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PURPOSE Cervical cancer (CC) is the most common cancer affecting women worldwide. Human papillomavirus (HPV) infection is a major contributing factor for the development of CC. The development of CC occurs progressively from precancer stages to cancerous stages (ie, invasive squamous cell carcinoma [ISCC] and adenocarcinoma [ADC]). ADC is a rare form of CC that develops from the mucinous endocervical epithelium. It is believed that the downstream targets of Notch signaling contribute to the etiology of CC. One such target is HES1, whose role in the modulation of ADC is unknown. The purpose of this study is to determine the role of HES1 protein in HPV-associated ADC subtype of CC and also to compare its expression in histologic subtypes of precancer and ISCC. PATIENTS AND METHODS A total of 148 patients (30 with precancers, 98 with ISCC, and 20 with ADC) and 40 normal control participants were analyzed for the expression of HES1 via immunohistochemistry, with results validated by immunoblotting. RESULTS The comparison between HPV-16 and HES1 expression was significant in precancer (cervical intraepithelial neoplasia grades 1 to 3; P = .013), ISCC (International Federation of Gynecology and Obstetrics stages I to IV; P = .001), and ADC ( P = .007). An overall significant mean difference was observed between HES1, JAG1, and Notch-3 proteins in precancer ( P = .001), ISCC ( P = .001), and ADC ( P = .001). Pairwise comparisons between HES1 and JAG1 and HES1 and Notch-3 were also found to be significant. CONCLUSION This study showed that among all HPV-16–positive precancers, the major HES1 positivity signal arises from cervical intraepithelial neoplasia grades 2 and 3 that develops into ISCC. Moreover, HPV-16–positive ADC also showed an association with HES1. The HES1, JAG1, and Notch-3 proteins showed their synergistic role in modulating HPV associated ADC along with histologic subtypes of precancer and ISCC of CC.

Since the inception of the COVID-19 pandemic, a large number of clinical trials on complementary and alternative medicine (CAM) for SARS-CoV-2 infection (COVID-19 disease) have been registered. The information is scattered at different resources making it difficult for researchers, scientists, health care professionals, and the general public to remain up-to-date on the latest CAM clinical trials being registered for COVID-19. The Complementary and Alternative Medicine Clinical Trials Database CoVAM (http://www.covam.in) is developed to provide the scientific community easy access to the latest information on the CAM clinical trials registered globally for COVID-19 disease. To develop the CoVAM, MySQL was used. API management was done through NodeJs (With Express), and Angular 11 was used as front end. The CoVAM is a single platform organized by ten CAM subtypes such as Acupuncture, Auricular point pressing, Ayurveda, Chiropractic, Homeopathy, Psychotherapy, Siddha, Traditional Chinese Medicine (TCM), Vitamins and dietary supplements, and Yoga/ Exercise. Each subtype has information on CAM medicines/ therapy being registered, the full title of the clinical trial, sponsor name, sponsor protocol number/ ID, age of the population, study type, actual number of participant enrolment for the trial, followed by the start date, phase, and status of the clinical trial. Each fact is linked to the clinical trial database/s from where the information was procured. Additionally, CoVAM is hyperlinked with PubMed for providing recent updates on COVID-19 and CAM research. To the best of our knowledge, CoVAM is a first-of-its-kind database that provides comprehensive information on globally registered CAMs related clinical trials conducted on COVID-19 disease.

Background: Coronavirus disease 2019 (COVID-19) is caused by coronavirus 2 (SARS-CoV-2). Symptoms include fever, cough, shortness of breath, muscle pain, pneumonia, and multi-organ failure. The infection spreads from one person to another via respiratory droplets. Alternative medicine (AMs) viz., Ayurveda, Homeopathy, Unani, and Traditional Chinese Medicine (TCM), are being promoted for the prevention of COVID-19. The aim of this systematic scoping review was to identify and summarize the scientific evidences promoting the use of AMs for the prevention of COVID-19. Methods: A comprehensive search of electronic search engines (PubMed and Web of Science) was performed. In addition, freewheeling searches of the government health ministries and government websites was done to retrieve the available information. Records available until 12th March 2020 were considered. Reports proposing the use of AMs for prevention or treatment of COVID-19 across all countries were included. Screening (primary and secondary) of the records and data extraction from the eligible studies were done by a single reviewer followed by a random quality check (10%) by the second reviewer. Results: Overall, 8 records (7 from China and 1 from India) exploring the use of AMs for the prevention or treatment of COVID-19 were identified. Different medicines were explored by different AM systems. Conclusions: Several AMs options are proposed for the prevention or treatment of COVID-19. However, their efficacy and safety still needs scientific validation through rigorous randomized controlled trials. This review may help inform decisions about the importance of research and development of AMs for COVID-19 prevention and treatment.

Laparoscopic CBD exploration (LCBDE) for Common bile duct (CBD) stones with laparoscopic cholecystectomy (LC) is an alternative to open CBD exploration, in patients with failed endoscopic retrograde cholangio-pancreatography (ERCP). It is being performed at few centres with adequate surgical expertise. Herein, we present our experience of LCBDE with LC over a period of 10 years.A retrospective analysis of prospectively recorded data of 121 consecutive patients was performed from February 2010 to November 2019, who underwent LC and LCBDE by choledochotomy in a single surgical unit. These included all patients with failed pre-operative ERCP.Out of 121 patients, LCBDE successfully cleared the CBD in 118, and three patients were converted to open surgery. All these patients underwent choledochotomy for adequate exploration of CBD. T-tube closure of CBD was performed in 103 patients and removed after a mean of 14.6 ± 2.4 days. Primary closure was performed in 15 patients. The mean hospital stay post-procedure was 3.4 ± 0.7 days. Complete ductal clearance was achieved in 115 patients, and residual stone fragments reported in three patients were removed by ERCP. None of the patients experienced biliary peritonitis, biliary fistula, pancreatitis or cholangitis. There was no 30-day mortality and no recurrent stones reported with a mean follow-up of 12.4 ± 3.9 months.With adequate surgical expertise, LCBDE is a feasible alternative to open surgery for CBD stones after failed ERCP with early recovery and low morbidity.

The majority of cervical cancer (CC) cases are attributable to HPV infection. Altered Notch pathway signals and HPV are believed to modify clinicopathogenesis of CC, however, the involvement of each molecular player and its mechanism is still not known. Jagged-1 (JAG1) is one of the ligands that induce Notch pathway. The involvement of JAG1 in the modulation of a disease condition is not very clear. Hence, this study aims to study the role of JAG1 in HPV-16/18 associated different histological sub-types of CC, especially ADC. 40 non-neoplastic cervical tissues, 30 precancer and 118 tumor specimens (total 188 tissue biopsies) were studied for the expression of the JAG1 protein through immunohistochemistry, immunoblotting and for HPV infection. Two folds increase of cytoplasmic (Mean ± S.E, 3.67 ± 0.33; p = 0.0001) and nuclear (3.70 ± 0.38, p = 0.0001) JAG1 expression was identified in normal (N) vs precancer and three folds cytoplasmic (4.44 ± 0.17, p = 0.0001) and nuclear (4.64 ± 0.17; p = 0.0001) in N vs. ISCC. Total 85% of ADC patients were found to be infected with HPV, which were 100% infected with HPV-16. These findings suggest the complex synergistic interplay between JAG1 and HPV in regulating clinicopathological progression of CC through its deregulation.

Areca Nut (AN), the seed of tropical palm tree Areca catechu, is a widely chewed natural product with estimated 600 million users across the world. Various AN products, thriving in the market, portray 'Areca nut' or 'Supari' as mouth freshener and safe alternative to smokeless tobacco. Unfortunately, AN is identified as a Group 1 human carcinogen by International Agency for Research on Cancer (IARC). Wide variation in the level of alkaloids, broadly ranging from 2 to 10 mg/gm dry weight, is observed in diverse variety of AN sold worldwide. For the first time, various factors influencing the formation of carcinogenic alkaloids in AN at various stages, including during the growth, processing, and storage of the nut, are discussed. Current review illustrates the mechanism of cancer induction by areca alkaloids in humans and also compiles dose-dependent pharmacology and toxicology data of arecoline, the most potent carcinogenic alkaloid in AN. Careful monitoring of the arecoline content in AN can potentially be used as a tool in product surveillance studies to identify the variations in characteristics of various AN sample sold worldwide. The article will help to generate public awareness and sensitize the government bodies to initiate campaigns against AN use and addiction.

Introduction Kiwam (Qiwam) is a partially fermented tobacco product consumed with Betal Quid (Paan). The major constituents of this product are tobacco, saffron (Zaffrani) and some additives. It contains Tobacco-Specific Nitrosamines (TSNA) which is considered as a cancer-causing agent. To elucidate the carcinogenic property of Kiwam, biochemical profiling of its constituents at different stages of processing is needed. The major processing steps involved in the formation of Kiwam and biochemical profiling/changes at each processing step is still unknown. Aim To describe the major processing steps and biochemical changes that occurs at each processing step during the preparation of Kiwam. Method Tobacco leaves and stems were boiled in water followed by filtering of the constituents to remove the leaves and stem residues. The filtrate is again boiled to form a thick paste residue. The resultant paste was partially fermented through sun curing, and lastly saffron along with some additives were added. The samples from each step were analyzed for biochemical profiling through Continuous Flow Autoanalyzer (CFA) using Flow View Solution 3700 Analyzer (version 1.2.2) software. Results The biochemical changes at TSNA levels were observed at each processing steps. The detailed chemical profiling will be presented during the meeting. Conclusions Processing of Kiwam involves four major steps i.e. (i) boiling of tobacco leaves and stem (ii) filtration of product (iii) re-boiling of the filtrate till the paste is formed (iv) partial fermentation through sun curing. Kiwam is rich in TSNA and hence its use should be avoided.

Purpose:The purpose of this study is to evaluate the clinical and radiologic outcomes after revision total hip arthroplasty (THA) using fourth-generation ceramic-on-ceramic (CoC) bearing surfaces.Methods:A total of 52 revision THAs (28 men and 19 women) using the fourth-generation CoC bearing surfaces were retrospectively evaluated. Both acetabular cup and femoral stem were revised in all cases. The mean follow-up period was 7.3 years (range, 4.0–9.9 years). The clinical results with Harris hip score (HHS), Western Ontario McMaster Osteoarthritis Index (WOMAC), and radiologic outcomes were evaluated.Results:At the final follow-up examination, the average HHS was 90.4 (range, 67–100). The average WOMAC pain and physical function score were 2.8 (range, 0–12) and 16.4 (range, 0–42), respectively. Complications were observed in 10 hips (19.2%). However, there were no bearing surface-related complications, and no cases of dislocation and squeaking. Retroacetabular pelvic osteolysis without cup loosening was observed in one hip at the final follow-up. However, no hip showed radiographic signs of cup loosening, vertical or horizontal acetabular cup migrations, and changes of inclinations during the follow-up period.Conclusion:Our data showed that clinical and radiologic outcomes after revision THA using fourth-generation CoC bearing were favorable. Hence, revision THA with the use of CoC bearing surfaces can be preferentially considered. Further studies with long-term follow-up data are warranted.

Oral cancer etiology is complex and controlled by multi-factorial events including genetic events. Candidate gene studies, genome-wide association studies, and next-generation sequencing identified various chromosomal loci to be associated with oral cancer. There is no available review that could give us the comprehensive picture of genetic loci identified to be associated with oral cancer by candidate gene studies–based, genome-wide association studies–based, and next-generation sequencing–based approaches. A systematic literature search was performed in the PubMed database to identify the loci associated with oral cancer by exclusive candidate gene studies–based, genome-wide association studies–based, and next-generation sequencing–based study approaches. The information of loci associated with oral cancer is made online through the resource “ORNATE.” Next, screening of the loci validated by candidate gene studies and next-generation sequencing approach or by two independent studies within candidate gene studies or next-generation sequencing approaches were performed. A total of 264 loci were identified to be associated with oral cancer by candidate gene studies, genome-wide association studies, and next-generation sequencing approaches. In total, 28 loci, that is, 14q32.33 (AKT1) , 5q22.2 (APC) , 11q22.3 (ATM) , 2q33.1 (CASP8) , 11q13.3 (CCND1) , 16q22.1 (CDH1) , 9p21.3 (CDKN2A) , 1q31.1 (COX-2) , 7p11.2 (EGFR) , 22q13.2 (EP300) , 4q35.2 (FAT1) , 4q31.3 (FBXW7) , 4p16.3 (FGFR3) , 1p13.3 (GSTM1-GSTT1) , 11q13.2 (GSTP1) , 11p15.5 (H-RAS) , 3p25.3 (hOGG1) , 1q32.1 (IL-10) , 4q13.3 (IL-8) , 12p12.1 (KRAS) , 12q15 (MDM2) , 12q13.12 (MLL2) , 9q34.3 (NOTCH1) , 17p13.1 (p53) , 3q26.32 (PIK3CA) , 10q23.31 (PTEN) , 13q14.2 (RB1) , and 5q14.2 (XRCC4) , were validated to be associated with oral cancer. “ORNATE” gives a snapshot of genetic loci associated with oral cancer. All 28 loci were validated to be linked to oral cancer for which further fine-mapping followed by gene-by-gene and gene–environment interaction studies is needed to confirm their involvement in modifying oral cancer.

Background Genome-wide association studies (GWAS) repeatedly identified 1q23 (FCER1A), 5q31 (RAD50-IL13 and IL4), and 12q13 (STAT6) as major susceptibility loci influencing the regulation of total serum IgE levels. As GWAS may be insufficient to capture causal variants, we performed fine-mapping and re-genotyping of the three loci using 1000 Genomes Project datasets. Methods Linkage disequilibrium tagging polymorphisms and polymorphisms of putative functional relevance were genotyped by chip technology (24 polymorphisms) or MALDI-TOF-MS (40 polymorphisms) in at least 1303 German children (651 asthmatics). The effect of polymorphisms on total serum IgE, IgE percentiles, and atopic diseases was assessed, and a risk score model was applied for gene-by-gene interaction analyses. Functional effects of putative causal variants from these three loci were studied in silico. Results Associations from GWAS were confirmed and extended. For 1q23 and 5q31, the majority of associations were found with mild to moderately elevated IgE levels, while in the 12q13 locus, single-nucleotide polymorphisms (SNPs) were associated with strongly elevated IgE levels. Gene-by-gene interaction analyses suggested that the presence of mutations in all three loci increases the risk for elevated IgE up to fourfold. Conclusion This fine-mapping study confirmed previous associations and identified novel associations of SNPs in 1q23, 5q31, and 12q13 with different levels of serum IgE and their concomitant contribution to IgE regulation.

Background Recently, three genome-wide association studies (GWAS) demonstrated FCER1A, the gene encoding a ligand-binding subunit of the high-affinity IgE receptor, to be a major susceptibility locus for serum IgE levels. The top association signal differed between the two studies from the general population and the one based on an asthma case–control design. In this study, we investigated whether different FCER1A polymorphisms are associated with total serum IgE in the general population and asthmatics specifically. Methods Nineteen polymorphisms were studied in FCER1A based on a detailed literature search and a tagging approach. Polymorphisms were genotyped by the Illumina HumanHap300Chip (6 polymorphisms) or MALDI-TOF MS (13 polymorphisms) in at least 1303 children (651 asthmatics) derived from the German International Study of Asthma and Allergies in Childhood II and Multicentre Asthma Genetics in Childhood Study. Results Similar to two population-based GWAS, the peak association with total serum IgE was observed for SNPs rs2511211, rs2427837, and rs2251746 (mean r2 > 0.8), with the lowest p-value of 4.37 × 10−6. The same 3 polymorphisms showed the strongest association in non-asthmatics (lowest p = 0.0003). While these polymorphisms were also associated with total serum IgE in asthmatics (lowest p = 0.003), additional polymorphisms (rs3845625, rs7522607, and rs2427829) demonstrated associations with total serum IgE in asthmatics only (lowest p = 0.01). Conclusions These data suggest that FCER1A polymorphisms not only drive IgE levels in the general population but that specific polymorphisms may also influence IgE in association with asthma, suggesting that disease-specific mechanisms in IgE regulation exist.

About 3.2 million people suffer from sickle-cell disease. Aim of this paper is to detect sickle cell anaemia and thalassaemia. The proposed method involves acquisition of the thin blood smear microscopic images, pre-processing by applying median filter, segmentation of overlapping erythrocytes using marker-controlled watershed segmentation, applying morphological operations to enhance the image, extraction of features such as metric value, aspect ratio, radial signature and its variance, and finally training the K-nearest neighbor classifier to test the images. The algorithm processes the infected cells increasing the speed, effectiveness and efficiency of training and testing. The K-Nearest Neighbour classifier is trained with 100 images to detect three different types of distorted erythrocytes namely sickle cells, dacrocytes and elliptocytes responsible for sickle cell anaemia and thalassemia with an accuracy of 80.6% and sensitivity of 87.6%.

Background: The burden of cancers caused due to tobacco-related carcinogenic products is increasing at an alarming rate in India. Unlike the western world, where cancer-causing products are mostly smoked (such as cigarettes), in India it is mostly consumed as such without combustion. Such products are produced for self-consumption or for selling in the local markets within specific geographical locations. There is very little information available in the public search engines (PubMed) about such products (i.e., dohra [mixture of areca nut, catechu, edible lime, peppermint, cardamom, and some flavoring agents], tuibur [tobacco water sipped and retained in mouth for 5-10 minutes and then spit out], kaddipudi [fine powder of tobacco plant used as such, or in processed form, as bricks and blocks made with jaggery and water], and hogesoppu [tobacco leaf used by women either as such or with betel]). Aim: To study the (i) geographical distribution, (ii) varieties, (iii) production and (iv) adverse health effects of unbranded chewable or drinkable carcinogenic products from India. Methods: The information on unbranded carcinogenic products was collected via study tour to different geographical areas of India, via group discussions or telephonic talks with community members of different age groups. Results: Dohra is found in the state of Uttar Pradesh, India. It majorly contains areca nut which contains a carcinogenic compound - arecoline known for causing histologic changes in the oral mucosa. Tuibur is found in the state of Manipur and Mizoram. Evidence suggests that it contains tobacco which is rich in N-nitroso compounds known for causing systemic tumors. Kaddipudi and hogesoppu are found in the state of Karnataka. Both of them contain tobacco. Conclusion: Dohra, Tuibur, kaddipudi and hogesoppu are unbranded cancer-causing products used at specific geographical locations in India. Since these products contain carcinogenic compounds, their use should be avoided.

Background: Alternate splicing (AS) is a regulatory process during gene expression that allows a single gene to code multiple proteins. Sequencing of RNA (RNA-Seq) is a high throughput technology, which has been used in various studies to identify AS mechanisms in head and neck cancer (HNC). Until date, there is no available review that could update us with the major outcomes from these studies. Aim: To perform a comprehensive literature search for AS studies on HNC via RNA-Seq. Methods: A systematic literature search was performed following PRISMA guidelines to give a complete picture of AS in HNC identified through RNA-Seq. In addition, comprehensive search was also performed to identify the bioinformatics softwares that analyses RNA-Seq data for finding AS in cancer. Results: Six studies were found that used RNA-Seq data for identifying AS events in HNC. Five softwares were used by these studies to identify AS events, of which Suppa and AltAnalyze can also categorize all four AS events to subtypes, i.e., cassette exon skipping (ES), intron retention (IR), mutually exclusive exon (MXE), and alternative 5′ and 3′ splice site (ASS). Additionally, SplAdder, ASprofile, JuncBASE, and MATS softwares have been used to identify and categorize AS events in cancers other than HNC. Conclusion: Alternate splicing in HNC is a complex regulatory process of gene expression. It can be studied through RNA-Seq using various bioinformatics softwares. SplAdder, ASprofile, JuncBASE, and MATS have been used to identify and characterize other cancers, but not implemented in HNC, and hence could be used for studying AS in HNC.

Background: Qiwam (Kimam) is a liquid tobacco preparation consumed with paan. It is mostly consumed in southeast Asian region. Evidences suggest that it causes potentially malignant disorders (PMD), oral cancer (OC) and decreases sperm count. Qiwam was mentioned in earlier research publications, however details are not known. It is produced for self-consumption as well as for commercial purpose. Aim: To study in detail the ingredients and processing steps involved in the production of Qiwam. In addition, also study the adverse health implication of this smokeless tobacco product on humans. Methods: The information on qiwam was collected via literature search study, study tour to different geographical areas of India, where group discussions with the people involved in the production of qiwam, paan vendors and with community members of different age group were done. Results: Qiwam is prepared by the user for his/her own consumption or by industry for sale. Tobacco leaves and tobacco roots are boiled for several hours then soaked in water flavored with varied spices and additives. The resultant mixture is mashed, strained, and finally dried into a thick paste. It is consumed mostly with paan. Conclusion: Processing of qiwam is a complex and time taking process which involves various steps and components that may influence the carcinogenic property of the product. The different processing steps gives different taste and texture to the product. Qiwam increases the risk of cancer and hence needs to be banned or better avoided.

Persistent infection with Helicobacter pylori confers an increased risk of peptic ulceration and gastric adenocarcinoma. Reactive oxygen and nitrogen species play a crucial role in the progression from normal gastric mucosa to cancer. The aim of the present study was to investigate the plasma malondialdehyde and nitric oxide levels in H. pylori related gastroduodenal diseases and associate their levels with gastric pathology and genotypes of H. pylori. Malondialdehyde and nitric oxide levels in plasma samples of 250 subjects were spectrophotometrically determined. Subsequently, genotypic and histopathological assessment was performed in gastric biopsies obtained during endoscopy. The levels of MDA and NO exceeded in subjects infected with genotype-1 of Hp than those with other genotypes suggesting more precise interaction of highly virulent strains of Hp in eliciting severe tissue damage. In conclusion, the study demonstrates close relationship between the plasma malondialdehyde and nitric oxide levels, gastric histopathology and genotypes of H. pylori.

BACKGROUND and AIM: Helicobacter pylori has been proven to be responsible for causing various gastrointestinal disorders including gastric adenocarcinoma. Several genes of pathogen (the genes of the cag-PAI, vacA, iceA, and babA) either in combination or independently have been reported to significantly increase the risk of ulceration/gastric carcinoma, with the cagA gene having the strongest predictive value. Pursuit to identify new genes which could serve as a marker of overt disease progression, lead to the discovery of hrgA gene. METHODS: Fifty-six indigenous strains of H. pylori from subjects with various gastric disorder were screened to assess the status of hrgA gene along with the cagA gene using simple polymerase chain reaction using specific oligonucleotide primers. Post-amplification, amplicons were subjected for sequencing to identify any strain specific variations in sequences from the H. pylori isolated from different disease manifestations. Histopathological analysis was done to ascertain any significant change in the histological scores of subjects infected with cagA+/hrgA+ and cagA-/hrg+ strains. RESULTS: All the 56 (100%) subjects amplified with the oligonucleotide primers specific to hrgA gene, whereas 81.71% subjects showed the presence of cagA gene. Sequencing of the amplimers showed 99% homology. Histology of the cagA+/hrgA+ and cagA-/hrg+ subjects did not show any significant difference. CONCLUSION: hrgA gene of Helicobacter pylori is not a ideal surrogate marker for identifying individuals with higher risk of developing overt gastro-duodenal diseases such as neoplasia of the stomach.

Mycobacterium w (Mw), is a cultivable, non-pathogenic mycobacterium and has been tried extensively as an immunomodulator in leprosy. This has been found to be safe and has shown beneficial immunoprophylactic effect in population based, double blind placebo controlled trials in North India. These effects were also observed in the vaccine trials in South India. Keeping in view these beneficial effects and its earlier reported protective effect against tuberculosis in animals, its protective efficacy was evaluated in a rural population of about 28,948 people belonging to 272 villages in Ghatampur, Kanpur (India). The population was vaccinated with two doses (1st dose of 1x10(9) heat killed organisms followed 6 months later with a 2nd dose of 5x10(8) organisms) of Mw 10-13 years ago originally to investigate its effect against leprosy. The vaccine/placebo was given to healthy contacts of leprosy patients who had no evidence of suffering from tuberculosis. Incidence and prevalence of pulmonary tuberculosis in the present study was assessed in a blind manner by an active field survey and also retrospectively by history of anti tuberculosis treatment received by the patient in the intervening period (since vaccination), which was also corroborated by scrutinizing the medical records. Diagnosis was confirmed by standard clinical and bacteriological criteria. A total of 69 patients were diagnosed to be suffering from pulmonary tuberculosis during the survey which included 17 new sputum smear positive cases and 52 previously partially treated but still active pulmonary tuberculosis cases. The difference in the new sputum positive cases between the vaccinated (5/17) and placebo groups (12/17) was significant at 5% level of significance for 1 tailed test (Z>1.64). As 75% (52/69) of the cases had been diagnosed as suffering from pulmonary tuberculosis but had not taken adequate therapy all the cases diagnosed during the intervening period were recorded and re-analysis done. The differences are more significant at 1% level of significance for 1 tail test (Z>2.59) when all cases were analysed as a group. A small proportion 12.85% (total number=3036) of the contacts in the study population had BCG scars. On analysis of results on protection against tuberculosis in this group, BCG did provide protection against tuberculosis (p

Objectives: To test susceptibilities of Mycobacterium tuberculosis (MTB) isolates to ethambutol by the Lowenstein-Jensen (LJ) proportion method and resazurin microtitre assay (REMA) and to evaluate REMA for the determination of ethambutol MICs for MTB and Mycobacterium avium isolates. Methods: A total of 50 MTB and 20 M. avium isolates were tested to determine the MICs of ethambutol by REMA and agar dilution method. MTB isolates were also tested by the LJ proportion method. Results: REMA provided ethambutol susceptibility results for all the isolates within 8-9 days. For MTB isolates, REMA showed 96.7% sensitivity, 100.0% specificity and 98.0% accuracy when LJ proportion results were taken as 'gold standard'. For both MTB and M. avium isolates, the MICs determined by REMA were lower than those determined in agar medium, indicating that MIC values determined by REMA are closer to the actual MICs for the isolates. Conclusions: REMA can be used as a rapid and inexpensive method for mycobacterial drug suscepti- bility testing against ethambutol. In comparison with the agar method, the MICs determined by REMA can more accurately be correlated with achievable plasma concentrations of antimycobacterial agents.

Tuberculosis continues to be a major killer disease, despite an all-out effort launched against it in the postgenomic era. We describe here the population structure of Mycobacterium tuberculosis strains, as revealed by a chromosome-wide scan of fluorescent amplified fragment length polymorphisms (FAFLPs), for more than 1,100 independent isolates from 11 different countries. The bacterial strains were genotyped based on a total of 136 ± 1 different FAFLP markers at the genome sequence interface, with details on IS 6110 profiles, drug resistance status, clinicopathological observations, and host status integrated into the analysis process. The strains were found to cluster with possible geographic affinities, including the parameters of host species type, IS 6110 profile, and drug susceptibility status. Of the five most commonly amplified fragment sets (or amplitypes), type A predominated in strains of mixed origin, deposited in The Netherlands; type B was exclusively observed for Indian isolates; type C was found mainly in strains from Peru and Australia; and types D and E predominated in European strains from France and Italy. The amplitypes were independent of certain large sequence polymorphisms representing two important deletions, TbD1 and Rd9. It appears that M. tuberculosis has a high genomic diversity with a possible geographic evolution. This may have occurred due to specific genomic deletions and synonymous substitutions selected rigorously against host defenses and environmental stresses on an evolutionary timescale. The genotypic data reported here are additionally significant for genotype-phenotype correlations and for determining whether pathogen diversity is a reflection f the host population diversity.

We investigated IS6110 polymorphism in clinical isolates of Mycobacterium tuberculosis from patients attending the outpatient department at various hospitals in northern India. DNA fingerprinting of 126 clinical isolates of M. tuberculosis was carried out using restriction fragment length polymorphism (RFLP) associated with the IS6110 element in M. tuberculosis genomes. A substantive degree of polymorphism was evident in the MDR M. tuberculosis isolates. The number of bands in the fingerprints varied from 0 to 19. However, the lack of common bands made it difficult to cluster the majority of these isolates. We were also unable to associate drug resistance with IS6110 copy number. Specific regions of the gyrA and katG genes from a representative number of these isolates were sequenced to determine the genotype. The majority of the isolates analyzed were found to belong to Group 1, indicating that these strains were evolutionarily older. We find no evidence of the W strain, prevalent in the US, in our study. The epidemiological patterns of the various strains in India seem to be very complex, as reflected by the presence of a large number of different strains (types) within north India.

Digoxigenin-labelled total, Leishmania donovani DNA was used as a probe to detect the parasite in Indian Phlebotomus argentipes. The probe was quite sensitive, detecting as little as 0.3 pg parasite DNA, equivalent to approximately 100 parasites. Positive signals could be detected in 12 (32%) of the 38 small (two- to 30-fly) pools of the wild-caught P. argentipes investigated and in a pool of 10 laboratory-bred P. argentipes fed on a patient with post-kala-azar dermal leishmaniasis. None of the wild-caught P. papatasi, P. sergenti or Sergentomyia sp. or of the newly emerged, laboratory-bred P. argentipes investigated were positive by this probe. The results indicate that such a probe may be very useful in screening sandflies for L. donovani, and so detecting the main vectors and transmission pathways of this parasite, in India and elsewhere.

The effect of trifluoperazine (TFP), a calmodulin antagonist, was investigated on in vitro ATP levels of human derived Mycobacterium leprae. M. leprae were obtained from biopsies from multi-bacillary forms of leprosy and were incubated in a modified Dubos medium system which supports limited in vitro synthesis of M. leprae. This incubation was carried out in the absence and presence of different concentrations of trifluoperazine. Samples for estimation of bacillary ATP levels were taken at day 0 and at 14 days of incubation. TFP inhibited ATP levels in M. leprae and this inhibitory effect was marginal at 2.5 Wg ml 31 (35% inhibition), highly significant at 5 W gm l 31 (87% inhibition) and almost total at 10 W gm l 31 (98.5% inhibition). This compound appears to have potential as an anti-leprotic drug and also as a broad spectrum anti-mycobacterial agent in view of its anti-tubercular activity reported earlier. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.

BackgroundThe phylogeny of the indigenous Indian-specific mitochondrial DNA (mtDNA) haplogroups have been determined and refined in previous reports. Similar to mtDNA superhaplogroups M and N, a profusion of reports are also available for superhaplogroup R. However, there is a dearth of information on South Asian subhaplogroups in particular, including R8. Therefore, we ought to access the genealogy and pre-historic expansion of haplogroup R8 which is considered one of the autochthonous lineages of South Asia.Methodology/principal findingsUpon screening the mtDNA of 5,836 individuals belonging to 104 distinct ethnic populations of the Indian subcontinent, we found 54 individuals with the HVS-I motif that defines the R8 haplogroup. Complete mtDNA sequencing of these 54 individuals revealed two deep-rooted subclades: R8a and R8b. Furthermore, these subclades split into several fine subclades. An isofrequency contour map detected the highest frequency of R8 in the state of Orissa. Spearman's rank correlation analysis suggests significant correlation of R8 occurrence with geography.Conclusions/significanceThe coalescent age of newly-characterized subclades of R8, R8a (15.4+/-7.2 Kya) and R8b (25.7+/-10.2 Kya) indicates that the initial maternal colonization of this haplogroup occurred during the middle and upper Paleolithic period, roughly around 40 to 45 Kya. These results signify that the southern part of Orissa currently inhabited by Munda speakers is likely the origin of these autochthonous maternal deep-rooted haplogroups. Our high-resolution study on the genesis of R8 haplogroup provides ample evidence of its deep-rooted ancestry among the Orissa (Austro-Asiatic) tribes.

In the present study, ethambutol (EMB) resistance-associated mutations were characterised in the embCAB genes of clinical isolates of Mycobacterium tuberculosis (MTB) collected in India. Thirty MTB isolates were tested for their susceptibility to first-line antitubercular drugs using the Lowenstein-Jensen proportion method, and EMB minimum inhibitory concentrations of MTB isolates were determined by the resazurin microtitre assay. Sequencing of various regions of the embCAB genes was performed to identify EMB resistance-associated mutations. Mutations of embB306 were detected in 15 of 23 EMB-resistant MTB isolates. Three EMB-resistant isolates had mutations at codon 270 of the embC gene, two of which also harboured embB306 mutations. No mutation was identified in the embA gene. All seven EMB-sensitive MTB isolates had the wild-type embCAB sequence. In summary, embB306 mutations were associated with EMB resistance, and mutation at codon 270 of the embC gene may contribute to high-level EMB resistance in some MTB isolates.

DNA fingerprinting using IS 6110 probe has been used all over the world quite successfully to characterize M. tuberculosis strains. The present study has been carried out to study the polymorphism among isolates of M. tuberculosis from Agra region from patients attending the clinics at SN Medical College and TBDTC, Agra. Sputa were collected in sterilized containers and brought to CJIL, Agra. Samples were processed and cultured on Lowenstein Jensen (LJ) slants. M. tuberculosis isolates were identified by standard biochemical tests. DNA from these isolates were purified by a physicochemical procedure, restricted with Pvu II enzyme and hybridized with PCR amplified and DIG labeled 245 bp IS 6110 probe. With a view to study IS 6110 polymorphism, M. tuberculosis isolates obtained from different geographical areas of Agra region were analyzed. Among the 60 isolates taken in study, 5 had no copy of IS 6110, 8 had 1-4 copies and 47 had multiple copies of IS 6110. DNA fingerprinting using this probe was found to be quite discriminating for typing of most of the strains (80%) which had multiple copies. RFLP profiles did not correlate with geographical areas, contacts or the resistance pattern of the strains. While this data shows the potential of IS 6110 based RFLP for strain characterization of M. tuberculosis in Agra, to understand the molecular epidemiology of tuberculosis in this region, a larger number of isolates from defined geographical areas need to be studied.

Pyrazinamide (PZA) is an important first-line antituberculosis drug because of its sterilizing activity against semidormant tubercle bacilli. In spite of its very high in vivo activity, its in vitro activity is not apparent unless an acidic environment is available, which makes PZA susceptibility testing difficult by conventional methods. The present study was, therefore, planned to assess the performance of the colorimetric BacT/ALERT 3D system and compare the results with those from conventional tests, i.e., the Löwenstein-Jensen (LJ) proportion method (pH 4.85) and Wayne's pyrazinamidase (PZase) assay, using 107 clinical isolates. The concordance among all of these tests was 89.71% after the first round of testing and reached 92.52% after resolution of the discordant results by retesting. Prolonged incubation of the PZase tube for up to 10 days was found to increase the specificity of the PZase test. The concordances between LJ proportion and BacT/ALERT 3D, LJ proportion and the PZase assay, and BacT/ALERT 3D and the PZase assay were found to be 99.06%, 93.46%, and 92.52%, respectively. Using the LJ results as the gold standard, the sensitivities of BacT/ALERT 3D and the PZase assay were 100 and 82.85%, respectively, while the specificity was 98.61% for both of the tests. The difference between the sensitivities of BacT/ALERT 3D and the PZase assay was significant ( P = 0.025). The mean turnaround times for the detection of resistant and susceptible results by BacT/ALERT 3D were 8.04 and 11.32 days, respectively. While the major limitations associated with the PZase assay and the LJ proportion method are lower sensitivity in previously treated patients and a longer time requirement, respectively, the BacT/ALERT 3D system was found to be rapid, highly sensitive, and specific.

Mutations in the metabolic enzymes IDH1 and IDH2 are frequently found in several tumors including glioma and acute myeloid leukemia (AML). Mutant IDH produces R-2-hydroxyglutarate (R2HG), which induces histone- and DNA-hypermethylation through inhibition of epigenetic regulators, thus linking metabolism to tumorigenesis. However, it is unknown whether R2HG alone is sufficient to recapitulate the biologic effects of mutant IDH1 in vivo. Recently, we have shown that IDH1mut cooperates with HoxA9 and induces a monocytic leukemia in mice. In order to evaluate the effects of R2HG independently of the mutated IDH1 protein and to determine whether the effects are specific to the R-enantiomer of 2HG, we treated mice transplanted with HoxA9 immortalised bone marrow cells with R2HG, S-2-hydroxyglutarate (S2HG), alpha-ketoglutarate (aKG) and phosphate buffered saline (PBS). The mice in the metabolite cohorts received an intraperitoneal dose of 1 mg per day. Mice treated with R2HG had higher engraftment levels at 16 and 20 weeks post transplantation than the mice treated with S2HG, αKG and PBS respectively (P We next compared mice receiving transplants of HoxA9+IDH1mut cells with mice receiving HoxA9 cells that were then treated with R2HG. Both cohorts developed monocytic leukemia, albeit with different kinetics. The Hoxa9+IDH1mut mice died with a median latency of 83 days while the R2HG cohort died with a median latency of 137 days post transplantation (P In order to rule out an influence of differential R2HG levels on the disease progression between the two cohorts, levels of R2HG were quantified. IDH1mut expressing and R2HG treated bone marrow cells from mice had similarly high ratios of R2HG/S2HG. The quantified R2HG levels were also comparable to that of primary AML patient cells harbouring mutated IDH1. In unsupervised hierarchical clustering mutated cells clustered together with R2HG and separated from PBS treated mice. 69 of the top 100 enriched Gene Ontology gene sets of HoxA9+IDH1mut were also found in HoxA9+R2HG, suggesting largely redundant but also non-overlapping functions of the mutant IDH1 protein and the oncometabolite R2HG. In summary, we show that R2HG, similar to the mutant IDH1 protein, promotes leukemogenesis in cooperation with HoxA9, although with delayed kinetics. Our data proves that R2HG acts as an oncometabolite in vivo in a murine model of leukemogenesis. Disclosures No relevant conflicts of interest to declare.