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Abstract Background While dealing with mixed in vitro lymphocyte cultures one is faced with the problem of relative contributions of different populations to the activity being studied. This is especially true in the controversy relating to the contributions of lymphocyte sub-populations to the Lymphokine Activated Killer (LAK) phenomenon. Flowcytometry can be used to highlight relative contributions of lymphocyte subpopulations towards LAK activity without resorting to difficult purification strategies. We set up long-term in vitro lymphocyte cultures, stimulated them with cytokines IL-2/IL-12, recorded their phenotypic changes and cytotoxic activity against U-937 tumor targets. Results The results indicated that natural killer cells (NK) constituted the predominant proliferating cell population in the cytokine stimulatedcultures. Flowcytometric evidence revealed that CD56+ T cells contributed little to LAK activity against U937 target cells as compared to cells with NK phenotype which were predominantly responsible for spontaneous killing of the tumor targets. The two cytokines, IL-2 and IL-12, had an additive effect on cell proliferation and spontaneous cytotoxicity. Conclusion Flowcytometry can be used to rapidly delineate phenotypic changes in immune cells after stimulation and simultaneously correlate them with corresponding functional activity. This approach may find application as a initial screening tool for studying different types of cells in mixed cultures and their respective activities under stimulatory / inhibitory conditions.

Introduction: Respiratory infections including Coronavirus Disease-2019 (COVID-19) infection spread through droplet infections. Hence standard precautionary measures like handwashing and use of masks are essential to prevent transmission of these infections in healthcare setting. Aim: To determine the effect of COVID-19 pandemic on awareness level of resident doctors on prevention of spread of infective respiratory secretions. Materials and Methods: This cross-sectional study was conducted among two subsequent batches (year 2019-20 and 2020-21) of resident doctors at a medical college hospital, from February 2020 to September 2020. The 2019-2020 batch of resident doctors worked as residents from March 2019 to March 2020 and were considered as pre-COVID-19 batch. The 2020-2021 batch of resident doctors had their training period from April 2020 to April 2021 and were considered the COVID-19 batch. A pretested semi-structured questionnaire consisting of 14 questions to evaluate the awareness on prevention of spread of infective respiratory secretions was administered. The responses were evaluated, marks awarded and summated. Results: The response rate of pre-COVID-19 batch was 85% and that of COVID-19 batch was 86%. The mean (standard deviation) score obtained by the COVID-19 batch was 9.91±3.42 which was significant higher than that obtained by the pre-COVID-19 batch which was 7.1±1.83. The score obtained by COVID-19 batch for 11/14 questions was significantly higher compared to the pre-COVID-19 batch. Conclusion: A significant improvement was seen in the knowledge level in prevention of spread of infective respiratory secretions among resident doctors after the onset of the COVID-19 pandemic.

Introduction Parotid gland squamous cell carcinoma is an uncommon aggressive neoplasm with poor prognosis. Aural polyps are usually the presenting features of chronic suppurative otitis media, tuberculous otitis media, and adenoma or carcinoma. The malignant aural polyp is very rare. Parotid gland carcinoma masquerading as an aural polyp has rarely been described in the literature. Case Report: We report a case study of parotid squamous cell carcinoma in a 29-year-old male masquerading as an ear polyp. Conclusion: Parotid gland primary squamous cell carcinoma is a rapidly advancing neoplasm which carries poor prognosis despite multimodality treatment. Diligent clinical and histopathological evaluation is imperative to discriminate this rare aggressive disease from the metastatic and other primary cancers of the parotid. A high index of suspicion is crucial in refractory aural polyps to arrive at early diagnosis.

Eagle syndrome is a rare condition caused by an elongated styloid process or abnormal calcification of the stylohyoid ligament complex. Patients typically present with recurrent throat pain, neck or facial pain, sensation of foreign body in the throat, or even dysphagia. The finding of an elongated styloid process is usually incidental, with patients having no related symptoms. Only patients with symptoms are diagnosed with Eagle syndrome. This condition may be difficult to diagnose due to its rarity and the nonspecific nature of signs and symptoms. Diagnosis is mainly by clinical assessment and confirmation with radiological evidence. We present a unique case of eagle syndrome in a patient who complained of acute neck pain after eating fish.

Rationale: The outcome of fully intermittent thrice-weekly antituberculosis treatment of various durations in HIV-associated tuberculosis is unclear. Objectives: To compare the efficacy of an intermittent 6-month regimen (Reg6M: 2EHRZ(3)/4HR(3) [ethambutol, 1,200 mg; isoniazid, 600 mg; rifampicin, 450 or 600 mg depending on body weight <60 or >/=60 kg; and pyrazinamide, 1,500 mg for 2 mo; followed by 4 mo of isoniazid and rifampicin at the same doses]) versus a 9-month regimen (Reg9M: 2EHRZ(3)/7HR(3)) in HIV/tuberculosis (TB). Methods: HIV-infected patients with newly diagnosed pulmonary or extrapulmonary TB were randomly assigned to Reg6M (n = 167) or Reg9M (n = 160) and monitored by determination of clinical, immunological, and bacteriological parameters for 36 months. Primary outcomes included favorable responses at the end of treatment and recurrences during follow-up, whereas the secondary outcome was death. Intent-to-treat and on-treatment analyses were performed. All patients were antiretroviral treatment-naive during treatment. Measurements and Main Results: Of the patients, 70% had culture-positive pulmonary TB; the median viral load was 155,000 copies/ml and the CD4(+) cell count was 160 cells/mm(3). Favorable response to antituberculosis treatment was similar by intent to treat (Reg6M, 83% and Reg9M, 76%; P = not significant). Bacteriological recurrences occurred significantly more often in Reg6M than in Reg9M (15 vs. 7%; P < 0.05) although overall recurrences were not significantly different (Reg6M, 19% vs. Reg9M, 13%). By 36 months, 36% of patients undergoing Reg6M and 35% undergoing Reg9M had died, with no significant difference between regimens. All 19 patients who failed treatment developed acquired rifamycin resistance (ARR), the main risk factor being baseline isoniazid resistance. Conclusions: Among antiretroviral treatment-naive HIV-infected patients with TB, a 9-month regimen resulted in a similar outcome at the end of treatment but a significantly lower bacteriological recurrence rate compared with a 6-month thrice-weekly regimen. ARR was high with these intermittent regimens and neither mortality nor ARR was altered by lengthening TB treatment. Clinical Trials Registry Information: ID# NCT00376012 registered at www.clinicaltrials.gov. [ABSTRACT FROM AUTHOR]

Nasal Myiasis is the infestation of the nasal cavity by dipterous larvae and which are commonly documented in developing countries where sanitation is a public problem. Other predisposing factors being poorly nourished patient with poor hygiene, atrophic rhinitis, and diabetic with purulent nasal discharge, midline granulomatous lesions or malignancy involving the nose. We have reported a case of a patient with underlying sinonasal tumour post excision and postoperative concurrent chemoradiation presenting with preseptal cellulitis and nasal myiasis.

Introduction: Atypical infratemporal fossa infections are rare and potentially fatal. Case Report: A case of an aspergillosis localized in the infratemporal fossa and another case of tuberculosis of the infratemporal fossa originating from the maxillary sinus, is described. The first patient was immunocompromised and showed symptoms of facial numbness; whereas the other was an immunocompetent man who complained of trigeminal neuralgia type pain. It was difficult to differentiate between infection and tumour despite the utilization of computed tomography scans and magnetic resonance imaging. Conclusion: These cases illustrate the need for a high index of suspicion; in addition to endoscopic confirmation and histopathology to establish precise diagnosis and early intervention.

Maintaining appropriate dose intensity is important not only in the curative setting but also in treatment with palliative intent. We evaluated the outcome of advanced non small cell lung cancer treated with doublet platinum based chemotherapy. Outcome was compared between patients treated by medical oncologists at a tertiary cancer center and those treated by non medical oncologists in the community. The dose intensity, overall response rate and overall survival was significantly better when patients were treated by trained qualified and experienced medical oncologists. Hence, even in the palliative setting, cancer directed systemic therapy will yield maximum benefit for the patients when treated by medical oncologists.

Matrix metalloproteinase 13 (MMP13) plays a central role in the MMP activation cascade that enables degradation of the extracellular matrix and basement membranes, and it is identified as a potential driver in oral carcinogenesis. Therefore, this study aims to determine the copy number, mRNA, and protein expression of MMP13 in oral squamous cell carcinoma (OSCC) and to associate these expressions with clinicopathological parameters. Copy number, mRNA, and protein expression analysis of MMP13 were determined using real-time quantitative PCR and immunohistochemistry methods in OSCC samples. The correlations between MMP13 expressions and clinicopathological parameters were evaluated, and the significance of MMP13 as a prognostic factor was determined. Despite discrepancies between gene amplification and mRNA and protein overexpression rates, OSCC cases showed high amplification of MMP13 and overexpression of MMP13 at both mRNA and protein levels. High level of MMP13 protein expression showed a significant correlation with lymph node metastasis (P=0.011) and tumor staging (P=0.002). Multivariate Cox regression model analysis revealed that high level of mRNA and protein expression of MMP13 were significantly associated with poor prognosis (P

To study the rate and pattern of epithelial migration in 18 dry, open mastoidectomy cavities. Methods: This was a prospective study of 19 patients who had undergone mastoidectomy at a secondary (state) hospital and a tertiary referral centre at least 6 months previously and whose mastoid cavities were dry and not infected. Their mastoidectomy cavities were cleaned and India ink dots applied in its medial aspect under microscope. The patients were followed up at 1 or 2 weekly intervals for a minimum of 3 months or until the dots disappeared, whichever came first. On follow-up, the cavities were examined under microscope. If migration was observed, the rate and pattern were noted. Results: Most (94.4%) of the cavities were noted to have epithelial migration laterally. No migration was observed in 5.6%. The start of migration of ink dots was noted in the 2nd week in 53% of the cavities, with a mean of 3.05 weeks. The mean rate of migration was 0.68 mm/week. Conclusion: Epithelial migration appears to be predominantly lateral (94.4%), with onset in the 2nd week (53%) at a rate of 0.68 mm/week. Epithelial migration might not play an important role in accumulation of wax in open mastoidectomy cavities.

Despite the advances in diagnosis and treatment of oral squamous cell carcinoma (OSCC), mortality and morbidity rates have not improved over the past decade. A major drawback in diagnosis and treatment of OSCC is the lack of knowledge relating to how genetic instability in oral cancer genomes affects oral carcinogenesis. Hence, the key aim of this study was to identify copy number alterations (CNAs) that may be cancer associated in OSCC using high-resolution array comparative genomic hybridization (aCGH). To our knowledge this is the first study to use ultra-high density aCGH microarrays to profile a large number of OSCC genomes (n = 46). The most frequently amplified CNAs were located on chromosome 11q11(52%), 2p22.3(52%), 1q21.3-q22(54%), 6p21.32(59%), 20p13(61%), 7q34(52% and 72%),8p11.23-p11.22(80%), 8q11.1-q24.4(54%), 9q13-q34.3(54%), 11q23.3-q25(57%); 14q21.3-q31.1(54%); 14q31.3-q32.33(57%), 20p13-p12.3(54%) and 20q11.21-q13.33(52%). The most frequently deleted chromosome region was located on 3q26.1 (54%). In order to verify the CNAs from aCGH using quantitative polymerase chain reaction (qPCR), the three top most amplified regions and their associated genes, namely ADAM5P (8p11.23-p11.22), MGAM (7q34) and SIRPB1 (20p13.1), were selected in this study. The ADAM5P locus was found to be amplified in 39 samples and deleted in one; MGAM (24 amplifications and 3 deletions); and SIRPB1 (12 amplifications, others undetermined). On the basis of putative cancer-related annotations, two genes, namely ADAM metallopeptidase domain 9 (ADAM9) and maltase-glucoamylase alpha-glucosidase (MGAM), that mapped to CNA regions were selected for further evaluation of their mRNA expression using reverse transcriptase qPCR. The over-expression of MGAM was confirmed with a 6.6 fold increase in expression at the mRNA level whereas the fold change in ADAM9 demonstrated a 1.6 fold increase. This study has identified significant regions in the OSCC genome that were amplified and resulted in consequent over-expression of the MGAM and ADAM9 genes that may be utilized as biological markers for OSCC.

Abstract Background Avahan, the India AIDS Initiative, implemented a large HIV prevention programme across six high HIV prevalence states amongst high risk groups consisting of female sex workers, high risk men who have sex with men, transgenders and injecting drug users in India. Utilization of the clinical services, health seeking behaviour and trends in syndromic diagnosis of sexually transmitted infections amongst these populations were measured using the individual tracking data. Methods The Avahan clinical monitoring system included individual tracking data pertaining to clinical services amongst high risk groups. All clinic visits were recorded in the routine clinical monitoring system using unique identification numbers at the NGO-level. Visits by individual clinic attendees were tracked from January 2005 to December 2009. An analysis examining the limited variables over time, stratified by risk group, was performed. Results A total of 431,434 individuals including 331,533 female sex workers, 10,280 injecting drug users, 82,293 men who have sex with men, and 7,328 transgenders visited the clinics with a total of 2,700,192 visits. Individuals made an average of 6.2 visits to the clinics during the study period. The number of visits per person increased annually from 1.2 in 2005 to 8.3 in 2009. The proportion of attendees visiting clinics more than four times a year increased from 4% in 2005 to 26% in 2009 (p Conclusions The programme demonstrated that acceptable and accessible services with marginalised and often difficult–to-reach populations can be brought to a very large scale using standardized approaches. Utilization of these services can dramatically improve health seeking behaviour and reduce STI prevalence.

Geographically, most tuberculosis (TB) cases in 2018 were reported from India. This TB burden is compounded by MDR-TB and XDR-TB. The strategies for the management and control of TB in the community depend on an understanding of the mode of spread of the different strains of TB isolates in the community. To determine the distribution and trends of M. tb strains over the time period in the community due to treatment, we carried out the present study on changes over two decades. Design/Methods . A total of 1218   M. tb isolates (year: 2001-2018) from Tiruvallur, India, were genotyped by spoligotyping after DNA extraction and subjected to anti-TB drug susceptibility testing for the first-line anti-TB drugs. Results . On analysis with the SpolDB4 database, majority (2001-2003: 53.32% and 2015-2018: 46.3%) of the isolates belonged to East African Indian (EAI) lineage, and the orphans designated in comparison to SpolDB4 stood 33% among 2001-2003 strain collection and 46.3% among 2015-2018 strain collection. 10.2% (2001-2003) and 9.26% (2015 to 2018) of isolates were monoresistant to isoniazid (H). MDR strains were less common among EAI strains (3.2%) compared to non-EAI strains (10.32%). Conclusions . EAI is the most predominant lineage in Tiruvallur, despite the presence of highly transmissible lineages like Beijing for the last two decades. The prevalence of MDR-TB is below the national average of 2-3% among the new TB cases in the last two decades. The reason can be attributed to the well-established nature of the locally circulating strains in this region which are not associated with drug resistance., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 S. Siva Kumar et al.)

Background: Shortening tuberculosis (TB) treatment duration is a research priority. This paper presents data from a prematurely terminated randomized clinical trial, of 4-month moxifloxacin or gatifloxacin regimens, in South India., Methods: Newly diagnosed, sputum-positive HIV-negative pulmonary TB patients were randomly allocated to receive gatifloxacin or moxifloxacin, along with isoniazid and rifampicin for 4 months with pyrazinamide for first 2 months (G or M) or isoniazid and rifampicin for 6 months with ethambutol and pyrazinamide for first 2 months (C). All regimens were administered thrice-weekly. Clinical and bacteriological assessments were done monthly during treatment and for 24 months post-treatment. The Data and Safety Monitoring Board recommended termination of the trial due to high TB recurrence rates in the G and M regimens., Results: Of 416 patients in intent-to-treat analysis, 6 (5%) of 124, 2 (2%) of 110 and 2 (2%) of 137 patients with drug-susceptible TB in the G, M and C arms respectively had unfavorable response at the end of treatment; during the next 24 months, 17 (15%) of 115, 11 (11%) of 104 and 8 (6%) of 132 patients respectively, had TB recurrence. Of 38 drug-resistant patients 1 of 8 and 3 of 26 in the G and C arms respectively had unfavourable response at the end of treatment; and TB recurrence occurred in 2 of 7 and 2 of 23 patients, respectively. The differences in TB recurrence rates between the G and C arms was statistically significant (p = 0.02). Gastro-intestinal symptoms occurred in 23%, 22% and 9% of patients in the G, M and C arms respectively, but most reactions were mild and manageable with symptomatic measures; 1% required regimen modification., Conclusions: 4-month thrice-weekly regimens of gatifloxacin or moxifloxacin with isoniazid, rifampicin and pyrazinamide, were inferior to standard 6-month treatment, in patients with newly diagnosed sputum positive pulmonary TB., Trial Registration: Clinical Trials Registry of India CTRI/2012/10/003060.

Setting: Tiruvallur district In Tamil Nadu where DOTS was implemented by the State Government as the tuberculosis control measure in 1999, and monitored by the National Institute for Research in Tuberculosis for over five years., Objective: To estimate trends in TB prevalence in a rural community with DOTS., Design: Surveys of pulmonary tuberculosis were undertaken in representative samples of subjects aged > or =15 years (N = 83,000 - 92,000), initially and after two and half, five and seven and half years of implementation of DOTS. Sputa were collected from those with abnormal radiograph and/or presence of chest symptoms, and examined by direct smear and culture., Results: The prevalence of culture-positive tuberculosis was 607, 454, 309 and 388 per 100,000 in the four surveys, and that of smear-positive tuberculosis was 326, 259, 168 and 180. In the first five years; annual decrease was 12.4% (95% CI 10.4 - 14.4%) for culture-positive tuberculosis, and 12.2% (95% CI 8.0-16.2) for smear-positive tuberculosis. This was, however, followed by a significant increase in the next two and half years. The average new smear-positive case-notification rate was 75 per 100,000 during first four years but declined to 49 in subsequent years. There were no methodological differences during this period and information on changes in socio-economic indicators and nutritional standards was unavailable., Conclusion: Despite the average annual success rate (78%) in this tuberculosis unit being lower than the expected rate of 85%, the implementation of DOTS was followed by a substantial decrease in the prevalence of pulmonary tuberculosis over the seven and half year period. Our findings suggest that sustaining the high effectiveness of DOTS programme needs vigilant supervision.

Background & Objectives: Resistance to nevirapine (NVP) has been described with single dose preventive regimens in other populations. Our aim was to study the pattern and prevalence of HIV drug resistance (DR) at baseline (during pregnancy) and after delivery among antenatal women exposed to single dose NVP for prevention of parent to child transmission (PPTCT)., Methods: HIV-infected, ART-naive primigravidae between 18-25 years of age, attending government antenatal clinics in Chennai, Vellore or Madurai were recruited. Drug resistance testing was carried out during pregnancy and after Sd-NVP treatment (one month after delivery) by Viroseq sequencing. HIV-1 testing by DNA PCR was done in newborns at 30 days., Results: Thirty one women were enrolled but only twenty six plasma specimens were analyzable (24 paired and two postnatal only). No major mutations were observed in any drug class at baseline though many polymorphisms were observed in both the reverse transcriptase and protease genes. Mutations to non-nucleoside reverse transcriptase inhibitors (NNRTI) were observed post-delivery in 33 per cent of women who were treated with Sd-NVP. None of the infants were HIV-positive., Interpretation & Conclusions: Among pregnant ART-naive women, baseline HIV drug resistance was not observed. A high rate of development of NNRTI class resistance among women treated with single-dose NVP was observed. Our results emphasize the need to implement more effective PPTCT regimens, minimizing emergence of drug resistance and thereby preserving long-term treatment options for HIV-infected women in India.

Background: Malnutrition in human immunodeficiency virus (HIV)-infected individuals is associated with faster disease progression, higher mortality rates, and suboptimal response to antiretroviral therapy (ART)., Methods: We conducted a prospective interventional study to evaluate the effects of an oral macronutrient supplement among HIV-infected adults in South India. Patients attending Tuberculosis Research Centre clinics from June 2005 through December 2007 had baseline nutritional assessment and laboratory investigations performed. Patients at 1 center received nutritional counseling and standard care, whereas patients at 2 centers additionally received a macronutrient providing 400 cal and 15 g of protein daily. Study outcomes were changes in anthropometry, body composition, blood chemistry, and immune status at 6 months., Results: In total, 636 ART-naive patients were enrolled in the study; 361 completed 6 months of follow-up (282 received supplements and 79 received standard care). Mean age +/- standard deviation (SD) was 31 +/- 7 years, mean weight +/- SD was 50 +/- 10 kg, and 42% were male. Significant increases in body weight, body mass index, midarm circumference, fat-free mass, and body cell mass were observed in the supplement group but not in the control group at 6 months; gains were greater in patients with CD4 cell counts <200 cells/microL. No changes were observed in lipid levels, whereas the CD4 cell count decreased in the control group. However, after adjusting for baseline differences, these changes were not statistically significantly different between the groups., Conclusions: Macronutrient supplementation did not result in significantly increased weight gain compared with standard care (including nutritional counseling) among patients with moderately advanced HIV disease. The effect of supplementation on specific subsets of patients and on preserving immune function needs further research.

Background: There is limited information on the relative proportion of reactivation and reinfection at the time of recurrence among human immunodeficiency virus (HIV)-infected and HIV-uninfected patients who are successfully treated for tuberculosis infection in India., Methods: HIV-infected and HIV-uninfected patients with sputum culture-positive pulmonary tuberculosis were treated with short-course regimens and followed up for 36 months at the Tuberculosis Research Centre, South India. Bacteriologic recurrences were documented, and typing of strains was performed using 3 different genotypic techniques: restriction fragment length polymorphism (RFLP) by IS6110, spoligotyping, and mycobacterial interspersed repeat unit (MIRU)-variable number tandem repeat (VNTR). DNA fingerprints of paired Mycobacterium tuberculosis isolates (baseline and recurrence) were compared., Results: Among 44 HIV-infected and 30 HIV-uninfected patients with recurrent tuberculosis during the period July 1999 to October 2005, 25 and 23 paired isolates, respectively, were typed using all 3 methods. Recurrence was due to exogenous reinfection in 88% of HIV-infected and 9% of HIV-uninfected patients (P<.05). Among recurrent isolates, the HIV-infected patients showed more clustering, as well as a higher proportion of drug resistance, including multidrug resistance., Conclusions: In India, a tuberculosis-endemic country, most recurrences after successful treatment of tuberculosis are due to exogenous reinfection in HIV-infected persons and endogenous reactivation in HIV-uninfected persons. Strategies for prevention and treatment of tuberculosis infection must take these findings into consideration.

Culture and drug susceptibility testing results of 2816 tuberculosis (TB) patients from across India who had failed repeated treatments from 2001 to 2004 were retrospectively analysed at the Tuberculosis Research Centre, Chennai. Of 1498 (53%) identified as having multidrug-resistant TB (MDR-TB), 671 (44.8%) were resistant to > or =1 second-line drugs (SLDs): 490 (32.7%) to ethionamide, 245 (16.4%) to ofloxacin and 169 (11.3%) to kanamycin; 69 (4.6%) were extensively drug-resistant TB (XDR-TB). Although from a highly select and non-representative patient group, such high SLD resistance levels, including XDR-TB, among MDR-TB patients is of concern. The prevention of MDR/XDR-TB through quality DOTS services, however, remains the priority. In addition, rapid scale-up of quality programmatic management under the RNTCP is needed, with more control and rational use of SLDs outside the programme.

A recombinant modified vaccinia Ankara virus vaccine candidate (TBC-M4) expressing HIV-1 subtype C env, gag, tat-rev, and nef-RT genes was tested in a randomized, double-blind, dose escalation Phase I trial in 32 HIV-uninfected healthy volunteers who received three intramuscular injections of TBC-M4 at 0, 1, and 6 months of 5 x 10(7) plaque-forming units (pfu) (low dosage, LD) (n = 12) or 2.5 x 10(8) pfu (high dosage, HD) (n = 12) or placebo (n = 8). Local and systemic reactogenicity was experienced by approximately 67% and 83% of vaccine recipients, respectively. The reactogenicity events were mostly mild in severity. Severe but transient systemic reactogenicity was seen in one volunteer of the HD group. No vaccine-related serious adverse events or events suggesting perimyocarditis were seen. A higher frequency of local reactogenicity events was observed in the HD group. Cumulative HIV-specific IFN-gamma ELISPOT responses were detected in frozen PBMCs from 9/11 (82%), 12/12 (100%), and 1/8 (13%) volunteers after the third injection of the LD, HD, and placebo groups, respectively. Most of the responses were to gag and env proteins (maximum of 430 SFU/10(6) PBMCs) persisting across multiple time points. HIV-specific ELISA antibody responses were detected in 10/11, 12/12, and 0/8 volunteers post-third vaccination, in the LD, HD, and placebo groups, respectively. No neutralizing activity against HIV-1 subtype C isolates was detected. TBC-M4 appears to be generally safe and well-tolerated. The immune response detected was dose dependent, modest in magnitude, and directed mostly to env and gag proteins, suggesting further evaluation of this vaccine in a prime-boost regimen.

There is limited information on the prevalence and pattern of HIV drug-resistant mutations (DRMs) among HIV-1-coinfected tuberculosis (TB) patients before and after antiretroviral treatment. Patients with HIV-1 and TB were recruited into a clinical trial from two different once-daily antiretroviral regimens and followed for a period of 6 months after ART initiation. Patients were treated with standard short-course anti-TB treatment (2EHRZ3/4RH3) and were randomized to receive ddI/3TC with either nevirapine or efavirenz, once daily. Genotypic drug resistance (DR) testing was carried out for the pol gene at baseline and at the time of virological failure. At baseline, major DRMs with respect to NNRTIs (G190GA) and TAMs (T215S and I) were observed in 3 out of 107 patients. Of 15 treatment failures, 14 had more than one major NRTI and NNRTI mutation. V106M was the major NNRTI mutation that emerged in EFZ and Y181C in the NVP group. Among NRTI mutations, M184V was the commonest followed by L74I/V. Primary drug resistance to antiretroviral drugs was low among HIV-1 co-infected TB patients in south India. A once-daily regimen of ddI/3TC/EFZ or NVP results in a specific pattern of NNRTI mutations and negligible thymidine analog mutations (TAMs).

Background & Objectives: Variability in the clinical outcome of persons exposed to and infected with HIV-1 and tuberculosis (TB) is determined by multiple factors including host genetic variations. The aim of the present study was to find out whether chemokine, chemokine receptor and DC-SIGN gene polymorphisms were associated with susceptibility or resistance to HIV and HIV-TB in south India., Methods: CCR2 V64I (G/A), monocyte chemoattractant protein-1 (MCP-1) -2518 A/G, stromal cell derived factor-1alpha; (SDF-1alpha) 3'UTR G/A and DC-SIGN gene polymorphisms were studied by polymerase chain reaction based methods in HIV-1 infected patients without TB (n=151), with pulmonary TB (PTB) (n=81) and extrapulmonary TB (n=31), 155 PTB patients without HIV and 206 healthy controls., Results: The genotype frequencies of CCR2 V64I, MCP-1 -2518 and DC-SIGN polymorphisms did not differ significantly between the study groups. A significantly increased frequency of GG genotype of SDF-1alpha polymorphism was observed among HIV+PTB+ patients compared to healthy controls (P=0.009, Pc=0.027)., Interpretation & Conclusions: Our data suggest that GG genotype of SDF-1alpha 3'UTR polymorphism may be associated with susceptibility to PTB in HIV-1 infected patients. A better understanding of genetic factors that are associated with TB could help target preventive strategies to those HIV patients likely to develop tuberculosis.

We report here that the Directly Observed Treatment, Short course (DOTS) is reaching all tuberculosis patients in the community irrespective of social classification based on the analysis from the tuberculosis prevalence survey and programme performance during 1999-2003 from a rural area in Tamilnadu, South India. New smear- positive cases treated under a DOTS programme were classified in two groups namely; scheduled caste living in colony and other population. The prevalence of smear- positive cases among the scheduled caste population was 1.9 times higher than the other population and this was reflected in the notification also. The successful treatment outcome was also similar in these two groups (75% and 78% respectively; overall 77%). From these findings it is concluded that people living in colony have equal access to DOTS as those in the village.

Background: Limited information about the prevalence of drug-resistant tuberculosis (TB) has been reported from India, the country with the world's highest burden of TB. We conducted a representative state-wide survey in the state of Gujarat (2005 population: 56 million)., Methods: Mycobacterium tuberculosis isolates from a representative sample of new and previously treated smear-positive pulmonary TB (PTB) cases were subjected to drug susceptibility testing (DST) against first-line drugs at a World Health Organization supranational reference laboratory. Isolates found to have at least both isoniazid (INH) and rifampicin (RMP) resistance (i.e., multidrug-resistant TB [MDR-TB]) were subjected to second-line DST., Results: Of 1571 isolates from new patients, 1236 (78.7%) were susceptible to all first-line drugs, 173 (11%) had any INH resistance and MDR-TB was found in 37 (2.4%, 95%CI 1.6-3.1). Of 1047 isolates from previously treated patients, 564 (54%) were susceptible to all first-line drugs, 387 (37%) had any INH resistance and MDR-TB was found in 182 (17.4%, 95%CI 15.0-19.7%). Among 216 MDR-TB isolates, 52 (24%) were ofloxacin (OFX) resistant; seven cases of extensively drug-resistant TB (XDR-TB) were found, all of whom were previously treated cases., Conclusion: MDR-TB prevalence remains low among new TB patients in Gujarat, but is more common among previously treated patients. Among MDR-TB isolates, the alarmingly high prevalence of OFX resistance may threaten the success of the expanding efforts to treat and control MDR-TB.

Background: Long term status of pulmonary tuberculosis (PTB) patients treated with short course chemotherapy (SCC) regimens remains unknown., Objective: To assess the clinical, bacteriological, radiological status and health related quality of life (HRQoL) of PTB patients 14-18 years after successful treatment with SCC., Methodology: In a cross-sectional study, cured PTB patients treated during 1986-1990 at the Tuberculosis Research Centre (TRC) were investigated for their current health status including pulmonary function tests (PFT). The St Georges respiratory questionnaire (SGRQ) was used to assess the HRQoL., Results: The mean period after treatment completion for the 363 eligible participants was 16.5 yrs (range 14-18 yrs., 84% coverage); 25 (7%) had been re-treated and 52 (14%) died. Among the investigated, 58 (29%) had persistent respiratory symptoms; 170 (86%) had radiological sequelae but none had active disease. Abnormal PFT was observed in 96 (65%) with predominantly restrictive type of disease in 66 (45%). The SGRQ scores for activity and impact were high implying impairment in HRQoL., Conclusion: Assessment of long term status of cured PTB patients showed an impairment of lung functions and HRQoL highlighting the need to address these issues in the management of TB that may provide added value to patient care.

We have shown earlier the association of human leucocyte antigen (HLA)-A11 with resistance and HLA-B40 and -DR2 with susceptibility to HIV and HIV-TB. In the present study, we have attempted to find out the HLA-DR2 subtypes and the possible HLA-A/-B/-DRB1 haplotype combinations that are associated with susceptibility or resistance to HIV and HIV with pulmonary tuberculosis (HIV+PTB+). HLA-DR2 subtyping was carried out by polymerase chain reaction-based sequence-specific oligonucleotide probe method. Overrepresentation of HLA-DRB1*1501 in HIV-positive PTB-negative (HIV+PTB-) patients (P = 0.004, P(c) = 0.06) and -DRB1*1502 in HIV-positive PTB-positive (HIV+PTB+) patients (P = 0.019) was observed as compared to healthy controls. Haplotype analysis revealed an increased frequency of HLA-A2-DRB1*1501 haplotype in HIV+PTB- patients (P = 0.008) and HLA-A2-DRB1*1502 among HIV+PTB+ patients (P = 0.01) compared to healthy controls. The haplotypes B40-DRB1*1501 and B40-DRB1*04 were found to be moderately increased in HIV+PTB(-) and HIV+PTB+ patients (P < 0.05). The study suggests that HLA-A2-DRB1*1501 haplotype may be associated with HIV infection while HLA-A2-DRB1*1502 haplotype might be associated with susceptibility to PTB in HIV patients. Moreover, HLA-B40-DRB1*1501 and HLA-B40-DRB1*04 haplotypes may be associated with susceptibility to HIV infection and to PTB in HIV patients.

Background & Objective: Mycobacterium tuberculosis infection has been shown to result in increased HIV replication and disease progression in HIV-infected individuals through increased immune activation. The objective of this study was to correlate plasma levels of immune activation markers with the presence of tuberculosis (TB) in HIV-infected and uninfected individuals, and to study the changes following anti-tuberculosis treatment., Methods: Plasma markers of immune activation - neopterin, beta-2-microglobulin (beta2M) and soluble tumour necrosis factor alpha receptor type I (sTNFalpha-RI) were measured by ELISA in 42 HIV positive TB patients (HIV+TB+) undergoing a six-month course of TB chemotherapy. Thirty seven HIV+ persons without active TB, 38 TB patients without HIV infection, and 62 healthy volunteers served as controls., Results: Plasma levels of all three markers were elevated in HIV+ individuals, more so in those with active TB. When HIV+ individuals were further categorized based on CD4+ T cell counts, HIV+TB+ patients with CD4+ T cells counts

Introduction: Vitamin D receptor (VDR) gene polymorphisms in the 5' regulatory region (Cdx2 and A-1012G), coding region (FokI), and 3' untranslated region (UTR; BsmI, ApaI, and TaqI) were studied to find out whether these polymorphisms are associated with susceptibility to or protection against HIV-1 and development of tuberculosis (TB) in human immunodeficiency virus (HIV)-1-infected patients., Study Subjects and Methods: The study was carried out in 131 HIV patients without TB (HIV+ TB-) and 113 HIV patients with TB (HIV+ TB+; includes 82 patients with pulmonary TB (HIV+ PTB+) and 31 with extra pulmonary TB), 108 HIV-negative pulmonary TB patients (HIV- PTB+), and 146 healthy controls., Results: Among the 5' regulatory and coding region polymorphisms, significantly increased frequency of G/A genotype of Cdx-2 was observed in HIV+ TB- group compared to controls (p = 0.012, odds ratio (OR) 1.89 95% confidence interval (CI) 1.14-3.15). In the 3' UTR genotypes, a decreased frequency of b/b genotype of BsmI in total HIV patients (p = 0.014, OR 0.54 95% CI 0.32-0.89) and increased frequencies of A/A genotype of ApaI in HIV+ TB+ patients (p = 0.041, OR 1.77 95% CI 1.02-3.06) and t/t genotype of TaqI in HIV+ PTB+ patients (p = 0.05, OR 2.32 95% CI 0.99-5.46) were observed compared to controls. Haplotype analysis revealed significantly increased frequencies of 3' UTR haplotype B-A-t in HIV+ TB+ and HIV+ PTB+ groups (Pc = 0.030, OR 1.75 95% CI 1.14-2.66) and decreased frequencies of b-A-T haplotype in total HIV patients (Pc = 0.012, OR 0.46 95% CI 0.27-0.77), HIV+ TB- (p = 0.031 OR 0.48 95% CI 0.25-0.89), and HIV+ PTB+ groups (Pc = 0.04, OR 0.47 95% CI 0.23-0.89) compared to controls., Conclusions: The results suggest that VDR gene 3' UTR haplotype b-A-T may be associated with protection against HIV infection while B-A-t haplotype might be associated with susceptibility to development of TB in HIV-1-infected patients.

Heat shock promoters of mycobacteria are strong promoters that become rapidly upregulated during macrophage infection and thus serve as valuable candidates for expressing foreign antigens in recombinant BCG vaccine. In the present study, a new heat shock promoter controlling the expression of the groESL1 operon was identified and characterized. Mycobacterium tuberculosis groESL1 operon codes for the immunodominant 10 kDa (Rv3418c, GroES/Cpn10/Hsp10) and 60 kDa (Rv3417c, GroEL1/Cpn60.1/Hsp60) heat shock proteins. The basal promoter region was 115 bp, while enhanced activity was seen only with a 277-bp fragment. No promoter element was seen in the groES-groEL1 intergenic region. This operon codes for a bicistronic mRNA transcript as determined by reverse transcriptase-PCR and Northern blot analysis. Primer extension analysis identified two transcriptional start sites (TSSs) TSS1 (-236) and TSS2 (-171), out of which one (TSS2) was heat inducible. The groE promoter was more active than the groEL2 promoter in Mycobacterium smegmatis. Further, it was found to be differentially regulated under stress conditions, while the groEL2 promoter was constitutive.

The gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay is used routinely to evaluate the potency of human immunodeficiency virus (HIV) vaccine candidates and other vaccine candidates. In order to compare candidates and pool data from multiple trial laboratories, validated standardized methods must be applied across laboratories. Proficiency panels are a key part of a comprehensive quality assurance program to monitor inter- and intralaboratory performance, as well as assay performance, over time. Seven International AIDS Vaccine Initiative-sponsored trial sites participated in the proficiency panels described in this study. At each laboratory, two operators independently processed identical sample sets consisting of frozen peripheral blood mononuclear cell (PBMC) samples from different donors by using four blind stimuli. PBMC recovery and viability after overnight resting and the IFN-gamma ELISPOT assay performance were assessed. All sites demonstrated good performance in PBMC thawing and resting, with a median recovery of 78% and median viability of 95%. The laboratories were able to detect similar antigen-specific T-cell responses, ranging from 50 to >3,000 spot-forming cells per million PBMC. An approximate range of a half log in results from operators within or across sites was seen in comparisons of antigen-specific responses. Consistently low background responses were seen in all laboratories. The results of these proficiency panels demonstrate the ability of seven laboratories, located across three continents, to process PBMC samples and to rank volunteers with differential magnitudes of IFN-gamma ELISPOT responses. These findings also illustrate the ability to standardize the IFN-gamma ELISPOT assay across multiple laboratories when common training methods, reagents such as fetal calf serum, and standard operating procedures are adopted. These results are encouraging for laboratories that are using cell-based immunology assays to test HIV vaccines and other vaccines.

We studied the immunomodulatory effect of 1,25(OH)(2)D(3) on single cell expression of IFN-gamma and TNF-alpha cytokines in T cell subsets of pulmonary tuberculosis (PTB) patients (n=22) and normal healthy subjects (n=22). Peripheral blood mononuclear cells (PBMCs) were cultured with live Mycobacterium tuberculosis (MTB) with or without 1,25(OH)(2)D(3) (10(-7)M) for 48 h. T cell subsets positive for IFN-gamma and TNF-alpha were enumerated by flow cytometry and the culture supernatants were assayed for both the cytokines using ELISA. In both NHS and PTB patients, a significantly reduced percentage of IFN-gamma and TNF-alpha expressing CD3+, CD3+CD4+ and CD3+CD8+ T cells were observed in cultures stimulated with live MTB and treated with 1,25(OH)(2)D(3) compared to cultures without 1,25(OH)(2)D(3) (NHS; CD3+ IFN-gamma+: p<0.0001; CD3+TNF-alpha+: p=0.0292 and PTB; CD3+ IFN-gamma+: p=0.0292; CD3+ TNF-alpha+: p=0.0028). The levels of IFN-gamma and TNF-alpha in the culture supernatants of 1,25(OH)(2)D(3) treated cultures were also found to be significantly decreased in both groups (NHS; IFN-gamma: p=0.0001; TNF-alpha: p<0.0001) and (PTB; IFN-gamma: p<0.0001; TNF-alpha: p<0.0001). A positive correlation was observed between IFN-gamma and TNF-alpha expressing CD3+CD8+ T cells in MTB stimulated cultures treated with or without 1,25(OH)(2)D(3) in NHS (p=0.0001; p=0.001, respectively) and PTB patients (p=0.002; p=0.005, respectively). The present study revealed the suppressive effect of 1,25(OH)(2)D(3) on single cell expression of IFN-gamma and TNF-alpha by CD3+CD4+ and CD3+CD8+ T cells in pulmonary tuberculosis. This suppressive effect of 1,25(OH)(2)D(3) on proinflammatory and Th1 cytokine positive cells might have a role in reducing inflammation at the site of infection.

Vitamin D receptor (VDR) genotypes have been shown to be associated with differential susceptibility or resistance to tuberculosis. The influence of FokI, BsmI, ApaI and TaqI variants of VDR gene on 1, 25(OH)(2) D(3) modulated granzyme A expression of cytotoxic lymphocytes induced by culture filtrate antigen (CFA) of Mycobacterium tuberculosis was studied in 40 pulmonary tuberculosis (PTB) patients and 49 normal healthy subjects (NHS) by flow cytometry. In both the study groups, addition of 1, 25(OH)(2) D(3) (10(-7)M) significantly reduced the percentage of granzyme A positive cells in both unstimulated (NHS, p<0.0001; PTB, p=0.02) and stimulated culture conditions (CFA, NHS, p<0.0001; PTB, p=0.0001) which correlated positively with the IFN-gamma levels (unstimulated, p=0.01; CFA stimulated, p=0.004) in NHS. The ApaI aa genotype and bbaaTT extended genotype were associated with a significantly decreased percentage of granzyme A positive cells in NHS (p<0.05). Our results suggest that 1, 25(OH)(2) D(3) suppresses granzyme A probably by down-regulating Th1 cytokine response. Moreover, the VDR gene variants might regulate cytotoxic T-cell response via 1, 25(OH)(2) D(3) mediated suppression of granzyme A expression in tuberculosis.

Background: Innovative schemes to ensure the participation of private practitioners (PPs) in the Revised National Tuberculosis Control Programme (RNTCP) are necessary to identify and treat all patients with tuberculosis (TB). We developed a novel public-private mix (PPM) model to encourage PPs to practise DOTS and participate in the RNTCP while retaining their patients., Methods: The Resource Group for Education and Advocacy for Community Health (REACH) developed and implemented the programme in partnership with the Chennai local health authority and the Tuberculosis Research Centre, Chennai, India. PPs were sensitised to the RNTCP and DOTS through a one-to-one approach or group meetings, and were assisted in referring patients. Surveys were carried out at baseline and at the completion of the study to assess changes in attitudes and practices., Results: Six hundred PPs underwent sensitisation about the RNTCP, after which the proportion of PPs adopting DOTS increased significantly (P < 0.001), and the majority (72.8%) used sputum testing for diagnosing TB. The proportion of PPs who used X-ray alone for diagnosis declined to 16.0% from a baseline of 45.4%., Conclusions: This PPM model, which emphasises sustained advocacy for DOTS and allows PPs to retain private patients, looks promising and needs to be tested at other sites.

Background & Objectives: Sputum acid-fast bacilli (AFB) microscopy services are not available in all health facilities. Alternative procedures are needed to transport sputum samples to the diagnostic centres for detection of AFB. The objective of the present study was to evaluate sputum smears made by pot-method with the direct smears made immediately after sputum collection by Ziehl-Neelsen (ZN) method., Methods: Ninety three sputum samples from 49 pulmonary tuberculosis suspects were studied. Their direct smears (ZN smears) were stained by hot ZN method. The samples were then mixed with phenol ammonium sulphate basic fuchsin solution and stored at ambient conditions. The smears (pot smears), made on day 7, were then, decolourized and counter-stained for detection of AFB (pot method). The ZN and pot smears were read blind. After excluding 18 samples for various reasons, the results of pot and ZN smears of 63 samples from smear positive (2 of 3 direct smears were positive) and 12 from smear negative (3 of 3 direct smears were negative) patients were analysed. ZN method was the gold standard., Results: Pot and ZN smears were positive in 61 of 63 samples from smear-positive patients and negative in 11 of 12 smear-negative patients (kappa = 0.87). The sensitivity and specificity of pot method were 96.8 and 91.7 per cent respectively., Interpretation & Conclusion: Sputum samples can be stored for up to seven days in the sputum container with phenol ammonium sulphate basic fuchsin solution. However, a comprehensive study needs to be done confirm the accuracy of the pot method for storage and transportation of sputum to microscopy centres for detection of AFB.

In this study, we compared several immune parameters in tuberculosis (TB) and nontuberculosis (NTB) pleurisy to gain an understanding of the mechanism behind enhanced Th1 apoptosis that occurs at sites of active Myobacterium tuberculosis (M. tuberculosis) infection. An initial evaluation of the accumulated cytokines in pleural fluid (PF) demonstrated that both TB and NTB pleurisy were associated with prointflammatory cytokines, while only TB pleurisy had augmented expression of interferon (IFN)-gamma and soluble Fas ligand (sFASL). Despite enhanced expression of the apoptosis-inducing molecule in TB pleurisy, T cells derived from both types of pleurisy exhibited significant apoptosis. In both groups, T-cell apoptosis correlated with low expression of CD80 on PF-derived macrophages and elevated accumulation of TGF-beta in the PF. A causative correlation between TGF-beta and low CD80 expression in the two groups was established by in vitro studies demonstrating TGF-beta inhibition of CD80 upregulation in a macrophage cell line. Together, the findings allude to the possibility that activation in the absence of appropriate CD80 costimulation is the mechanism that leads to T-cell apoptosis at sites of active M. tuberculosis infection. Furthermore, the findings also indicate that T-cell apoptosis is perhaps a host regulatory mechanism to limit inflammation, rather than a pathogen-induced immune deviation.

We report here that the Directly Observed Treatment, Short course (DOTS) is reaching all tuberculosis patients in the community irrespective of social classification based on the analysis from the tuberculosis prevalence survey and programme performance during 1999-2003 from a rural area in Tamilnadu, South India. New smear- positive cases treated under a DOTS programme were classified in two groups namely; scheduled caste living in colony and other population. The prevalence of smear- positive cases among the scheduled caste population was 1.9 times higher than the other population and this was reflected in the notification also. The successful treatment outcome was also similar in these two groups (75% and 78% respectively; overall 77%). From these findings it is concluded that people living in colony have equal access to DOTS as those in the village.