The frequency of HIV-I drug resistance mutations among treatment-naive individuals at a tertiary care centre in south India.

Antiretroviral treatment (ART) use in India requires information on baseline drug resistance mutations and polymorphisms in the protease (Pr) and reverse transcriptase (RT) genes of HIV-1 strains from treatment-naïve individuals. We report resistance predictor mutations and polymorphisms in the Pr and the RT sequence of non-clade B HIV-1 strains from ART naïve individuals. The genotypic resistance assay was done on 93 treatment-naïve individuals. The sequences were analysed by Stanford HIV drug resistance data for genotypic drug resistance analysis and REGA HIV-1 subtyping tool. Phylogenetic tree was generated with MEGA 4 for quality control. Ninety-two strains belonged to clade C and one to clade A (A1). Amino acid substitutions were seen at positions associated with drug resistance in Pr gene--10, 24, 74 (each 3%) and position 82 (11%). Substitutions were seen at positions 41 (1%), 100 (1%), 101 (6%), 103 (2%), 179 (6%) and 181 (1%) of the RT sequence known to confer drug resistance in clade B. Polymorphisms in HIV-1 pol gene among treatment-naïve individuals were similar when compared with previous data. One strain each had Y181C substitution, T74S and E35G substitutions in the Pr and one had A98G, K101R and L210FL substitutions in RT.

High rates of regimen change due to drug toxicity among a cohort of South Indian adults with HIV infection initiated on generic, first-line antiretroviral treatment.

OBJECTIVES: To determine the rates, reasons and predictors of treatment change of the initial antiretroviral treatment (ART) regimen in HIV-infected south Indian adults. METHODS: In this prospective cohort study, ART-naive adults initiated on generic, fixed dose combination ART as per the National AIDS Control Organization guidelines were followed up at an academic medical center. Treatment change was defined as any event which necessitated a change in or discontinuation of the initial ART regimen. RESULTS: Two hundred and thirty persons with HIV infection (males 74.8% and median age 37 years) were followed up for median duration of 48 weeks. The majority (98.7%) had acquired HIV infection through the heterosexual route. Most (70.4%) had advanced IV infection (WHO clinical stage 3 or 4) and 78% had CD4+ T-lymphocyte counts below 200 cells/microL. The initial ART regimens used were: Lamivudine (3TC) with Stavudine (d4T) (in 76%) or Azidothymidine (AZT) and Nevirapine (NVP) (in 86%) or Efavirenz (EFV). The cumulative incidence of treatment change was 39.6% (91 patients). Drug toxicity (WHO grade 3 or 4) was the reason for treatment change among 62 (27%) (incidence rate 35.9/100 person-years). The most common toxicities were attributable to the thymidine analogue nucleoside reverse transcriptase inhibitors (NRTIs), d4T and AZT [lactic acidosis (8.7%), anemia (7%) and peripheral neuropathy (5.2%)]. The other toxicities were rash (3.9%) and hepatitis (1.3%) due to NVP. The mortality (4.6/100 person-years) and disease progression rates (4.1/100 person-years) were low. CONCLUSIONS: The ART regimens used in this study were effective in decreasing disease progression and death. However, they were associated with high rates of drug toxicities, particularly those attributable to thymidine analogue NRTI. As efforts are made to improve access to ART, treatment regimens chosen should not only be potent, but also safe.

Hantaviruses: an emerging public health threat in India? A review.

The emerging viral diseases haemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) are a cause of global concern as they are increasingly reported from newer regions of the world. The hantavirus species causing HFRS include Hantaan virus,Seoul virus, Puumala virus, and Dobrava-Belgrade virus while Sin Nombre virus was responsible for the 1993 outbreak of HCPS in the Four Corners Region of the US. Humans are accidental hosts and get infected by aerosols generated from contaminated urine,feces and saliva of infected rodents. Rodents are the natural hosts of these viruses and develop persistent infection. Human to human infections are rare and the evolution of the virus depends largely on that of the rodent host. The first hantavirus isolate to be cultured, Thottapalayam virus,is the only indigenous isolate from India,isolated from an insectivore in 1964 in Vellore, South India. Research on hantaviruses in India has been slow but steady since 2005. Serological investigation of patients with pyrexic illness revealed presence of anti-hantavirus IgM antibodies in 14.7% of them. The seropositivity of hantavirus infections in the general population is about 4% and people who live and work in close proximity with rodents have a greater risk of acquiring hantavirus infections. Molecular and serological evidence of hantavirus infections in rodents and man has also been documented in this country. The present review on hantaviruses is to increase awareness of these emerging pathogens and the threats they pose to the public health system.

Development and evaluation of a rapid IgM capture ELISA (JEV-Chex) for the diagnosis of Japanese encephalitis.

BACKGROUND: The IgM capture ELISA has been the most widely used diagnostic method for Japanese encephalitis. However, the lack of availability of validated commercial kits as well as the short shelf life of the kit reagents has limited the use of this technique to very few centres in Asia. OBJECTIVES: Development and evaluation of a rapid IgM capture ELISA (JEV Chex) in comparison to the conventional IgM capture ELISA. Produce key reagents such as cell culture derived JEV antigen and biotinylated monoclonal antibody which are stable at room temperature. STUDY DESIGN: The conventional IgM capture ELISA was modified to reduce the total assay time and two key reagents used in the assay JEV antigen and biotinylated anti-JEV monoclonal antibodies were rendered stable at room temperature using a special procedure. A multi-centric evaluation of this rapid ELISA was carried out using well characterized stored CSF and serum samples. Long term stability of the key reagents was also assessed over a period of 6 months. RESULTS: The rapid IgM capture ELISA developed by us showed complete concordance with the results obtained using the conventional ELISA at all the three centres where it was evaluated. In addition, the stability studies carried out with the inactivated cell culture antigen and the biotinylated monoclonal antibodies stored at room temperature for a period of 180 days revealed that both these reagents yielded consistent optical density values in the ELISA. CONCLUSIONS: The rapid ELISA format of the IgM capture ELISA (JEV-Chex) developed by us as well as the stability of reagents achieved by us in this study is what renders this rapid IgM capture ELISA very robust and user friendly since reagents can be stored at 4 degrees C by peripheral labs.

Rapid identification of Burkholderia pseudomallei in blood culture supernatants by a coagglutination assay.

In total, 309 blood culture supernatants were tested for the presence of Burkholderia pseudomallei antigen using an in-house coagglutination test prepared by sensitising Cowan I staphylococcal cells with B. pseudomallei polyclonal antiserum. The coagglutination test gave a sensitivity, specificity, positive predictive value and negative predictive value of 100% in comparison with blood culture. A subset of 102 supernatants was also tested for B. pseudomallei antigen using a monoclonal antibody-based latex agglutination test. The sensitivity, specificity, positive predictive value and negative predictive values of this test were 100%, 90%, 75% and 100%, respectively.

Molecular epidemiology of HIV.

The phylogenic analysis of the nucleotide sequences of the env gene has enabled classification of HIV-1 into three groups. The group M of HIV-1 infection has been classified into 9 different genetic subtypes A-K, with E and I being classified as circulating recombinants forms (CRFs). The groups O and N are less frequently encountered in human infections. Presently group M of HIV-1 globally causes 99.6 per cent of all human infections. The epidemiological trends suggest that subtype C strains would dominate the HIV pandemic in the coming years. The geographic spread of subtype C strains is also very diverse with prevalence in Africa, Latin America and Asia. Data from India show a high prevalence of subtype C. In north and western India, 78.4 and 96 per cent of HIV-1 strains respectively were shown to be subtype C. Among female sex workers in Kolkata 95 per cent of the HIV-1 strains were subtype C. The south Indian subtype data are very similar to the data from the rest of India. The HIV-2 groups (subtypes) recognized are A-H. Unlike HIV-1, HIV-2 strains are predominantly found in Africa. The Indian HIV-2 strains identified till date are subtype A. This is also the predominant strain circulating in western African countries. This group (subtype) is estimated to cause 0.11 per cent of all HIV infections in humans.

Subtype & cytokine profiles of HIV infected individuals from south India.

BACKGROUND & OBJECTIVE: The global surveillance of human immunodeficiency virus (HIV) subtypes (clades) helps understand the global distribution and incidence of different HIV subtypes. As knowledge about subtypes circulating in an area is needed for developing a candidate vaccine, prevalence of the subtypes HIV-1 and HIV-2 were studied in south India. The profile of cytokines interleukin 10 (IL10) and interferon gamma (IFNgamma) in both types of infection were also analysed as these are considered indicators of disease progression. METHODS: Patients who belonged to the 4 south Indian States i.e. Tamil Nadu, Kerala, Karnataka and Andhra Pradesh were included. HIV-1 subtyping was carried out by the heteroduplex mobility analysis (HMA) while that of HIV-2 was done by direct sequencing. The quantitation of IFNgamma and IL-10 was carried out using commercial ELISA kits. RESULTS: Among the 82 HIV-1 infected individuals subtyped, 78 (95.1%) were subtype C while all 12 HIV-2 strains were subtype A. IL-10 concentration was significantly higher among HIV infected individuals compared to normal healthy controls. IFNgamma was significantly higher among symptomatic and AIDS groups compared to asymptomatic HIV-1 infected individuals. INTERPRETATION & CONCLUSION: HIV-1 subtype C and the HIV-2 subtype A are the major subtypes circulating in south India. The study showed a trend towards a shifting of the cytokine profile from Th1 to Th2/Th0 in HIV-1, HIV-2 infections, and HIV-1 and HIV-2 dual infected individuals as the disease progresses. This trend observed is not unlike that reported from the West, despite the difference in subtype profile.

A pilot study for serological evidence of hantavirus infection in human population in south India.

BACKGROUND AND OBJECTIVES: Hantaviruses are rodent-borne viruses of the family Bunyaviridae that have been identified as aetiological agents of two human diseases, haemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). There are no reports of hantavirus infections in humans from India, hence this pilot study was undertaken to provide the serological evidence of hantavirus infections in humans in south India. METHODS: Serum samples were obtained from individuals with acute febrile illness and from voluntary blood donors, majority of whom were from south India. Serum samples were tested for anti-hantavirus IgM using a commercial enzyme immunoassay (EIA). Samples found positive by the EIA were tested by an indirect immunofluorescence assay (IFA) using slides coated with Seoul virus (SEOV) infected cells as substrate. RESULTS: Of the 152 serum samples from individuals with pyrexic illness, 23 (14.7%) were positive for anti-hantavirus IgM by EIA. In contrast, only 5.7 per cent of healthy blood donors were positive by this assay. Eighteen of the 22 (82%) EIA-positive samples from patients were positive by the IFA assay. In contrast, only 2 of the 5 (40%) blood donor EIA positive samples were positive in the IFA assay. INTERPRETATION AND CONCLUSION: The finding of this study indicated the possible presence of hantavirus infections in the human population of India presenting both as asymptomatic and symptomatic infections. Further studies need to be done to confirm the findings on a larger sample using molecular techniques.

Community prevalence of antibodies to human immunodeficiency virus in rural and urban Vellore, Tamil Nadu.

BACKGROUND: Human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS) are becoming increasingly common in India. Currently, antenatal prevalence is a surrogate marker for HIV prevalence in the community. The association between antenatal and community prevalence of HIV needs to be validated so that estimates can be verified or adjusted appropriately. METHODS: A probability proportional to size cluster survey was conducted in the Kaniyambadi block of Vellore district and in the urban wards of Vellore town to estimate the prevalence of antibodies to rubella from August 1999 to February 2000. All personal identifier data from the serum samples were removed to yield a collection for which only the age and sex were known. Estimation of antibodies to HIV in sera from individuals between 15 and 40 years of age, was carried out by one screening ELISA and the reactive sera were further subjected to a supplementary test. RESULTS: We tested 1512 serum samples from subjects residing in rural areas and 1358 samples from those residing in urban areas. The seropositivity among rural samples was 0.66% and among urban samples 1.4%. The prevalence was almost equal among men and women and the youngest infected individual was 15 years old. CONCLUSION: The prevalence of HIV during the period of study was similar to the national surveillance data for Tamil Nadu based on antenatal women. HIV prevalence differs in urban and rural Tamil Nadu, with urban areas having a higher burden of the disease.

A peptide enzyme linked immunosorbent assay (ELISA) for the detection of human immunodeficiency virus type-2 (HIV-2) antibodies: an evaluation on polymerase chain reaction (PCR) confirmed samples.

BACKGROUND: HIV-1 and HIV-2 infections differ in prognosis, and may also require different prevention and/or treatment approaches. Thus, estimating the true prevalence of HIV-1 and HIV-2 infections, as well as co-infections, is a critical step in controlling the disease. There are a few commercial ELISA and immunoblot kits, which can differentiate between HIV-1 and HIV-2 infections. However, some of these assays overestimate the prevalence of dual infection. Hence, it is necessary to develop assays capable of discriminating between the two infections. OBJECTIVES: To develop a synthetic HIV-2 env based peptide ELISA for the detection of HIV-2 specific antibodies and evaluate its performance on samples from HIV positive individuals previously tested by HIV-1 and HIV-2 PCR and HIV seronegative individuals. STUDY DESIGN: We studied 45 HIV seronegative and 63 HIV infected individuals, including 30 HIV-1 PCR and immunoblot positives, 19 HIV-2 PCR and immunoblot positives, five HIV-1 and two PCR and dual immunoblot positives, two PCR negative but positive for HIV-2 by immunoblot and seven dual immunoblot positives who were only positive for HIV-1 by PCR. RESULTS: All 24 HIV-2 PCR positive samples tested were positive by the peptide assay. Among 30 HIV-1 PCR and immunoblot positive samples, only one (3.3%) showed an absorbance value above the cut off level. The seven dual positive samples by immunoblot (only positive for HIV-1 by PCR) were negative by the HIV-2 peptide ELISA. There was a 100% concordance between HIV-2 PCR and peptide ELISA. The sensitivity, specificity, and the likelihood ratio for the peptide ELISA were 100,94.9, and 19.5, respectively when compared against the PCR findings. CONCLUSIONS: This ELISA, using a specific immunodominant epitope (11 amino acids) from the transmembrane (gp36) portion of the HIV-2 envelope glycoprotein showed a high concordance with PCR findings. This can be considered as a highly sensitive, specific and economically feasible assay for the discrimination of HIV-1 and HIV-2, and may serve as an alternative to HIV-2 PCR in epidemiological studies.

Peripheral CD4+/CD8+ T-lymphocyte counts estimated by an immunocapture method in the normal healthy south Indian adults and HIV seropositive individuals.

BACKGROUND: Flow cytometry is the standard method for the estimation of CD4/CD8 counts, but the high initial investment for this instrument and costly reagents make it unaffordable to most of the centers in a developing country like India. OBJECTIVES: To evaluate the feasibility of an alternate system for the estimation of CD4 and CD8 counts in normal south Indian adults and validate the usefulness of this assay to monitor the counts in HIV seropositive individuals. STUDY DESIGN: Forty-six normal healthy adults and 68 HIV seropositive individuals both belonging to south Indian linguistic groups were enrolled in this cross-sectional study. The HIV seropositive individuals included 54 HIV-1, 9 HIV-2 and 5 HIV 1&2 infected individuals serologically confirmed by one of the commercial Immunoblot kits. The Capcellia CD4/CD8 whole blood assay, an immuno-capture ELISA based kit from Sanofi DIAGNOSTICS Pasteur, (France) was used with a few modifications in the procedure to measure the CD4 and CD8 counts. RESULTS: The mean CD4 cell counts were 1048 (central 95 centile only), 746 and 424 for the normal healthy adults, asymptomatic HIV seropositives and symptomatic HIV patients, respectively, and the mean CD8 counts were 595, 889 and 732, respectively. Statistically significant differences were observed in the CD4 cell counts between HIV seronegative healthy adults and asymptomatic (P < 0.001) as well as asymptomatic and symptomatic (P < 0.05) HIV infected individuals. The mean CD4 counts of asymptomatic HIV-2 infected individuals was significantly higher than the counts of asymptomatic HIV-1 infected individuals (P < 0.05). CONCLUSIONS: This is an user friendly test and can be an alternate to flow cytometry for the estimation of peripheral T-lymphocyte subsets in developing countries. The assay system has certain limitations inherent to ELISA techniques.

Evaluation of modified passive haemagglutination assay for Vi antibody estimation in Salmonella typhi infections.

A simple passive haemmagglutination assay (PHA) was developed to detect Vi antibodies, to improve the diagnosis of typhoid fever by small laboratories. The Vi capsular antigen of Salmonella typhi was extracted by alternate alcohol and acetone precipitation. Formalin fixed, sheep red blood cells treated with chromium chloride were sensitised with this Vi antigen and antibodies detected and measured by PHA. The test had a sensitivity of 83.3% among 30 cases of typhoid fever confirmed by culture. The specificity of the test was 94%, making it suitable for use in laboratories without facilities for IFAT or ELISA.

Improved sensitivity of reverse transcriptase polymerase chain reaction for hepatitis C virus using random hexamer primers.

The use of specific primers and random primers for the detection of hepatitis C virus RNA by reverse transcriptase-polymerase chain reaction was compared. The cDNA obtained from using both methods was amplified by the same protocol. Of the 30 plasma specimens tested, 6 (20%) were hepatitis C virus RNA-positive using specific primers and 19 (63%) were positive using random primers, demonstrating that the use of random hexamer-primed reverse transcriptase-polymerase chain reaction significantly improves the sensitivity (p < 0.01).

Comparison of two different methodologies for the detection of hepatitis B virus DNA in plasma.

In this study, two different methodologies were compared for the detection of hepatitis B virus (HBV) DNA in the plasma of 28 patients and 36 controls. Method 1 was a nested polymerase chain reaction (PCR) followed by product detection in an ethidium bromide stained gel, whereas method II was a commercial single step PCR with digoxigenin labeled product captured by a probe and then detected in a digoxigenin-antidigoxigenin enzyme-linked immunosorbent assay (DIG ELISA). The results indicate that both methods are comparable showing a concordance of 98.4%, there was no statistically significant difference in the detection rates. We feel that any one of these assays may be suitable in a clinical laboratory setting, though the commercial assay may offer some advantages to laboratories without sufficient skilled staff in trouble-shooting PCR related problems.

A simple method to prepare Salmonella (A-E) polyvalent antiserum.

The preparation of Salmonella O serogroup (A-I) polyvalent antiserum is laborious. In an alternate method, rabbit antiserum raised to cell surface proteins of Salmonella serotype typhi was compared with a commercial Salmonella polyvalent (A-I) antiserum; 51 Salmonella (A-E) and 16 strains of related organisms tested gave comparable agglutination with both sera.

Vi-specific latex agglutination for early and rapid detection of Salmonella serotype typhi in blood cultures.

Latex particles coated with rabbit antisera against Salmonella serotype typhi (S. typhi) Vi and O (STO) antigens were used in slide agglutination tests for the rapid identification of S. typhi in blood culture broths as soon as Gram-negative bacilli (GNB) were detected in them. Among 231 consecutive blood cultures showing GNB tested for Vi, and a subset of 163 tested for STO, by latex agglutination (LA), 125 and 32, respectively, were positive. The GNB in 127 blood cultures were confirmed by conventional methods as S. typhi, 125 (98.4%) of which had been identified by the Vi LA test. In the subset of 163, 81 grew S. typhi, of which only 32 (39.5%) had been identified by the STO LA tests. Thus, the sensitivity of the Vi and STO LA tests was 98.4% and 39.5%, respectively, whereas the specificity was 100% for both tests. Of the S. typhi isolates, 38 (30.4%) were detected by the Vi LA test on day 2 and 73 (58.4%) on day 3, day 1 being the date of inoculation of the blood culture broths. Thus, the Vi LA test is suitable for the early and rapid confirmation of S. typhi in blood culture.

Serotypes of Streptococcus pneumoniae causing meningitis in southern India. Use of new direct latex agglutination antigen detection tests in cerebrospinal fluid.

The utility of a new latex agglutination (LA) test to directly determine serotypes of Streptococcus pneumoniae in cerebrospinal fluid (CSF) was assessed in prospective evaluation at a referral hospital in southern India. Samples from 18 ill patients with Gram-positive organisms in CSF were tested. The presence of C polysaccharide or specific serotype antigen (types 1, 3, 4, 5, 6, 12, 14, 18, 19, and 23) of S. pneumoniae was detected by slide LA test. Pneumococcal antigen was detected in 17 (94%) of 18 CSF specimens; in 14 (78%) the serotype was determined directly. Serotypes 1, 5, 6, 19, 23, 7, 10, 34, and 38 were found in these patients. A quellung test of the cultured isolates confirmed the serotypes. Information regarding the serotype distribution of pneumococci in varied geographic locations is important for the design and evaluation of pneumococcal vaccines. The slide LA tests seemed useful in detecting S. pneumoniae antigens and in determining the serotype, and have promise in simplifying the gathering of serotype data.

Monovalent latex agglutination reagents for the diagnosis of nonmeningitic pneumococcal infection.

The pneumococcus is a leading cause of serious bacterial infection worldwide. Given the difficulties with available assays for the diagnosis of invasive nonmeningitic pneumococcal infection, we evaluated monovalent slide latex agglutination reagents among patients with blood culture-confirmed pneumococcal infection and control patients in Baltimore, Maryland, USA; São Paulo, Brazil; and Cairo, Egypt. Among 50 patients with invasive nonmeningitic pneumococcal infection, 23 had a positive urine test for a sensitivity of 46% (95% confidence intervals of 32% and 61%). Among 39 healthy children, 36 had a negative assay, for a specificity of 92% (95% confidence intervals of 78% and 98%). Among 80 children with pneumonia without a positive blood culture for Streptococcus pneumoniae, the specificity was 88% (95% confidence intervals of 78% and 94%). Although the assay was fairly specific, the positive predictive value using optimistic assumptions was only 73%-83%. This study suggests that this assay has a sensitivity and positive predictive value that may limit its value in some settings.

Invasive properties of south Indian strains of Streptococcus pyogenes in a HEp-2 cell model.

The objective of this study was to consider the invasive properties of Streptococcus pyogenes in human pharyngeal epithelial cells, and to correlate these with their clinical significance. Clinical isolates of S. pyogenes obtained from blood cultures over a period of 10 years, and throat and skin isolates from a community-based study, were used in this investigation. The S. pyogenes isolates were inoculated in HEp-2 cells and subsequently treated with antibiotics to kill the extracellular bacteria. The cells were then lyzed, and a colony count was carried out to check for invasion. The throat and skin isolates had 45.7%, 25.7% and 28.5% of low, intermediate and high invasion efficiencies, respectively, while 80%, 8.6% and 11.4% of the blood isolates had low, intermediate and high invasion efficiencies. We concluded that the throat and the skin isolates from superficial infections were more invasive than the blood isolates, which is an interesting and paradoxical feature.