General diagnosis of viruses

Specimens collection and transport:

        Collection and transport of an appropriate sample to the laboratory is a vital factor in successful diagnosis (Smith and Yassin, 2000).

Specimens collection

Collection of samples that contain the highest titer of virus is most desirable. Preservation of the viral titer and viral infectivity until cell cultures can be inoculated is essential. Body sites and collection methods vary according to the type of infection and viral etiology e.g. clinical samples collected from body sites such as skin and the genital tract, which is usually contaminated with microbial flora, are collected with

dacron or polyester swab and placed in viral transport medium (VTM) but CSF which is expected to be free of contamination, is collected in sterile containers (Leland and Ginocchio, 2007).

Table (1): shows Specimens useful for viral studies (Specter and Bendinelli, 2005).

Specimen type	Disease category (example)
Nasopharyngeal swab or aspirate	Respiratory tract infection, certain exanthemas, CNS infection (especially Enteroviruses).
Throat wash or swab	Respiratory tract infection.
Sputum	Respiratory infection (HSV, CMV).
Bronchoalveolar lavage	Lower respiratory infection (CMV, Influenza virus, PIV).
Rectal swab or stool	Gastroenteritis and Enteroviruses.
CSF	Meningitis, encephalitis.
Brain biopsy	Meningitis, encephalitis (HSV, Rabis virus).
Blood	CMV, HIV, HBV, HCV.
Vesicle fluid or lesion scraping	HSV, VZV.
Endocervical swab	Genital infections (HSV).
Eye swab	Ocular infections (Adenovirus, HSV).
Urine	CMV, BK virus, Mumps virus.
Serum	Antibody studies (most viruses).

Transport:

            Several VTM are commercially available. Most VTM consist of buffered isotonic solution with some types of protein as albumin, gelatin or serum to protect less stable viruses. Antibacterial and antifungal agents are added to prevent overgrowth of bacteria and fungi (Nauschuetz and Learmonth, 2007).

Ideally, all specimens collected for viral detection should be placed in ice and transported to the laboratory at once. If a delay is unavoidable, the specimen should be refrigerated not frozen until processing occurs. Every attempt should be made to process the specimen within 12 to 24 hours of collection. Under unusual circumstances, specimens may need to be held for days before processing. For storage up to 5 days, hold specimen at 4°C. Storage for 6 or more days should be at -70°C. Specimens for freezing should first be diluted in VTM. Significant loss of viral infectivity may occur during prolonged storage, especially for the more labile enveloped viruses (Forbes et al, 2007).                   

Methods of laboratory diagnosis:

1. Direct Microscopy :

         Electron microscope (EM) with negative staining methods can be used to directly examine specimens for the presence of viral particles. EM is particularly helpful to detect non cultivatable or fastidious viruses. The major limitation are low sensitivity and specificity, the expertise needed, successful detection requires the presence of 10⁵  or greater particles/ml. EM is effective for identification of virus morphologically by family, which may be sufficient clinically in many cases, and can be enhanced by use of antibodies (immune EM), which permits identification of specific viruses (Petric and Szymanski, 2000).

2. Virus isolation:

              It is still the gold standard method for virus detection. Traditionally, three methods are used for virus isolation; cell culture, animal inoculation and embryonated eggs. Animal inoculation is extremely costly and used only in research laboratories e.g. certain Coxsakie A viruses are isolated in suckling mice and embryonated eggs are rarely used e.g. Influenza viruses. Cell culture is the most commonly used by clinical virology laboratories (Nauschuetz and Learmonth, 2007).

Traditional cell culture

            There are three basic types of cell cultures. Primary cultures are obtained from tissue removed from an animal. The tissue is finely minced and then treated with trypsin to disperse cells then the cells seeded            onto a surface to form monolayer, as flask or test tube. Primary cell lines can only be passaged a few times e.g. primary monkey kidney cell (Nauschuetz and Learmonth, 2007).

            Diploid cell lines are secondary cultures which have undergone a change that allows their limited culture (up to 50 passages); with increasing passage diploid cells become more insensitive to viral infection e.g. human neonatal lung culture. Continous cell lines have variable number of chromosomes (haploid) and are capable of more prolonged  perhaps indefinite growth that have been derived from diploid cell lines or from malignant tissues e.g. HEP2(derived from human laryngeal carcinoma). The type of cell culture used for viral cultivation depends on the sensitivity of the cells to a particular virus (Knipe, 2001).

Table (2): shows Different cell lines sensitive to common viruses (Specter and Bendinelli, 2005).

Cell line	Viruses
A549 cells	Adenovirus, HSV.
HeLa cells	HSV.
HEP-2 cells	Adenovirus, HSV, RSV.
Human embryonic kidney	Adenovirus, BK virus, HSV.
Human diploid fibroblasts	CMV, HSV, VZV, Enteroviruses, Rhinoviruses.
Madin-Darby canine kidney (MDCK)	Influenza viruses.
NCI-H 929	Adenovirus, Enteroviruses, Mumpes,
Primary monkey kidney (PMK)  cells	Measles virus, PIV, RSV.
Primary rabbit kidney cells	Enteroviruses, Influenza viruses, PIV. HSV.

Centrifugation-Enhanced shell vial culture

       This technique is simple method that more rapidly identifies the virus than the traditional viral culture. Cells are grown on a round coverslip in a shell vial. The shell vial is inoculated with the clinical sample and then centrifugated to promote viral absorption. The shell vial is incubated for 24-48 hours, after which the cells are scraped from the coverslip, and the DFA technique is performed using a varity of Abs (Nauschuetz, 2000).

Identification of viruses detected in cell culture:

            Detection of virus in cell culture depend on cytopathic effects (CPE) which are morphological changes noted as a result of viral replication. CPE of cells may be clumping, destruction, granulation, rounding or vaculation, giant cell or syncytia formation, or retractile cells. In some circumstances, viruses will not cause a visible CPE and it is necessary to resort to other methods to detect their presence. Mumps, Influenza and Parainfluenza viruses will not normally cause CPE or   little CPE but addition of guinea pig red blood cells (RBCs) to infected primary monkey kidney cells will result in adherence of the RBC to the cells (hemadsorption) and Rubella virus may be detected by interference with echovirus 11 to replicate and cause cell destruction of African green monkey kidney cells (Smith and Yassin, 2000).                                   

3. Antigen detection:

          Immunological methods are highly effective for detection of viral antigens (Ag). They offer high degree of sensitivity and specificity, are rapid, and the costs are reasonable. Virtually all methods use antibodies that are tagged with a fluorochrome, enzyme, or radiolabel (Schutzbank and McGuire, 2000).

A) Immunofluorescence (IF):

           The direct staining of clinical specimens using monoclonal or polyclonal antibodies (Abs) bound to fluorescent dye. This may be direct  or indirect techniques. In direct IF, a single fluorochrome-labeled Ab is used. Indirect IF uses two Abs, one specific for the Ag and a second fluorochrome-labeled Ab to the immunoglobulin (Madeley and Peiris, 2002).

B) Enzyme Immunoassay (EIA):

Enzyme Immunoassay can perform to detect antigens in clinical specimens. This versatility has resulted in wide spread use of EIA in diagnostic virology. Most commonly these assays are performed using a solid phase format, as ELISA, with the particular target reagent bound to plastics in a microtiter well. EIA are generally high specific and sensitive, and rapid, within minutes to hours (Leland, 2000).

C) Radioimmunoassays (RIA):

Radioimmunoassays have fallen out of use for most viral assays due to exposure to radioisotope and the high cost of disposal of radioactivity.  RIA rivals EIA in specificity and sensitivity (Mushahwar and Brawner, 2000).

4. Serology:

            This approach is currently the most widely used. Typically during acute primary infection, virus specific IgM Abs appear in serum between 5-10 days after initiation of infection, reach peak concentration at 2-4 weeks, and then decline to undetectable level at 2-4 months, although they may last considerably longer in some infections. In          the course of persistent infections, IgM Ab is usually negative, although they can reappear during episodes of reactivation of virus. Thus, detecting IgM to a given virus, with or without the corresponding IgG, even in a single serum sample obtained during the acute phase of illness, denotes current or very recent primary infection or reactivation of persistent infection by that virus. By contrast, the presence of virus specific IgG but not IgM signifies past infection (Specter and Bendinelli, 2005).

5. Molecular methods (nucleic acid detection):

        A wide variably of commercial assays are available to detect viral nucleic acid directly or after amplification. These procedures are rapidly becoming the standards for diagnostic virology and it is more sensitive than antigen detection and cultures include polymerase chain reaction (PCR), Reverse -Transcriptase PCR and others. These procedures allow detection of viruses (e.g. Enteroviruses) as well as quantitation of the viruses (e.g. CMV, HIV) which guide antiviral therapy.  The principle is the hyperdization of nucleic acid probe (single strand DNA or RNA) to specific nucleic acid sequence in the specimen followed by detection of the paired hybrid. The probe is labeled with enzyme, chemiluminescent molecule, or radioisotopes to facilitate detection of the hyperdization product .

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Conventional Methods for Diagnosis of Infectious Diseases

Infectious diseases are common diseases all over the world. A recent World Health Organization report indicated that infectious diseases are now the world’s biggest killer of children and young adults. Infectious diseases in non-industrialized countries caused 45% in all and 63% of death in early childhood.

Causes of infectious diseases:

The microbial causes of human diseases are classified into theses groups:

1-    Bacteria

2-    Viruses

3-    Fungi

4-    Protoza

5-    Chlamydiae

6-    Rickettsiae

7-    Mycoplasmas.

Infection may be endogenous or exogenous.

1-    Endogenous infections:  the microorganism (usually a bacterium) is a component of the patient's endogenous flora. Endogenous infections can occur when the microorganism is aspirated from the upper to the lower respiratory tract or when it penetrates the skin or mucosal barrier as a result of trauma or surgery.

2-    Exogenous infections: the microorganism is acquired from the environment (e.g., from soil or water) or from another person or an animal.

The ability to control such microbial infections is largely dependent on the ability to detect these aetiological agents in the clinical microbiology laboratory (Millar et al., 2003)

Diagnoses of Infectious diseases

       Diagnosis of infectious diseases in clinical microbiological laboratory through

- Conventional methods

Laboratory tests may identify organisms directly (eg, visually, using a microscope, growing the organism in culture) or indirectly (eg, identifying antibodies to the organism

-molecular methods

Specimen Selection, Collection and Processing

A- Specimen quantity

Specimens selected for microbiologic examination should reflect the disease process and be collected in sufficient quantity to allow complete microbiologic examination. The number of microorganisms per milliliter of a body fluid or per gram of tissue is highly variable, ranging from less than 1 to 10⁸ or 10¹⁰ colony-forming units (CFU).

Swabs, although popular for specimen collection, frequently yield too small a specimen for accurate microbiologic examination and should be used only to collect material from the skin and mucous membrane

Specimen collection is important. The wrong type of swab can produce false-negative results. Wooden-shafted swabs are toxic to some viruses. Cotton-tipped swabs are toxic for some bacteria and chlamydiae. Blood cultures require decontamination and disinfection of the skin (eg, povidone iodine swab, allowed to dry, removed with 70% alcohol).      Multiple samples, each from a different site are generally used; they are taken nearly simultaneously with fever spikes if possible. Normal flora of skin and mucous membranes that grow in only a single blood sample are usually interpreted as contamination.

If a blood specimen is obtained from a central line, a peripheral blood specimen should also be obtained to help differentiate systemic bacteremia from catheter infection. Cultures from infected catheters generally turn positive more quickly and contain more organisms than simultaneously drawn peripheral blood cultures. Some fungi, particularly molds (eg, Aspergillus sp), usually cannot be cultured from blood.

The specimen must be transported rapidly, in the correct medium, and in conditions that limit growth of any potentially contaminating normal flora. For accurate quantification of the pathogen, additional pathogen growth must be prevented; specimens should be transported to the laboratory immediately or, if transport is delayed, refrigerated (in most cases). Certain cultures have special considerations.

B-Prevention of contamination of a specimen

Because skin and mucous membranes have a large and diverse endogenou flora, every effort must be made to minimize specimen contamination during collection. Contamination may be avoided by various means:

-         The skin can be disinfected before aspirating or incising a lesion.

-         The contaminated area may be bypassed altogether. Examples of such approaches are trans tracheal puncture with aspiration of lower respiratory secretions or supra pubic bladder puncture with aspiration of urine.

It is often impossible to collect an uncontaminated specimen, and decontamination procedures, cultures on selective media, or quantitative cultures must be used.

      Specimens collected by invasive techniques, particularly those obtained intra operatively, require special attention. Enough tissue must be obtained for both histopathologic and microbiologic examination.

C-Time of collection

If possible, specimens should be collected before the administration of antibiotics. Above all, close communication between the clinician and the microbiologist is essential to ensure that appropriate specimens are selected and collected and that they are appropriate

The specimen must be transported rapidly, in the correct medium, and in conditions that limit growth of any potentially contaminating normal flora. For accurate quantification of the pathogen, additional pathogen growth must be prevented; specimens should be transported to the laboratory immediately or, if transport is delayed, refrigerated (in most cases). Certain cultures have special considerations.

 Laboratory procedures used in confirming a clinical diagnosis of infectious disease with a bacterial etiology.

1- Laboratory methods for diagnosis of infectious diseases

A-Direct method:

They are Laboratory tests may identify organisms directly (eg, visually, using a microscope, growing the organism in culture)

1- Microscopy

Microscopy can be done quickly, but accuracy depends on the experience of the microscopist and quality of equipment. Regulations often limit physicians' use of microscopy for diagnostic purposes outside a certified laboratory (Siqueira, J. Fet al., 2005)

Most specimens are treated with stains that color pathogens, causing them to stand out from the background, although wet mounts of unstained samples can be used to detect fungi, parasites (including helminth eggs and larvae), vaginal clue cells, motile organisms (eg, Trichomonas), and syphilis (via darkfield microscopy). Visibility of fungi can be increased by applying 10% potassium hydroxide (KOH) to dissolve surrounding tissues and nonfungal organisms (Fredricks and Relman, 1999).

The clinician orders a stain based on the likely pathogens, but no stain is 100% specific. Most samples are treated with Gram stain and, if mycobacteria are suspected, an acid-fast stain. However, some pathogens are not easily visible using these stains; if these pathogens are suspected, different stains or other identification methods are required. Because microscopic detection usually requires a microbe concentration of about 1 × 10⁵/mL, most body fluid specimens (eg, CSF) are concentrated (eg, by centrifugation) before examination.

Types of stains which commonly used.

Gram stain: The Gram stain classifies bacteria according to whether they retain crystal violet stain (gram-positive—blue) or not (gram-negative—red) and highlights cell morphology (eg, bacilli, cocci) and cell arrangement (eg, clumps, chains, diploids). Such characteristics can direct antibiotic therapy pending definitive identification. To do a Gram stain, technicians heat-fix specimen material to a slide and stain it by sequential exposure to Gram's crystal violet, iodine, decolorizer, and counterstain (typically safranin).

Acid-fast and moderate (modified) acid-fast stains: These stains are used to identify acid-fast organisms (Mycobacterium sp) and moderately acid-fast organisms (primarily Nocardia sp). These stains are also useful for staining Rhodococcus and related genera, as well as oocysts of some parasites (eg, Cryptosporidium).

Although detection of mycobacteria in sputum requires only about 5, 000 to 10, 000 organisms/mL, mycobacteria are often present in lower levels, so sensitivity is limited. Usually, several mL of sputum are decontaminated with Na hydroxide and concentrated by centrifugation for acid-fast staining. Specificity is better, although some moderately acid-fast organisms are difficult to distinguish from mycobacteria.

Fluorescent stains: These stains allow detection at lower concentrations (1 × 10⁴ cells/mL). Examples are acridine orange (bacteria and fungi), auramine-rhodamine and auramine O (mycobacteria), and calcofluor white (fungi, especially dermatophytes).

Coupling a fluorescent dye to an antibody directed at a pathogen (direct or indirect immunofluorescence) should theoretically increase sensitivity and specificity. However, these tests are difficult to read and interpret, and few (eg, Pneumocystis and Legionella direct fluorescent antibody tests) are commercially available and commonly used.

India ink (colloidal carbon) stain: This stain is used to detect mainly Cryptococcus neoformans and other encapsulated fungi in a cell suspension (eg, CSF sediment). The background field, rather than the organism itself, is stained, which makes any capsule around the organism visible as a halo. In CSF, the test is not as sensitive as cryptococcal antigen. Specificity is also limited; leukocytes may appear encapsulated.

Wright's stain and Giemsa stain: These stains are used for detection of parasites in blood, Histoplasma capsulatum in phagocytes and tissue cells, intracellular inclusions formed by viruses and chlamydia, trophozoites of Pneumocystis jiroveci, and some intracellular bacteria.

Trichrome stain (Gomori-Wheatley stain) and iron hematoxylin stain:

These stains are used to detect intestinal protozoa.

The Gomori-Wheatley stain is used to detect microsporidia. It may miss helminth eggs and larvae and does not reliably identify Cryptosporidium. Fungi and human cells take up the stain.

The iron hematoxylin stain differentially stains cells, cell inclusions, and nuclei. Helminth eggs may stain too dark to permit identification.

Disadvantages of Microscopic methods :

(a)    Microscopy may suggest an etiologic agent, but it rarely provides definitive evidence of infection by a particular species.

(b)    Microscopic findings regarding bacterial morphology may be misleading, because many species can be pleomorphic and conclusions can be influenced by subjective interpretation of the investigator.

(c)     Limited sensitivity is because a relatively large number of microbial cells are required before they are seen under microscopy (e.g. 104 bacterial cells/ml of fluid) (Fredricks & Relman, 1999). Some micro-organisms can even require appropriate stains and/or approaches to become visible.

(d)    Limited specificity is because our inability to speciate micro-organisms based on their morphology and staining patterns.

2-Culture methods

Culture is microbial growth on or in a nutritional solid or liquid medium; increased numbers of organisms simplify identification. Culture also facilitates testing of antimicrobial susceptibility (Relman DA., 2002)

Communication with the laboratory is essential. Although most specimens are placed on general purpose media (e g, blood or chocolate agar), some pathogens require inclusion of specific nutrients and inhibitors or other special conditions (Wade W., 2002)

For more than a century, cultivation using artificial growth media has been the standard diagnostic test in infectious diseases. The microbiota associated with different sites in the human body has been extensively and frequently defined by studies using cultivation approaches.

 The success in cultivation of important pathogenic bacteria probably led microbiologists to feel satisfied with and optimistic about their results and to recognize that there is no death of known pathogens (Relman, 1992 and Wade W, 2002).

But should we be so complacent with what we know about human pathogens? Making micro-organisms grow under laboratory conditions presupposes some knowledge of their growth requirements. Nevertheless, very little is known about the specific growth factors that are utilized by innumerous micro-organisms to survive in virtually all habitats, including within the human body (Wade, 2002).

 A huge proportion of the microbial species in nature are difficult to be tamed in the laboratory. Certain bacteria are fastidious or even impossible to cultivate. Some well-known human pathogens, such as Mycobacterium leprae and Treponema pallidum continue to defy scientists regarding their cultivation under laboratory conditions (Fredricks and Relman, 1999)

Culture Anaerobic bacteria

Should not be cultured from sites where they are normal flora because differentiation of pathogens from normal flora may be impossible. Specimens must be shielded from air, which can be difficult. For swab specimens, anaerobic transport media are available. Specimens collected with a syringe (eg, abscess contents) should be transported in the syringe.

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Sepsis

The word sepsis has its origin with the Greeks, who postulated two fundamental forms of tissue breakdown: pepsis was a process exemplified by the fermentation of wine or the digestion of food and was associated with life and good health: sepsis, on the other hand, described the processes of putrefaction and decay and was associated with death and disease (Majno, 1991). With the recognition that microorganisms were agents of putrefaction, the word (sepsis) was applied to the clinical condition resulting from bacterial infection, and the neologism septicemia denoted the presence of these organisms in the blood stream.


Sepsis has traditionally been considered synonymous with "disseminated infection" because microbial toxins were thought to be a prerequisite for the development of fever, tachycardia, and tachypnea, the clinical signs of serious infection.

The classic clinical syndrome of sepsis was originally described in patients with disseminated gram-negative bacterial infections (Maclean et al., 1967). Later it became apparent that an identical syndrome developed in patients with gram positive bacterial (Wiles et al., 1980) or Viral; (Deutschmann et al., 1987) infection in association with salicylate intoxication (Leatherman et al., 1991), and even in normal volunteers who received an infusion of sterile stress hormones (Watters et al., 1986).

Sepsis is one of the main causes of morbidity and mortality in the intensive care units (ICU) (Bone et al., 1989). A delay in making the diagnosis and instituting appropriate therapy has been associated with increased mortality. Moreover, its diagnosis is frequently difficult since clinical signs of sepsis are often misleading and/or absent and, in addition, traditional markers of infection such as body temperature (BT) and white blood cell count (WBC) can remain unchanged, mainly in the early stage of the process (Springer and Verlag, 1998). Septicemia is a clinical syndrome characterized by fever, chills, malaise, tachycardia, hyperventilation and toxicity or prostration, (Failure to thrive) may indicate chronic septicemiae in infants (Shanson, 1999).

In the early stages, the clinical features may be very varied, especially in infants. Prompt recognition of septicemia and immediate treatment based on the knowledge of the likely causative organisms is essential. Complications include septic shock, disseminated intravascular coagulation (DIC) and acute renal failure. The mortality rate depends on the age, the underlying condition and the treatment given, in many series the mortality rate varies between 15% and 35% (Shanson, 1999).


1.2 How bacteremia occurs:

The blood is normally sterile, bacteremia occurs when microorganisms enter the blood stream. Bacteremia may be transient, intermittent or continuous (Koneman et al., 1997).

Transient bacteremia may occur when organisms, often comprising the normal flora, are introduced into the blood (e.g., following brushing of teeth) (Koneman et al., 1997).

Intermittent bacteremia occurs when bacteria from an infected site are released into the blood from extravascular abscesses, empyemic cavities, or diffuse infections, cellulites, peritonitis and septic arthritis (Koneman et al., 1997).

Continuous bacteremia usually occurs in cases where organisms have direct access to the blood stream such as subacute bacterial endocarditic, infected arteriovenous fistulas, intra-arterial catheter, or indwelling cannulae (Kaneman et al., 1997).

Also bacteremia was classified as primary if it occurs in the absence of an apparent portal entery, and secondary if a portal of entery was identified (Koneman et al., 1997).

Several mechanisms play a role in the removal of microorganisms from the blood stream. In healthy and immuno competent hosts, a sudden influx of bacteria is usually cleared from the blood within 30 to 45 minutes. The liver and spleen play the primary role in clearing bacteria ; intravascular phagocytes play only a minor role. Encapsulated bacteria are more difficult to clear ; however, the presence of specific antibodies promotes clearance (Koneman et al., 1997). Patients with debilitating or immuno deficiency diseases are at higher risk because the circulating bacteria may not be cleared from the blood for hours (Eykyn, 1998).

1.3. Causative organisms:

The relative incidence of the different causative organisms varies between hospitals according to the specialties practiced, the incidence of hospital infection and the type of community served by each hospital hence, the most frequent organisms causing bacteremia varies, both nationally and internationally (Shanson, 1999).

The most common causative organisms of bacteria are discussed below:

1.3.1. Gram negative septicemia:

The organisms most commonly isolated from blood are Gram-negative rods including Enterobacteriacease and Pseudomonas species E.Coli is by far the most frequent cause of this condition. The majority of these episodes results from urinary tract infection, other occur with biliary infections (Eykyn, 1998).

¬The release of endotoxin from Gram-negative organisms may result in septic shock (Crichton, 1993).

Immunocompromised patients tend to be colonized with bacteria that are relatively antibiotic resistant such as serratia marcescens, Enterobacter species and Pseudomonas aruginosa (Bailey and Scott, 1994.

Salmonella bacteremia frequently develops in patients with severe salmonella gastro-intestinal tract infection particularly in infants, elderly and debilitated patients leading to high mortality rate. Metastatic infective complications may occur such as meningitis, skin infection, splenic abscess, pyelonephritis, and very rarely Pneumoniae and Endocarditic (Shanson, 999).

Haemophilus influenzae can be isolated from blood particularly as an etiologic agent of endocarditic (Shanson, 1999).

Many factors predispose to Gram-negative septicemia including instrumentation and surgery on the gastrointestinal and urinary tracts, and neutropenia in oncology or transplanted patients Fig. (1). The incidence of Gram-negative sepsis in hospitals and also the effects of widespread use of broad-spectrum antibiotics, such as ampicillin, in promoting infections due to antibiotic-resistant strains (Shanson, 1999).



Fig.1. Venn diagram showing the possible interactions of four important groups of factors predisposing for Gram-negative septicemia (Shanson, 1999).



NO OBVIOUS LOCALIZED SEPSIS

e.g.,Gram-negative bacilli invade blood from a site with normal flora, such as the gut

SURGERY OR INSTRUMENTATION

e.g., Surgery on carcinoma of the colon Cystoscopy Intravenous infusion therapy IMPAIRED HOST DEFENCES

e.g., Severe neutropenia and immunosuppressive therapy low birth weight and prematurity.



PRE-EXISTING LOCALIZED SEPSIS

e.g., Urinary tract infection Gram-negative pneumonia Gram-negative infection of burns



1.3.2. Gram positive septicemia:

Staphylococci and streptococci cause the majority of Gram positive septicemia which usually complicate infections of the skin, soft tissue, bones, joints and lungs (Shanson, 1999).

Staph aureus infection at any site of the body can result in staphylococcal bacteremia which is always symptomatic, high grade and potentially lethal. Staph. aureus bacteremia has its highest frequency in the very young and the very old with serious underlying diseases and intravenous drug abusers (Dalton and Nottebart, 1986).

The septic shock and disseminated intravascular coagulation that can complicate staphylococcal septicemia may be due to the effect of its protein A (Roberts and Gaston, 1987).

An increasing proportion of blood stream infections due to coagulase negative staphylococci, enterococci has been documented in recent years. It reflects changing in, hospital people population and increasing use of invasive devices (Eykyn, 1998).

Coagulase negative staphylococci are especially important in the pediatric age group and were responsible for 43% of bacteremia in Great Ormond hospital, London. It is the third most common blood stream pathogen after group B streptococci and staph aureus in a Swedish neonatal unit (Eykyn, 1998).

The -hemolytic streptococci or viridans streptococci, comprise a heterogenous collection of species of which streptococuus sanguis I and II, streptococcus mutans and streptococcus mitis are most frequently isolated from cases with endocarditis. These organisms are normal inhabitants of the oral cavity and gastrointestinal tract (GI, II) often gaining enterence to the blood stream be cause of gingivitis or dental manipulations (Shanson, 1999).

Heart vavles especially that previously damaged convenient surfaces for attachment of the bacteria. The resulting vegetations uttimately seed bacteriae into the blood at a slow but constant rate (Scheld and Sande, 1990).

Group A B-hemolytic streptococci was a frequent cause of endocarditis during the pre-antibiotic era. It may occasionally cause fulminating septicemic infections, generally in previously healthy often young people (Dalton and Nottebart, 1986).

Adult group B-streptococcal bacteremia is associated usually with underlying disease as diabetes mellitus, neoplasm and urinary tract infection (Shanson, 1999).

Group D streptococci may be -- or non hemolytic and are separated into two divisions: enterococci and non enterococci. Of the enterococci, streptococcus faecalis is the most frequent human pathogen among debilitated and immuno suppressed adults. They are normal inhabitants of the gastrointestinal and genitourinary tracts of man (Dalton and Nottebart, 1986).

Septicemia occurs in 25-30%of patients with penumococcal pneumonia and this makes the prognosis worse. Because the organism produce autolytic enzyme, early subculture of broth (before 18 hour) is necessary (Bailey and Scoot, 1994).

Listeria monocytogenus is a cause of septicemia in immuno suppressed patients, alcoholics, and pregnant women. The bacterium may resemble a diphtheroid or coccus in Gram stain, but grows on 5% sheep blood agar as a translucent weakly B-hemolytic colony resembling B streptococci, so listeria must be distinguished from diptheroids and from group B and D streptococci. It displays a characteristic tumbling motility at room temperature but not at 35oc (Bailey and Scott, 1994).

Corynebacterium group IK should be suspected if a diptherid like bacterium is isolated from blood cultures usually of debilitated and immunocompromised patients and displays an unusual broad antibiotic resistance pattern, often being sensitive only to vancomycin (Shanson, 1999).

Members of this group of organisms exist as components of the normal skin flora especially at axillary, inguinal and rectal regions (Bailey and Scott, 1994).

The clinical features and complications of septicemia due to Gram-positive organisms may be indistinguishable from those due to Gram-negative organisms the septic shock and disseminated intravascular coagulation that can complicate staphylococcal septicemia may possibly due to the effects of protein A of staph aureus. Metastatic abscesses and acute infective endocarditis affecting a previously healthy arotic valve can also complicate Staph. aureus septicemia (Shanson, 1999).

1.3.3. Anaerobic septicemia:

Anaerobic bacteria originate from the normal flora of the mucosal surfaces (Collee et al., 1996).

Organisms of the genus peptostreptococcus have been reported from most review of blood culture. It may occasionally invade the blood from an infected female genital tract or oral sepsis (Bailey and Scott, 1994).

Bacteroides fragilis and other bacteroides are the main causes of anaerobic septicemia. These species can clinically cause septicemia that is indistinguishable from that produced by aerobic Gram-negative septicemia. Abdominal or gynaecological sepsis is usually present and bacteroides may be mixed with E.coli or other coliforms in the blood (Shanson, 1999).

Clostridium perfringens can cause rapidly fatal disease with severe toxaemia, hemolysis, shock, jaundice and acute renal failure. It must be presumptively identified by Gram stain appearance, gas and hemolysis in broth so that the clinician can be alerted to the possibility of the presence of this virulent bacterium in the blood stream (Bailey and Scott, 1994).

1.3.4. Other bacteria such as:

Campylobacter species especially campylobacter fetus, Alealigenes species, Eikenella Corrodens and flavobacterium species. If these less common organisms are suspected by the clinician, the laboratory can be alerted to hold the blood culture media for an extended period of time past the first week (Biley and Scott, 1994).



1.3.5. Rickettsia species:

Rickettsai species (causing typhus fever) and coxiella burnetti are rarely isolated from the blood and in that case serological tests may be helpful in the diagnosis. Animal inoculation techniques may be necessary (Bailey and Scott, 1994).

1.3.6. Mycoplasma species:

The isolation of Mycoplasma hominis and ureaplasma urealyticum from blood has been repeatedly encountered in patient with post partum fever (Dalton and Nottebart, 1986).

1.3.7. Fungi:

The large size and sterol-containing cell walls of fungi make them particularly insensitive to the primary host defenses, antibody and phagocytic cells. Fungi in the blood stream can be carried to all organs of the host where they may grow, invade normal tissue and elaborate toxic products. Fungi gain enterance to the circulatory system via loss of integrity of the gastrointestinal tract or other organs or by means of intravascular catheters (Baily and Scott, 1994).

Those fungi include:

1- Candida albicans which is the most frequently isolated fungus from the blood cultures (vented broths containing glucose are desirable for the isolation of fungi) (Shanson, 1999).

2- Other Candida species include Cryptococcus neoformans, Histoplasma capsulatum and Aspergillus species which are rarely isolated from blood cultures (Shonson, 1999).

1.3.8. Viruses:

Arboviruses and viral causes of haemorrhagic fevers such as lassa fever virus ; animal inoculation techniques or suitable tissue cultures in designated reference laboratories are required (Shanson, 1999).

1.4. Predisposing factors for septicemia:

Factors predispose for septicemia are all listed in table (3) (Shanson, 1999).

Table. 1. Predisposing factors for septicaemia:

1. Impairment of general host defences



ii. Examples by treatment Immunosuppression

- Steroids

- Radiotoxic drugs

- Radiotherapy

- Severe neutropenia

- Cytotoxic drugs

i. Examples by disease

Diabetes mellitus.

Age extremes.

Premature babies

Very elderly

Debilitation

Malignancy

Uraemia

Hepatic failure

Leukaemia, reticulosis

Aplastic anaemia

Myeloma

Malabsorption

Nephrotic syndrome

Congenital immunodeficiency

2. Instruonentation and surgery

examples:

surgery on the 4 urinary tract or large blowel

intravenous therapy central venous lines

urinary catheterization cytoscopy, trans-rectal prostatic biopsy

tracheostomy plus intermittent positive pressure ventilation and humidifiction

baby incubator and resuscitation equipment

radiological invasive techniques

3. factors that may encourage spread of spread of bacteria in hospital these include:

i. poor hospital hyginen and inadequate handwashing by staff between touching patients

ii. Aseptic technique lapes.

iii. Disinfectant policy not followed.

iv. Antibiotic overusage.

v. Isolation policy not practiced.

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