Old Questions: TU: MSC 2nd Year: Bacteriology: 2063

BACTERIOLOGY

TU 2063

GROUP A

(3*15)

1.      Define infective endocarditis. Describe the principle and procedure for diagnosis of bacterial endocarditis.

2.      Describe urinary tract infection. Explain the procedures for laboratory diagnosis of urinary tract infection.

Old Questions: TU: MSC 2nd Year: Bacteriology: 2062

T.U. 2062 BACTERIOLOGY

GROUP A(3*15)

1. Define enteric fever. Describe the pathogenesis and laboratory diagnosis of typhoid fever.
2. Describe respiratory tract infection. Explain the laboratory diagnostic method for diagnosis of bacterial pneumonia.
   

Old Questions: TU: MSC 2nd Year: Bacteriology: 2060

TU. 2060 BACTERIOLOGY

GROUP A(3*15)

1. Describe the mechanisms of action of antibiotics in detail.
2. Describe laboratory diagnosis of Mycobacterium tuberculosis.
3. Define UTI. Describe laboratory diagnosis of urinary tract infection ( UTI) using both rapid and conventional methods.
   

Laboratory diagnosis of dengue infection

TU 2063

Laboratory diagnosis of dengue infection

The clinical diagnosis of dengue, both the uncomplicated dengue fever form and DHF(dengue hemorrhagic fever)/DSS( dengue shock syndrome), is often unreliable. Dengue fever may resemble clinically a variety of acute febrile illnesses, although the severe muscle and bone pain is suggestive of dengue.

Similarly, DHF may resemble other causes of haemorrhagic fever, although thrombocytopaenia with haemoconcentration and signs of a moderate consumptivecoagulopathy is suggestive of dengue.

The most widely used serological test is the HI test, detecting antibodies as early as 4 days post-onset. A specific diagnosis of dengue can be made early in primary infections, but cross-reactions with other flaviviruses occur in late primary or secondary infections. The IgM antibody capture ELISA (MAC ELISA) is being used during outbreaks of dengue, and there are now rapid assays available for the detection of dengue IgG and IgM, although crossreaction may occur with the IgG assay. The IgM antibodies may persist for over 3 months, so the test is also useful for retrospective studies, but this persistence may cause diagnostic problems in areas where dengue is endemic. A combined IgG and IgM assay, detecting high levels of IgG indicating secondary infection, is useful in dengue endemic areas. Immunofluorescent assays have been used successfully to detect dengue IgG and IgM antibodies. The CF test is more specific than the HI test, but the antibodies detected by this assay appear later and disappear earlier. The NT and PRNT tests are the most specific and sensitive but are difficult to perform and thus tend to be used only for specific purposes.

Virus isolation, the only definitive way of being able to type isolates, is difficult and if mice are used, a number of blind passages are usually required. Intracerebral inoculation of adult or larval Toxorhynchites spp. is a sensitive and rapid method for the isolation of dengue virus, giving results in 2–3 days. The use of a rapid centrifugation method may increase sensitivity and reduce time scales. Intrathoracic inoculation of mosquitoes is easier and just as sensitive, but head squashes cannot be made or tested for specific dengue antigen for at least 7 days post-inoculation.

The most commonly used system of virus isolation is the inoculation of mosquito cell lines, viz. A. albopictus (C6-36), A. pseudoscutellaris (AP-61) and Toxorhynchites

ambionensis (TRA-248), which are almost as sensitive as mosquito inoculation, which allows specific results to be obtained within 2–3 days using fluorescent- labelled monoclonal antibodies. A combination of MAC ELISA and RT-PCR on peripheral blood

Leukocytes have been shown to give high levels of sensitivity and specificity.

Laboratory diagnosis of dengue infection

(JAWETZ)

Reverse transcriptase- polymerase chain reaction based methods are available for rapid identification and serotyping of dengue virus in acute- phase serum, roughly during the period of fever. Isolation of the virus is difficult. The current favored approach is inoculation of a mosquito cell line with patient serum, coupled with nucleic acid assay to identify a recovered virus.

Serological diagnosis is complicated by cross- reactivity of IgG antibodies to heterologus falvivirus antigens. A variety of methods are available; the most commonly used methods are E/M viral protein- specific capture IgM or IgG ELISA and the hemagglutination inhibition test. IgM antibodies develop within a few days of illness. Neutralizing and hemagglutination- inhibiting antibodies appear within a week after onset of dengue fever. Analysis of paired acute and convalescent sera to show a significant rise in antibody titer is the most reliable evidence of an active dengue infection.

LABORATORY DIAGNOSIS OF MEASLES VIRUS

T.U 2063

LABORATORY DIAGNOSIS OF MEASLES VIRUS

Typical measles is reliably diagnosed on clinical grounds; laboratory diagnosis may be necessary in cases of modified or atypical measles.

Antigen detection

Measles antigens can be detected directly in epithelial cells in respiratory secretions and urine. Antibodies to the nucleoprotein are useful because that is the most aboundant viral protein in infected cells.

Isolation and identification of virus

Nasopharyngeal and conjunctival swabs, blood samples, respiratory secretions, and urine collected form a patient during the febrile period are appropriate sources for viral isolation. Monkey or human kidney cells or a lympho- blastoid cell line (B95-a) are optimal for isolation attempts. Measles virus grows slowly; typical cytopathic effects ( multinucleated giant cells containing both intra- nuclear and intra- cytopalsmic inclusion bodies) take 7-10 days to develop. Shell vial culture tests be completed in 2-3 days using fluorescent antibody staining to detect measles antigens in the inoculated cultures. However, virus isolation is technically difficult.

Serology

Serologic confirmation of measles infection depends on a fourfold rise in antibody titer between acute phase and convalescent phase sera or on demonstration of measles specific IgM antibody in a single serum specimen drawn between 1 and 2 weeks after the onset of rash. ELISA,HI, Nt tests all may be used to measure measles antibodies, though ELISA is the most practical method.

The major part of the immune response is directed against the viral nucleoprotein. Patients with subacute sclerosing panencephalitis(SSPE) display an exaggerated antibody response, with titers 10 to 100 fold higher than those seen in typical convalescent sera.

Practical approach

Antigen-capture measles-specific IgM antibody assays (ELISA) have been developed, and when used appropriately are highly sensitive and specific. The test becomes positive within 48 hours after rash and may remain positive for up to 30 days after the onset of illness. The standard method for confirming the diagnosis is demonstration of a 4-fold rise in IgG measles antibodies in acute and convalescent sera. Haemagglutination–inhibition tests or ELISA antibody assays are most practical, but plaque reduction neutralization tests are the most sensitive and specific. The virus has been isolated from respiratory tract secretions and rarely from urine or circulating lymphocytes during the prodromal phase of illness or within a few days after the rash onset. Immunofluorescence staining of nasal or throat secretions or urine has been successful, but is not widely available. SSPE is confirmed based on characteristic EEG patterns and demonstration of measles antibody in the cerebrospinal fluid (CSF) with an increased CSF to serum measles antibody ratio, or by demonstration of virus in brain tissue.

Very high measles antibody titres aside from acute infection and SSPE (Subacute sclerosing panencephalitis) are regularly seen in autoimmune chronic active hepatitis and occasionally in systemic lupus erythematosus.

LABORATORY DIAGNOSIS OF RABIES VIRUS

T U.2060

LABORATORY DIAGNOSIS OF RABIES VIRUS

The diagnosis of human or animal rabies has to be based on the following

findings:

1. Demonstration of virus antigens or nucleic acid from brain, spinal cord, salivary glands, saliva, cornea or skin by means of immunofluorescence or PCR, respectively.
2. Postmortem demonstration of Negri bodies in brain tissue.
3. Isolation of virus from brain tissue and/or saliva.
4. Antibodies cannot usually be demonstrated before clinically manifest disease, and in many cases serological tests are also negative throughout the clinical course.

LABORATORY DIAGNOSIS (JAWETZ)

Rabies antigens or nucleic acid

Tissues infected with rabies virus are currently identified most rapidly and accurately by means of immunofluorescence or immunoperoxidase staining using anti rabies monoclonal antibodies. A biopsy specimen is usually taken from the skin of the neck at the hairline. Impression preparations of brain or cornea tissue may be used.

 A definitive pathologic diagnosis of rabies can be based on the findings of negri bodies in the brain or the spinal cord. They are sharply demarcated, more or less spherical, and 2-10µm in diameter, and they have a distinctive internal structure with basophilic granules in an eosinophilic matrix. Negri bodies contain rabies virus antigens and can be demonstrated by immunofluorescence. Both negri bodies and rabies antigen can usually be found in animals or humans infected with rabies, but they are rarely found in bats.

Reverse transcription- polymerase chain reaction testing can be used to amplify parts of a rabies virus genome from fixed or unfixed brain tissue. Although unusual as a diagnostic test, sequencing of amplified products allows identification of the infecting virus strain.

Virus isolation

Available tissue is inoculated intracerebrally into suckling mice. Infection in mice results in encephalitis and death. The central nervous system of the inoculated animal is examined for Negri bodies and rabies antigen. In specialized laboratories, hamster and mouse cell lines can be inoculated for rapid (2 to 4 days) growth of rabies virus;

This is much faster than virus isolation in mice. An isolated virus is identified by fluorescent antibody tests with specific antiserum. Virus isolation takes too long to be useful in making a decision about whether to give vaccine.

Serology

Serum antibodies to rabies can be detected by immunofluorescence or Neutralisation( Nt) tests. Such antibodies develop slowly in infected persons or animals during progression of the disease but promptly after vaccination with cell derived vaccines. Antibodies in cerebrospinal fluid are produced in rabies- infected individuals but not in response to vaccination.

Animal observation

All animals considered rabid or suspected rabid should be sacrificed immediately for laboratory examination of neural tissues. Other animals should be held for observation for 10 days. If they show any signs of encephalitis, rabies, or unusual behavior, they should be killed humanely and the tissues examined in the laboratory. If they appear normal after 10 days, decisions must be made on an individual basis in consultation with public health officals.

LABORATORY DIAGNOSIS OF HIV VIRUS

T.U 2059,2060,2062,2063

LABORATORY DIAGNOSIS OF HIV VIRUS

Tests to identify HIV infection can be divided into different categories: virus cultivation, antigen detection and viral genome amplification (PCR).

Virus cultivation

Virus can be isolated from infected persons in most phases of the infection. Peripheral blood mononuclear cells (PBMC) can be co-cultivated with activated PBMC from HIV-negative donors in the presence of IL-2. A positive result is recognized by appearance of virus antigen (p 24) or reverse transcriptase activity in the culture medium.

Antigen/Antibody detection

Antibodies usually become detectable from 3 to 12 weeks after infection. As a rule, an infected person remains antibody-positive for life, but antibody titres often fall in patients with AIDS. The most widely applied tests are the indirect and the competitive ELISA, using mostly a mixture of viral antigens. It is recommended that confirmatory tests are carried out to exclude the possibility of false positive results. These are either variations of ELISA tests or Western blot analysis of antibody specificity.

Viral genome amplification (PCR).

The PCR technique represents a major advance in the diagnosis of HIV infection. This powerful technique can amplify target DNA present in minute amounts. It is therefore useful for early detection of HIV in infants born to infected mothers, since the presence of maternal IgG antibodies excludes serological testing during the first months after birth. Quantitative determination of plasma viraemia (virion RNA) by reverse transcription PCR (RT-PCR) has become a major tool to follow the progression of HIV infection in untreated patients and to monitor the effects of antiviral chemotherapy in patients.

CD4+ lymphocyte count

A hallmark of chronic HIV infection is the depletion of CD4+ lymphocytes and loss of these cells is closely associated with acquisition of the characteristic opportunistic infections. The monitoring of CD4+ lymphocyte count is therefore an important determinant for clinical staging, initiation of antiviral therapy and PCP prophylaxis. However, the present knowledge that HIV is actively replicating in lymphatic organs throughout the infection raises the question of whether the peripheral blood, containing 2% of the total T-cell population, gives a representative picture of the pathogenic process. Plasma viraemia, together with CD4 counts, has therefore come to play a more important role in deciding when to initiate antiviral therapy.

STRUCTURE CLINICAL FEATURE AND LAB DIAGNOSIS OF HERPES SIMPLEX VIRUS

T.U 2058, 2060

STRUCTURE CLINICAL FEATURE AND LAB DIAGNOSIS OF HERPES SIMPLEX VIRUS

These viruses are members of the Alphaherpesvirinae subfamily of human

herpesviruses together with varizella-zoster virus, also called human herpesvirus. HSV is a large virus with a core containing double-stranded DNA within a coat, an icosahedron with 162 capsomeres. The envelope which surrounds the ‘naked’ particle is partly nuclear membrane derived, partly virally coded, with glycoprotein spikes. The diameter of a complete particle is 120–200 nm. The ‘naked’ virion measures about 100 nm.

Salk and sabin vaccine (T.U 2063)

Salk and sabin vaccine (T.U 2063)

Two types of vaccine are available against poliomyelitis, inactivated vaccine (IPV, Salk) and live attenuated oral vaccine (OPV, Sabin). Both vaccine formulations contain all three polio types.

Sabin or OPV

OPV is the most widely used vaccine for prevention of poliomyelitis. It is composed of attenuated strains of the three poliovirus types, and is administered orally.