Pratibha Narang

Persistently elevated T cell interferon-γ responses after treatment for latent tuberculosis infection among health care workers in India: a preliminary report

BackgroundT cell-based interferon-γ (IFN-γ) release assays (IGRAs) are novel tests for latent tuberculosis infection (LTBI). It has been suggested that T cell responses may be correlated with bacterial burden and, therefore, IGRAs may have a role in monitoring treatment response. We investigated IFN-γ responses to specific TB antigens among Indian health care workers (HCWs) before, and after LTBI preventive therapy.MethodsIn 2004, we established a cohort of HCWs who underwent tuberculin skin testing (TST) and a whole-blood IGRA (QuantiFERON-TB-Gold In-Tube [QFT-G], Cellestis Ltd, Victoria, Australia) at a rural hospital in India. HCWs positive by either test were offered 6 months of isoniazid (INH) preventive therapy. Among the HCWs who underwent therapy, we prospectively followed-up 10 nursing students who were positive by both tests at baseline. The QFT-G assay was repeated 4 and 10 months after INH treatment completion (i.e. approximately 12 months and 18 months after the initial testing). IFN-γ responses to ESAT-6, CFP-10 and TB7.7 peptides were measured using ELISA, and IFN-γ ≥0.35 IU/mL was used to define a positive QFT-G test result.ResultsAll participants (N = 10) reported direct contact with smear-positive TB patients at baseline, during and after LTBI treatment. All participants except one started treatment with high baseline IFN-γ responses (median 10.0 IU/mL). The second QFT-G was positive in 9 of 10 participants, but IFN-γ responses had declined (median 5.0 IU/mL); however, this difference was not significant (P = 0.10). The third QFT-G assay continued to be positive in 9 of 10 participants, with persistently elevated IFN-γ responses (median 7.9 IU/mL; P = 0.32 for difference against baseline average).ConclusionIn an environment with ongoing, intensive nosocomial exposure, HCWs had strong IFN-γ responses at baseline, and continued to have persistently elevated responses, despite LTBI treatment. It is plausible that persistence of infection and/or re-infection might account for this phenomenon. Our preliminary findings need confirmation in larger studies in high transmission settings. Specifically, research is needed to study T cell kinetics during LTBI treatment, and determine the effect of recurrent exposures on host cellular immune responses.

Sensitivity of a Whole-Blood Interferon-Gamma Assay Among Patients with Pulmonary Tuberculosis and Variations in T-Cell Responses During Anti-Tuberculosis Treatment

Background:Interferon-γ (IFN-γ) assays are new tests for tuberculosis (TB) infection, and T-cell responses may be correlated with antigen burden. However, it is unclear if IFN-γ assays can be used to monitor response to TB treatment.Materials and Methods:We measured T-cell responses to TB specific antigens in 60 Indian patients with microbiologically confirmed active pulmonary tuberculosis, before, during, and after TB treatment. Most patients were hospitalized and had moderate to advanced disease. IFN-γ responses were measured using the commercial whole-blood Quanti-FERON-TB Gold In Tube (QFT-G) assay at three time-points: at diagnosis (N = 60), after 2 months of intensive treatment (N = 47), and at 6 months (treatment completion) (N = 39).Results:At baseline, 44 of 60 (73%) patients were positive by QFT-G. At the second time-point, 38 of 47 (81%) patients were positive. At treatment completion, 31 of 39 (79%) patients were positive. Changes in IFN-γ responses over time were highly inconsistent - some individuals showed increases, while others showed decreases or no changes. Although the average IFN-γ levels decreased slightly during treatment (not significant), the QFT-G sensitivity remained mostly unchanged during therapy.Conclusions:Our data suggest that the QFT-G assay has modest sensitivity in patients with moderate to advanced pulmonary disease, but our results do not show a clear correlation between antigen burden and T-cell responses. Further research is needed to understand the kinetics of Tcell responses during TB treatment.

Evaluation of Diagnostic Accuracy, Feasibility and Client Preference for Rapid Oral Fluid-Based Diagnosis of HIV Infection in Rural India

Background Oral fluid-based rapid tests are promising for improving HIV diagnosis and screening. However, recent reports from the United States of false-positive results with the oral OraQuick® ADVANCE HIV1/2 test have raised concerns about their performance in routine practice. We report a field evaluation of the diagnostic accuracy, client preference, and feasibility for the oral fluid-based OraQuick® Rapid HIV1/2 test in a rural hospital in India. Methodology/Principal Findings A cross-sectional, hospital-based study was conducted in 450 consenting participants with suspected HIV infection in rural India. The objectives were to evaluate performance, client preference and feasibility of the OraQuick® Rapid HIV-1/2 tests. Two Oraquick® Rapid HIV1/2 tests (oral fluid and finger stick) were administered in parallel with confirmatory ELISA/Western Blot (reference standard). Pre- and post-test counseling and face to face interviews were conducted to determine client preference. Of the 450 participants, 146 were deemed to be HIV sero-positive using the reference standard (seropositivity rate of 32% (95% confidence interval [CI] 28%, 37%)). The OraQuick test on oral fluid specimens had better performance with a sensitivity of 100% (95% CI 98, 100) and a specificity of 100% (95% CI 99, 100), as compared to the OraQuick test on finger stick specimens with a sensitivity of 100% (95% CI 98, 100), and a specificity of 99.7% (95% CI 98.4, 99.9). The OraQuick oral fluid-based test was preferred by 87% of the participants for first time testing and 60% of the participants for repeat testing. Conclusion/Significance In a rural Indian hospital setting, the OraQuick® Rapid- HIV1/2 test was found to be highly accurate. The oral fluid-based test performed marginally better than the finger stick test. The oral OraQuick test was highly preferred by participants. In the context of global efforts to scale-up HIV testing, our data suggest that oral fluid-based rapid HIV testing may work well in rural, resource-limited settings.

Inducible clindamycin resistance in Staphylococcus aureus isolated from clinical samples

PURPOSE Clindamycin is commonly used in the treatment of erythromycin resistant Staphylococcus aureus causing skin and soft tissue infections. In vitro routine tests for clindamycin susceptibility may fail to detect inducible clindamycin resistance due to erm genes resulting in treatment failure, thus necessitating the need to detect such resistance by a simple D test on routine basis. MATERIALS AND METHOD 247 Staphylococcus aureus isolates were subjected to routine antibiotic susceptibility testing including oxacillin (1ìg) by Kirby Bauer disc diffusion method. Inducible clindamycin resistance was detected by D test, as per CLSI guidelines on erythromycin resistant isolates. RESULTS 36 (14.5%) isolates showed inducible clindamycin resistance, nine (3.6%) showed constitutive resistance while remaining 35 (14.1%) showed MS phenotype. Inducible resistance and MS phenotype were found to be higher in MRSA as compared to MSSA (27.6%, 24.3% and 1.6%, 4% respectively). CONCLUSION Study showed that D test should be used as a mandatory method in routine disc diffusion testing to detect inducible clindamycin resistance.

Evaluation of different methods for diagnosis of P. falciparum malaria

Rapid diagnosis is a prerequisite for institution of effective treatment and reducing the mortality and morbidity of falciparum malaria. This study was taken up to compare the efficacy of various rapid methods viz, acridine orange, Plasmodium falciparum histidine rich protein II antigen detection and Fields stain with traditional microscopy i.e, Leishman stain for diagnosing falciparum malaria. Thick and thin blood films of 443 consecutive patients with history of fever with chills and rigors were examined by Leishman and Fields method. Acridine orange stained wet mounts of blood were examined under fluorescence microscopy. All films were examined by two independent microbiologists. Plasmodium falciparum histidine rich protein II antigen was detected using commercially available kit, Paracheck Pf. Out of the 443 subjects examined for P.falciparum 18.28% were detected by Leishman stain, 6.32% by Fields stain, 18.28% by acridine orange and 18.1% by antigen based technique. Fields stain missed 53 (65.4%), while Paracheck Pf was negative in 6(7.4%) of the Leishman positive samples. All Fields stain and acridine orange positives were positive by Leishman, but five Paracheck Pf positives were negative. Leishman stain is cost effective but if facilities are available one should use acridine orange for screening. The antigen detection kits are rapid, simple and are useful but to rule out false negatives in clinically suspected cases, Leishman stain is reliable.

CANDIDA COLONIZATION IN PRETERM BABIES ADMITTED TO NEONATAL INTENSIVE CARE UNIT IN THE RURAL SETTING

PURPOSE Candida colonization in neonates results in significant morbidity and mortality. The purpose of this study was to determine colonization of Candida spp. in preterm babies and identify the risk factors. METHODS Swabs from oral, rectum, groin and umblicus of 103 preterm and 100 term neonates were obtained within 24 hours of birth, day three, day five, day seven and thereafter every week till the neonate was admitted in the neonatal intensive care unit (NICU). Swabs were also collected from the mothers vagina prior to delivery. Twice every month, air of the NICU was sampled by settle plate and swabs were collected from the hands of health care workers and inanimate objects of NICU. Identification and speciation was done by standard methods. Antibiotic sensitivity was studied against amphotericin B, ketoconazole and fluconazole by disk diffusion method. RESULTS Colonization with Candida was significantly higher in preterms. Earliest colonization was of oral mucosa and 77.1% of the preterms had colonised at various sites by the first week of life. Significant risk factors in colonized versus non-colonized preterms were male sex, longer duration of rupture of membranes (DROM), administration of steroids and antibiotics and vaginal colonization of mothers, whereas those in preterms versus terms were low birth weight and gestational age. C. albicans was the commonest species, both in the colonized preterms (45.9%) and vagina of mothers. Resistance was seen to fluconazole and ketoconazole only. No Candida spp. was isolated from health care personnel or environment. CONCLUSIONS Colonization of preterms by Candida is a significant problem in NICU and the significant risk factors observed in colonized preterms were male sex, longer DROM, administration of steroids and antibiotics and vaginal colonization of mothers.

STATUS OF HIGH LEVEL AMINOGLYCOSIDE RESISTANT ENTEROCOCCUS FAECIUM AND ENTEROCOCCUS FAECALIS IN A RURAL HOSPITAL OF CENTRAL INDIA

Considering the emergence of high level aminoglycoside resistance (HLAR) in enterococci this study was undertaken to determine their status in a rural setting. HLAR by disc diffusion and agar dilution, beta lactamase by nitrocefin disc and vancomycin resistance by agar dilution was determined in 150 enterococcal isolates, as per NCCLS guidelines. Only two species, Enterococcus faecalis (85.5%) and Enterococcus faecium (14.7%) were recovered, mostly from blood. Forty six percent showed HLAR. Multi drug resistance and concomitant resistance of HLAR strains to beta lactams were quite high. None showed beta lactamase activity or vancomycin resistance.

Keratitis due to Colletotrichum dematium--a case report.

Colletotrichum dematium has been rarely reported from India before. The present case, a farmer, developed peripheral corneal ulcer five days following trauma with plant. At presentation his visual acuity was 6/60 (unaided) and 6/24P with pinhole. Slit lamp and fluorescent stain examination revealed paracentral corneal ulcer with irregular margins, stromal infiltration and multiple epithelial defects. Microbiological examination of corneal samples confirmed the initial diagnosis of fungal corneal ulcer and the fungus was identified as C.dematium. Patient was treated with topical natamycin and ciprofloxacin. Patient left against medical advice and was lost to follow up. This report emphasizes that Colletotrichum keratitis may not be rare. Early diagnosis may help in institution of specific therapy early in the disease.

Isolation and identification of nontuberculous mycobacteria from water and soil in central India.

Nontuberculous mycobacteria (NTM), important organisms in the Genus Mycobacterium and commonly present in the environment, are known to cause disseminated disease in AIDS patients. In this study, NTM were isolated from environment (soil and water) of the AIDS patients with disseminated NTM disease to know the prevalence of environmental NTM species and their correlation with clinical isolates from patients of the same area. Paraffin baiting technique was used to isolate NTM from environmental samples. Once isolated, subcultures were made on Lowenstein Jensen and Middlebrook 7H10 media and the species were identified using phenotypic and genotypic techniques. A total of 26 NTM isolates belonging to seven different species could be identified. Mycobacterium avium was the only species isolated from both clinical and environmental samples of the same patient; but the isolates did not match using PCR for IS 1311 and IS 1245 spacer sequences.

EVALUATION OF RAPID MTT TUBE METHOD FOR DETECTION OF DRUG SUSCEPTIBILITY OF MYCOBACTERIUM TUBERCULOSIS TO RIFAMPICIN AND ISONIAZID

PURPOSE To evaluate MTT method for detection of drug resistance to rifampicin and isoniazid in M.tuberculosis . This method utilises the ability of viable mycobacterial cells to reduce MTT( 3-4,5-dimethylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide). METHODS The method was standardised with known resistant and sensitive strains of M.tuberculosis and was then extended to 50 clinical isolates. An inoculum of 10 7 cfu/mL was prepared in Middlebrook 7H9 medium supplemented with oleic acid, albumin, dextrose and catalase. For each drug three tubes were used, one with INH(0.2microg/mL) or RIF(1microg/mL), another as inoculum control and third as blank control. These were incubated at 37 degrees C for four and seven days respectively for RIF and INH after which MTT assay was performed. Results were read visually and by colorimeter at 570 nm. Relative optical density unit (RODU) of 0.2 was taken as cut off. Results were compared with drug sensitivity obtained by proportion method using LJ medium. RESULTS For rifampicin, concordance with proportion method was 90% by visual and 94% by RODU. Sensitivity and specificity was 86.8% and 100% respectively by visual method and 95.2% and 87.5% respectively by RODU. For Isoniazid, concordance was 94% and sensitivity and specificity was 94.7 and 91.7% respectively by both visual and RODU. CONCLUSIONS MTT assay proved to be rapid and cheap method for performing drug sensitivity of M.tuberculosis.