Investigation of apoptotic markers among human immunodeficiency virus (HIV-1) infected individuals.

BACKGROUND & OBJECTIVES: Apoptosis causes a decline in the counts of uninfected bystander CD4+ T cells in HIV infection. The rate of disease progression of HIV infection is considered to be faster in the developing countries. The present study was carried out to investigate certain markers for apoptosis in immunopathogensis of disease in HIV infected south Indian population. METHODS: Soluble Fas (sFas) antigen and Fas ligand levels in plasma samples from 39 antiretroviral treatment naïve patients was estimated and compared with T cell subsets and HIV-1 viral load. RESULTS: The mean sFas antigen levels among controls and the CDC A, B and C clinical stages were 2.77, 3.08, 3.26 and 3.28 ng /ml respectively, higher though not significantly among HIV-1 infected individuals compared to controls. The mean sFas ligand levels in CDC A, B and C stages were 0.138, 0.125 and 0.117 ng/ml respectively were higher (P<0.001) than controls (0.073 ng/ml) and positively correlated with total lymphocyte % (r=0.43, P =0.007). sFas antigen levels were negatively correlated with total WBC count (r=-0.34, P=0.04), CD4% (r=-0.4, P=0.01) and CD4:CD8 ratio (r=-0.37, P=0.02). There was an increase in plasma levels of sFas antigen and Fas ligand over time in asymptomatics. INTERPRETATION & CONCLUSION: The high levels of sFas antigen and Fas ligand seen in HIV infected individuals suggest increased activation and apoptosis of T cells, due to constant stimulation of the immune system by inter-current infections of HIV infected individuals in south India.

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.

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.

Frequency of extended spectrum ß-lactamase producing urinary isolates of Gram-negative bacilli among patients seen in a multispecialty hospital in Vellore district, India.

Extended-spectrum beta-lactamase (ESBL) producing strains of Coliform bacilli are on the rise and present a major threat especially in India. We assessed the frequency of ESBL producers among urinary isolates from patients presenting urinary tract infections. ESBL screening was done using Double Disk Synergy Test (DDST) and confirmed using E-test and Polymerase Chain Reaction (PCR). With E-test, 92.2% were positive for ESBL. In PCR, 100% strains were positive for any of the three gene targets tested. CTX-M was positive in majority of the strains followed by TEM and SHV. Two (3.22%) strains were positive for all the three genes; 21% strains were positive for both TEM and CTX-M genes. There was no statistically significant difference in the findings of E-test and PCR testing in the determination of ESBL producers (Fisher exact test P = 0.15). The strength of agreement between them was 'fair' (k = 0.252). Continuous monitoring of ESBL producers among Indian strains is important to rationalize the antibiotic policy to be followed.

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.

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.

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.

Transducer related Enterobacter cloacae sepsis in post-operative cardiothoracic patients.

The use of intra-arterial pressure monitoring devices in patients undergoing major cardiac surgery is on the increase. Here we document an outbreak of Enterobacter cloacae septicaemia among seven post-operative cardiothoracic patients. Detailed investigations revealed the source of this nosocomial problem to be contaminated transducer heads. The need to follow strict aseptic measures in handling intra-arterial devices is emphasized, in order to minimize the morbidity and mortality in otherwise low-risk post-operative patients.

Dissemination of the TEM-I beta-lactamase gene into disparate plasmids in faecal strains of Escherichia coli isolated in Vellore, south India.

The genetic basis for ampicillin resistance in commensal strains of Escherichia coli isolated in Vellore, south India has been examined. Of the 58 strains tested, 41% could transfer ampicillin resistance to a standard E. coli host strain. With the exception of one isolate, transferable ampicillin resistance was shown to result from the presence of the TEM-I beta-lactamase which was found on a wide variety of plasmid types.

Coagglutination test in the diagnosis of typhoid fever.

Staphylococcal coagglutination (CoA) test for the detection of Salmonella typhi O (factor 9) antigen was evaluated as a diagnostic test in typhoid fever. Supernatants from 106 blood cultures with Gram negative bacilli were subjected to CoA test. The sensitivity of the CoA test for the detection of S. typhi O antigen was 88 per cent and the specificity 97 per cent.

A simplified procedure for rapid screening of vibrios for cholera toxin production.

Conventional methods for the detection of cholera toxin (CT) production by vibrios are not readily available to most laboratories. A modification is described here of a simplified method standardised earlier and based on the degradation of nicotinamide adenine dinucleotide (NAD) by CT; this is simple and can be carried out in small laboratories also. It is also easy to perform, and gives reproducible results.

Resistance of Vibrio cholerae 01 to nalidixic acid.

Until 1987 all isolates of V. cholerae 01 at a tertiary care hospital in south India were susceptible to drugs commonly used to treat gastroenteritis including cholera. Since July 1987 strains resistant to co-trimoxazole have been encountered and since October 1995 strains resistant to nalidixic acid are being isolated. In this study the latter strains were examined by determining minimum inhibitory concentration levels of nalidixic acid as well as norfloxacin, the fluoroquinolone extensively used to treat diarrhoea. No cross resistance to norfloxacin was found in any of the nalidixic acid resistant V. cholerae 01 strains.

The appearance & spread of Vibrio cholerae 0139 in India.

A new clone of non-01 V. cholerae designated as serogroup 0139, which produces cholera toxin, was detected first in south India in September 1992 and has spread to many parts of India since then. It was identified in Bangladesh in December 1992 and in Thailand in April 1993. By May 1993 it was found in Haryana and Punjab. Its clinical manifestations are typical of cholera, occurring in outbreaks. This clone has largely replaced the previously prevalent 01 V. cholerae in several cholera endemic areas indicating that a new cholera pandemic has begun.

Aeromonas species in human septicaemia & diarrhoea.

An increase was observed in the isolation of Aeromonas sp. from clinical specimens, especially faeces, from patients with diarrhoeal disease and blood cultures from patients with suspected septicaemias. The isolation rate from diarrhoeal patients was 0.2 per cent in 1978-79 and this increased to 5.0 per cent in 1986-87. It is noteworthy that 13 patients of septicaemia due to Aeromonas species were encountered, predominantly in adults with lowered resistance due to underlying disease states including chronic diseases of the liver and kidney.

Plasmid mediated multidrug resistance in Salmonella typhi.

Between September 1989 and February 1991, strains of S. typhi showing multiple drug resistance were isolated from blood cultures of patients with typhoid fever. A total of 283 isolates were obtained over a period of 18 months. Forty four (11%) of these isolates were resistant to chloramphenicol, ampicillin, co-trimoxazole and tetracycline, the first three being drugs currently used in treating typhoid fever. Forty of the 44 multi-resistant strains tested showed transfer of resistance 'en block' to recipient Escherichia coli K 12 (J62-2). All 44 multi-resistant strains were sensitive to ciprofloxacin and ofloxacin. Since the resistance is plasmid-mediated, the problem is likely to get aggravated.

Transferable trimethoprim resistance of shigellae encountered in Vellore (south India).

Shigella flexneri and Sh. shigae which are the two most common shigellae encountered in Vellore (south India) were found to exhibit resistance to trimethoprim and trimethoprim-sulphamethoxazole. Eighty four per cent of this was high level resistance. Transfer studies conducted with these strains indicated that this high level resistance is plasmid mediated.