Validation of an indigenous assay for rapid molecular detection of rifampicin resistance in presumptive multidrug-resistant pulmonary tuberculosis patients.

BACKGROUND & OBJECTIVES: There is a need for an affordable, easy, high-sensitivity test usable at the peripheral health facility for diagnosis of drug-resistant (DR) tuberculosis (TB) to interrupt disease transmission. Nucleic acid amplification tests (NAATs) for early detection of DR-TB are ideal to bring testing near to the patient. TruenatTM MTB (Mycobacterium tuberculosis) and TruenatTM MTB-RIF (rifampicin) is an indigenous chip-based real-time polymerase chain reaction (PCR) based test for detection of multidrug-resistant (MDR) TB. The test involves extraction of DNA using automated, battery operated Trueprep instrument and real-time PCR performed on the Truelab analyzer. We report here multicentric validation of Truenat MTB-RIF for detection of DR-TB in suspected DR-TB patients. METHODS: Consecutive patients aged 18-65 yr, with symptoms suggestive of TB and with a history of previous treatment, reporting to the National TB Elimination Programme (NTEP) clinics under four national institutes, namely AIIMS (All India Institute of Medical Sciences, New Delhi), NITRD (National Institute of Tuberculosis and Respiratory Diseases, New Delhi), NIRT (National Institute for Research in Tuberculosis, Chennai) and ICMR-National JALMA Institute for Leprosy and other Mycobacterial Diseases, Agra, were included in the study. Two sputum samples (one spot and one morning) were collected from each patient, after obtaining informed written consent. The samples were subjected to smear, GeneXpert and MGIT 960 culture (and drug susceptibility testing to RIF) (surrogate for MDR-TB) to serve as reference tests. The samples were coded to ensure blinding and subjected to Truenat MTB-RIF. Truenat MTB-RIF Version 1.5 was used for testing 1084 samples for RIF resistance, while Version 2.0 was used to test another 1201 samples. RESULTS: Truenat MTB-RIF Version 1.5 in comparison with comprehensive laboratory reference standards yielded sensitivity and specificity of 76.2 and 94.7 per cent, respectively for the detection of RIF resistance in 1084 samples, collected across four sites. Based on the analysis of discordant samples, Version 2.0 of Truenat was developed by the manufacturer and this was further tested on additional 1201 samples, yielding a sensitivity of 87.5 per cent and specificity of 99.5 per cent. INTERPRETATION & CONCLUSIONS: Multicentric trial of TruenatTM MTB-RIF demonstrated a great potential of this point of care NAAT for detection of MDR-TB. The test would be useful in limited resource settings and inaccessible areas without need for any additional infrastructure.

Multicentric validation of indigenous molecular test Truenat™ MTB for detection of Mycobacterium tuberculosis in sputum samples from presumptive pulmonary tuberculosis patients in comparison with reference standards.

BACKGROUND & OBJECTIVES: : Early case detection is essential to interrupt transmission and to prevent further spread of tuberculosis (TB) in high endemic settings. Nucleic acid amplification tests (NAATs) with visual read-outs are ideal as point-of-care tests. Truenat™ MTB is an indigenous chip-based NAAT for detection of Mycobacterium tuberculosis, which involves extraction of DNA and real-time polymerase chain reaction (PCR) using portable, automated, battery-operated instruments. The current multicentric study was aimed to evaluate Truenat for detection of MTB in sputum samples obtained from patients with presumptive pulmonary TB with reference to culture as gold standard and Xpert as a comparator. METHODS: : The study was conducted at four sites, namely ICMR-National Institute for Research in Tuberculosis, Chennai; All India Institute of Medical Sciences, New Delhi; ICMR-National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra; and National Institute of TB and Respiratory Diseases, New Delhi. Patients suspected to have TB were screened for eligibility. Two sputum samples were collected from each patient. Tests included smear, Xpert and Truenat directly from the sputum sample and culture by Lowenstein-Jensen (L-J) medium and MGIT960 from decontaminated pellets. Sample used for Truenat assay was coded. Resolution of Truenat false positives was done using an in-house PCR with TRC4 primers. RESULTS: : The study enrolled 2419 presumptive TB patients after screening 2465 patients, and 3541 sputum samples were collected from the enrolled patients. Results of 2623 samples were available for analysis. Truenat showed a positivity rate of 48.5 per cent as compared to 37.0 per cent by Xpert. The sensitivities of Truenat and Xpert were was 88.3 and 79.7 per cent, respectively in comparison with culture. INTERPRETATION & CONCLUSIONS: : Truenat MTB identified more positives among culture-confirmed samples than Xpert and had higher sensitivity. In addition, other advantageous operational features of Truenat MTB were identified which would be useful in field settings.

Prevalence of human papillomavirus in women without cervical cancer: a population-based study in Eastern India.

Despite the high incidence of cervical cancer, population-based data on prevalence of human papillomavirus (HPV) are limited in India. This study aimed to evaluate the prevalence of any HPV type and type-specific prevalence of HPV 16/18 in women without cervical cancer. HPV viral load was measured and correlated with cytologic abnormalities of the cervix. A total of 2501 women between 25 and 65 years of age and without cervical cancer were screened by pap smear cytology. HPV DNA was detected from cervical scrapes by nested polymerase chain reaction. Detection of HPV 16/18 was carried out by polymerase chain reaction using type-specific primers and was confirmed by Southern hybridization. Viral load was determined by absolute real-time polymerase chain reaction. Population prevalence of any HPV was found to be 9.9%. The risk of HPV infection was higher in women aged 25 to 34 years (odds ratio, 1.11), in married women below 20 years of age (odds ratio, 1.80), and in women with parity ≥4 (odds ratio, 1.04). Prevalence of HPV 18 (1.4%) was greater than that of HPV 16 (0.6%) in the overall screened population. High-grade squamous intraepithelial lesion cytology was more frequent in women infected with HPV 16 than in those infected with HPV 18 and other types. A gradual increase in HPV copy numbers was associated with progressive cytologic severity. In this study, HPV prevalence is comparable to HPV prevalence reported by other studies among Indian and Asian women. Although the prevalence of HPV 18 was more than that of HPV 16, type 16 infection was associated with higher oncogenicity.

Funding for cancer research by an Indian funding agency, DBT.

Cancer is a group of diseases with major societal impact and accounts for approximately 55 percent of mortality in India. The Indian population is increasing in size and gradually ageing. As a result, the number of people diagnosed with and dying of cancer are increasing. Government funding agencies such as the Department of Biotechnology (DBT) has a clear definitive role in the management and control of cancer. Through Research and Development programs and multi-institutional networking programs, DBT has provided resources to individual investigators and to institutions, to carry out basic, applied, translational and clinical research and to develop new methods to prevent and treat disease and to conduct research especially in challenging areas pertaining to different types of cancer. This article summarizes the funding provided by DBT for different cancer research programs.

Region-wise distribution of high-risk human papillomavirus types in squamous cell carcinomas of the cervix in India.

HYPOTHESIS: Assessment of the prevalence and type distribution of human papillomavirus (HPV) in squamous cell carcinomas (SCC) of the cervix across India was undertaken to estimate the impact of available prophylactic HPV-L1 vaccines in the country and to find out additional types that might be needed to be incorporated in second-generation vaccines. METHODS: High-risk (HR) HPVs were genotyped from 667 histopathologically confirmed cases of SCC from 6 different centers representing 4 regions across India: Advanced Centre for Treatment, Research and Education in Cancer, Mumbai; All India Institute of Medical Sciences, New Delhi; Cancer Foundation of India, Kolkata; Christian Medical College, Vellore; Kidwai Memorial Institute of Oncology, Bangalore; and Regional Cancer Center, Thiruvananthapuram. Human papillomaviruses in tumor biopsies were analyzed by Xcytonscreen HPV based on PGMY09/11 multiplex polymerase chain reaction and reverse dot blot assay. RESULTS: Overall viral prevalence across India was not different; 92.1% of 667 cases harbored HPV; 8% were negative. Infection with single HR type was seen in 86.8%: predominant types being HPV-16 followed by HPV-18, -45, -73, -31, -56, -52, -58, -59, -33, -68, -51, -35, -26, and -39. Human papillomavirus types 16/18-positive fraction formed 79.6%; other types comprised 12.4%. CONCLUSIONS: Prophylactic HPV-16/18-L1 vaccines would provide greater than 75% protection against SCC in India. Ranking and frequencies of non-16/18 types were different from earlier reports. Hence, considering the possibility of promotion of persistence of nonvaccine types in the vaccinees due to original antigenic sin and the lack of organized screening programs in India, a broad-based vaccine approach would be appropriate.

Molecular variants of HPV-16 associated with cervical cancer in Indian population.

Human papilloma virus is a causative factor in the etiology of cervical cancer with HPV16 being the most prevalent genotype associated with it. Intratype variations in oncogenic E6/E7 and capsid L1 proteins of HPV 16 besides being of phylogenetic importance, are associated with risk of viral persistence and progression. The objective of this multicentric study was to identify HPV-16 E6, E7 and L1 variants prevalent in India and their possible biological effects. Squamous cell cervical cancer biopsies were collected from 6 centres in India and examined for the presence of HPV 16. Variants of HPV-16 were characterized by full length sequence analysis of L1, E6 and E7 genes in 412 samples. Similar distribution of the variants was seen from the different centres/regions, with the European variant E350G being the most prevalent (58%), followed by American Asian variant (11.4%). Fifty six changes were seen in E6 region, 31 being nonsynonymous. The most frequent being L83V (72.3%), Q14H (13.1%) and H78Y (12.1%). Twenty-nine alterations were seen in E7 region, with 12 being nonsynonymous. The most frequent being F57V (9%). L1 region showed 204 changes, of which 67 were nonsynonymous. The most frequent being 448insS (100%), and 465delD (100%), H228D (94%), T292A (85%). The identified variants some new and some already reported can disrupt pentamer formation, transcriptional regulation of the virus, L1 protein interface interaction, B and T cell epitopes, p53 degradation, and thus their distribution is important for development of HPV diagnostics, vaccine, and for therapeutic purpose.

Human papilloma virus genotyping, variants and viral load in tumors, squamous intraepithelial lesions, and controls in a north Indian population subset.

A study of human papilloma virus (HPV) types and variants is important for developing preventive protocols and appropriate intervention targets. The presence of HPV types, their variants, and viral load in a population subset from North India was studied. Polymerase chain reaction (PCR) and line blots were used for HPV genotyping; HPV 16 and 18 viral loads were measured using real-time PCR. Variant analysis was done by sequencing of the PCR-amplified E6/E7 regions of HPV 16 and the long control region and E6/E7 regions of HPV 18. The 93.6%, 78.6%, and 10% of tumors, squamous intraepithelial lesions (SILs), and controls were HPV-positive, respectively. The most commonly observed type was HPV 16. Human papilloma virus 73 which is uncommonly observed was seen in 2 tumors. Multiple infections were more common in controls and SILs than tumors. The majority (86.4%) of the HPV 16-positive and all of the HPV 18-positive samples belonged to the European variant class. Five novel nonsynonymous changes were seen in the HPV 16-positive and 2 in HPV 18-positive samples. There was a significant increase in viral loads from controls through SILs to tumors, but no significant differences in viral loads were observed between different stages of cancer. In tumors, a significant increase in HPV 16 viral loads was seen with increasing age. The study shows a similar HPV type and variant distribution to European studies, with some differences in type distribution. Viral load does not appear to be good marker for stage wise progression and intralesional variability may affect its use as a differentiating parameter between high-grade squamous intraepithelial lesion and low-grade squamous intraepithelial lesions.

Efficacy and Safety of Mycobacterium indicus pranii as an adjunct therapy in Category II pulmonary tuberculosis in a randomized trial.

Prolonged treatment of tuberculosis (TB) often leads to poor compliance, default and relapse, converting primary TB patients into category II TB (Cat IITB) cases, many of whom may convert to multi-drug resistant TB (MDR-TB). We have evaluated the immunotherapeutic potential of Mycobacterium indicus pranii (MIP) as an adjunct to Anti-Tubercular Treatment (ATT) in Cat II pulmonary TB (PTB) patients in a prospective, randomized, double blind, placebo controlled, multicentric clinical trial. 890 sputum smear positive Cat II PTB patients were randomized to receive either six intra-dermal injections (2 + 4) of heat-killed MIP at a dose of 5 × 108 bacilli or placebo once in 2 weeks for 2 months. Sputum smear and culture examinations were performed at different time points. MIP was safe with no adverse effects. While sputum smear conversion did not show any statistically significant difference, significantly higher number of patients (67.1%) in the MIP group achieved sputum culture conversion at fourth week compared to the placebo (57%) group (p = 0.0002), suggesting a role of MIP in clearance of the bacilli. Since live bacteria are the major contributors for sustained incidence of TB, the potential of MIP in clearance of the bacilli has far reaching implications in controlling the spread of the disease.

A Randomized Double-Blind Placebo-Controlled Phase IIB Trial of Curcumin in Oral Leukoplakia.

Oral leukoplakia is a potentially malignant lesion of the oral cavity, for which no effective treatment is available. We investigated the effectiveness of curcumin, a potent inhibitor of NF-κB/COX-2, molecules perturbed in oral carcinogenesis, to treat leukoplakia. Subjects with oral leukoplakia (n = 223) were randomized (1:1 ratio) to receive orally, either 3.6 g/day of curcumin (n = 111) or placebo (n = 112), for 6 months. The primary endpoint was clinical response obtained by bi-dimensional measurement of leukoplakia size at recruitment and 6 months. Histologic response, combined clinical and histologic response, durability and effect of long-term therapy for an additional six months in partial responders, safety and compliance were the secondary endpoints. Clinical response was observed in 75 (67.5%) subjects [95% confidence interval (CI), 58.4-75.6] in the curcumin and 62 (55.3%; 95% CI, 46.1-64.2) in placebo arm (P = 0.03). This response was durable, with 16 of the 18 (88.9%; 95% CI, 67.2-96.9) subjects with complete response in curcumin and 7 of 8 subjects (87.5%) in placebo arm, demonstrating no relapse after 6 months follow-up. Difference in histologic response between curcumin and placebo was not significant (HR, 0.88, 95% CI, 0.45-1.71; P = 0.71). Combined clinical and histologic response assessment indicated a significantly better response with curcumin (HR, 0.50; 95% CI, 0.27-0.92; P = 0.02). Continued therapy, in subjects with partial response at 6 months, did not yield additional benefit. The treatment did not raise any safety concerns. Treatment of oral leukoplakia with curcumin (3.6 g for six months), thus was well tolerated and demonstrated significant and durable clinical response for 6 months. Cancer Prev Res; 9(8); 683-91. ©2016 AACR.

Development of chimeric candidate vaccine against HPV18: a proof of concept.

Human papillomaviruses (HPVs) are prerequisite for the development of cervical cancer, with HPV16 and HPV18 being the most prevalent. Despite the fact that two prophylactic vaccines against HPVs are in the market, wide-scale application of the vaccine in developing countries is a major problem as far as cost of the vaccine and lack of therapeutic efficacy are concerned. Hence, the aim of our study was to develop HPV18 L1E7 chimeric virus-like particles (CVLPs) vaccine candidate possessing both, prophylactic and therapeutic potential against HPV18-associated cervical cancer. In this study, we have developed a potential candidate vaccine against HPV18 involving HPV18 L1E7 CVLPs, which was expressed in E. coli and assembled in vitro. These CVLPs were able to induce a neutralizing antibody response as well as a cell-mediated immune response in mice.

First International Workshopson Provocative Questions (PQ) in Cancer Research, October-November 2014, New Delhi, Bengaluru, and Thiruvananthapuram, India.

In 2011, the National Cancer Institute (NCI, USA) introduced the Provocative Questions (PQ) Initiative, a new approach allowing active researchers to define major unsolved or neglected problems in oncology unaddressed by existing funding. Last year, the U.S. NCI teamed up with the Indian Department of Biotechnology (DBT) to pilot the PQ approach in three cities in India. Workshop outcomes includedthe generation of fundable "PQs" (perplexing questions understudied by the international scientific community), as well as the identification of several non-PQ projects and research-related issues of importance to DBT and other Indian funding groups. The workshops clearly indicated the need to expand beyond crafting "PQs" when considering the best areas for research funding in international settings. Nonetheless, the first set of PQ workshops provided a forum to discuss key issues regarding cancer research in India, including the paucity of cancer research funding, and the lack of relevant human resource training and technology sharing platforms. Continued open debate between researchers, funders and policymakers will be essential to effectively strengthen the cancer research portfolio in India.

Safety and immunogenicity of a live oral recombinant cholera vaccine VA1.4: a randomized, placebo controlled trial in healthy adults in a cholera endemic area in Kolkata, India.

BACKGROUND: A live oral cholera vaccine VA 1.4 developed from a non-toxigenic Vibrio cholerae O1 El Tor strain using ctxB gene insertion was further developed into a clinical product following cGMP and was evaluated in a double-blind randomized placebo controlled parallel group two arm trial with allocation ratio of 1∶1 for safety and immunogenicity in men and women aged 18-60 years from Kolkata, India. METHOD: A lyophilized dose of 1.9×109 CFU (n = 44) or a placebo (n = 43) reconstituted with a diluent was administered within 5 minutes of drinking 100 ml of a buffer solution made of sodium bicarbonate and ascorbic acid and a second dose on day 14. RESULT: The vaccine did not elicit any diarrhea related adverse events. Other adverse events were rare, mild and similar in two groups. One subject in the vaccine group excreted the vaccine strain on the second day after first dose. The proportion of participants who seroconverted (i.e. had 4-folds or higher rise in reciprocal titre) in the vaccine group were 65.9% (95% CI: 50.1%-79.5%) at both 7 days (i.e. after 1st dose) and 21 days (i.e. after 2nd dose). None of the placebo recipients seroconverted. Anti-cholera toxin antibody was detected in very few recipients of the vaccine. CONCLUSION: This study demonstrates that VA 1.4 at a single dose of 1.9×109 is safe and immunogenic in adults from a cholera endemic region. No additional benefit after two doses was seen. TRIAL REGISTRATION: Clinical Trials Registry-India, National Institute of Medical Statistics (Indian Council of Medical Research) CTRI/2012/04/002582.

Clearance of cervical human papillomavirus infection by topical application of curcumin and curcumin containing polyherbal cream: a phase II randomized controlled study.

Curcumin and curcumin containing polyherbal preparations have demonstrated anti-microbial and anti- viral properties in pre-clinical studies. Till date no therapeutic intervention has been proved to be effective and safe in clearing established cervical human papillomavirus (HPV) infection. The present study evaluated the efficacy of Basant polyherbal vaginal cream (containing extracts of curcumin, reetha, amla and aloe vera) and of curcumin vaginal capsules to eliminate HPV infection from cervix. Women were screened by Pap smear and HPV DNA test by PCR. HPV positive women without high grade cervical neoplasias (N=287) were randomized to four intervention arms to be treated with vaginal Basant cream, vaginal placebo cream, curcumin vaginal capsules and placebo vaginal capsules respectively. All subjects were instructed to use one application of the assigned formulation daily for 30 consecutive days except during menstruation and recalled within seven days of the last application for repeat HPV test, cytology and colposcopy. HPV clearance rate in Basant arm (87.7%) was significantly higher than the combined placebo arms (73.3%). Curcumin caused higher rate of clearance (81.3%) than placebo though the difference was not statistically significant. Vaginal irritation and itching, mostly mild to moderate, was significantly higher after Basant application. No serious adverse events were noted.

Human papillomavirus 16 L1-E7 chimeric virus like particles show prophylactic and therapeutic efficacy in murine model of cervical cancer.

Cervical cancer is found to be associated with human papillomavirus (HPV) infection, with HPV16 being the most prevalent. An effective vaccine against HPV can thus, be instrumental in controlling cervical cancer. An ideal HPV vaccine should aim to generate both humoral immune response to prevent new infection as well as cell-mediated immunity to eliminate established infection. In this study, we have generated a potential preventive and therapeutic candidate vaccine against HPV16. We expressed and purified recombinant HPV16 L1(ΔN26)-E7(ΔC38) protein in E. coli which was assembled into chimeric virus like particles (CVLPs) in vitro. These CVLPs were able to induce neutralizing antibodies and trigger cell-mediated immune response, in murine model of cervical cancer, exhibiting antitumor efficacy. Hence, this study has aimed to provide a vaccine candidate possessing both, prophylactic and therapeutic efficacy against HPV16 associated cervical cancer.