A multiplex nested PCR for the simultaneous detection of Salmonella typhi, Mycobacterium tuberculosis, and Burkholderia pseudomallei in patients with pyrexia of unknown origin (PUO) in Vellore, South India.

BACKGROUND AND OBJECTIVES: Salmonella typhi, Mycobacterium tuberculosis, and Burkholderia pseudomallei are among the most important monocyte-tropic bacterial agents causing pyrexia of unknown origin (PUO), with a significant number of endemic infections in both South and Southeast Asian regions. These infections pose a major risk to travelers to these regions as well. METHODS: We developed and evaluated a multiplex nested polymerase chain reaction (PCR) for the simultaneous detection of the three pathogens in 305 patients' buffy coat samples. RESULTS: The assay for S. typhi and B. pseudomallei was able to detect down to 1 colony forming unit/5 µL PCR input and M. tuberculosis was detected down to 20 genome copies/5 µL PCR input. S. typhi was detected in 10 (3.3 %) individuals, B. pseudomallei in 10 individuals (3.3 %), and M. tuberculosis in 18 individuals (5.9 %). Co-infections of M. tuberculosis and B. pseudomallei were detected in three individuals and S. typhi and B. pseudomallei in two individuals. CONCLUSION: This protocol is efficient for PUO diagnosis especially in Asian countries.

Risk factors for tuberculosis among health care workers in South India: a nested case-control study.

OBJECTIVE: The epidemiology of tuberculosis (TB) among health care workers (HCWs) in India remains under-researched. This study is a nested case-control design assessing the risk factors for acquiring TB among HCWs in India. STUDY DESIGN AND SETTINGS: It is a nested case-control study conducted at a tertiary teaching hospital in India. Cases (n = 101) were HCWs with active TB. Controls (n = 101) were HCWs who did not have TB, randomly selected from the 6,003 subjects employed at the facility. Cases and controls were compared with respect to clinical and demographic variables. RESULTS: The cases and controls were of similar age. Logistic regression analysis showed that body mass index (BMI) <19 kg/m(2) (odds ratio [OR]: 2.96, 95% confidence interval [CI]: 1.49-5.87), having frequent contact with patients (OR: 2.83, 95% CI: 1.47-5.45) and being employed in medical wards (OR: 12.37, 95% CI: 1.38-110.17) or microbiology laboratories (OR: 5.65, 95% CI: 1.74-18.36) were independently associated with increased risk of acquiring TB. CONCLUSION: HCWs with frequent patient contact and those with BMI <19 kg/m(2) were at high risk of acquiring active TB. Nosocomial transmission of TB was pronounced in locations, such as medical wards and microbiology laboratories. Surveillance of high-risk HCWs and appropriate infrastructure modifications may be important to prevent interpersonal TB transmission in health care facilities.

Chemokine profile among human immunodeficiency virus-1 (HIV-1) infected individuals from southern India.

BACKGROUND & OBJECTIVE: Individuals infected with HIV-1 have higher levels of chemokine producing cells compared to uninfected individuals. It is important to know the changes in chemokine levels associated with rate of progression of disease. There is a paucity of information on the plasma chemokines in HIV-1 infected individuals from India. We therefore carried out this study to estimate the levels of three chemokines namely macrophage inflammatory protein alpha (MIP1alpha), MIP1beta and RANTES, in relation to disease status in HIV-1 infected individuals and compared with uninfected individuals. METHODS: RANTES and MIP1alpha were estimated using ELISA in 114 HIV-1 infected and 30 controls, whereas MIP1beta was estimated in 101 HIV infected individuals only and 30 controls. The values were compared to the T cell subsets, HIV-1 viral loads and plasma cytokines (interferon gamma and interleukin-10). RESULTS: Compared to controls the mean MIP1alpha and RANTES level among the HIV-1 infected individuals was higher while MIP1beta level was lower in HIV infected individuals except CDC C groups. There was a significant positive correlation for MIP1á with HIV-1 viral load and IFNgamma, for MIP1alpha with viral load and IL10. There was a significant negative correlation between MIP1alpha with CD4 count and CD4: CD8 ratio and MIP1beta with CD4 count and CD8 count. There was a negativecorrelation between RANTES values and CD8 per cent. INTERPRETATION & CONCLUSION: In conclusion, our study showed a significantly higher level of beta chemokines in south Indian HIV-1 infected individuals compared to controls. These beta chemokines may have the inhibitory effect on HIV-1 only during the initial period and with the progression of disease this inhibitory effect wanes as shown by the positive correlation of beta chemokines with HIV-1 viral load.

Primary oral tuberculosis: report of two cases.

Oral lesions of tuberculosis though uncommon, are seen in both the primary and secondary stages of the disease. In secondary tuberculosis, the oral manifestations may be accompanied by lesions in the lungs, lymph nodes, or in any other part of the body and can be detected by a systemic examination. Primary oral tuberculosis may present as a diagnostic challenge for the clinician. Here we report two patients with primary tuberculosis in the oral cavity who presented to the dental department, were diagnosed and referred for medical management.

Toxigenicity testing of clinical isolates of non-typhoidal salmonellae in Vero cell culture & Caenorhabditis elegans model.

The non-typhoidal salmonellae (NTS) are recognized agents of gastroenteritis worldwide. Some of the NTS do not produces cytotoxic changes in tissue culture and not much is known about the endotoxicity of the clinical isolates of NTS (mostly Salmonella enterica serotype Typhimurium and Salmonella enterica serotype Enteritidis). We examined the exotoxic (cytotoxin) and endotoxic activity of clinical isolates of NTS in two assay models namely Vero cell culture and the nematode, Caenorhabditis elegans. Bacteria-free culture supernatants of 40 isolates NTS were tested in 96 well microtitre plate containing confluent monolayers of Vero cells. For the effects on C. elegans, the worms were exposed to bacteria free culture supernatants in 24 well microtitre plate for 24 h and then transferred to OP50 Escherichia coli lawn culture. The endotoxic activity of the live bacterium was studied by feeding the worms in the lawn culture of NTS separately. No cytopathic effect was observed with NTS tested in Vero cell culture assay. Likewise, the worms exposed to the bacteria-free culture supernatants were found active up to 7 days. In the co-culture killing assay, worms were found dead with characteristic stiff and straight appearance by 16(th) day. The worms were alive up to 21 days in OP50 E. coli. Bacteria-free culture supernatants did not have any deleterious effect on worms or in Vero cell culture, suggesting that there is no soluble toxic factor (diffusible toxin) in the culture supernatants. However, live NTS were found to be lethal to the worms; indicating that direct interaction between viable NTS and C. elegans is necessary for killing.

Prospective evaluation of a rapid diagnostic test Typhidot for typhoid fever.

BACKGROUND & OBJECTIVE: Typhoid fever still continues to be a major public health problem around the world. A simple, reliable and affordable rapid diagnostic test has been a long felt need of the clinicians. We therefore prospectively evaluated the sensitivity and specificity of Typhidot test. METHODS: The study was carried out between January 2002 and December 2003, on a total of 563 samples from patients clinically suspected to have typhoid fever; blood culture as well as serum for Typhidot test were received. RESULTS: Of the 563 samples, Typhidot test and blood culture were positive in 36 patients, both the tests were negative for 503 patients. Typhidot test was positive for 9 patients with S. Paratyphi A infection. The sensitivity and specificity of the test using blood culture as gold standard were 92.3 and 98.8 per cent respectively for the typhoid fever. INTERPRETATION & CONCLUSION: Typhidot test is rapid, easy to perform and reliable test for diagnosing typhoid fever, useful for small less equipped laboratories as well as for those with better facilities.

Comparison of two methods to detect carbapenemase & metallo-beta- lactamase production in clinical isolates.

BACKGROUND & OBJECTIVE: Bacterial resistance has greatly hampered effective treatment of patients in clinical settings. Non-fermenting Gram-negative bacilli (NFGNB) are common nosocomial pathogens. In this study we attempted to develop a convenient test for early detection of carbapenemase and metallo-beta-lactamase (MbetaL) production in NFGNB. Lack of sufficient reports from India in this area indicated the need for this study. METHODS: A total of 50 imipenem resistant NFGNB were speciated, and their resistance reconfirmed by disk diffusion and minimum inhibitory concentration (MIC) determination by agar dilution. Two different methods namely modified Hodge and EDTA disk synergy tests were evaluated for carbapenemase and metallo-beta-lactamase (MbetaL) production. RESULTS: Of the 50 imipenem resistant NFGNB, 48 and two respectively fell in the resistant and intermediate range in MIC using agar dilution. Majority of these were Pseudomonas aeruginosa (n=28), followed by Burkholderia cepacia (n=9). The modified Hodge test could detect 28 strains as carbapenemase and MbetaL producers, while the EDTA disk synergy test was able to detect an additional 8 strains producing MbetaL and carbapenemase. INTERPRETATION & CONCLUSION: Pseudomonas aeruginosa was found to be the predominant NFGNB in our hospital setting and EDTA disk synergy could detect more carbapenemase and metallo-beta- lactamase producers compared to modified Hodge test.

Cytotoxin testing of environmental Aeromonas spp. in Vero cell culture.

BACKGROUND & OBJECTIVES: Aeromonas spp. are known to cause a variety of infections in humans and this organism has been isolated from a variety of sources including environmental sources. The pathogenicity of the environmental isolates in and around Vellore has not been studied. This study was conducted to determine the cytotoxicity of the Aeromonas spp. isolated from water bodies, soil sediments, plankton and sewers in and around Vellore. METHODS: Aeromonas spp. isolated from environmental sources were identified by standard procedures. Representative isolates of Aeromonas spp. were tested for cell free cytotoxic factor in tissue culture system. Undiluted and diluted cell free filtrates of isolates and known toxigenic and non-toxigenic bacteria were added to Vero cell monolayer in microtitre plates. After appropriate incubation in 5 per cent CO2 atmosphere, the microtitre plate was examined for cytopathic effect. Cell detachment and shrinkage of Vero cells were recorded as toxic changes. RESULTS: All 36 environmental isolates demonstrated cytopathic effect of which 41.7, 50 and 8.3 per cent belonged to A. hydrophila, A. veronii biotype sobria and A. caviae respectively. INTERPRETATION & CONCLUSION: The results demonstrated the presence of potentially pathogenic environmental aeromonads in and around Vellore and they produced cytotoxin.

Treatment failure in typhoid fever with ciprofloxacin susceptible Salmonella enterica serotype Typhi.

Fluoroquinolones are considered the most effective drugs for the treatment of typhoid fever and hence are widely used in the empiric treatment of acute undifferentiated febrile illnesses in India. Recent reports of Salmonella enterica serotype Typhi (S. Typhi) strains with increasing minimum inhibitory concentration (MIC) of ciprofloxacin have raised the fear of potential treatment failures. In this case series of 109 consecutive patients hospitalized with typhoid fever (S. Typhi grown from blood), we documented clinical failure (fever persisting >6 days) in 25 of 46 (54.3%) adults who could be evaluated. Among these, eight (17.4%) had microbiological failure (S. Typhi recovered from blood after 6 days of ciprofloxacin therapy) despite adequate serum ciprofloxacin levels, and all required alternative drugs for treatment. These 8 S. Typhi strains, although susceptible to ciprofloxacin (MIC < 1 microg/mL) (NCCLS 2000), had MICs (median MIC 0.5 microg/mL) that were increased 15-fold compared to S. Typhi strains from patients with typhoid fever seen at our center in 1995 (median MIC 0.032 microg/mL), and were nalidixic acid resistant S. Typhi (NARST) (MIC > or =32 microg/mL). The poor treatment outcomes with ciprofloxacin therapy in patients infected with NARST strains that exhibit an increased ciprofloxacin MIC call for a need to revise the ciprofloxacin breakpoints for S. Typhi.

Detection of virulence attributes of Burkholderia pseudomallei.

BACKGROUND & OBJECTIVES: Melioidosis caused by Burkholderia pseudomallei is an emerging disease in India. This study examined the toxin activity of bacteria-free culture filtrate in three different cell lines (cytotoxic assay) and its effect on Caenorhabditis elegans (nematode toxicity assay). Endotoxic activity of the viable bacteria was also studied in C. elegans (co-culture killing assay). METHODS: For toxin studies, serial doubling dilutions of unheated, heated crude and ultra filtrate of bacteria-free culture supernatants of B. pseudomallei were tested in 96-well microtitre plate containing confluent mono layers of McCoy, Hep-2 and HeLa cell lines. For the effects on C. elegans, the worms were exposed to heated and unheated bacteria-free culture supernatants in 24-well microtitre plate for 24h and then transferred to OP50 Escherichia coli lawn culture. The endotoxic activity of the live bacterium was studied by feeding the worms in the lawn culture of B. pseudomallei. RESULTS: All the clinical isolates (n=38) produced cytotoxic changes in all the cell lines. No difference was observed in the cytotoxicity of unheated, heated and ultra-filtered culture supernatant. The septicaemic isolates were observed to produce cytotoxic changes in high dilutions (1:160) of culture filtrate. None of the unheated and heated crude filtrate had deleterious effect on C. elegans, while all the live bacteria were found to be lethal to the nematode. INTERPRETATION & CONCLUSION: The culture supernatants, though produced cytopathic effect in various tissue cultures, failed to have any deleterious effect on the worms. However, live bacteria were lethal to the worms B. pseudomallei. Use of C. elegans model to detect virulence attributes of B. pseudomallei is recommended as an alternative to tissue culture methods as this can be carried out in laboratories where a tissue culture set up is not available.

Indirect immunofluorescent antibody test for the rapid diagnosis of melioidosis.

Culture is the only reliable method available at present for the diagnosis of melioidosis. Though serological tests have been described, their value in routine diagnosis is controversial. All indirect immunofluorescent assay (IFA) was therefore evaluated to determine its use in the diagnosis of melioidosis. Whole cell antigen prepared from a laboratory isolate of Burkholderia pseudomallei was used to assay IgG and IgM antibodies. Fourteen of the 22 (63.6%) culture proven cases had IgM antibodies while only 10 (45.5%) had IgG antibodies. Negative predictive value of IgM assay was 92 per cent. Positive predictive value was 100 per cent if both IgM and IgG were considered together. The present study done on a limited number of samples suggests that IFA may be useful in routine diagnosis of melioidosis.

Septicaemic melioidosis in a tertiary care hospital in south India.

BACKGROUND & OBJECTIVES: Melioidosis and the causative organism Burkholderia pseudomallei are being recognized gradually in various centres in India. In the septicaemic form, melioidosis is a serious and life threatening condition which requires early detection and specific treatment to avoid case fatality. A review of patients with septicaemic melioidosis at a tertiary care hospital in south India was carried out with a view to define the clinical features, predisposing conditions, if any, and the outcome. METHODS: A total of 28 patients with culture proven septicaemic melioidosis during December 1993 to December 2002 were included. Information on clinical details and outcome was obtained and antibiotic susceptibility of the isolates studied. RESULTS: Of the 28 patients of blood culture proven septicaemic melioidosis, the organism was also isolated from pus in two patients. The presenting clinical features were varied, most presenting as pyrexia of unknown origin or visceral abscesses, or septic arthiritis. Associated/predisposing conditions were present in 50 per cent of the patients, and diabetes mellitus was the commonest one. mortality was 58 per cent in our series. INTERPRETATION & CONCLUSION: Melioidosis is an emerging infection in India. The magnitude of the problem can only be assessed by increasing awareness, both of its existence in the clinical setting and its identification in the laboratory.

HIV-2 subtype circulating in India (south).

The genetic types and subtypes of HIV vary from region to region. This study looked at the relative prevalence of HIV-2 subtypes in south India. Nucleotide sequencing of the HIV-2 envelope V3 region of strains from 11 individuals infected with HIV-2 and one individual who had dual infection with HIV-1 and HIV-2 was performed. Phylogenetic analysis showed that all individuals were infected with HIV-2 subtype A. These strains were from individuals who live in different regions of south India. In all, up to present, 17 strains inclusive of the authors' 12 have been sequenced, and subtype A of HIV-2 is seen in both monoinfections and dual infections with HIV-1 in India.

Shigella isolation in Vellore, south India (1997-2001).

Shigellosis has been a major cause of dysentery for many years at Vellore, south India. In the last two years the number of Shigella being isolated from samples of faeces from patients with diarrhoea has decreased (5% isolation rate in 1997 to 3.9% in 2001), although the microbiological methods and media used have not changed. Also, the nalidixic acid (NA) resistance has increased for S. sonnei (now 94%). This is noteworthy, since NA has been recommended for the empirical treatment of patients suspected to have shigellosis and this concept needs to be reconsidered based on available data.

Non-01 Vibrio cholerae septicemia and meningitis in a neonate.

Non-O1 Vibrio cholerae is known to cause diarrhoea as well as extra-intestinal infections in adults and children. However meningitis in children is a rare occurrence. We report a neonate who developed septicemia and meningitis due to Non-O1 Vibrio cholerae.

Rapid particle agglutination test for human immunodeficiency virus: hospital-based evaluation.

The performance of a rapid particle agglutination test for human immunodeficiency virus (HIV) (Capillus HIV type 1 [HIV-1]/HIV-2) on hospital samples is compared with enzyme-linked immunosorbent assays. The test had a sensitivity and specificity of 99 and 98.9%, respectively. In addition, the test was reactive on plasma samples from all individuals infected with HIV-1 subtype C. This test can safely be used for voluntary counseling and testing in India.

Increased number of CCR5+ CD4 T cells among south Indian adults probably associated with the low frequency of X4 phenotype of HIV-1 in India.

BACKGROUND & OBJECTIVE: The shift of the human immunodeficiency virus (HIV) from nonsyncytium inducing strains (NSI/R5) to syncytium inducing strains (SI/X4) seen in subtype B infections during progression to acquired immunodeficiency syndrome (AIDS) is less frequently reported in subtype C. NSI and SI strains differ in the co-receptor they utilize to infect a T-cell. We postulated that a larger pool of CD4 T cells expressing CCR5 would be present among individuals in the Indian population. To validate this hypothesis, we estimated the percentage of CD4 cells expressing CCR5 or CXCR4 molecules among healthy south Indian adults and HIV infected individuals. METHODS: HIV-1 infected and uninfected adult volunteers, belonging to the four southern states of India with Tamil/Malayalam/Kannada or Telugu as their spoken language were prospectively recruited. A two colour flowcytometry examination of the blood sample was done using the following monoclonals; anti-CD45 (FITC)/CD14 (PE), anti IgG1 (FITC)/IgG2a (PE), anti-CD3 (FITC)/CD4 (PE), anti-CD3 (FITC)/CD8 (PE), anti-CD4 (FITC) and anti CCR5 (PE) or anti CXCR4 (PE). RESULTS: In the healthy population (n = 30) studied, 24.6 per cent of CD4 T cells expressed CCR5 and the percentage of CD4 T cells expressing CXCR4 was 80.4. Among the HIV infected individuals (n = 51) the percentage of CD4 T cells expressing CCR5 and CXCR4 was 26.8 and 78.7 per cent respectively. INTERPRETATION & CONCLUSION: The percentage of CD4 cells expressing CCR5 and CXCR4 in both the HIV uninfected and infected adults was significantly higher in the south Indian population than in the West. The larger pool of CCR5 positive CD4 cells probably allows for the R5 HIV strain to have a replication advantage over X4 HIV strains. This may explain the lack of shift in the viral phenotype during disease progression and also the perceived rapid progression of the disease in India compared to the West.

Quinolone susceptibility of Vibrio cholerae O1 & O139 isolates from Vellore.

BACKGROUND & OBJECTIVES: Vellore is an endemic area for cholera. The relative prevalence of clinical cases of Vibrio cholerae O1 and O139 has been fluctuating. Few studies have examined the susceptibility of local isolates to quinolones. The objective of the present study was to look at quinolone susceptibility and determine MIC of ciprofloxacin to representative clinical isolates of V. cholerae O1 and O139 in Vellore, obtained between 1997 and 1999. METHODS: Antimicrobial susceptibility testing of V. cholerae strains was performed by disc diffusion technique and MIC determination by E test. RESULTS: Five of 30 O1 and all the O139 serogroup isolates were susceptible to nalidixic acid. All isolates of both serogroups were sensitive to norfloxacin. All isolates of both serogroups gave MIC results in the susceptible range to ciprofloxacin; the MICs being lower for V. cholerae O139 (MIC50 = 0.004 microgram/ml and MIC90 = 0.047 microgram/ml) than for O1 serogroup (MIC50 = 0.38 microgram/ml and MIC90 = 0.5 microgram/ml). INTERPRETATION & CONCLUSION: V. cholerae O1 and O139 show differences in quinolone susceptibility, the reason for this is not clear. This could be because of longer exposure of the O1 serogroup to quinolone antimicrobials as compared to the O139 serogroup.