolysis of fluorogenic substrates, pH changes following substrate utilization, production of specific metabolic by-products, or evaluation of the rate of production of specific metabolic by-products after 2.5 h of incubation (O’Hara, et al, 2000). These panels can be processed only on a WalkAway instrument, as their opaque color prevents a visual read of the wells. The bacterial suspensions must be made from 18 to 24 h colonies grown on Mac-Conkey agar plates with lactose and crystal violet ( Caroline M and O’Hara., 2005).

The current database is LabPro 1.51, which contains 44 genera and 125 species of both Enterobacteriaceae and oxidase-positive glucose-fermenting and non-fermenting gram-negative bacilli. The database includes Y. pestis, V. cholerae, and E. coli O157:H7. There have been several reports indicating the usefulness and accuracy of direct bacterial identification with inocula from positive blood culture bottles. A study by Waites et al. indicated 99%

concordance between gram-negative identifications when blood was concentrated and the bacterial pellet was used to directly inoculate the panels and identifications resulting from standard biochemical methods (Waites, et al, 1998).

Cephalosporins

The first cephalosporins, cephalothin and cephaloridine, modestly expanded the spectrum of ampicillin and possessed relative stability to staphylococcal β-lactamase. Some later derivatives offered little, if any improvement, but Cephazolin exhibited the unusual characteristic of achieving enhanced concentrations in bile and Cephamandole offered partial resistance to some enterobacteria β-lactamases (Greenwood, 1995).

The first generation agents were active against Methicillin-susceptible S. aureus, Penicillin-susceptible S. pneumoniae and other common Gram positive bacteria as well as many community acquired Gram negative bacilli (Reese et al., 2000).

The second generation agent’s cephamandole and later cefuroxime were developed because of their activity against ampicillin-susceptible and resistant H. influenzae and many S. pneumoniae, while cefoxitin was the first cephalosporin to be active against anaerobes (Reese et al., 2000).

The third generation agents were initially touted as having enhanced Gram negative activity, including activity against hospital acquired pathogens and P. aeruginosa. In addition, the once daily ceftriaxone agent has significant Gram positive activity (Resse et al., 2000).

Cefepime has sometimes been called a fourth generation agent, but it
may be better summarized as a hybrid of cefotaxime and ceftazidime (Marshall and Blair, 1999).

The cephalosporins resemble the penicillins both in their structure and their mode of action. They inhibit mucopeptide synthesis, cause the accumulation of cell wall precursors, and the formation of morphologically aberrant forms of bacteria. They are generally resistant to the β-lactamases produced by most Gram-positive bacteria, although they are themselves effective inducers of these enzymes. Cephaloridine is, however, slowly hydrolysed by the penicillinase of s. aureus (Benner et al. 1965, Ridley and Phillips 1965).

Sepsis

Is systemic inflammatory response (SIR) to infection (Rackow EC., 1986). It represents progressive stages of the same illness in which a systemic response to an infection mediated by endogenous mediators may lead to a generalized inflammatory reaction in organs remote from the initial insult and eventually to end organ dysfunction and /or failure (Bone RB, et al., 1998). Sepsis remains an important and life-threatening problem as it is the most common cause of death in the intensive care unit (Parrillo JE, et al., 1990). Also it is possible that many deaths due to sepsis are attributed to underlying diseases when mortality statistics are complied (Young, L.s., 1990).

The use of the term sepsis is not restricted to a systemic inflammatory syndrome secondary to bacterial infection, but to this syndrome resulting from any microorganisms and/ or its products (toxins). The term sepsis is applicable only when the systemic response is clinically relevant, which can manifest in a variety of situations of increasing complexity such as : (a) severe sepsis, understood as sepsis associated with organ failure, hypoperfusion (which includes, but is not limited to lactic acidosis, oliguria or an acutely altered state of consciousness) and hypotension; (b) septic shock, understood as sepsis associated with hypoperfusion alterations, but with persistent hypotension even after suitable volumetric resuscitation, and (c) multiple organ failure syndrome (MOFS), which may represent the final stage of the sever systemic inflammatory response. However, the limits which separate sepsis from severe sepsis and this from septic shock are not easily detected in clinical at ICUs, or

even from a conceptual point of view. (Levy MM, et al, 2003; Despond O, et al, 2001).

Diagnosis of sepsis

Diagnosis of sepsis is based on a high level of suspicion, which demands a minutely detailed collection of information on present status and medical history of the patient, a good clinical evaluation, certain laboratory tests, in addition to rigorous clinical monitoring of the patient. There are three key difficulties associated with the diagnosis of infection in patients who have sepsis: і) establishing infection as the primary cause which is the first important step in the diagnosis, this will exclude non-infective causes of SIRS ((M.LIewelyn, et al., 2001). іі) localizing the site of infection: The identification of the primary site of infection is a critical part of the work-up of the septic patient. Together with the gram stain of specimens obtained from any site suspected of infection, it is probably the single most important information in guiding the choice of antibiotic therapy (Sands KE, et al., 1997; Boillot A, et al., 1995; Bernard GR, et al., 1997). ііі) interpreting the microbiological findings (M.LIewelyn, et al., 2001).

The International Sepsis Definitions Conference amplified the list of possible clinical and laboratory signs of sepsis which may allow for more efficacious suspicion and management. (Paulo R, et al., 2003). The symptoms and signs that should lead to suspect sepsis are as follow:

General variables:
- Fever (core temperature > 38.3 °C)

- Hypothermia (core temperature < 36 °C) - Heart rate > 90 min-1 or > 2 SD above the normal value for age
- Tachypnea
- Altered mental status
- Significant edema or positive fluid balance (> 20 ml/kg over 24 hrs)
- Hyperglycemia (plasma glucose > 120 mg/dl or 7.7 mmol/l) in the absence of diabetes

Inflammatory variables:
- Leukocytosis ( WBC count >12,000/mm3)
- Leukopenia (WBC count < 4,000/ mm3) - Normal WBC count with > 10 % immature forms
- Plasma C- reactive protein > 2 SD above the normal value
- Plasma procalcitonin > 2 SD above the normal value

Hemodynamic variables:
- Arterial hypotension (SBP < 90 mm Hg, MAP < 70, or an SBP decrease > 40 mm Hg in adults or <2 SD below normal for age) - Mixed venous oxygen saturation SvO2 > 70 %
- Cardiac index > 3.5 1/min-1/M -23

Organ dysfunction variables:
- Arterial hypoxemia (PaO2/FIO2< 300) - Acute oliguria (urine output < 0.5 ml/kg -1/hr -1 or 45 mmol/1 for at least 2 hrs) - Creatinine increase > 0.5 mg/dl
- Coagulation abnormalities ( INR >1.5 or aPTT > 60 secs)
- Ileus (absent bowel sounds)

- Thrombocytopenia (platelet count < 100,000/mm3) - Hyperbilirubinemia (plasma total bilirubin > 4 mg/dl or 70 mmol/l)

Tissue perfusion variables:
- hyperlactatemia (>1 mmol/l)
- Decreased capillary refill or mottling
(Levy MM, et al., 2003)

Causative organisms:

Sepsis and septic shock, caused by gram-negative, gram positive bacteria, fungi, viruses, and parasites, have become increasingly important over the past decades (Glauser, et al., 1991). In the United States, the septicemia rates more than doubled between 1979 and 1987 causing up to 250,000 deaths annually (Opal, et al., 1999 ; Parillo, et al., 1993). The proportion of infections due to gram-negative bacteria varied between 30 and 80% and that of infections due to gram-positive bacteria varied between 6 and 24% of the total number of cases of sepsis, with the remainder being accounted for by other pathogenic organisms (Glauser, et al., 1991).

Gram-negative sepsis

Was a relatively rare clinical diagnosis only a few decades ago, but today it is the most important infectious disease problem in hospitals. Nearly 80 % of all documented epidemics were caused by gram-negative bacilli. ( Roger C and Bone ., 1993). Estimated mortality from sepsis of gram-negative etiology ranges from 20 to 50 % of the overall total number of septic death. (Wenzel, R. P., 1988; Young, L. S., 1990).

Most gram negative infections were caused by Enterobacteriaceae with Escherichia coli which is the most commonly isolated pathogen, followed by klebseilla and enterobacter species. Although pseudomonas species were encountered somewhat less frequently, pseudomonas aeruginosa has consistently been associated with the highest mortality rate among all causes of bacteremic infection. (Young, L. S., 1990)

Applications of Molecular Diagnostic Identification of Pathogens

Molecular identification should be considered in three scenarios, namely (a) for the identification of an organism already isolated in pure culture, (b) for the rapid identification of an organism in a diagnostic setting from clinical specimens or (c) for the identification of an organism from non-culturable specimens, e.g. culture negative endocarditis (Millar et al., 2003).

Molecular Diagnosis of Viral Infections
The diagnosis of viral infections has been hampered for many years due to the cost, laboratory time and skilled personnel required for the cell culture systems used, together with the generally low sensitivity and slow growth of many viruse in artificial media (Takeuchi, Y et al., 2001)
Serology is often unhelpful in the early stages of infection, specific antisera for the serology tests can be difficult to obtain, and the clinical detection of antibodies is relatively insensitive for a number of viruses.
Recent advances in molecular biology have made possible the detection and characterization of viral nucleic acids. Methods such as PCR enable the amplification of specific regions of interest
The implementation of molecular methods has resulted in progress regarding the diagnosis of many viruses and the monitoring of antiviral therapy, especially HIV-1 (Human Immunodeficiency Virus type 1), HBV (Hepatitis B Virus) and HCV (Human Cytomegalovirus). It has also led to the development of amplification assays on nearly all human viruses, including those that are more easily be cultivated, such as HSV-1 and HSV-2 (Herpes Simple Virus type 1 and 2) (Espy, M.J et al., 2000)
1- Herpes simplex virus (HSV)
Encephalitis is a serious infection but diagnosis previously required brain biopsy in certain cases due to the low sensitivity of cerebrospinal fluid (CSF) culture and serology (Gilbert. GL, et al., 1999) PCR now allows the detection of HSV DNA from CSF with 95% sensitivity (Lakeman. FD et al., 1995) thus avoiding invasive brain biopsy.
Viral meningitis, commonly caused by either enteroviruses or HSV, is more reliably detected by PCR when compared to culture (van Vliet.KE et al., 1998) and in a shorter time (one versus up to five days). HSV PCR can be multiplexed with other pathogens responsible for meningitis (Read.SJ et al., 1997)
Genital ulceration due to HSV, usually due to HSV type 2 infection, is now routinely detected by PCR in many clinical microbiology laboratories due to its increased sensitivity over viral culture.
2-Blood borne virus
The detection of blood borne virus infection is also improved by both PCR and non-PCR molecular methods.
1- HCV
Active hepatitis C virus (HCV) infections are diagnosed by the presence of HCV RNA since the detection of antibody to HCV cannot distinguish between past and present infection.
In terms of infectiousness only those with detectable HCV RNA have a significant risk of transmitting HCV by transfusion, organ transplantation, needle-stick injury or vertically to the child (Dore.GJ et al., 1997)
2- HIV
Although infection with the human immunodeficiency virus (HIV) is routinely diagnosed by serology, early HIV infection can be detected by HIV pro-viral DNA detection before HIV antibodies are confirmed by Western Blot serology (Dax EM.2004) Vertical transmission of HIV infection is also detected in the infant using HIV pro-viral DNA detection (Luzuriaga. K, Sullivan.JL, 1994)
3-Intrauterine viral infection of the foetus
Intrauterine infection of the foetus with viruses e.g:
- cytomegalovirus (CMV) (Palasanthiran P, Jones C, Garland S, 2002)
- rubella (Nourse C. Rubella, 2002) and
- varicella zoster virus (Heuchan A, Isaacs D, 2002) can be detected by PCR testing of amniocentesis fluid.
4- Respiratory viral infection
Molecular detection of respiratory viral pathogens from both upper respiratory specimens such as nasopharyngeal aspirates or throat swabs and lower respiratory specimens such as sputum or bronchoalveolar lavage fluid is cost-effective due to the prevention of hospitalisation, decreasing unnecessary testing and procedures, directing specific therapy, and reducing unnecessary antibiotic use (Henrickson KJ, 2005).
Large multiplex or tandem PCR assays testing for all the common respiratory viruses along with fastidious bacterial causes of pneumonia are now feasible providing a thorough yet cost-effective alternative to conventional detection methods.
Uncommon yet significant respiratory viruses such as severe acute respiratory syndrome (SARS) -coronavirus (SARS-CoV) and -influenza A/H5N1 (avian influenza) virus can also be incorporated into these assays thus acting as an in-built early detection system.
- SARS-CoV
During the SARS epidemic due to the SARS-CoV, PCR testing of respiratory specimens for other respiratory viruses was crucial to exclude a number of suspected cases which fulfilled the case definition for SARS. PCR detection was most helpful due to the ability to rapidly screen for many respiratory viruses.
Subsequently a specific SARS CoV PCR has been developed for the early detection of SARS-CoV infection with a sensitivity of 50-87% early in the disease (Ng EK et al., 2003) Serology for SARS-CoV is up to 100% sensitive but of limited diagnostic value early in the disease when the risk of transmission is greatest (Ho PLet al., 2005)
-H5N1
The recent avian influenza (H5N1) outbreaks in South East Asia and beyond have also illustrated the need for rapid viral diagnosis. Molecular detection methods were developed following the 1997 Hong Kong outbreak (Yuen KY et al., 1998) and have the advantage of being rapid and able to be performed in many clinical microbiology laboratories. Specific serology needs live virus for the microneutralisation assay which is currently classed as a Biosafety Level 4 organism in Australia. Likewise direct immunofluorescence detection requires influenza type A/H5-specific monoclonal antibodies (. World Health Organisation 2005)
-H1N1
With the relative global lack of immunity to the pandemic influenza A/H1N1/2009 virus that emerged in April 2009 as well as the sustained susceptibility to infection, rapid and accurate diagnostic assays are essential to detect this novel influenza A variant.
Among the molecular diagnostic methods that have been developed to date, most are in tandem monoplex assays targeting either different regions of a single viral gene segment or different viral gene segments. We describe a dual-gene (duplex) quantitative real-time RT-PCR method selectively targeting pandemic influenza A/H1N1/2009 .(Lee HK et al.,2010)
A new nucleic acid amplification-based rapid test for diagnosis of pandemic influenza (H1N1) 2009 virus was developed. The molecular test for pandemic H1N1, SAMBA (simple amplification-based assay), is based on isothermal amplification and visual detection on a dipstick characterized by high sensitivity, high specificity, a short turnaround time, and minimal technical requirements.( Wu LT.,2010)
5- Viral diarrhoeal disease
Viruses cause more infectious diarrhoea worldwide than bacteria and other pathogens. The diagnosis of viral diarrhoeal disease has improved with the development of PCR detection. The method of choice for microbiological diagnosis of rotavirus from stool samples is PCR. Norovirus, a calicivirus formerly known as Norwalk virus and responsible for large outbreaks both in the community and health care facilities, can be diagnosed by electron microscopy, enzyme immunoassay and PCR but PCR is the most sensitive and rapid method. PCR is also the most sensitive method for the diagnosis of astroviruses and enteric adenoviruses (serotypes 40 and 41) (Clark B, McKendrick M.2004)

Nucleic acid analysis without Amplification (Nucleic acid probe technology) Part I

A nucleic acid probe is a labeled sequence of single stranded DNA or RNA that can hybridise specifically with its complementary sequence (Smith, 2002).
Nucleic acid hybridization technique
Fluorescent in situ hybridization (FISH) is a tool that today is widely used for identification, visualization and localization of microorganisms in many fields of microbiology (Thurnheer et al., 2004 & Amann et al., 2001).
FISH was mainly applied in connection with environmental samples during the first years but it became clear that the method also has advantages in diagnostic microbiology for rapid identification and direct visualization of bacteria (Kempf et al., 2000).
Nucleic acid hybridization is a technique was first described in 1961 by Marmur and Doty. Most molecular diagnostics testing procedures use the basic concept of nucleic acid hybridization.
Nucleic hybridization refer to formation of hydrogen bonds between nucleotides of single stranded DNA and/or RNA molecules that are complementary to each other (Marras et al., 2006). This form a stable double stranded nucleic acid molecule. The resulting double stranded hybrids may be DNA:DNA; DNA:RNA, or RNA:RNA.
This hybridization process called duplex formation. This process is key of component for many testes including blotting methods, PCR, and other molecular based techniques.
The two single stranded nucleic acid molecules used in hybridization techniques are referred to by different terms. One of the strands is known as the target.
The target strand is the DNA or RNA sequence that will be identified by the employed molecular diagnostics method. The target is referred to as the template it can be either immobilized on a solid support mechanism or is suspended in solution.The other strand is called the probe (Jensen TK, et al., 2001) (see figure, 4)
The probe is usually a single stranded DNA or RNA oligonucleotide that is labeled with an attached reporter chemical or a radionucleotide that can be detected either visually, by film, or by an instrument. The probe is produced synthetically to detect a specific target (Mothershed E.A., Whitney A.M., 2006)
Hybridization reaction variables
Several variables affect the outcome of a given hybridization reaction. These variable include;
-Temperature
The stability of a given hybrid can be calculated by determining the melting temperature (™) of a probe. The Tm is the temperature at which 50% of hybrids have formed and 50% of the single stranded nucleic acid molecules are still dissociated. Tm is dependent on the G+C ratio because three hydrogen bonds form between G and C, instead of the two hydrogen bonds that form between adenine (A) and (T);The G\C bond pair is more thermodynamically stable than the A\T bond pair.
-Length of the probe
Another aspect that affects the Tm is the length of the probe ; in general the Tm is lower for a shorter probe.Hybridization reactions tend to occur more rapidly for shorter probes than for longer probes
-Probe Concentration
Higher probe concentrations typically lower the reaction time by saturating all of the available probe target sequences. However, excessive probe concentrations promote nonspecific binding of the probe to non target sequences
-Salt concentration
The rate of a hybridization reaction will increase as the salt concentration increases, up to a threshold; past 1.2 M Nacl, the rate of the reaction become constant.
-PH
Neutral PH is preferable for most hybridization reactions
-Probe selection
Selection of proper probe for nucleic acid hybridization reaction is important as the hybridization method itself. probes may be either DNA or RNA based, and are either radiolabeled or non isotopically labeled.
Radiolabeled probes are rarely used due to have short half lives and un desirable waste.radiolabeled probes have been replaced by nonisotopic labels, including biotin digoxigenin (DIG), and fluorescein, nonisotopic labels have resolution and sensitivity that approache .A probe may also be end labeled or continuously labeled
Hybridization Formats
Hybridization reaction may occur in a solid support mechanism, in situ or in solution
A-Solid support hybridization
Technique often called (blotting) the target nucleic acid is transferred and immobilized to a membrane, composed of either nitrocellulose or nylon. Labeled probe is then hybridized to the immobilized nucleic acid washing steps are used to remove excess probe. Two examples of solid support hybridization techniques are:
1-Southern Blot
The southern blot was first described in 1975 by E.M southern; he described a technique whereby chromosomal DNA has digested with a restriction enzyme then separated by agarose gel electrophoresis, then transferred and immobilized to a nitro cellulose membrane, then labeled probe is hybridized to the specific target DNA sequences. [Figure, 5]The southern blot takes more than one day to perform, it can be used to identified micro organism, to detect mutation, to type strains for epidemiological investigation, and for other purposes (Vanrompay.d., 2000)

2- Northern Blot
A northern blot was first described by Alwine et al 1977; northern blot used to determine the size of particular RNA transcript. It has the same procedure of the southern blot but with difference, the restriction enzyme is not used to digest RNA before separation due to RNA is small enough to be separated by agaros gel electrophoresis. Like southern blotting, northern blotting is not often used in clinical microbiology laboratories (see figure, 6)

3-In Situ Hybridization
In Situ Hybridization (ISH) first described in 1969 by Pardue and Gall, is also not used in clinical microbiology laboratories.This is a method of hybridization wherein DNA or RNA transcript can be detected directly in the tissue with labeled probes.ISH may used to detected low level of viruses in tissue specimen such as human papillomaviruses (HPV) (Vanrompay.d., 2000)
B-In Solution Hybridization
In solution hybridization is the type of hybridization reaction most often used by clinical microbiology laboratories.Hybridization between a labeled probe and target nucleic acids in a liquid solution in tubes or in microtiter wells, usually detection methods are chemiluminescent based. It used to rapidly identify infectious disease organisms.

Nucleic acid analysis without Amplification (Nucleic acid probe technology) Part I

A nucleic acid probe is a labeled sequence of single stranded DNA or RNA that can hybridise specifically with its complementary sequence (Smith, 2002).
Nucleic acid hybridization technique
Fluorescent in situ hybridization (FISH) is a tool that today is widely used for identification, visualization and localization of microorganisms in many fields of microbiology (Thurnheer et al., 2004 & Amann et al., 2001).
FISH was mainly applied in connection with environmental samples during the first years but it became clear that the method also has advantages in diagnostic microbiology for rapid identification and direct visualization of bacteria (Kempf et al., 2000).
Nucleic acid hybridization is a technique was first described in 1961 by Marmur and Doty. Most molecular diagnostics testing procedures use the basic concept of nucleic acid hybridization.
Nucleic hybridization refer to formation of hydrogen bonds between nucleotides of single stranded DNA and/or RNA molecules that are complementary to each other (Marras et al., 2006). This form a stable double stranded nucleic acid molecule. The resulting double stranded hybrids may be DNA:DNA; DNA:RNA, or RNA:RNA.
This hybridization process called duplex formation. This process is key of component for many testes including blotting methods, PCR, and other molecular based techniques.
The two single stranded nucleic acid molecules used in hybridization techniques are referred to by different terms. One of the strands is known as the target.
The target strand is the DNA or RNA sequence that will be identified by the employed molecular diagnostics method. The target is referred to as the template it can be either immobilized on a solid support mechanism or is suspended in solution.The other strand is called the probe (Jensen TK, et al., 2001) (see figure, 4)
The probe is usually a single stranded DNA or RNA oligonucleotide that is labeled with an attached reporter chemical or a radionucleotide that can be detected either visually, by film, or by an instrument. The probe is produced synthetically to detect a specific target (Mothershed E.A., Whitney A.M., 2006)
Hybridization reaction variables
Several variables affect the outcome of a given hybridization reaction. These variable include;
-Temperature
The stability of a given hybrid can be calculated by determining the melting temperature (™) of a probe. The Tm is the temperature at which 50% of hybrids have formed and 50% of the single stranded nucleic acid molecules are still dissociated. Tm is dependent on the G+C ratio because three hydrogen bonds form between G and C, instead of the two hydrogen bonds that form between adenine (A) and (T);The G\C bond pair is more thermodynamically stable than the A\T bond pair.
-Length of the probe
Another aspect that affects the Tm is the length of the probe ; in general the Tm is lower for a shorter probe.Hybridization reactions tend to occur more rapidly for shorter probes than for longer probes
-Probe Concentration
Higher probe concentrations typically lower the reaction time by saturating all of the available probe target sequences. However, excessive probe concentrations promote nonspecific binding of the probe to non target sequences
-Salt concentration
The rate of a hybridization reaction will increase as the salt concentration increases, up to a threshold; past 1.2 M Nacl, the rate of the reaction become constant.
-PH
Neutral PH is preferable for most hybridization reactions
-Probe selection
Selection of proper probe for nucleic acid hybridization reaction is important as the hybridization method itself. probes may be either DNA or RNA based, and are either radiolabeled or non isotopically labeled.
Radiolabeled probes are rarely used due to have short half lives and un desirable waste.radiolabeled probes have been replaced by nonisotopic labels, including biotin digoxigenin (DIG), and fluorescein, nonisotopic labels have resolution and sensitivity that approache .A probe may also be end labeled or continuously labeled
Hybridization Formats
Hybridization reaction may occur in a solid support mechanism, in situ or in solution
A-Solid support hybridization
Technique often called (blotting) the target nucleic acid is transferred and immobilized to a membrane, composed of either nitrocellulose or nylon. Labeled probe is then hybridized to the immobilized nucleic acid washing steps are used to remove excess probe. Two examples of solid support hybridization techniques are:
1-Southern Blot
The southern blot was first described in 1975 by E.M southern; he described a technique whereby chromosomal DNA has digested with a restriction enzyme then separated by agarose gel electrophoresis, then transferred and immobilized to a nitro cellulose membrane, then labeled probe is hybridized to the specific target DNA sequences. [Figure, 5]The southern blot takes more than one day to perform, it can be used to identified micro organism, to detect mutation, to type strains for epidemiological investigation, and for other purposes (Vanrompay.d., 2000)

2- Northern Blot
A northern blot was first described by Alwine et al 1977; northern blot used to determine the size of particular RNA transcript. It has the same procedure of the southern blot but with difference, the restriction enzyme is not used to digest RNA before separation due to RNA is small enough to be separated by agaros gel electrophoresis. Like southern blotting, northern blotting is not often used in clinical microbiology laboratories (see figure, 6)

3-In Situ Hybridization
In Situ Hybridization (ISH) first described in 1969 by Pardue and Gall, is also not used in clinical microbiology laboratories.This is a method of hybridization wherein DNA or RNA transcript can be detected directly in the tissue with labeled probes.ISH may used to detected low level of viruses in tissue specimen such as human papillomaviruses (HPV) (Vanrompay.d., 2000)
B-In Solution Hybridization
In solution hybridization is the type of hybridization reaction most often used by clinical microbiology laboratories.Hybridization between a labeled probe and target nucleic acids in a liquid solution in tubes or in microtiter wells, usually detection methods are chemiluminescent based. It used to rapidly identify infectious disease organisms.

Innate Immune Response to Pathogens and Recent Advances in Microbiology Researches

An immune system is a system of biological structures and processes within an organism that protects against disease by identifying and killing pathogens and tumor cells. It detects a wide variety of agents, from viruses to parasitic worms, and needs to distinguish them from the organism's own healthy cells and tissues in order to function properly. Detection is complicated as pathogens can evolve rapidly, and adapt to avoid the immune system and allow the pathogens to successfully infect their hosts.
To survive this challenge, multiple mechanisms evolved that recognize and neutralize pathogens. Even simple unicellular organisms such as bacteria possess enzyme systems that protect against viral infections. Other basic immune mechanisms evolved in ancient eukaryotes and remain in their modern descendants, such as plants and insects. These mechanisms include antimicrobial peptides called defensins, phagocytosis, and the complement system. Jawed vertebrates, including humans, have even more sophisticated defense mechanisms. The typical vertebrate immune system consists of many types of proteins, cells, organs, and tissues that interact in an elaborate and dynamic network. As part of this more complex immune response, the human immune system adapts over time to recognize specific pathogens more efficiently. This adaptation process is referred to as "adaptive immunity" or "acquired immunity" and creates immunological memory. The following chapters will discuss various mechanisms of immune response to pathogens based on laboratory evidence studies.
Innate Immune Response to Pathogens and Recent Advances in Microbiology Researches [Kindle Edition]

Chapter One: Immune response to Pathogen
Chapter Two: Interleukin 6 response in Children with Echerichia coli 6 associated Urinary tract Infection, One centre Study.
Chapter Three: Natural Killer Activity and Mycobacterium tuberculosis Infection
Chapter Four: Study of Interleukin 12-Gamma interferon Axis and Natural Killer Cells in Acute Viralhepatitis
Chapter Five: Study of antioxidants' Enzymes Kinetics, Superoxide

dismutase, Glutathione peroxidase and Catalase, in Neonatal Sepsis
Chapter Six: TNFα and IFNγ, EARLY KINETICS DURING FEVER IN 64 PATIENTS WITH HAEMATOLOGICAL MALIGNANCIES AND NEUTROPENIA

Nosocomial pneumonia

Ventilator-associated pneumonia (VAP) is the most common intensive care unit (ICU)-acquired infection. VAP prevalence varies from 8 to 28 cases per 100 patients depending on population studied, type of ICU and diagnostic criteria used (Meyer et al., 2009).
Patients who experience delayed initiation of appropriate antibiotic therapy for suspicion of VAP and those who receive empirical antibiotic treatment but are subsequently found to be infected with an antibiotic-resistant organism have a higher mortality than patients who receive timely appropriate initial antibiotics (Iregui et al., 2002).
Bacteria most frequently isolated from hospital acquired pneumonia are: Pseudomonas species (41%), Klebsiella species (12.7%) and Escherichia coli (12%) (Pugh et al., 2010).

HAIs and resistant superbugs

Superbugs are common microbes that have been genetically modified to become multiple-drug-resistant strains. Bacteria have developed resistance to almost all existing antibiotics known today and this has been a major issue over the last few decades (Palanychamay and Kaliappan, 2010).
The prevalence of superbugs, such as vancomycin-resistant Staphylococcus aureus (VRSA) and methicillin-resistant Staphylococcus aureus (MRSA), is increasing at a rapid rate both in the hospital sector and in the community. Surveys revealed that resistance rates were higher in bacterial isolates derived from in-patients when compared with those from out-patients or from general practice (Shannon and French, 2004).
Multidrug resistance is worrisome since it corresponds to the addition of unrelated mechanisms of resistance that are difficult if not impossible to reverse once gathered in single genetic resistance structure (transposon, integron, plasmid), these latter structures contributing to co-selection of resistances (Nordmann et al., 2007).
The great genetic plasticity of bacteria has permitted the transfer of resistance genes on plasmids and integrons between bacterial species allowing an unprecedented dissemination of genes leading to broad-spectrum resistance (Gootz, 2006).
Recent evidence suggests that antibiotic resistance genes in human bacterial pathogens originate from a multitude of bacterial sources, indicating that the genomes of all bacteria can be considered as a single global gene pool into which most, if not all, bacteria can dip for genes necessary for survival (Bennett, 2008).
Causes of superbugs prevalence in HAIS:
It is notable that the majority of infection control problems in the hospital are due to increasingly resistant bacteria. It is important to reflect on the role that antibiotic use may have as a selecting force for evolution of these superbugs. Selecting force of antibiotics, combined with lapses in infection control techniques make from these resistant organisms resident flora in hospitals and lead to their spread from person to person (Gould, 2009).
A-Selective pressure of antibiotic use:
Current policies to shorten length of stay and curtail costs encourage empiric use, often of unnecessarily broad-spectrum antibiotics. Combination therapy is often used for a number of reasons including broadening spectrum to accommodate increasing antibiotic resistance. This over-use and sometimes misuse inevitably leads to evolution and spread of superbugs (Gould, 2009).
Evolution of antibiotic resistance is the result of two essential forces: variability (chance) and selection (necessity). Variability is created by random mutation; variants with a mutation in the antibiotic target become resistant. These variants are selected by antibiotic use and consequently they increase the frequency of resistance. If the variability (as in a hyper-mutable strain) increased or the intensity of selection (antibiotic hyper-consumption) increased, the result is more resistance (Baquero and Cantón, 2009).
Manual of Antibiotics: Method of Actions, Mechanisms of Resistance and Relations to Health Care associated Infections [Kindle Edition]

Antiviral Drugs: Introduction

Antiviral Drugs: Introduction

Compared with the number of drugs available to treat bacterial infections, the number of antiviral drugs is very small. The major reason for this difference is the difficulty in obtaining selective toxicity against viruses; their replication is intimately involved with the normal synthetic processes of the cell. Despite the difficulty, several virus-specific replication steps have been identified that are the site of action of effective antiviral drugs
Another limitation of antiviral drugs is that they are relatively ineffective because many cycles of viral replication occur during the incubation period when the patient is well. By the time the patient has a recognizable systemic viral disease, the virus has spread throughout the body and it is too late to interdict it. Furthermore, some viruses, e.g., herpesviruses, become latent within cells, and no current antiviral drug can eradicate them.

Another potential limiting factor is the emergence of drug-resistant viral mutants. At present, this is not of major clinical significance. Mutants of herpesvirus resistant to acyclovir have been recovered from patients, but they do not interfere with recovery.

Inhibition of Early Events

Amantadine (-adamantanamine, Symmetrel) is a three-ring compound (Figure 35–1) that blocks the replication of influenza A virus. It prevents replication by inhibiting uncoating of the virus by blocking the "ion channel" activity of the matrix protein (M2 protein) in the virion. Absorption and penetration occur normally, but transcription by the virion RNA polymerase does not because uncoating cannot occur. This drug specifically inhibits influenza A virus; influenza B and C viruses are not affected.
Despite its efficacy in preventing influenza, it is not widely used in the United States because the vaccine is preferred for the high-risk population. Furthermore, most isolates have become resistant to amantadine. The main side effects of amantadine are central nervous system alterations such as dizziness, ataxia, and insomnia. Rimantadine (Flumadine) is a derivative of amantadine and has the same mode of action but fewer side effects.

Enfuvirtide (Fuzeon) is a synthetic peptide that binds to gp41 on the surface of the virion, thereby blocking the entry of human immunodeficiency virus (HIV) into the cell. It is the first of a new class of anti-HIV drugs known as "fusion inhibitors," i.e., they prevent the fusion of the viral envelope with the cell membrane.

Maraviroc (Selzentry) blocks the binding of HIV to CCR-5—an important coreceptor for those strains of HIV that use CCR-5 for entry into the cell. The drug binds to CCR-5 and blocks the interaction of gp120, an HIV envelope protein, to CCR-5 on the cell surface.

Inhibition of Viral Nucleic Acid Synthesis

Inhibitors of Herpesviruses

Nucleoside Inhibitors

These drugs are analogues of nucleosides that inhibit the DNA polymerase of one or more members of the herpesvirus family. For example, acyclovir inhibits the DNA polymerase herpes simplex virus types 1 and 2 (HSV-1 and -2) and varicella-zoster virus but not cytomegalovirus (CMV).

Acyclovir—Acyclovir (acycloguanosine, Zovirax) is a guanosine analogue that has a three-carbon fragment in place of the normal sugar, ribose, that has five carbons (Figure 35–1). The term "acyclo" refers to the fact that the three-carbon fragment does not have a sugar ring structure (a = without, cyclo = ring).

Acyclovir is active primarily against HSV-1 and -2 and varicella-zoster virus (VZV). It is relatively nontoxic, because it is activated preferentially within virus-infected cells. This is due to the virus-encoded thymidine kinase, which phosphorylates acyclovir much more effectively than does the cellular thymidine kinase. Because only HSV-1, HSV-2, and VZV encode a kinase that efficiently phosphorylates acyclovir, the drug is active primarily against these viruses. It has no activity against CMV. Once the drug is phosphorylated to acyclovir monophosphate by the viral thymidine kinase, cellular kinases synthesize acyclovir triphosphate, which inhibits viral DNA polymerase much more effectively than it inhibits cellular DNA polymerase. Acyclovir causes chain termination because it lacks a hydroxyl group in the 3' position.

To recap, the selective action of acyclovir is based on two features of the drug. (1) Acyclovir is phosphorylated to acyclovir monophosphate much more effectively by herpesvirus-encoded thymidine kinase than by cellular thymidine kinase. It is therefore preferentially activated in herpesvirus-infected cells and much less so in uninfected cells, which accounts for its relatively few side effects. (2) Acyclovir triphosphate inhibits herpesvirus-encoded DNA polymerase much more effectively than it does cellular DNA polymerase. It therefore inhibits viral DNA synthesis to a much greater extent than cellular DNA synthesis (Figure 35–2).

Topical acyclovir is effective in the treatment of primary genital herpes and reduces the frequency of recurrences while it is being taken. However, it has no effect on latency or on the rate of recurrences after treatment is stopped. Acyclovir is the treatment of choice for HSV-1 encephalitis and is effective in preventing systemic infection by HSV-1 or VZV in immunocompromised patients. It is not effective treatment for HSV-1 recurrent lesions in immunocompetent hosts.

Acyclovir-resistant mutants have been isolated from HSV-1- and VZV-infected patients. Resistance is most often due to mutations in the gene encoding the viral thymidine kinase. This results in reduced activity of or the total absence of the virus-encoded thymidine kinase.

Acyclovir is well-tolerated and causes few side effects—even in patients who have taken it orally for many years to suppress genital herpes. Intravenous acyclovir may cause renal or central nervous system toxicity.

Derivatives of acyclovir with various properties are now available. Valacyclovir (Valtrex) achieves a high plasma concentration when taken orally and is used in herpes genitalis and in herpes zoster. Penciclovir cream (Denavir) is used in the treatment of recurrent orolabial herpes simplex. Famciclovir (Famvir) when taken orally is converted to penciclovir and is used to treat herpes zoster and in herpes simplex infections.

Ganciclovir—Ganciclovir (dihydroxypropoxymethylguanine, DHPG, Cytovene) is a nucleoside analogue of guanosine with a four-carbon fragment in place of the normal sugar, ribose (Figure 35–1). It is structurally similar to acyclovir but is more active against CMV than is acyclovir. Ganciclovir is activated by a CMV-encoded phosphokinase in a process similar to that by which HSV activates acyclovir. Isolates of CMV resistant to ganciclovir have emerged, mostly due to mutations in the UL97 gene that encodes the phosphokinase.

Ganciclovir is effective in the treatment of retinitis caused by CMV in AIDS patients and may be useful in other disseminated infections, such as colitis and esophagitis, caused by this virus. The main side effects of ganciclovir are leukopenia and thrombocytopenia as a result of bone marrow suppression. Valganciclovir, which can be taken orally, is also effective against CMV retinitis.

Cidofovir—Cidofovir (hydroxyphosphonylmethoxypropylcytosine, HPMPC, Vistide) is a nucleoside analogue of cytosine that lacks a ribose ring. It is useful in the treatment of retinitis caused by CMV and in severe human papillomavirus infections. It may be useful in the treatment of severe molluscum contagiosum in immunocompromised patients. Kidney damage is the main side effect.

Vidarabine—Vidarabine (adenine arabinoside, ara-A) is a nucleoside analogue with arabinose in place of the normal sugar, ribose. On entering the cell, the drug is phosphorylated by cellular kinases to the triphosphate, which inhibits the herpesvirus-encoded DNA polymerase more effectively than the cellular DNA polymerase. Vidarabine is effective against HSV-1 infections such as encephalitis and keratitis but is less effective and more toxic than acyclovir.

Iododeoxyuridine—Iododeoxyuridine (idoxuridine, IDU, IUDR) is a nucleoside analogue in which the methyl group of thymidine is replaced by an iodine atom (Figure 35–1). The drug is phosphorylated to the triphosphate by cellular kinases and incorporated into DNA. Because IDU has a high frequency of mismatched pairing to guanine, it causes the formation of faulty progeny DNA and mRNA. However, because IDU is incorporated into normal cell DNA as well as viral DNA, it is too toxic to be used systemically. It is clinically useful in the topical treatment of keratoconjunctivitis caused by herpes simplex virus, but in the United States, trifluorothymidine (see below) is the drug of choice.

Trifluorothymidine—Trifluorothymidine (trifluridine, Viroptic) is a nucleoside analogue in which the methyl group of thymidine contains three fluorine atoms instead of three hydrogen atoms. Its mechanism of action is probably similar to that of IDU. Like IDU, it is too toxic for systemic use but is clinically useful in the topical treatment of keratoconjunctivitis caused by herpes simplex virus.
Nonnucleoside Inhibitors

These drugs inhibit the DNA polymerase of herpesviruses by mechanisms distinct from the nucleoside analogues described above. Foscarnet is the only approved drug in this class at this time.

Foscarnet—Foscarnet (trisodium phosphonoformate, Foscavir), unlike the previous drugs, which are nucleoside analogues, is a pyrophosphate analogue. It binds to DNA polymerase at the pyrophosphate cleavage site and prevents removal of the phosphates from nucleoside triphosphates (dNTP). This inhibits the addition of the next dNTP and, as a consequence, the extension of the DNA strand. Foscarnet inhibits the DNA polymerases of all herpesviruses, especially HSV and CMV. Unlike acyclovir, it does not require activation by thymidine kinase. Foscarnet also inhibits the reverse transcriptase of HIV. It is useful in the treatment of retinitis caused by CMV, but ganciclovir is the treatment of first choice for this disease. Foscarnet is also used to treat patients infected with acyclovir-resistant mutants of HSV-1 and VZV.

Inhibitors of Retroviruses

Nucleoside Inhibitors

The selective toxicity of azidothymidine, dideoxyinosine, dideoxycytidine, d4T, and 3TC is based on their ability to inhibit DNA synthesis by the reverse transcriptase of HIV to a much greater extent than they inhibit DNA synthesis by the DNA polymerase in human cells. The effect of these drugs on the replication of HIV is depicted in Figure 45–3.

Azidothymidine—Azidothymidine (zidovudine, Retrovir, AZT) is a nucleoside analogue that causes chain termination during DNA synthesis; it has an azido group in place of the hydroxyl group on the ribose (Figure 35–1). It is particularly effective against DNA synthesis by the reverse transcriptase of HIV and inhibits the growth of the virus in cell culture. The main side effects of AZT are bone marrow suppression and myopathy.

Dideoxyinosine—Dideoxyinosine (didanosine, Videx, ddI) is a nucleoside analogue that causes chain termination during DNA synthesis; it is missing hydroxyl groups on the ribose. The administered drug ddI is metabolized to ddATP, which is the active compound. It is effective against DNA synthesis by the reverse transcriptase of HIV and is used to treat patients with AIDS who are intolerant of or resistant to AZT. The main side effects of ddI are pancreatitis and peripheral neuropathy.

Dideoxycytidine—Dideoxycytidine (zalcitabine, Hivid, ddC) is a nucleoside analogue that causes chain termination during DNA synthesis; it is missing hydroxyl groups on the ribose. The administered drug ddC is metabolized to ddCTP, which is the active compound. It is effective against DNA synthesis by the reverse transcriptase of HIV and is used to treat patients who are intolerant of or resistant to AZT. The main side effects of ddC are the same as those of ddI but occur less often.

Stavudine—Stavudine (d4T, Zerit) is a nucleoside analogue that causes chain termination during DNA synthesis. It inhibits DNA synthesis by the reverse transcriptase of HIV and is used to treat patients with advanced AIDS who are intolerant of or resistant to other approved therapies. The molecular name of stavudine is didehydrodideoxythymidine. The main side effect is peripheral neuropathy.

Lamivudine—Lamivudine (3TC, Epivir) is a nucleoside analogue that causes chain termination during DNA synthesis by the reverse transcriptase of HIV. When used in combination with AZT, it is very effective both in reducing the viral load and in elevating the CD4 cell count. The molecular name of lamivudine is dideoxythiacytidine. Lamivudine is also used in the treatment of chronic hepatitis B. It is one of the best-tolerated of the nucleoside inhibitors, but side effects such as neutropenia, pancreatitis, and peripheral neuropathy do occur.

Abacavir—Abacavir (Ziagen) is a nucleoside analogue of guanosine that causes chain termination during DNA synthesis. It is available through the "expanded access" program to those who have failed currently available drug regimens. Abacavir is used in combination with either a protease inhibitor or AZT plus lamivudine.

Tenofovir—Tenofovir (Viread) is an acyclic nucleoside phosphonate that is an analogue of adenosine monophosphate. It is a reverse transcriptase inhibitor that acts by chain termination. It is approved for use in patients who have developed resistance to other reverse transcriptase inhibitors and in those who are starting treatment for the first time. It should be used in combination with other anti-HIV drugs.

Nonnucleoside Inhibitors

Unlike the drugs described above, the drugs in this group are not nucleoside analogues and do not cause chain termination. The nonnucleoside reverse transcriptase inhibitors (NNRTI) act by binding near the active site of the reverse transcriptase and inducing a conformational change that inhibits the synthesis of viral DNA. NNRTIs should not be used as monotherapy because resistant mutants emerge rapidly. Strains of HIV resistant to one NNRTI are usually resistant to others as well. NNRTIs are typically used in combination with one or two nucleoside analogues.

Nevirapine—Nevirapine (Viramune) is usually used in combination with zidovudine and didanosine. There is no cross-resistance with the nucleoside inhibitors of reverse transcriptase described above. The main side effect of nevirapine is a severe skin rash (Stevens-Johnson syndrome). Nevirapine is a member of a class of drugs called the dipyridodiazepinones; its precise name is beyond the scope of this book.

Delavirdine—Delavirdine (Rescriptor) is effective in combination with either zidovudine or zidovudine plus didanosine. Delavirdine is a member of a class of drugs called bisheteroarylpiperazines; its precise name is beyond the scope of this book.

Efavirenz—Efavirenz (Sustiva) in combination with zidovudine plus lamivudine was more effective and better tolerated than the combination of indinavir, zidovudine, and lamivudine. The most common side effects are referable to the central nervous system, such as dizziness, insomnia, and headaches. Efavirenz is a member of a class of drugs called benzoxazin-2-ones; its precise name is beyond the scope of this book.

Inhibitors of Hepatitis B Virus

Adefovir

Adefovir (Hepsera) is a nucleotide analogue of adenosine monophosphate that inhibits the DNA polymerase of hepatitis B virus. It is used for the treatment of chronic active hepatitis caused by this virus.

Entecavir

Entecavir (Baraclude) is a guanosine analogue that inhibits the DNA polymerase of hepatitis B virus (HBV). It has no activity against the DNA polymerase (reverse transcriptase) of HIV. It is approved for the treatment of adults with chronic HBV infection.

Lamivudine

Lamivudine is described under section "Inhibitors of Retroviruses."

Telbivudine

Telbivudine (Tyzeka) is a thymidine analogue that inhibits the DNA polymerase of HBV but has no effect on the reverse transcriptase of HIV. It is useful in the treatment of chronic HBV infection.

Inhibitors of Other Viruses

Ribavirin

Ribavirin (Virazole) is a nucleoside analogue in which a triazole-carboxamide moiety is substituted in place of the normal purine precursor aminoimidazole-carboxamide (Figure 35–1). The drug inhibits the synthesis of guanine nucleotides, which are essential for both DNA and RNA viruses. It also inhibits the 5' capping of viral mRNA. Ribavirin aerosol is used clinically to treat pneumonitis caused by respiratory syncytial virus in infants and to treat severe influenza B infections. Ribavirin is also used in combination with -interferon (peg-interferon) for the treatment of hepatitis C.

Inhibition of Integrase

Raltegravir (Isentress) is an integrase inhibitor, i.e., it blocks the HIV-encoded integrase that mediates the integration of the newly synthesized viral DNA into host cell DNA.

Inhibition of Cleavage of Precursor Polypeptides

Members of the protease inhibitor (PI) class of drugs, such as saquinavir (Invirase, Fortovase), indinavir (Crixivan), ritonavir (Norvir), lopinavir/ritonavir (Kaletra), azatanavir (Reyataz), tipranavir (Aptivus), amprenavir (Agenerase), darunavir (Prezista), and nelfinavir (Viracept) inhibit the protease encoded by HIV (Figure 35–3). The protease cleaves the gag and pol precursor polypeptides to produce several nucleocapsid proteins and enzymatic proteins, e.g., reverse transcriptase, required for viral replication. These inhibitors contain peptide bonds that bind to the active site of the viral protease, thereby preventing the protease from cleaving the viral precursor. These drugs inhibit production of infectious virions but do not affect the proviral DNA and therefore do not cure the infection. The effect of protease inhibitors on the replication of HIV is depicted in Figure 45–3.

The Normal Flora

The Normal Flora

In a healthy human, the internal tissues, e.g. blood, brain, muscle, etc., are normally free of microorganisms. However, the surface tissues, i.e., skin and mucous membranes, are constantly in contact with environmental organisms and become readily colonized by various microbial species. The mixture of organisms regularly found at any anatomical site is referred to as the normal flora, except by researchers in the field who prefer the term "indigenous microbiota". The normal flora of humans consists of a few eucaryotic fungi and protists, but bacteria are the most numerous and obvious microbial components of the normal flora.

(1) The staphylococci and corynebacteria occur at every site listed. Staphylococcus epidermidis is highly adapted to the diverse environments of its human host. S. aureus is a potential pathogen. It is a leading cause of bacterial disease in humans. It can be transmitted from the nasal membranes of an asymptomatic carrier to a susceptible host.

S. epidermidis. Scanning EM. CDC.
(2) Many of the normal flora are either pathogens or opportunistic pathogens, The asterisks indicate members of the normal flora a that may be considered major pathogens of humans.

S. aureus. Gram stain.

(3) Streptococcus mutans is the primary bacterium involved in plaque formation and initiation of dental caries. Viewed as an opportunistic infection, dental disease is one of the most prevalent and costly infectious diseases in the United States.

Streptococcus mutans. Gram stain. CDC

(4) Enterococcus faecalis was formerly classified as Streptococcus faecalis. The bacterium is such a regular a component of the intestinal flora, that many European countries use it as the standard indicator of fecal pollution, in the same way we use E. coli in the U.S. In recent years, Enterococcus faecalis has emerged as a significant, antibiotic-resistant, nosocomial pathogen.

Vancomycin Resistant Enterococcus faecalis. Scanning E.M. CDC

(5) Streptococcus pneumoniae is present in the upper respiratory tract of about half the population. If it invades the lower respiratory tract it can cause pneumonia. Streptococcus pneumoniae causes 95 percent of all bacterial pneumonia.

Streptococcus pneumoniae. Direct fluorescent antibody stain. CDC.

(6) Streptococcus pyogenes refers to the Group A, Beta-hemolytic streptococci. Streptococci cause tonsillitis (strep throat), pneumonia, endocarditis. Some streptococcal diseases can lead to rheumatic fever or nephritis which can damage the heart and kidney.

Streptococcus pyogenes. Gram stain.

(7) Neisseria and other Gram-negative cocci are frequent inhabitants of the upper respiratory tract, mainly the pharynx. Neisseria meningitidis, an important cause of bacterial meningitis, can colonize as well, until the host can develop active immunity against the pathogen.

Neisseria meningitidis. Gram stain.

(8) While E. coli is a consistent resident of the small intestine, many other enteric bacteria may reside here as well, including Klebsiella, Enterobacter and Citrobacter. Some strains of E. coli are pathogens that cause intestinal infections, urinary tract infections and neonatal meningitis.

E. coli. Scanning E.M. Shirley Owens. Center for Electron Optics. Michigan State University.

(9) Pseudomonas aeruginosa is the quintessential opportunistic pathogen of humans that can invade virtually any tissue. It is a leading cause of hospital-acquired (nosocomial) Gram-negative infections, but its source is often exogenous (from outside the host).

Colonies of Pseudomonas aeruginosa growing on an agar plate. The most virulent Pseudomonas species produce mucoid colonies and green pigments such as this isolate.


(10) Haemophilus influenzae is a frequent secondary invader to viral influenza, and was named accordingly. The bacterium was the leading cause of meningitis in infants and children until the recent development of the Hflu type B vaccine.

Haemophilus influenzae. Gram stain.

(11) The greatest number of bacteria are found in the lower intestinal tract, specifically the colon and the most prevalent bacteria are the Bacteroides, a group of Gram-negative, anaerobic, non-sporeforming bacteria. They have been implicated in the initiation colitis and colon cancer.

Bacteroides fragilis. Gram stain.

(12) Bifidobacteria are Gram-positive, non-sporeforming, lactic acid bacteria. They have been described as "friendly" bacteria in the intestine of humans. Bifidobacterium bifidum is the predominant bacterial species in the intestine of breast-fed infants, where it presumably prevents colonization by potential pathogens. These bacteria are sometimes used in the manufacture of yogurts and are frequently incorporated into probiotics.

Bifidobacterium bifidum. Gram stain

(13) Lactobacilli in the oral cavity probably contribute to acid formation that leads to dental caries. Lactobacillus acidophilus colonizes the vaginal epithelium during child-bearing years and establishes the low pH that inhibits the growth of pathogens.

Lactobacillus species and a vaginal squaemous epithelial cell. CDC

(14) There are numerous species of Clostridium that colonize the bowel. Clostridium perfringens is commonly isolated from feces. Clostridium difficile may colonize the bowel and cause "antibiotic-induced diarrhea" or pseudomembranous colitis.

Clostridium perfringens. Gram stain.

(15) Clostridium tetani is included in the table as an example of a bacterium that is "transiently associated" with humans as a component of the normal flora. The bacterium can be isolated from feces in 0 - 25 percent of the population. The endospores are probably ingested with food and water, and the bacterium does not colonize the intestine.

Clostridium tetani. Gram stain.

(16) The corynebacteria, and certain related propionic acid bacteria, are consistent skin flora. Some have been implicated as a cause of acne. Corynebacterium diphtheriae, the agent of diphtheria, was considered a member of the normal flora before the widespread use of the diphtheria toxoid, which is used to immunize against the disease.


Corynebacterium diphtheriae. No longer a part of the normal flora.
Associations Between Humans and the Normal Flora

E. coli is the best known bacterium that regularly associates itself with humans, being an invariable component of the human intestinal tract. Even though E. coli is the most studied of all bacteria, and we know the exact location and sequence of 4,288 genes on its chromosome, we do not fully understand its ecological relationship with humans.

In fact, not much is known about the nature of the associations between humans and their normal flora, but they are thought to be dynamic interactions rather than associations of mutual indifference. Both host and bacteria are thought to derive benefit from each other, and the associations are, for the most part, mutualistic. The normal flora derive from their host a steady supply of nutrients, a stable environment, and protection and transport. The host obtains from the normal flora certain nutritional and digestive benefits, stimulation of the development and activity of immune system, and protection against colonization and infection by pathogenic microbes.

While most of the activities of the normal flora benefit their host, some of the normal flora are parasitic (live at the expense of their host), and some are pathogenic (capable of producing disease). Diseases that are produced by the normal flora in their host may be called endogenous diseases. Most endogenous bacterial diseases are opportunistic infections, meaning that the the organism must be given a special opportunity of weakness or let-down in the host defenses in order to infect. An example of an opportunistic infection is chronic bronchitis in smokers wherein normal flora bacteria are able to invade the weakened lung.

Sometimes the relationship between a member of the normal flora an its host cannot be deciphered. Such a relationship where there is no apparent benefit or harm to either organism during their association is referred to as a commensal relationship. Many of the normal flora that are not predominant in their habitat, even though always present in low numbers, are thought of as commensal bacteria. However, if a presumed commensal relationship is studied in detail, parasitic or mutualistic characteristics often emerge.

Tissue specificity


Most members of the normal bacterial flora prefer to colonize certain tissues and not others. This "tissue specificity" is usually due to properties of both the host and the bacterium. Usually, specific bacteria colonize specific tissues by one or another of these mechanisms.

1. Tissue tropism is the bacterial preference or predilection for certain tissues for growth. One explanation for tissue tropism is that the host provides essential nutrients and growth factors for the bacterium, in addition to suitable oxygen, pH, and temperature for growth.


Lactobacillus acidophilus, informally known as "Doderlein's bacillus" colonizes the vagina because glycogen is produced which provides the bacteria with a source of sugar that they ferment to lactic acid.


2. Specific adherence Most bacteria can colonize a specific tissue or site because they can adhere to that tissue or site in a specific manner that involves complementary chemical interactions between the two surfaces. Specific adherence involves biochemical interactions between bacterial surface components (ligands or adhesins) and host cell molecular receptors. The bacterial components that provide adhesins are molecular parts of their capsules, fimbriae, or cell walls. The receptors on human cells or tissues are usually glycoprotein molecules located on the host cell or tissue surface.

Figure 2. Specific adherence involves complementary chemical interactions between the host cell or tissue surface and the bacterial surface. In the language of medical microbiologist, a bacterial "adhesin" attaches covalently to a host "receptor" so that the bacterium "docks" itself on the host surface. The adhesins of bacterial cells are chemical components of capsules, cell walls, pili or fimbriae. The host receptors are usually glycoproteins located on the cell membrane or tissue surface.

Some examples of adhesins and attachment sites used for specific adherence to human tissues are described in the table below.
3. Biofilm formation
Some of the indigenous bacteria are able to construct biofilms on a tissue surface, or they are able to colonize a biofilm built by another bacterial species. Many biofilms are a mixture of microbes, although one member is responsible for maintaining the biofilm and may predominate.
The classic biofilm that involves components of the normal flora of the oral cavity is the formation of dental plaque on the teeth. Plaque is a naturally-constructed biofilm, in which the consortia of bacteria may reach a thickness of 300-500 cells on the surfaces of the teeth. These accumulations subject the teeth and gingival tissues to high concentrations of bacterial metabolites, which result in dental disease.

Coated from Kenneth Todar, Ph.D. Todar online Text book of bacteriology
Occupational Health Hazards in Hospitals, What Health Care Workers Should Know?

Healthcare workers acquired hepatitis C virus

Virology:
HCV is a small, enveloped, single-stranded, positive sense RNA virus. There are six major genotypes of the HCV, which are indicated numerically (e.g., genotype 1, genotype 2, etc.) (Tarantola et al., 2006).
Incidence:
HCV is not transmitted efficiently through occupational exposures to blood. The average incidence of anti-HCV sero-conversion after accidental percutaneous exposure from an HCV-positive source is 1.8% (range: 0%–7%). Transmission rarely occurs from mucous membrane exposures to blood and no transmission in HCWs has been documented from intact or non-intact skin exposures to blood. Data are limited on survival of HCV in the environment. In contrast to HBV, the epidemiologic data for HCV suggest that environmental contamination with blood containing HCV is not a significant risk for transmission in the healthcare setting (Manns et al., 2007).


Laboratory diagnosis:
Hepatitis C testing begins with serological blood tests used to detect antibodies to HCV. Anti-HCV antibodies can be detected in 80% of patients within 15 weeks after exposure, in >90% within 5 months after exposure and in >97% by 6 months after exposure. Overall, HCV antibody tests have a strong positive predictive value for exposure to the hepatitis C virus, but may miss patients who have not yet developed antibodies (seroconversion), or have an insufficient level of antibodies to detect (Watanabe et al., 2003).
Rarely, people infected with HCV never develop antibodies to the virus and therefore, never test positive using HCV antibody screening. Because of this possibility, RNA testing should be considered when antibody testing is negative but suspicion of HCV is high (e.g. because of elevated transaminases in someone with risk factors for HCV) (Scott et al., 2006).
Anti-HCV antibodies indicate exposure to the virus, but cannot determine if ongoing infection is present. All persons with positive anti-HCV antibody tests must undergo additional testing for the presence of the HCV itself to determine whether current infection is present. The presence of the virus is tested by using molecular nucleic acid testing methods such as PCR, transcription mediated amplification (TMA), or branched DNA (b-DNA) (Watanabe et al., 2003).
Determining the ALT level is useful for monitoring the effectiveness of therapy for HCV infection. Because ALT levels can fluctuate, a single value in the reference range does not rule out active infection, progressive liver disease, or cirrhosis. ALT normalization with therapy is not proof of cure (Watanabe et al., 2003).

Fig. (1): Diagnosis of HCV (Watanabe et al., 2003).

Genotyping is helpful for predicting the likelihood of response and duration of treatment. Patients with genotypes 1 and 4 are generally treated for 12 months, whereas 6 months of treatment is sufficient for other genotypes. Genotyping can be performed by direct sequence analysis, reverse hybridization to genotype-specific oligonucleotide probes or restriction fragment length polymorphisms (Manns et al., 2007).

Role of PB19 in Acute Leukemia (AL)

There are three current hypotheses concerning infectious mechanisms in the aetiology of childhood leukaemia: exposure in utero or around the time of birth, delayed exposure beyond the first year of life to common infections and unusual
population mixing. Till now, no specific virus has been definitively linked with childhood leukaemia and there is no evidence to date of viral genomic inclusions within leukaemic cells. Nevertheless, case–control and cohort studies have revealed equivocal results. Maternal infection during pregnancy has been linked with increased risk whilst protective roles were determined to be breast feeding and day care attendance in the first year of life.
There is debate from studies on early childhood infectious exposures, vaccination and social mixing. Some supportive evidence for an infectious aetiology is provided by the findings of space-time clustering and seasonal variation of acute leukemia new cases presentations..
Spatial clustering suggests that higher incidence is associated with specific areas with increased levels of population mixing, particularly in previously isolated populations. Ecological studies have also shown excess incidence with higher population mixing.
The marked childhood peak in resource-rich countries and an increased incidence of the childhood peak in acute lymphoblastic leukaemia (ALL) (occurring at ages 2–6 years predominantly with precursor B-cell ALL) is supportive of the concept that reduced exposure to early infection may play a role.
In addition, genetically determined individual response to infectious agents may be critical in the proliferation of preleukaemic clones as evidenced by the human leucocyte antigen class II polymorphic variant association with precursor B-cell and T-cell ALL (McNally1and Eden ,2004).
Previous data led by Greaves (1999) has demonstrated that for certain types of acute leukemia as infant ALL with MLL rearrangements, mostly in the precursor B-cell ALL (certainlyTEL-AML1 and hyperdiploid ALL) and childhood AML with t(8;21) translocations, the first genetic event in childhood leukaemia involves production of a preleukaemic clone in utero. Greaves (1999) hypothesized that whatever the cause of the gene rearrangements in utero, postnatal events including infection are almost certainly required to promote the development of clinical leukaemia.
These data has clearly defined the need for at least two and possibly multiple postnatal events. Therefore, when considering aetiology the initiating events occurring most frequently in utero and subsequent events occurring postnatally are important. Whether infection plays a part at either or both time-points is unclear.
It is claimed that the first ‘genetic’ event might well be initiated by infection. However, all the studies to date have shown no evidence of viral genomic inclusion in leukaemic cells. Thus, the suggestion that maternal infection during pregnancy may be associated with an increased risk of childhood leukaemia would be supportive of such a relationship (Smith et al., 1997).
The hypothesis includes the possibility that infection might occur in utero or near to birth. The space time clustering data based on time and place of birth would clearly be consistent with events in utero (Kinlen’s et a. 1988 and 1995).
The findings from space-time clustering studies from the UK and Sweden (Gustafsson & Carstensen, 2000, McNally et al, 2002) support the in utero exposure, clearly in the most recent time period.
Obviously there is quite a lot of supportive data to suggest that the later postnatal events are indeed related to infections and/or the body’s response to them. Evidence from the geographical incidence and temporal increases in the incidence of childhood leukemia is consistent with lack of immune stimulation during the first few years of life, particularly in more affluent communities and societies.
The limited and protective effect of breast feeding and more significantly, the apparent benefit of increased social contact because of nursery attendance in the first year of life would again be supportive of the concept that leukemia is more likely if there is an absence of infectious exposure early in life and that leukemia results from some form of delayed and possibly abnormal response to infection at a later age.
The host factor represented by HLA class II allele linkage to precursor B-cell ALL is further corroborative evidence. Space-time clustering studies centered on time and place of diagnosis are also consistent with the delayed infection hypotheses, as are those based on time of diagnosis and place of birth and also Kinlen’s studies on unusual population mixing.
It is very important to realize that the population mixing hypotheses are not mutually exclusive. Elements of both may be involved in individual cases. Infection may initiate a preleukemic clone but that may not lead to overt leukemia in the absence of delayed later infection and/or abnormal response to such infection. Controversely to benefits of population mixing this may increase the chance of infectious exposure in susceptible people at any stage.
At the present time, in the absence of definitive evidence regarding either specific single or multiple infectious culpable agents at either time-point, it is beneficial to continue to investigate events and exposures around the two time-points and the molecular events that result from such exposures.
Greater clarification as to likely etiological agents may emerge. For
example, in infant leukemia, where very characteristic rearrangement of the MNLL gene occurs, there has been both epidemiological and molecular evidence to suggest that exposure to naturally occurring topoisomerase II inhibitors and the inability by the mother and fetus to metabolize them rapidly significantly increases the risk of developing leukaemia.
Topoisomerase II reduces DNA damage that is inappropriately repaired. A combination of exposure, DNA damage and inappropriate repair plus failure to metabolize the inhibitors all contributes to the initiation of a rare leukemia.
So we can say that there may be a multiple pathways involved in the development of any childhood leukemia that will always involve initial breakage and inaccurate repair of DNA in response to infection, chemicals, low level irradiation or other, as yet unknown, environmental exposures. Then one or more subsequent events convert a preleukaemic clone into an overt malignant population. Two genetic events and possibly also proliferative stimuli and/or suppression of bone marrow are all required to produce an overt leukaemia.
Another important issue is the genetic susceptibility. It looks likely in terms of not only the response to infection, but also the recognition and repair of DNA.
The study of the molecular events associated with leukemia transformation may help us to better understand the changes.
However, at present we are still some time away from measures that would enable us to subtly target therapy or indeed apply preventative measures based on a clear understanding of causation, including immune modulation of response to infection.
One of the implicated virus in development of acute leukemia is PB19. It is suggested that the interaction between PB19 and host immune response has a role. Acute parvovirus infection is associated with a significant cytokine cascade, which is associated with a degree of disturbed haematopoiesis and/or suppression of normal marrow function, which may allow ‘release’ of low level malignant clones or indeed, induce acute leukemia. Serum concentrations of TNFα and IFNγ are typical of symptomatic acute B19 infection.8 Greatly raised serum concentrations of IL-6 and GM-CSF have previously been documented at the onset of acute leukaemia.9,10 The chemokine MCP-1 is released by leukaemic cells; it is chemotactic for monocytes and induces the tumoricidal activity of monocytes, and is thus a possible host defence against neoplasia.16 B19 virus infection is associated with the suppression of erythroid elements in the bone marrow, along with immune cell proliferation and upregulation of key mediators, such as GM-CSF. Such mechanisms may play an important role in the conversion of preleukaemic clones to an overt leukaemia.

Parvovirus and hematological disorders in children

Role of PB19 in Acute Leukemia (AL

There are three current hypotheses concerning infectious mechanisms in the aetiology of childhood leukaemia: exposure in utero or around the time of birth, delayed exposure beyond the first year of life to common infections and unusual
population mixing. Till now, no specific virus has been definitively linked with childhood leukaemia and there is no evidence to date of viral genomic inclusions within leukaemic cells. Nevertheless, case–control and cohort studies have revealed equivocal results. Maternal infection during pregnancy has been linked with increased risk whilst protective roles were determined to be breast feeding and day care attendance in the first year of life.
There is debate from studies on early childhood infectious exposures, vaccination and social mixing. Some supportive evidence for an infectious aetiology is provided by the findings of space-time clustering and seasonal variation of acute leukemia new cases presentations..
Spatial clustering suggests that higher incidence is associated with specific areas with increased levels of population mixing, particularly in previously isolated populations. Ecological studies have also shown excess incidence with higher population mixing.
The marked childhood peak in resource-rich countries and an increased incidence of the childhood peak in acute lymphoblastic leukaemia (ALL) (occurring at ages 2–6 years predominantly with precursor B-cell ALL) is supportive of the concept that reduced exposure to early infection may play a role.
In addition, genetically determined individual response to infectious agents may be critical in the proliferation of preleukaemic clones as evidenced by the human leucocyte antigen class II polymorphic variant association with precursor B-cell and T-cell ALL (McNally1and Eden ,2004).
Previous data led by Greaves (1999) has demonstrated that for certain types of acute leukemia as infant ALL with MLL rearrangements, mostly in the precursor B-cell ALL (certainlyTEL-AML1 and hyperdiploid ALL) and childhood AML with t(8;21) translocations, the first genetic event in childhood leukaemia involves production of a preleukaemic clone in utero. Greaves (1999) hypothesized that whatever the cause of the gene rearrangements in utero, postnatal events including infection are almost certainly required to promote the development of clinical leukaemia.
These data has clearly defined the need for at least two and possibly multiple postnatal events. Therefore, when considering aetiology the initiating events occurring most frequently in utero and subsequent events occurring postnatally are important. Whether infection plays a part at either or both time-points is unclear.
It is claimed that the first ‘genetic’ event might well be initiated by infection. However, all the studies to date have shown no evidence of viral genomic inclusion in leukaemic cells. Thus, the suggestion that maternal infection during pregnancy may be associated with an increased risk of childhood leukaemia would be supportive of such a relationship (Smith et al., 1997).
The hypothesis includes the possibility that infection might occur in utero or near to birth. The space time clustering data based on time and place of birth would clearly be consistent with events in utero (Kinlen’s et a. 1988 and 1995).
The findings from space-time clustering studies from the UK and Sweden (Gustafsson & Carstensen, 2000, McNally et al, 2002) support the in utero exposure, clearly in the most recent time period.
Obviously there is quite a lot of supportive data to suggest that the later postnatal events are indeed related to infections and/or the body’s response to them. Evidence from the geographical incidence and temporal increases in the incidence of childhood leukemia is consistent with lack of immune stimulation during the first few years of life, particularly in more affluent communities and societies.
The limited and protective effect of breast feeding and more significantly, the apparent benefit of increased social contact because of nursery attendance in the first year of life would again be supportive of the concept that leukemia is more likely if there is an absence of infectious exposure early in life and that leukemia results from some form of delayed and possibly abnormal response to infection at a later age.
The host factor represented by HLA class II allele linkage to precursor B-cell ALL is further corroborative evidence. Space-time clustering studies centered on time and place of diagnosis are also consistent with the delayed infection hypotheses, as are those based on time of diagnosis and place of birth and also Kinlen’s studies on unusual population mixing.
It is very important to realize that the population mixing hypotheses are not mutually exclusive. Elements of both may be involved in individual cases. Infection may initiate a preleukemic clone but that may not lead to overt leukemia in the absence of delayed later infection and/or abnormal response to such infection. Controversely to benefits of population mixing this may increase the chance of infectious exposure in susceptible people at any stage.
At the present time, in the absence of definitive evidence regarding either specific single or multiple infectious culpable agents at either time-point, it is beneficial to continue to investigate events and exposures around the two time-points and the molecular events that result from such exposures.
Greater clarification as to likely etiological agents may emerge. For
example, in infant leukemia, where very characteristic rearrangement of the MNLL gene occurs, there has been both epidemiological and molecular evidence to suggest that exposure to naturally occurring topoisomerase II inhibitors and the inability by the mother and fetus to metabolize them rapidly significantly increases the risk of developing leukaemia.
Topoisomerase II reduces DNA damage that is inappropriately repaired. A combination of exposure, DNA damage and inappropriate repair plus failure to metabolize the inhibitors all contributes to the initiation of a rare leukemia.
So we can say that there may be a multiple pathways involved in the development of any childhood leukemia that will always involve initial breakage and inaccurate repair of DNA in response to infection, chemicals, low level irradiation or other, as yet unknown, environmental exposures. Then one or more subsequent events convert a preleukaemic clone into an overt malignant population. Two genetic events and possibly also proliferative stimuli and/or suppression of bone marrow are all required to produce an overt leukaemia.
Another important issue is the genetic susceptibility. It looks likely in terms of not only the response to infection, but also the recognition and repair of DNA.
The study of the molecular events associated with leukemia transformation may help us to better understand the changes.
However, at present we are still some time away from measures that would enable us to subtly target therapy or indeed apply preventative measures based on a clear understanding of causation, including immune modulation of response to infection.
One of the implicated virus in development of acute leukemia is PB19. It is suggested that the interaction between PB19 and host immune response has a role. Acute parvovirus infection is associated with a significant cytokine cascade, which is associated with a degree of disturbed haematopoiesis and/or suppression of normal marrow function, which may allow ‘release’ of low level malignant clones or indeed, induce acute leukemia. Serum concentrations of TNFα and IFNγ are typical of symptomatic acute B19 infection.8 Greatly raised serum concentrations of IL-6 and GM-CSF have previously been documented at the onset of acute leukaemia.9,10 The chemokine MCP-1 is released by leukaemic cells; it is chemotactic for monocytes and induces the tumoricidal activity of monocytes, and is thus a possible host defence against neoplasia.16 B19 virus infection is associated with the suppression of erythroid elements in the bone marrow, along with immune cell proliferation and upregulation of key mediators, such as GM-CSF. Such mechanisms may play an important role in the conversion of preleukaemic clones to an overt leukaemia.

Parvovirus and hematological disorders in children

Parvovirus and Myocarditis

Human parvovirus B19 has been linked to a variety of cardiac diseases. A causal association between viral infection and cardiac disease was frequently postulated following the detection of B19 DNA by PCR in endomyocardial biopsy specimens. Several authors have postulated a causative role of B19 in cardiac disease, such as acute myocarditis (Schowengerdt, et al.,1997, Dettmeyer et al., 2003, Kühl et al.,2005,), dilative cardiomyopathy or idiopathic left ventricular dysfunction (Donosoet al., Klein 2004, et al,2004 ), and peripartum cardiomyopathy (Bultmannet al., 2005, Schenk et al., 2009).
There have been two fatal cases of PB19-associated myocarditis reported. A 1 year-old child, developed cardiac insufficiency following serologically confirmed erythema infectiosum. There was a temporary response to digoxin and diuretics, but the child died two weeks later. On autopsy there was an active myocarditis with a mononuclear inflammatory infiltrate and severe myocyte necrosis. PB19 capsid proteins were detected in myocardial tissue sections. The myocardial findings were similar to those seen in fetuses infected in utero by PB19 (Saint-Martin et al., 1990). In a second case, a 3 year-old child died of myocarditis following PB19 infection. Although PB19 could be detected in liver and spleen tissues, no PB19 DNA was detected in the myocardial tissue. The role of PB19 in the pathogenesis of myocarditis needs further investigations, particularly as P antigen is found on fetal myocardial cells and PB19 appears to cause myocarditis in the fetus (Young and Brown, 2004).
Parvovirus and Hematological disorders in children
PB19-associated inflammatory cardiomyopathy is characterized by infection of intracardiac endothelial cells of small arterioles and veins, which may be associated with endothelial dysfunction, impairment of myocardial microcirculation, penetration of inflammatory cells, and secondary myocyte necrosis. Recent observations showed that B19 is involved in intracellular calcium regulation by the viral phospholipase. B19-induced caspase activation can lead to proinflammatory/proapoptotic processes through dysregulation of STAT signaling. These cellular interactions may contribute to mechanisms by which B19 establishes persistent infection in endothelial cells and play a critical role in viral pathogenesis of inflammatory cardiomyopathy (Kandolf et al., 2008).