Female genital tuberculosis (GTB) contributes significantly to infertility in low- and middle-income countries. Dissemination of infection from pulmonary and extrapulmonary sites is the major reason for causation of GTB. Additionally, sexual transmission of GTB from male partners has been reported. We selected 81 couples desiring babies from an in vitro fertilization clinic. We used multiplex-PCR for mycobacterial detection in semen of males, in the endometrium of their female counterparts and in the products of conception (POC) from miscarriage. Data interpretation shows that these pregnancies failed owing to sexual transmission of mycobacteria. We noticed by multiplex PCR that mycobacterial infestation in the female can take place in either endometrium or POC from asymptomatic males harbouring mycobacteria in their semen. Therefore, we propose sexual transfer of mycobacteria to be a probable cause of miscarriage. Thus, we suggest multiplex PCR based screening of semen for all males of the couples attempting successful childbirth.

BACKGROUND:Polycystic ovary syndrome (PCOS) is associated with insulin resistance and elevated risk of cardiovascular disease and diabetes. Chronic inflammation has been observed in PCOS in several studies but there is also opposing evidence and a dearth of research in Indians.OBJECTIVE:To estimate chronic inflammation in PCOS and find its relationship with appropriate anthropometric and biochemical parameters.MATERIALS AND METHODS:Chronic inflammation was assessed in 30 women with PCOS (Group A) and 30 healthy controls (Group B) with highly sensitive C-reactive protein (hsCRP), interleukin-6 (IL-6), tumour necrosis factor alpha (TNFα), and platelet microparticles (PMP). In group A, the relationship of chronic inflammation with insulin resistance, waist hip ratio (WHR) serum testosterone, and serum glutamate pyruvate transaminase (SGPT) were examined.RESULTS:In group A, the hsCRP, TNFα, and PMP were significantly elevated compared to group B. However, IL-6 level was similar between the groups. In group A, PMP showed a significant positive correlation with waist-hip ratio and serum testosterone. IL-6 showed a significant positive correlation with insulin sensitivity and significant negative correlation with insulin resistance and serum glutamate pyruvate transaminase.CONCLUSION:PCOS is associated with chronic inflammation and PMP correlates positively with central adiposity and biochemical hyperandrogenism in women with PCOS.

The present study aimed to explore the early predictive marker of diabetic retinopathy (DR) and to elucidate the associated demographic, metabolic, and ocular factors. We enrolled 43 type 2 diabetic subjects with mild non-proliferative retinopathy (MNPDR), 30 diabetic subjects with no retinopathy (DNR), and 35 healthy controls (HC). The study groups showed no significant alteration in central macular thickness (CMT) and visual acuity (VA). The contrast sensitivity (CS) score was found to be significantly lower among DNR and MNPDR subjects compared to HCs (p < 0.0001). Between MNPDR and DNR subjects, the CS score was significantly lower in the former (p = 0.0036). CS score discriminated DNR subjects from HC, with 74% accuracy for the optimal threshold 0.71. The associated area under the ROC curve (AUC) is 0.82 (p < 0.0001) while the discrimination rule has 66% sensitivity and 80% specificity. The CS score also discriminated MNPDR subjects from DNR with 64% accuracy for the optimal threshold 0.53. The associated AUC is 0.65 (p < 0.023) and the rule has 86% sensitivity and 33% specificity. According to best subset regression analysis, not only glycaemic parameters but also lipid parameters [low-density lipoprotein cholesterol (LDL-C) (p = 0.045) and triglycerides (TG) (p = 0.0005)] were found to be significant predictors of CS. CMT (p = 0.058) was another marginally significant predictor of CS. CS may be used as an early predictive marker for DR. So, not only hyperglycemia, but also hyperlipidemia seems to significantly affect retinal CS function in diabetes.

Aim: To explore the possibility of risk factor between latent genital tuberculosis with association of low secretion anti-mullerian hormone(AMH) and low level of Vitamin D for infertility. Design: Retrospective study was done in the reproductive medicine unit of Calcutta Fertility Mission, Kolkata-700019, and India Result: From January 2017 to January 2019, total 150 Subjects (n= 75 TB-PCR positive and n=75 TB-PCR negative cases as control) were included in this study on the basis of inclusion and exclusion criteria. We have found that mean value of AMH and Vitamin D were higher in PCR negative group than positive group. Various level of AMH and Vitamin D concentration for PCR positive and negative cases were statistically significant (p-value < 0.01). Conclusion: Our finding is that vitamin D deficiency is highly prevalent not only in active tuberculosis but also in latent genital tuberculosis subjects and serum Vitamin D level may help in treatment of latent genital tuberculosis. Elevated level of Vitamin D in serum may rule out latent tuberculosis.

BACKGROUND: Nowadays, one of the most common vulnerable sites of extrapulmonary tuberculosis is female genital tract tuberculosis (FGTB) leading to infertility. As FGTB produce clinical symptom quite late, its detection is very difficult to the health-care providers and according to some experts in this field; FGTB has no confirmatory investigation procedure. FGTB can cause other form of reproductive failure through ectopic pregnancy, tubal block, and implantation failure. Their presence may alter the cytokines level in endometrium. METHODS: A total of 300 cases in our clinic had undergone polymerase chain reaction (PCR) for detection of tubercle bacillus (TB-PCR). Among of them, 91 individuals fulfilling all inclusion and exclusion criteria were included in this study. In our study, we measured cytokines, two from pro-inflammatory group (interleukin-6 [IL-6] and IL-10) and three from inflammatory group (IL-2, interferon gamma [IFNγ], and tumor necrosis factor alpha [TNFα]). RESULTS: Out of 91 participants, 45 (49.46%) cases were TB-PCR positive, and 46 (50.55%) cases were TB-PCR negative. It has been observed that the value of IL-10 and TNFα did not fit any statistical parameter. The result showed that pro-inflammatory indicators IL-6 and inflammatory indicators IL-2 and IFNγ had significant different values between TB-PCR- positive and TB-PCR-negative groups. P value of this cytokines has statistically significant. CONCLUSION: From our study, we conclude that cytokine study was undertaken, of which IFNγ showed a possibility to become an important clinical indicator of endometrial hostility followed by IL-2.

Genital tuberculosis (GTB) is a potent contributor to irreversible damage to the reproductive system and infertility in females. As no gold standard diagnostic tool is yet available, clinical suspicion and relatively insensitive approaches such as histopathology, laparoscopy and hysterosalpingogram are currently critical determinants in the diagnosis of GTB. Although a polymerase chain reaction (PCR)-based assay using endometrial tissue seems promising, sampling does require an invasive procedure.

Introduction: Mycobacterium leprae has a small genome and a tendency of persisting as a very low-grade infection. The authors have shown earlier, that the changes in TTC repeats, in M. leprae genome may contribute to the restriction of the pathogenicity of the bacterium and its survival strategy in case of pure neural Hansen's disease. We suspect, that a similar genomic reduction if happens in treated cases of Hansen's disease, can be a determining factor for developing persisters and relapse. Aim: The present study aimed to find out if there was any evidence of genomic reduction in treated cases of Hansen's disease that showed microbiological nonresponse. Methods: Skin biopsies were taken from treated cases of Hansen's disease at tertiary centers in Kolkata and at Raipur who had bacterial index (BI) unchanged or increased compared to their pretreatment BI. Analysis for the mutation in rpoB gene and folP1 gene were done to rule out rifampicin and dapsone resistance, respectively. The entire TTC repeat region of the bacteria was amplified by polymerase chain reaction and was subjected to sequencing. The obtained sequences were then analyzed by CLUSTALW. Results: A total of 127 patients were included in the study of which in 52 the BI remained same and 75 had an increase in BI, even after 6 months of completion of multidrug therapy. Among the samples, 2 had positive rpoB gene mutation. No mutation was found in the folP1 gene. The TTC repeat of both the rpoB-resistant samples was found to have 17 copies, which matched their pretreatment copy number. In other 125 cases, 60 cases showed no change from their pretreatment TTC number. Of those 65 samples that showed evidence of genomic reduction, 11 samples showed one copy, 41 showed 2 copies, and 13 showed 3 copies deletion. We also observed a significant regional variation. Conclusion: We concluded that there was evidence of genomic reduction, which might lead to microbiological nonresponse in treated cases of Hansen's disease. This indicated a possibility of future persistence and relapse.

Purpose: Despite the disease having similar glycemic status and duration microangiopathy in some patients develop within few years whereas it doesn't appear as a health complication in some diabetics for a considerable period. This study is undertaken to assess the hyperglycemia-induced biochemical background behind the development of diabetic retinopathy (DR) in type 2 diabetes mellitus (DM). Methods: Following proper diagnosis, 100 patients of type 2 DM of 10–12 years duration having no DR, and 42 patients of type 2 DM of the same duration and glycemic status as the second group, with mild retinopathy were recruited in the study. Lactic acid, glutamate, malondialdehyde (MDA), nitrate, advanced glycation end-products (AGEs), peripheral blood mononuclear cell reactive oxygen species (ROS), vascular endothelial growth factor (VEGF), and its receptor 2 (VEGFR2) in these two groups were produced in an assay following standard methodology. Results: Biochemical markers of anaerobic glycolysis, lipid peroxidation, AGEs, glutamate concentration, oxidative stress, and expression of VEGF and its VEGFR2 with significantly elevated markings were found in them who developed earliest stage of DR rather than them who had not. Conclusion: Hyperglycemia-induced anomalous glucose metabolism, lipid peroxidation, advanced glycation, glutamate toxicity, and oxidative stress create a background where apoptosis of retinal capillary endothelial cells invite increased expression of VEGF and VEGFR2, these being the crucial factors behind the development of diabetic microangiopathy.

Background: Pure neural leprosy (PNL) still remains a diagnostic challenge because of the absence of sine qua non skin lesions of leprosy and a confirmatory diagnostic method. The authors had earlier described a simple yet objective technique of combining fine needle aspiration cytology (FNAC) coupled with a multiplex polymerase chain reaction (PCR) in a pilot study, wherein the technique showed promise of a reliable diagnostic tool. In the pursuit of further evidence, the authors carried out a 4-year study with PNL cases to find the efficacy and reliability of the said method in a larger sample size. Aim: This study was conducted to find the efficacy, reliability, and reproducibility of FNAC coupled with multiplex PCR and Ziehl-Neelsen (ZN) staining in identifying the cases of PNL. Materials and Methods: All cases that were suspected to be suffering from PNL, following evaluation by two independent observers were included in the study and were subjected to FNAC from the affected nerve, and the aspirates were evaluated for cytology, ZN staining, and multiplex PCR for Mycobacterium leprae genome. In addition, serum anti-PGL1 levels were also performed in all the study subjects. Fifteen non-PNL cases were also included in the control arm. Results: A total of 47 cases were included in the test arm and subjected to FNAC. Conventional ZN staining could demonstrate acid-fast bacilli (AFB) in only 15 out of 47 cases (31.91%) while M. leprae DNA could be elicited in 37 (78.72%) cases by the multiplex PCR. Only 13 (27.65%) out of 47 cases showed anti-PGLI-1 antibody positivity. On cytological examination of the nerve aspirates, only 11 (23.40%) cases showed epithelioid cells whereas nonspecific inflammation was seen in 26 (75.60%) cases. Conclusion: The results of this study conducted over a larger sample size corroborate with the findings of our pilot study. In a resource poor set up, FNAC in combination with ZN staining and multiplex PCR is a rapid, simple, and easily performed test, which can give a reproducible and objective diagnosis in cases of PNL.

Background: Metallo beta lactamases in Pseudomonas aeruginosa are often detected phenotypically. In our institution we frequently encountered such incidence but no report about the genotypic character of such isolates was available. Objective: Thus present study aimed at characterization of the MBL encoding gene in imipenem nonsusceptible multi drug resistant Pseudomonas aeruginosa ,isolated from clinical samples. Materials & methods: 98 Non-duplicate consecutive imipenem non susceptible strains of Pseudomonas aeruginosa were selected from different clinical samples. Combined disc diffusion test with imipenem EDTA (10μg+750μg)combined disc was used to screen for MBLs activity. PCR for identification of blaVIM &blaIMP gene was performed. Result: Combined disc test phenotypically screened 51 out of 98 imipenem non-susceptible isolates of Pseudomonas aeruginosa with MBL activity and 47 as MBL negative . blaVIM gene was detected in 9 out of 98 isolates by PCR; no other MBL encoding gene was detected. The existence of blaVIM gene explained imipenem non-susceptibility in multidrug resistant Pseudomonas aeruginosa and its prevalence was 9.18%(9 out of 98) in our healthcare set up at the time of study.

Background: Group A Streptococcus strains causing wide variety of diseases, recently became noticeable in eastern India, are not amenable to standard treatment protocol thus enhancing the possibility of disease morbidity by becoming antibiotic resistance.Methods: The association of Lancefield group A Streptococcal variation with degree of vir architectural diversity was evaluated using emm typing and restriction fragment length polymorphism analyses. The antibiotic sensitivity patterns were examined by modified Kirby-Bauer method of disk diffusion. Percentage calculations, 95% confidence interval and one-way ANOVA were used to assess differences in proportions.Results: Our observations revealed 20 different emm types and 13 different HaeIII vir typing patterns. A 1.2 kb fragment was found in all HaeIII typing pattern. Fragments of 1.2 kb and 550 bp were conserved in majority of the isolates. HinfI digestion was found proficient in differentiating the strains of same vir typing patterns. Strong predominance of speC (85%) and speF (80%) genes have been observed encoding exotoxins production. 4 isolates were found to be erythromycin resistant and were of genotype emm49. High degree of tetracycline resistance was shown by 53.57% isolates which belonged to 12 different emm genotypes.Conclusions: These findings suggested that in addition to emm typing, sequential application of HaeIII and HinfI restriction enzymes in vir typing analysis is an effective tool for group A streptococcal molecular characterization associated with antibiotic resistance.

We studied the roles of vitamin D and its receptor, VDR, in the progression of leprosy. The majority of individuals with leprosy from Kolkata, India, with a type 1 or type 2 reaction have low levels of vitamin D3 in serum samples. Interestingly, individuals with a type 2 reaction associated with neuritis/erythema nodosum leprosum had very low VDR mRNA expression levels, ranging from 5% to 10%, compared to that of healthy control subjects; these patients also had a high bacilli index, ranging from 3+ to 5+. This is the first report to indicate that VDR expression levels may determine the complexity and severity of the progression of leprosy.

Background:Chlamydia trachomatisis recognized as one of the most common sexually transmitted pathogen in the world. 50-80% of infected females are asymptomatic. These untreated women are at risk of developing chronic sequelae leading to tubal pathology causing infertility. Infertility is defined as 1 year of unprotected intercourse without pregnancy. It may be primary or secondary.Aim:To find out the association of genitalChlamydia trachomatisinfection with female infertility.Materials and Methodology:This case control study has been carried out in collaboration with R. G. Kar Medical College and Institute of Post Graduate Medical Education & Research, India, between July 2012 and June 2013. 40 infertile and 40 pregnant women were enrolled by purposive sampling as per inclusion and exclusion criteria. ELISA test was performed to detect serum IgG and IgA antibody against recombinant analogs of MOMP and 3 different PCR assays were done targeting MOMP and rRNA DNA from DNA extracted from first void urine.Results:IgG seropositivity was significantly higher (15%vs0%,P=.0255) in cases than controls, though there was no significant difference in the proportion of IgA seropositivity among 2 groups (12.5%vs2.5%,P=0.2007). Out of 80 samples 2 samples showed the production of amplicons with R1 – R2 primers. Only 1 sample gave positive result with production of amplicons with all the 3 primers used (R1 – R2, CT0005 – CT06 and JM15 – JM16).Conclusion:PersistentC. trachomatisinfection must be recognized as a risk factor of infertility in this region of India. The low PCR positivity in FVU sample helps to conclude the diagnostic utility of serological tests in screening of infertile women.

Background: Genomic reduction helps obligate intracellular microbes to survive difficult host niches. Adaptation of Mycobacterium leprae in cases of pure neural leprosy (PNL) in the intracellular niche of peripheral nerves can be associated with some gene loss. Recently, a stable but variable number of tandem repefzats (TTC) have been reported in strains of M. leprae. FolP and rpoB genes are the two common mutation sites which deal with the susceptibility of the bacteria to drugs. Aim: We attempted to find if genomic reduction of M. leprae in context of these TTC repeats or mutations in folP1 and rpoB can be the reason for the restriction of M. leprae in the nerves in PNL. Materials and Methods: DNA extracts taken from fine needle aspiration of affected nerves of 24 PNL cases were studied for tandem repeats with 21TTC primer in multiplex-PCR. Mutations were also studied by PCR Amplification of SRDR (Sulphone Resistance Determining Region) of the folP1 and multiple primer PCR amplification refractory mutation system (MARS) of the rpoB. Results: Of the 24 PNL, only 1 patient showed mutation in the rpoB gene and none in the folp1 gene. Studying the mutation in TTC region of the M. leprae gene we found that all the cases have a loss of a few bases in the sequence. Conclusion: We can conclude that there is consistent loss in the bases in the TTC region in all cases of pure neural Hansen and we postulate that it may be an adaptive response of the bacteria to survive host niche resulting in its restriction to peripheral nerves.

The present study was aimed to investigate the relation between nuclear factor kappa beta (NFκB) activation and downstream up-regulation of vascular endothelial growth factor (VEGF) in diabetic retinopathy (DR). Moreover the study was intended to evaluate the role of VEGF gene single nucleotide polymorphisms (SNPs) in DR occurrence and to investigate the functional relevance of VEGF gene SNPs in terms of VEGF expression in DR. Serum level of VEGF, VEGF R1 (receptor 1), VEGF R 2 (receptor 2) and NFκB (p50/65) activity was measured by enzyme linked immune sorbent assay. Genotyping and allelic composition of different SNPs i.e., rs2010963, rs3025039, rs1570360 and rs 2071559 were investigated by Taqman SNP genotyping assay. VEGF, NFκB p50/p65, and VEGF R1 & R2 gene expressions were quantified by real time quantitative polymerase chain reaction. Increased NFκB p50/p65 activity and expressions were observed in non proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR) subjects compared to type 2 diabetes mellitus without retinopathy (DNR) group. Significantly elevated levels of serum VEGF and highest VEGF expression were found among PDR subjects compared to DNR or NPDR subjects. CC genotype and C allele of rs2010963 and TT genotype and T allele of rs3025039 were significantly over represented among PDR subjects compared to DNR group. Increased activation of NFκβ in NPDR and PDR subjects might involve increased up regulation of VEGF. VEGF SNPs i.e., rs2010963 C allele and rs3025039 T allele might be associated with PDR occurrence and in turn regulates VEGF expression among PDR subjects.

BACKGROUND:The current strategy for leprosy control depends mainly on early case detection and providing the recommended multidrug therapy (MDT) dosage. Understanding the molecular mechanisms of drug resistance to each of these drugs is essential in providing effective treatment and preventing the spread of resistant strains in the community. The progress of molecular biology research provides a very efficient opportunity for the diagnosis of drug resistance by in vitro method.AIM:We aimed to investigate the point mutations within the rpoB gene region of the Mycobacterium leprae genome, which are responsible for resistance to rifampicin, in order to determine the emergence of drug resistance in leprosy in the Kolkata region of West Bengal.METHODS:A total of 50 patients with a relapse of leprosy were enrolled in the study. Skin smears were obtained for estimation of bacillary index and biopsies were obtained in 70% alcohol for extraction of DNA. The extracted DNA was amplified by M. leprae-polymerase chain reaction (PCR) targeting rpoB gene region. Every single nucleotide base in the sequence is aligned to reference sequence and identity gaps were determined by NCBI - BLAST. Later in-silico analysis was done to identify the changes in the translated protein sequences.RESULTS:A mutation at the base pair position 2275405 where G is replaced by C in the M. leprae genome, which corresponds to the coding region of rpoB gene (279 bp - 2275228 to2275506), was observed in two patients. This missense mutation in CAC codon brings about a glutamic acid to histidine change in the amino acid sequence of RNA polymerase beta subunit at the position 442 (Glu442His), a region specific for rifampicin interaction, which might be responsible for unresponsiveness to rifampicin by manifesting a stable bacteriological index in these 2 patients even after completion of 24 months of multibacillary multi-drug therapy (MB-MDT).LIMITATIONS:The major limitations of multiple-primer PCR amplification refractory mutation system (MARS) assay is that it capable of detecting mutation at codon 425 and cannot distinguish any silent amino acid changes.CONCLUSION:The study indicates the existence of rifampicin drug resistance in Eastern India.

Sir, South Asian population is known to have an increased predisposition to type 2 diabetes mellitus (T2DM), which turns out to be an important health concern in this Region1. As per an earlier report about 11.7 per cent people of Kolkata, West Bengal, suffered from T2DM and the prevalence was on a rise2. Anaemia is also present in the Region as a significant public health problem, having a prevalence of greater than 40 per cent in South Asia3. However, the actual status of iron remains unclear because of extensive prevalence of haemoglobinopathies, possible genetic mutations contributing to iron overload, indiscriminate over-the-counter use of iron pills and traditional formulations containing undefined concentrations of iron. Higher heme iron intake and increased body iron stores were found to be significantly associated with a greater risk of T2DM4,5. Patients suffering from haemoglobinopathies or undergoing repeated blood transfusions also suffer from secondary iron loading disorder6. Insulin influences the iron uptake and storage in cells by increasing the cell surface transferrin receptors7. Whether a patient with diabetes has excess iron due to increased insulin resistance still remains an unanswered question. Insulin resistance has been shown to have an association with chronic kidney diseases8,9. Iron accumulation has been reported in the proximal renal tubules in diabetic nephropathy10. Iron in its free form11,12 i.e. in non-transferrin bound form is known to induce oxidation of biomolecules and in the formation of reactive oxygen species. We, therefore, studied the free iron status in patients with T2DM and compared with healthy individuals and to find a suitable biocompatible reagent which can bind the free iron. A cross-sectional, pilot study was conducted on consecutive patients attending the General Medicine outpatients department of M.R. Bangur Hospital, Kolkata, India, between August 2012 and February 2014. Fasting blood and urine samples (10 ml each) were collected from 111 patients with T2DM (53.7 ± 12.4 yr, M:F 60: 51) and 30 healthy controls (75.6 ± 2.5 yr, M:F 8:7). Approval of ethical committee of Institute of Post Graduate Medical Education & Research (IPGMER), Kolkata, was obtained prior to the study. Those (i) having anaemia, (ii) suffering from any form of haemoglobinopathy, (iii) who were on iron therapy within one year, (iv) having non-diabetic kidney disease, (v) having febrile illness, (vi) having benign prostatic hypertrophy or prostatic cancer, (vii) having urinary tract infection, or (viii) with uncontrolled hypertension, were excluded. The total iron analysis was done by Ferrozine method13 and free iron by HPLC14. Fasting plasma glucose was estimated by glucose oxidase-peroxidase (GOD-POD) method15, serum insulin by ELISA monobind kit and creatinine analysis was done by a kinetic assay15 of Jaffe's involving alkaline solution of sodium picrate16. The plasma creatinine clearance or estimated glomerular filtration rate (eGFR) were estimated as per Cockroft and Gault formulae in ml/min17. Insulin resistance was calculated by homeostatic model assessment - insulin resistance (HOMA-IR) formula18. Urinary microalbumin analysis was done by immunoturbidimetry19. Conarachin I was extracted from peanut and purified20, and was used as a complexing agent for free iron. Conarachin I was characterized by molecular weight determination and absorption spectrometry20. A 15 per cent resolving sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gel was used for molecular weight determination and confirmation of protein purification from crude peanut protein20. Presence of band at about 18 kD (Figure, lane 1) showed the presence of conarachin I in the purified protein fraction as shown earlier19. Conarachin I (0.1 ml) was mixed with 0.4 ml of the diluted serum, injected to HPLC column and analyzed to compare the amount of free Fe2+ and Fe3+ of the same serum samples20. Figure Purified conarachin I fraction along with its precursors subjected to sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE). Lane 1, pure conarachin 1; Lanes 2 and 3, protein part before purification; Lane 4, mol.wt.marker. Qualitative data were grouped and compared by chi square test. Yates correction was done when the cell frequency was below five21. Quantitative data were subjected to comparison by their differences in mean by unpaired t test. A HOMA value of 2.4 was taken as comparator as the value exhibited evidence of diabetic kidney disease in an Indian study22. Serum iron value of 150 µg/dl was taken as upper reference limit for both sexes considering 145 and 160 as upper reference limit (URL) for females and males, respectively as per the kit insert. Insulin resistance was not found to be significantly associated with total iron. Serum total iron values 150 µg/dl were compared with HOMA values 2.4 for both sexes. No association was seen when the means of total free iron were compared with HOMA 2.4 (15.7±1.64 vs16.02±2.56 ppm); insulin resistance was not related to Fe3+/Fe2+ ratio either in males or in females. Obesity, as body mass index (BMI in kg/m2) >2523 was compared with HOMA, the association was significant at (P<0.01). Free iron has been known to cause damage by generation of free radicals. As obesity with raised amount of adipose tissue contributes to more serum iron and serum iron is known to contribute to insulin resistance, we decided to multiply the BMI with the ferric-ferrous ratio, and took the product (BMI×Fe3+/Fe2+) as the index value. The median value of the distribution was around 0.37, the mode was 0.3, and HOMA was compared with values above and below 0.3 in females and males. Iron contributes to insulin resistance (IR) by hindering its action on liver. It also retards the catabolism of insulin leading to hyperinsulinaemia. Insulin contributes to iron overload by generating more transferrin receptors, more ferritin and entry of iron into the fat cells24. So with more number of fat cells, more iron will be present in the body. This will contribute to insulin resistance. A significant difference was obtained in females (P<0.001) but not in males when the index was compared to HOMA of 2.4. Also, concordance of our index values between females and males showed significance (P<0.001) (Table I). Thus, one can assume that the product of free iron ratios and BMI bears a stronger association with IR in females than in males or BMI alone. A mean free iron value of 15.82±1.38 ppm (n=111) was found in patients with diabetes, whereas it was 9.28±1.21 ppm in healthy controls (n=30) (P<0.001). Table I Comparison of sex concordance to index The stronger relationship with the index in females may be due to lesser amount of iron stores in them25. Ferric form contributes more to IR possibly as the origin of free iron is supposed to be from transferrin which contains iron in its ferric form. Ferric form may represent the initial active redox state of iron before being reduced to ferrous form. This led us to hypothesize that with more iron in ferric form, there would be more free iron turnover and hence more damage. Though higher amounts of both free and total iron were found amongst patients with microalbuminuria and lower eGFR, the results were not significant in our population which may be due to the presence of lesser amount of nephropathy in patients. Free iron was considerably reduced in the serum of most patients with diabetes upon addition of conarachin I with a few showing increase whereas serum samples of healthy subjects showed increase in the free iron concentration upon addition of conarachin I. The difference between the two groups was significant (P<0.001) (Table II). Patients with diabetes (mean fasting plasma glucose = 164.77±16.84 mg/dl) had a higher level of free iron than healthy individuals possibly due to more generation of non-transferrin bound iron (NTBI) by glycation of apotransferrin which does not bind iron avidly26. It was interesting to observe that the higher plasma glucose level in patients with diabetes renders the medium reducing so as the initial Fe3+/Fe2+ equilibrium in plasma is maintained even after being exposed to aerial oxidation for up to 72 h27. The serum of patients with diabetes having higher mean fasting plasma glucose levels might enhance the complexing ability of conarachin I and reduce the free iron level. In healthy controls with normal glucose levels, addition of the protein conarachin I from outside probably disturbs the equilibrium of transferrin bound iron and releases some bound iron free thus increasing the free iron level. The results indicate that peanut proteins may serve to design therapeutics to reduce excess free iron in patients with diabetes and hence control sugar levels especially in insulin resistant female patients. Table II Comparison between effect of conarachin I in serum of patients with and without diabetes In conclusion, free iron was significantly raised in serum of patients with T2DM when compared with healthy subjects. The calculated index of the product of BMI with the ferric-ferrous ratio may be important in assessing insulin resistance, particularly in females or BMI at any level of glycaemia. A peanut protein, conarachin I binds with the free iron in the serum of patients with diabetes and may contribute to the reduction of insulin resistance. The limitation of the study was that serum ferritin and total iron binding capacity (TIBC) were not measured. As serum ferritin is falsely raised in inflammatory states, thus may contribute as a confounding factor. The use of peanut protein to bind serum free iron is a subject of further investigations in animal models.

Based on the evidence of distinct association between hfe mutants, iron overload, onset of nephropathy and type 2 diabetes mellitus, we proceeded to know its genetic prevalence in the eastern part of India focusing on Southern part of West Bengal, which has a large number of diabetic population. Our pilot study involved 37 diabetic cases and 37 non-diabetic controls whose allele frequency for hfe mutations, onset of nephropathy (by estimating eGFR and urinary ACR), insulin resistance (by estimating HOMA-IR) and iron status (in form of serum ferritin, percentage saturation, and free iron status in forms of ferric and ferrous ions) were studied. Chi square and unpaired t test were used for statistical analyses. A higher number of mutants among diabetics were found whereas C282Y mutants had an earlier onset of T2DM than H63D mutants though neither had any statistical significance. However, a significant difference was found regarding percentage transferrin saturation amongst mutants and non-mutants. The means of ferric to ferrous ratio were significantly different in mutant diabetics and non-mutant diabetics probably due to a higher reducing environment in mutant diabetics. Over the counter use of iron medications should be restricted as India contains a high number of iron loading mutations amongst diabetics.

Background: Worldwide highest number of new pulmonary tuberculosis (PTB) cases, was reported from India in 2012. Adverse treatment outcomes and emergence of drug resistance further complicated the prevailing scenario owing to increased duration, cost and toxicity associated with the treatment of drug-resistant cases. Hence to reinforce India's fight against TB, identification of the correlates of adverse treatment outcomes and drug resistance, seemed critical.Methods: To estimate the associations between diagnostic findings, patient types (based on treatment outcomes), drug resistance and socio-demographic characteristics of PTB patients, a cross-sectional study was conducted in two tertiary-care hospitals in Kolkata between April 2010 and March 2013. Altogether, 350 consenting Mycobacterium tuberculosis sputum-culture positive PTB patients were interviewed about their socio-demographic background, evaluated regarding their X-ray findings (minimal/moderately advanced/far advanced/cavities), sputum-smear positivity, and treatment history/outcomes (new/defaulter/relapse/treatment-failure cases). Multiple-allele-specific polymerase chain reaction (MAS-PCR) was conducted to diagnose drug resistance.Results: Among all participants, 31.43% were newly diagnosed, while 44%, 15.43% and 9.14% patients fell into the categories of relapsed, defaulters and treatment-failures, respectively. 12.29% were multi-drug-resistant (MDR: resistant to at least isoniazid and rifampicin), 57.71% had non-MDR two-drug resistance and 12% had single-drug resistance. Subjects with higher BMI had lower odds of being a relapse/defaulter/treatment failure case while females were more likely to be defaulters and older age-groups had more relapse. Elderly, females, unmarried, those with low BMI and higher grade of sputum-smear positivity were more likely to have advanced X-ray features. Higher grade of sputum-smear positivity and advanced chest X-ray findings were associated with relapse/treatment-failures. Elderly, unmarried, relapse/defaulter/treatment-failure cases had higher odds and those with higher BMI and moderately/far advanced X-ray findings had lower odds of having MDR/non-MDR two-drug resistant PTB.Conclusion: Targeted intervention and appropriate counseling are needed urgently to prevent adverse treatment outcomes and development of drug resistance among PTB patients in Kolkata.

Vitamin-D supplementation in vitamin-D insufficient/deficient prediabetes individuals is associated with significantly lower progression to diabetes (6/55 vs. 13/49; p=0.04) and higher reversal to normoglycemia (23/55 vs. 10/49; p=0.02), associated with decreased insulin resistance and systemic inflammation (TNFα and IL6). Baseline vitamin-D and 2h blood glucose independently predicted progression to diabetes.