Evaluation of IgM ELISA using a sonicate and a lipopolysaccharide antigen for the serodiagnosis of melioidosis.

Melioidosis, caused by Burkholderia pseudomallei, has variable manifestations. The disease can present as an acute or a chronic form or localized or disseminated or can remain latent for many years. Acute septicaemic melioidosis has a high fatality rate when untreated and therefore, an early diagnosis is critical. Lack of testing facilities and of an awareness of the manifestations of the disease makes it likely that it is underreported in India. A sonicate and a lipopolysaccharide (LPS) antigen were evaluated by an IgM enzyme immunoassay in patients with culture-confirmed melioidosis (n = 29), fever of unknown origin (n = 214) and healthy controls (n = 109). Patients with melioidosis had significantly higher optical density values than both control categories, but the sensitivity of both tests was low (25% for sonicate, 62% for LPS). These data highlight the problems with serodiagnosis in endemic settings, where high cut-off values are required for specificity, and result in low sensitivity.

A molecular method for typing Herpes simplex virus isolates as an alternative to immunofluorescence methods.

BACKGROUND: Typing of Herpes simplex virus (HSV) isolates is required to identify the virus isolated in culture. The methods available for this include antigen detection by immunofluorescence (IF) assays and polymerase chain reaction (PCR). This study was undertaken to standardize a molecular method for typing of HSV and compare it with a commercial IF reagent for typing. OBJECTIVES: To compare a molecular method for typing HSV isolates with a monoclonal antibody (MAb) based IF test. STUDY DESIGN: This cross-sectional study utilized four reference strains and 42 HSV isolates obtained from patients between September 1998 and September 2004. These were subjected to testing using an MAb-based IF test and a PCR that detects the polymerase ( pol ) gene of HSV isolates. RESULTS: The observed agreement of the MAb IF assay with the pol PCR was 95.7%. Fifty four point eight percent (23/42) of isolates tested by IF typing were found to be HSV-1, 40.5% (17/42) were HSV-2, and two (4.8%) were untypable using the MAb IF assay. The two untypable isolates were found to be HSV-2 using the pol PCR. In addition, the cost per PCR test for typing is estimated to be around Rs 1,300 (USD 30), whereas the cost per MAb IF test is about Rs 1,500 (USD 35) including all overheads (reagents, instruments, personnel time, and consumables). CONCLUSION: The pol PCR is a cheaper and more easily reproducible method for typing HSV isolates as compared to the IF test. It could replace the IF-based method for routine typing of HSV isolates as availability of PCR machines (thermal cyclers) is now more widespread than fluorescence microscopes in a country like India.

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.

Aciclovir resistance among Indian strains of Herpes simplex virus as determined using a dye uptake assay.

Resistance to aciclovir (ACV) among Herpes simplex virus (HSV) isolates is increasingly being reported in the literature particularly in immunocompromised patients. However, there is only limited data available from India despite widespread use of ACV in our hospital. A cross-sectional study was hence conducted to determine the aciclovir (ACV) susceptibility of HSV 1 and 2 isolates using a dye uptake (DU) assay. This study showed a 3.0% prevalence of ACV resistance among HSV-1 strains (2/66, median IC 50 0.098 microg/mL) while in HSV-2 strains, it was 7.8% (5/64, median IC 50 0.195 microg/mL). The IC 50 for the HSV-1 and HSV-2 strains resistant to ACV was greater than or equal to 6.25 microg/mL.

Isolated liver abscesses in melioidosis.

Melioidosis is a suppurative chronic infection caused by a gramnegative bacterium, Burkholderia pseudomallei. We report two patients who presented with isolated liver abscesses caused by this pathogen. Both patients presented with high-grade fever and abdominal pain. On examination they were toxic and had tender hepatomegaly. Investigations showed leucocytosis and a shift to the left. Early diagnosis of melioidosis was made by culture and growth of Burkholderia pseudomallei from aspirated pus from the abscesses and the patients were treated with ceftazidime and co-trimoxazole. Despite institution of antibiotics both the patients succumbed to their illness. Melioidosis is an emerging infection in the Indian subcontinent and can cause isolated liver abscesses.

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.

Non tuberculous mycobacteria isolated from clinical specimens at a tertiary care hospital in South India.

PURPOSE: This is a retrospective analysis of the isolation rates of nontuberculous mycobacteria (NTM) from various clinical specimens and their antimicrobial susceptibility patterns. METHODS: All NTM isolated between 1999 and 2004 at Christian Medical College, Vellore, South India, were identified with various biochemical tests. Antimicrobial susceptibility test for all NTM was performed by standard methods. RESULTS: A total of 32,084 specimens were received for culture, of which 4473 (13.9%) grew acid fast bacilli (AFB). Four thousand three hundred (96.1%) of the AFB were M. tuberculosis while 173 (3.9%) were NTM. Of the 173 NTM, 115 (66.5%) were identified to the species level. Pus, biopsy specimens and sputum specimens yielded most of the NTM of which M. chelonae (46%) and M. fortuitum (41%) accounted for majority of them. M. chelonae and M. fortuitum, showed highest susceptibility to amikacin (99.2%). NTM were repeatedly isolated from seven sputum specimens, 15 biopsy and pus specimens, two CSF and two blood cultures. Six were isolated from patients with AIDS and five from post transplant patients. CONCLUSIONS: The isolation of NTM from various clinical specimens is reported in this study to highlight the associated diseases and therapeutic options in these infections.

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.

Mycobacterium fortuitum bacteraemia in an immunocompromised patient.

A case of Mycobacterium fortuitum bacteraemia in an immunocompromised patient confirmed by four positive serial blood cultures is reported here. The patient was a known case of acute lymphoblastic leukemia (ALL) on intensive chemotherapy. The source of bacteraemia was most probably a peripherally inserted vascular catheter. After initiation, of treatment with amikacin to which the strain was sensitive and clarithromycin and removal of the central line the patient's fever defervesced and repeat blood cultures were negative. This is the first time we have encountered an immunocompromised patient with M. fortuitum septicaemia in our hospital. The possibility of an infection with rapidly growing mycobacteria is important to consider when conventional organisms are not isolated in culture especially in the context of patients with malignancy.

The immunological and virological response in human immunodeficiency virus type-1 (HIV-1) infected Indian individuals on haart therapy: A one-year follow up study.

Currently, antiretroviral therapy has become more affordable even in developing countries and it is being used in India. Fifteen HIV-1 infected individuals, who were on highly active antiretroviral therapy (HAART), were followed up for an average period of one year. The plasma viral load and CD4+ T cell estimation done at mean intervals of 5 months and 11 months after initiation of therapy showed a good response to therapy in 14 (93%) individuals.

Resistance of Mycobacterium tuberculosis to the first line anti tubercular drugs--a twenty year review.

Tuberculosis and more so the multi drug resistant variety has been thrust into the forefront as a serious and life threatening illness in recent years. The advent of AIDS contributes to this substantially, especially in the developed world where it had become practically non- existent. We reviewed our data over the past 20 years with a view to determine when drug resistance began to manifest in the strains.

Clinical characteristics and treatment response among patients with multidrug-resistant tuberculosis: a retrospective study.

BACKGROUND: This retrospective study was conducted to evaluate the characteristics and therapeutic response among patients with multidrug-resistant tuberculosis (MDR TB). METHODS: One hundred subjects with isolates resistant to isoniazid and rifampicin were included over a three-year period (1997-1999). There were 82% males with a mean age of 36 years, mean duration of symptoms of 29 months, and a previous history of tuberculosis in 85% (pulmonary 96% and extrapulmonary 4%). RESULTS: HIV ELISA test was positive in two out of 28 (7%) patients while diabetes was diagnosed in 16 percent. Mean time to diagnose MDR TB was 5.5 months. Subjects had received a mean of 3.2 anti-TB drugs before the diagnosis of MDR TB was made. Forty-five patients were lost to follow-up. The rest had a median follow-up of 13.5 months (range 1-37 months). Follow-up AFB smear and culture results were available in 49 out of 55 and 26 out of 55 patients, respectively. Sputum smear became negative for AFB in 26 out of 49 (53%) and culture converison occurred in 16 out of 26 (61.5%) patients. Clinical and radiological response was noted in 31 (56%) and 13 (32.5%) out of 40 patients respectively. A mean of 5.5 drugs was used in those who achieved sputum conversion. Combination therapy containing ofloxacin in the regimen was noted to have a favourable response. CONCLUSION: Only a limited number of patients with MDR tuberculosis have a favourable response.

A study on some phenotypic virulence markers of enteropathogenic Escherichia coli.

BACKGROUND & OBJECTIVES: The problem of enteropathogenic Escherichia coli (EPEC) causing diarrhoea in infants exists in India. But often the enteropathogenic status is not based on adequate characterization. Hence there is a need for evaluating the serotyping being used to identify EPEC for its validity in the light of recent knowledge on phenotypic markers of virulence. This study was done to evaluate the EPEC isolates for two potential virulence factors namely entero-adhesiveness with subsequent actin accumulation and verotoxin production. METHODS: Fifty consecutive EPEC strains identified by serotyping from stool samples of children with diarrhoea during January 1997 to June 1999 were studied for HEp-2 cell adherence, the fluorescent actin staining (FAS) characteristics of Hep-2 cells and vero cytotoxin production. RESULTS: Serotypes O55, O125 and O126 accounted for most of the isolates. In the Hep-2 assay, 72 per cent of the strains showed localised pattern of adherence and 22 per cent showed a mixed pattern of localised and diffuse adherence. In the FAS test 96 per cent strains showed typical staining while none of the strains produced verotoxin. INTERPRETATION & CONCLUSION: 'O' serogrouping appears to be still the simplest and an useful test for presumptive identification of EPEC. The FAS test for confirmation of EPEC was found to be very consistent in indicating EPEC.

An indirect ELISA for the diagnosis of melioidosis.

BACKGROUND & OBJECTIVES: Very little information is available on melioidosis in India. This disease caused by Burkholderia pseudomallei is not often considered as a differential diagnosis and patients are not usually investigated for it. Thus we are unaware of its prevalence in India. This study was undertaken to detect the presence of melioidosis in patients presenting with pyrexia of unknown origin (PUO) using an indirect ELISA. METHODS: The well established ELISA technique was adapted to detect melioidosis in patients attending the Christian Medical College and Hospital, Vellore and to provide a serological test using reagents with a reasonable shelf-life. The ELISA is designed to detect IgG antibodies to B. pseudomallei in serum samples. RESULTS: A cut-off optical density (OD) of 0.36 (mean +/- 2 SD of healthy controls) was chosen as diagnostic criterion for the diseased group. The mean OD values in the sera of patients with culture proven melioidosis was significantly (P < 0.001) higher than that of healthy controls. INTERPRETATION & CONCLUSION: The indirect ELISA was simple to perform and may be recommended as a diagnostic serological test when melioidosis is considered as a differential diagnosis.

Systemic bacterial infections in bone marrow transplant patients.

The clinical microbiology department at CMC&H, Vellore in conjunction with the haematology department carries out routine surveillance of patients admitted to the hematology department. Since 1994 in a sample population of 55 patients with various underlying clinical conditions who have had bone marrow transplant, sepsis was observed in 16 patients (29%). The predominant Gram negatives associated with sepsis were non-fermenting Gram negative bacilli and all the 5 Gram positives were coagulase negative staphylococci. These organisms were susceptible to most of the routinely used antimicrobial agents. Continued surveillance is needed to determine changing trends with respect to organisms causing systemic infections and their susceptibility to antimicrobials.

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.

Cytotoxicity of non O1, non O139 Vibrios isolated from fresh water bodies in Vellore, south India.

The samples of plankton, soil sediment and water from a pond, a lake and a moat respectively in and around Vellore were studied for environmental vibrios. Vibrios were isolated from all these specimens after enrichment in alkaline peptone water and subculture on selective media. Non O1, non O139 Vibrio cholerae, Aeromonas spp. and Plesiomonas spp. were isolated. There were no isolates of V. cholerae serogroup O1 and O139. Representative strains of non O1 and non O139 V. cholerae from environmental sources were tested for toxin production in Chinese hamster ovary (CHO) and Vero cell monolayer in microtitre plates. Thirty-three (91.7%) of the 36 strains tested demonstrated cytopathic effect (CPE) in both cell lines indicating their toxigenicity. PCR done on representative strains of non O1 and non O139 V. cholerae showed that none of the strains were positive for ctx A, tcp A-E and tcp C genes. These results indicate that these non agglutinating environmental vibrios produced cytotoxins other than the cholera toxin.