Evaluation of a one-tube RT-PCR system for detection of enteroviruses.

BACKGROUND: A highly sensitive PCR assay for early and rapid detection of enteroviral (EV) RNA in CSF is necessary to investigate the role of EV in acute neurological illnesses. OBJECTIVES: To evaluate and compare two PCR protocols (Titan one-tube RT-PCR and random primed RT-PCR) for detection of enteroviral RNA in CSF. STUDY DESIGN: The PCR protocols were evaluated for lower limit of input detection using log dilutions of five stock EV strains and an isolate of enterovirus-71 in minimum essential medium and three EV stock strains in CSF. The tests were also applied on 77 CSF samples, 46 from patients with suspected acute EV neurological illness and 31 from 'disease controls'. RESULTS: Even though in the initial virus titration assays there was no statistically significant difference in the limit of input detection by Titan system and the random primed two-step PCR, the latter had a higher positivity rate when used on CSF samples from patients (20/46 vs. 10/46, P<0.01). CONCLUSIONS: Random primed RT-PCR assay is superior to Titan one-tube RT-PCR for detection of EV RNA in CSF.

Three manual immunoassays for the rapid detection of hepatitis B surface antigen.

We have evaluated 3 commercial test kits for the detection of hepatitis B surface antigen (HBsAg) which do not require elaborate instrumentation and are relatively inexpensive. These included the Immunocomb-II (Orgenics, Israel), Austragen latex agglutination (Span, India) and Daina Screen (Abbott, USA) tests. A panel of 98 sera tested by IMx HBsAg assay (Abbott, USA) were used to evaluate the tests. Sensitivities of 96.6%, 86.2% and 96.6%, and specificities of 95.7%, 94.2% and 100%, respectively, were obtained. We therefore recommend the Immunocomb-II or Daina Screen tests for HBsAg detection in small laboratories where facilities to conduct automated enzyme-linked immunosorbent assays are not available.

Characterization of antibody response to human cytomegalovirus in Indian renal transplant patients.

BACKGROUND & OBJECTIVES: Cytomegalovirus (CMV) disease in seroendemic transplant populations is due to reactivation of the virus, or reinfection. In this context, the antibody response is likely to influence presentation, clinical severity and outcome of the disease, and may provide a diagnostic and prognostic marker. This study was carried out in Indian renal transplant patients and healthy adults to characterize the antibody response to cytomegalovirus. METHODS: Thirty three transplant recipients with CMV illness (symptomatology with IgM and/or nPCR positive status), 20 recipients who were asymptomatic in the 6 months of follow up after transplantation and 62 healthy controls were investigated for markers of CMV infection. These individuals were tested for IgG avidity and neutralizing antibody by ELISA techniques. RESULTS: All 53 transplant recipients were found to have an IgG avidity index of > 50 per cent. Antibody to a CMV envelope glycoprotein gB/AD-1 (putative neutralizing antibody) was expressed as S/N ratio and was > or = 5 in asymptomatic (65%) and symptomatic (27%) immunosuppressed renal transplant recipients. However, none of the 53 CMV IgG positive healthy controls were positive for neutralizing antibodies S/N ratio > or = 5 (S/N ratio = sample mean OD/mean OD of 3 negative controls in each run). We observed the simultaneous presence of CMV PCR signal in leukocytes and neutralizing antibody (S/N ratio > or = 5) in the plasma in 22 (41.5%) of the 53 renal transplant recipients. INTERPRETATION & CONCLUSIONS: In this study among the immunosuppressed transplant patients we observed an association between symptomatic disease and the relative absence of neutralizing antibodies. The neutralizing antibodies are less frequently demonstrable among controls; while appearance in a higher proportion of asymptomatic recipients especially in association with high IgG avidity (> 90%) is suggestive of its role in control of CMV disease despite reactivation as evidenced by DNAemia while on immunosuppressive therapy.

A pilot study on the role of cytomegalovirus & human herpesvirus-6 infections in Indian bone marrow transplant recipients.

BACKGROUND & OBJECTIVES: Studies from Western transplant centers have shown the importance of cytomegalovirus (CMV) in infections among immunosuppressed post-transplant patients (both solid and bone marrow transplant recipients). Human herpesvirus-6 (HHV-6) infection is also important. Since such data are lacking from India, we carried out a pilot study to investigate the role of these two viruses in infections among Indian allogeneic bone marrow transplant (BMT) recipients. METHODS: A total of 21 BMT patients who developed acute graft versus host disease (GVHD), two patients who developed chronic GVHD, and eight recipients who did not develop GVHD but had skin rash/elevated liver enzymes, persistent cytopaenia or interstitial pneumonitis with a high clinical suspicion of possible CMV association were studied for markers of CMV and HHV-6 infections. RESULTS: CMV DNAemia was documented in 9 (42.8%) and CMV IgM in 4(19%) of the 21 patients with acute GVHD. HHV-6 DNAemia was not seen in any patient with acute GVHD but 2 (9.5%) had HHV-6 IgM. Of the 2 patients with chronic GVHD, 1 was positive for CMV DNA and IgM, and both were negative for HHV-6 markers. The lower incidence of CMV DNAemia in our recipients may be attributable to the presence of neutralizing antibody (anti gB/AD-1) among the 17 CMV and HHV-6 DNAemia negative recipients, 4(23.5%) had neutralizing antibodies (S/N ratio > or = 5). Of the 13 CMV DNAemia positive recipients, only one (7.7%) was positive for neutralizing antibodies. Among the 5 neutralizing antibody (S/N ratio > or = 5) positive recipients, 4 (80%) were negative for CMV DNAemia. The one nPCR positive was revealed only at high DNA (> 0.1 microgram) input indicating low CMV signal strength. INTERPRETATION & CONCLUSION: The present study shows the use of DNAemia in detecting CMV infections among BMT recipients. All recipients had high avidity CMV IgG (AI > 50%) confirming CMV reactivation or reinfection in these patients. There was evidence from this study suggesting that neutralizing antibodies may play a role in controlling CMV reactivation. We found no significant HHV-6 association with GVHD in Indian allogeneic BMT recipients.

Vaccinia reactive antibodies in a south Indian population.

The prevalence of vaccinia virus antibodies was determined in both urban and rural populations in southern India. The study sample consisted of 211 adults and 52 children. The antibody titre was measured in all sera by virus neutralisation and by indirect immunofluorescence assay (IFA). A small panel of sera was tested by Western blotting. There was no significant difference in detection rates between the tests. Generally, seropositivity correlated with a previous history of vaccination. All children were negative for vaccinia antibodies. Among adults overall, 54% had neutralising antibodies whereas 60% were positive for antibodies detected by IFA, however, the prevalence of vaccinia antibody by either method was significantly higher (P < 0.001) among rural subjects than in urban subjects. This higher antibody prevalence among the rural population could be due to exposure to other indigenous orthopoxviruses, possibly buffalopox.

Cytomegalovirus infection in a seroendemic renal transplant population: a longitudinal study of virological markers.

BACKGROUND/AIMS: The detection of viremia by polymerase chain reaction (PCR) in cytomegalovirus (CMV) infection in renal allograft recipients has been shown to have a predictive value for disease. However, its diagnostic utility in a population with high background seropositivity has not been defined. This prospective study was undertaken to assess the relationship of CMV DNAemia, and/or IgM seropositivity to CMV disease in a seroendemic transplant population. METHODS: Consecutive patients undergoing renal transplantation between August 1997 and February 1998 were enrolled. Blood was sampled before transplantation from the donors and recipients for CMV serology and nested PCR for CMV DNA, and after transplantation from the recipients only at monthly intervals until 6 months. Patients were observed for the development of any CMV-like illness during follow-up. CMV DNA was quantitated using limiting dilution PCR on samples obtained from symptomatic patients at the time of illness and from asymptomatic patients at the end of their 6-month follow-up. RESULTS: A total of 57 recipient-donor pairs were recruited. Immunosuppression was cyclosporine-based in 55 of 57 (95. 6%). The CMV serologic status was D+R+ in 55 of 57 and D+R- in 2 of 57 pairs. PCR positivity indicating viremia increased from 5% before transplantation to 95% at 6 months after transplantation. Similarly IgM positivity reached 80% at 3 months and thereafter; positivity for any marker was 100% by 6 months. Viremia was sustained in over half the patients. The incidence of CMV-attributable disease peaked at 3 months, and was predominantly mild and self-limiting. Tissue-invasive disease appeared later in 4 patients (7%). Asymptomatic viremia was seen in 60-70% of patients at each sampling point. The positive predictive value (PPV) of PCR positivity for disease was 35-40%, and the negative predictive value (NPV), 90-100%. However, the high NPV was of use only in the early post-transplant period, negativity for markers declining rapidly with time. Quantitative assay showed significantly higher levels of CMV DNA in symptomatic patients (p = 0.01). A cutoff of 0.001 microg had a specificity of 95% and a PPV of 92.3% for symptomatic CMV disease. CONCLUSION: Qualitative tests to detect CMV DNAemia and IgM, although useful markers of viremia and active infection, have limited utility for the diagnosis of disease in a seroendemic transplant population. Quantitation of CMV DNAemia may play an important role in diagnosis in such a setting.