Comparison of indirect cyanmethaemoglobin method and TrueHb haemometer point of care test with direct cyanmethaemoglobin (gold standard) method for strengthening anaemia detection at primary health care level.

Introduction: The most reliable indicator for anaemia diagnosis at the population level is haemoglobin (Hb) estimation. The direct cyanmethaemoglobin method is considered the gold standard method for haemoglobin estimation. However, for resource constraint areas like primary health care (PHC) level, either blood samples are transported on filter paper for Hb testing (indirect cyanmethaemoglobin method) in laboratory or point of care testing is commonly used. Therefore, a comparative analysis of haemoglobin estimation of direct with indirect cyanmethaemoglobin method and also with TrueHb (Wrig Nanosystems Pvt. Ltd.) haemometer was done to strengthen anaemia diagnosis at the PHC level. Materials and Methods: This was a cross-sectional study. A total of 90 participants above 9 years of age, who visited the outpatient department (OPD) of health centre, Kheri and gave consent were included. Comparative analysis was done between Hb concentration assessed by indirect cyanmethaemoglobin method and TrueHb haemometer device against the gold standard method. Results: The mean Hb value estimated by direct method, TrueHb haemometer and indirect methods (filter paper A, B and C) was 11.42 ± 1.59 g/dl, 11.52 ± 1.54 g/dl, 10.66 ± 1.52 g/dl, 9.84 ± 1.50 g/dl and 10.19 ± 1.62 g/dl, respectively. There was no significant difference found between the mean Hb concentration estimated by the direct method and the TrueHb haemometer device. However, there was a significant difference in mean Hb values between the direct method and the indirect method. Therefore, regression analysis was done to estimate the correction factor for the indirect method. Conclusion: TrueHb metre device gave promising results in comparison to the gold standard method and can be used if resource permits in PHC centres. Indirect methods of haemoglobin estimation can be an alternative in resource-constraint settings, specifically for surveys. However, further studies are required for the validation of the indirect method.

Comprehensive assessment of nutritional and functional status of patients with ulcerative colitis and their impact on quality of life.

INTRODUCTION: Patients with ulcerative colitis (UC) are likely to have poor nutritional intake and increased gut losses. This study was designed to study the prevalence and predictors of nutritional deficiencies in patients with UC and their impact on the quality of life (QOL). METHODS: A prospective study was conducted among consenting patients with UC (cases) and healthy relatives of the cases (controls) visiting a university teaching hospital. They were assessed for clinical, demographic, endoscopic (Mayo score) and histological profile (Robart's score). They were assessed for the presence of macronutrient and micronutrient deficiency, anthropometry, functional status (muscle strength by dynamometer and sit-to-stand test) and the quality of life (short inflammatory bowel disease questionnaire [SIBDQ]). A SIBDQ score of ≤ 50 was considered poor QOL. RESULTS: We studied 126 cases and 57 healthy controls (age [mean ± SD] 37.7 ± 13.2 years vs. 34.40 ± 11.05 years; [p = 0.10] females [38.1% vs. 38.7%]; p = 0.94). Cases more often were underweight (28% vs. 3.5%; p < 0.001), had low mid arm circumference (45% vs. 12%; p < 0.0001), lower functional status in the form of weaker hand grip strength (67% vs. 45.6%; p = 0.007) and weaker lower limb strength (80% vs. 42%; p < 0.0001). Cases more often had the evidence of macronutrient deficiencies: total serum protein deficiency (31% vs. 3.5%; p < 0.0001), serum albumin deficiency (25.4% vs. 0.00%; p < 0.0001) and cholesterol deficiency (63% vs. 28%; p < 0.0001). Micronutrient deficiencies were highly prevalent among cases: calcium (44%), phosphate (21%), magnesium (11%), zinc (76%), iron (87%), folate (16%), vitamin B12 (10%) and vitamin D (81%). Most cases had a poor quality of life (85/126; 67.5%). Factors associated with poor QOL were low hemoglobin, serum albumin, zinc and vitamin D levels and histologically active disease. On multi-variate analysis, low vitamin D levels (odds ratio [OR] = 6.1; 95% confidence interval [CI]: 1.9-19.7) and histologically active disease (OR = 4.0; 95% CI: 1.6-9.9) were identified as independent predictors of poor QOL. CONCLUSIONS: Macronutrient deficiency, micronutrient deficiency, lower functional status and poorer QOL are highly prevalent among patients with UC. The independent predictors of poor QOL were histologically active disease and low serum vitamin D levels. Identifying and correcting the deficiencies may help in improving the QOL of patients with UC.

Decreased myocardial expression of dystrophin and titin mRNA and protein in dilated cardiomyopathy: possibly an adverse effect of TNF-alpha.

BACKGROUND AND AIMS: While the molecular basis of dilated cardiomyopathy (DCM) remains uncertain, concrete evidence is emerging that sarcomeric and cytoskeleton gene expression of myocardium isolated from failing versus non-failing patients differ dramatically. The central aim to this work was to find out the possible role of dystrophin and titin along with the TNF-alpha in the pathogenesis of cardiomyopathy. PATIENTS AND METHODS: mRNA levels and protein expression of a cytoskeletal protein, dystrophin and a sarcomeric protein, titin in endomyocardial biopsies of DCM patients were examined using RT-PCR and immunohistochemistry, respectively. Further, we examined the effect of TNF-alpha on myocardial expression of titin and dystrophin in vitro in rat cardiac myoblast cell line (H9c2). RESULTS: We observed significantly decreased mRNA and protein levels of dystrophin and titin in endomyocardial biopsy of DCM patients as compared to control group. The decreased levels of these proteins correlated with the severity of the disease. Plasma levels of both TNF-alpha and its soluble receptors TNFR1 and TNFR2 were found to be significantly higher in patients as compared to control group. Treatment of H9c2 cells with TNF-alpha resulted in a dose- and time-dependent decrease in mRNA levels of dystrophin and titin. Pretreatment of these cells with MG132, an inhibitor of nuclear factor kappa B (NF-kappaB) pathway, abolished TNF-alpha-induced reduction in mRNA levels of dystrophin and titin. CONCLUSION: Our results suggest that reduced expression of dystrophin and titin is associated with the pathophysiology of DCM, and TNF-alpha may modulate the expression of these proteins via NF-kappaB pathway.

Circulating proinflammatory cytokines and N-terminal pro-brain natriuretic peptide significantly decrease with recovery of left ventricular function in patients with dilated cardiomyopathy.

BACKGROUND: Increased levels of TNF-alpha, IL-6, their soluble receptors, and NT-proBNP have been observed in patients with dilated cardiomyopathy (DCM). In the present study, we assessed the possible involvement of proinflammatory cytokines and their soluble receptors with and without recovery of LV function in DCM patients. METHODS AND RESULTS: Forty patients with DCM were enrolled and divided into two groups: Group I consisted of DCM patients (n = 30) whose left ventricular ejection fraction (LVEF) had not recovered on follow up and Group II comprised DCM patients (n = 10) whose LVEF had recovered. Ten healthy subjects were included as controls (Group III). TNF-alpha, IL-6,TNFR1, TNFRII, gp130, and NT-proBNP levels were significantly increased in Group I and were significantly lower in patients with LVEF recovery as compared to those without recovery of LVEF (P < 0.05). CONCLUSION: Circulating TNF-alpha, IL-6, and NT-proBNP appear to correlate with the LV function recovery of patients with DCM and could be used as prognostic biomarkers.

Expression of accessory colonization factor subunit A (ACFA) of Vibrio cholerae and ACFA fused to cholera toxin B subunit in transgenic tomato (Solanum lycopersicum).

In earlier study from our group, cholera toxin B subunit had been expressed in tomato for developing a plant-based vaccine against cholera. In the present investigation, gene for accessory colonization factor (acf) subunit A, earlier reported to be essential for efficient colonization in the intestine, has been expressed in Escherichia coli as well as tomato plants. Gene encoding for a chimeric protein having a fusion of cholera toxin B subunit and accessory colonization factor A was also expressed in tomato to generate more potent combinatorial antigen. CaMV35S promoter with a duplicated enhancer sequence was used for expression of these genes in tomato. Integration of transgenes into tomato genome was confirmed by PCR and Southern hybridization. Expression of the genes was confirmed at transcript and protein levels. Accessory colonization factor A and cholera toxin B subunit fused to this protein accumulated up to 0.25% and 0.08% of total soluble protein, respectively, in the fruits of transgenic plants. Whereas protein purified from E. coli, in combination with cholera toxin B subunit can be used for development of conventional subunit vaccine, tomato fruits expressing these proteins can be used together with tomato plants expressing cholera toxin B subunit for development of oral vaccine against cholera.

Resveratrol regulates body weight in healthy and ovariectomized rats.

OBJECTIVE: The elevated body weight in post-menopausal state attributes to the reduced estrogen levels which is alleviated by resveratrol (RES) but its role in control rats is not well understood. The main objective of the study was to explore the effects of RES on the body weight of ovariectomized (OVX) female rats with controls and to relate their biochemical parameters. METHODS: Female Wistar rats weighing 200-300 g underwent bilateral ovariectomy (OVX) and were fed soya free diet (n = 8 rats per group). In all groups: (Control, Control + Resveratrol, OVX and OVX + Resveratrol) resveratrol was administered orally at a dose of 5 mg/kg/day for 1 month. Glucose and other biochemical parameters were examined. RESULTS: Significant reduction in the gain of body weight was observed in the control rats treated with resveratrol. Ovariectomy caused an escalation in gain of body weight due to loss of estrogen which was brought down with resveratrol. There was a slight dip in the blood glucose levels after resveratrol treatment. CONCLUSION: Resveratrol significantly reduced the gain of body weight in the control rats and in OVX rats showing its antiobesogenic effects.

Telomere mean length in patients with diabetic retinopathy.

Telomere regression has been shown to be associated with several complex disorders like diabetes mellitus, cancer, cataract etc. Diabetic retinopathy develops as a complication of chronic hyperglycemia leading to increased oxidative stress that may potentially lead to shortening of telomeres. We sought to determine whether there is any association between telomere mean length (TML) of peripheral blood monocytes with the presence and severity of diabetic retinopathy. The study involved 120 subjects, comprising 27 non-insulin dependent diabetes mellitus (NIDDM) without any diabetic retinopathy (NDR), 45 NIDDM subjects with non-proliferative diabetic retinopathy (NPDR), 12 NIDDM subjects with proliferative diabetic retinopathy (PDR) and 36 healthy controls. Determination of TML of the study subjects was performed by Southern hybridization using telomere probe. Among the biochemical parameters, HBA1c showed a negative correlation with shortened telomeres in the PDR subjects. However, telomere length was positively correlated with high density lipo protein (HDL) in the control subjects. The control group had significantly greater TML as compared to the rest of the groups and the NDR subjects with NPDR and PDR had substantially decreased TML than the NIDDM subjects without retinopathy.

Construction of a recombinant live oral vaccine from a non-toxigenic strain of Vibrio cholerae O1 serotype inaba biotype E1 Tor and assessment of its reactogenicity and immunogenicity in the rabbit model.

The disease cholera is an important cause of mortality in many developing countries. Though it can be controlled through improved sanitation, this goal is not easily attainable in many countries. Development of an efficacious vaccine offers the best immediate solution. A new oral candidate vaccine has been constructed from a non-toxigenic strain of Vibrio cholerae E1 Tor, Inaba, which is not only devoid of the cholera toxin (CT) virulence cassette but also is completely non-reactogenic in rabbit ileal loop assay. The strain, however, had toxR and tcpA genes. Through a series of manipulations, the ctxB gene of V. cholerae, responsible for the production of the 'B' subunit of the cholera toxin (CTB) was introduced into the cryptic hemolysin locus of the strain. The resulting strain, named vaccine attempt 1.3 (VA1.3), was found to be able to produce copious amounts of CTB. In the RITARD model this strain was found to be non-reactogenic and provided full protection against the challenge doses of both V. cholerae O1, classical and E1 Tor. In the immunized rabbit it invoked significant levels of anti-bacterial and anti-toxin immunity.

Comparison of antibiogram, virulence genes, ribotypes and DNA fingerprints of Vibrio cholerae of matching serogroups isolated from hospitalised diarrhoea cases and from the environment during 1997-1998 in Calcutta, India.

This study identified 17 matching serogroups of Vibrio cholerae belonging to serogroups other than O1 and O139 isolated from human cases and from the environment during a concurrent clinical and environmental study conducted in Calcutta, a cholera endemic area. Isolates within these matching serogroups were compared by various phenotypic and genotypic traits to determine if the environment was the source of the organisms associated with the disease. Clinical strains of V. cholerae were resistant to a greater number of drugs and exhibited multi-drug resistance compared with their environmental counterparts. Except for the presence of the genes for the El Tor haemolysin and the regulatory element ToxR in most of the strains of V. cholerae examined, non-O1, non-O139 V. cholerae strains lacked most of the other known virulence traits associated with toxigenic V. cholerae O1 or O139. Restriction fragment-length polymorphism of virulence-associated genes, ribotypes and DNA fingerprints of strains of matched serogroups showed considerable diversity, although some gene polymorphisms and ribotypes of a few strains of different serogroups were similar. It is concluded that despite sharing the same serogroup, environmental and clinical isolates were genetically heterogeneous and were of different lineages.

Strategies for production of a potential candidate vaccine for cholera.

First attempt at cholera vaccination was made by Jaime Ferran in 1884. Since then, a variety of strategies and methods have been evolved to create a safe, efficacious vaccine against cholera. For the first few years emphasis was on the development of parenteral vaccines. However, as a result of accumulation of a tremendous amount of knowledge, not only on Vibrio cholerae-the causative agent, but also on its interaction with the host, emphasis has shifted towards the development of oral vaccines. Two such vaccines, one killed, a whole cell/B subunit combination vaccine and the other a live attenuated one, have shown promise. The combination vaccine in its present state of development confers only a transient protection in young children, while the live attenuated one produces adverse reaction. To combat these, various strategies are being evolved. In one attempt, a potential candidate vaccine strain has been constructed from a non-reactogenic clinical isolate of V. cholerae, which is devoid of all known major virulence genes and is also a good colonizer. In animal studies this construct has shown considerable promise. This review discusses the various strategies that have been employed so far in the quest for an ideal cholera vaccine.

Multilocus microsatellite typing for Rhizopus oryzae.

Rhizopus oryzae is the most frequent causative agent of zygomycosis. Although zygomycosis causes considerable morbidity and mortality in immunocompromised patients, the epidemiology of the disease is not well studied and no standard molecular typing method has been described for any of the causative agents. Here we describe a multilocus microsatellite typing (MLMT) method for R. oryzae. R. oryzae genome sequences were downloaded from the Fungal Genome Initiative database (Broad Institute). The intergenic regions and ORFs of approximately 5.7 Mb were screened for repeat regions with the help of the online repeat search tool Repeat Masker. Of the 30 microsatellite loci identified, 3 microsatellites [RO3, (CCT)(n); RO4, (TA)(n); and RO8, (GAA)(GGA)(n)] were selected after validation of the ability to amplify them and their size variation in 8 randomly selected clinical isolates of R. oryzae. Nucleotide sequence analysis of these loci demonstrated polymorphism in the microsatellite repeat number. The capabilities of these microsatellite loci were assessed for strain differentiation on 30 clinical isolates, based on fragment size determination in an automated capillary electrophoresis using fluorescent labelled primers. These three polymorphic microsatellite loci were found to have good discriminatory power (D) (RO3, D=0.846; RO4, D=0.747; RO8, D=0.742; with a combined D=0.986) and stability for seven subcultures. It was also confirmed that the MLMT method may be applied to both R. oryzae and Rhizopus delemar (a proposed new species), although MLMT analysis could not differentiate them into two clusters. The MLMT system, described here for what is believed to be the first time for a zygomycotic fungus, holds promise as a powerful tool for the strain typing of R. oryzae.

Randomized placebo controlled human volunteer trial of a live oral cholera vaccine VA1.3 for safety and immune response.

A live oral cholera vaccine developed from a non-toxigenic Vibrio cholerae O1 El Tor strain VA1.3 was tested in a double-blind randomized placebo controlled study for safety and immunogenicity in 304 men aged between 16 and 50 years from Kolkata, India. A dose of 5 x 10(9)CFU (n=186) or a placebo (n=116) containing the diluent buffer was administered. The vaccine did not elicit adverse events except in two vaccine recipients with mild diarrhoea and vomiting. None excreted the vaccine strain. Vibriocidal antibody response developed in 105/186 (57%) and 5/116 (4%) in vaccine and placebo recipients, respectively. In a subgroup, anti-CT antibody rose (> or =2-folds) in 23/30 (77%) and 6/19 (32%) in vaccine and placebo recipients, respectively. These studies demonstrate that VA1.3 at a dose of 5 x 10(9) is safe and immunogenic in adults from a cholera endemic region.

Expression of toxin co-regulated pilus subunit A (TCPA) of Vibrio cholerae and its immunogenic epitopes fused to cholera toxin B subunit in transgenic tomato (Solanum lycopersicum).

For protection against cholera, it is important to develop efficient vaccine capable of inducing anti-toxin as well as anti-colonizing immunity against Vibrio cholerae infections. Earlier, expression of cholera toxin B subunit (CTB) in tomato was reported by us. In the present investigation, toxin co-regulated pilus subunit A (TCPA), earlier reported to be an antigen capable of providing anti-colonization immunity, has been expressed in tomato. Further, to generate more potent combinatorial antigens, nucleotides encoding P4 or P6 epitope of TCPA were fused to cholera toxin B subunit gene (ctxB) and expressed in tomato. Presence of transgenes in the tomato genome was confirmed by PCR and expression of genes was confirmed at transcript and protein level. TCPA, chimeric CTB-P4 and CTB-P6 proteins were also expressed in E. coli. TCPA protein expressed in E. coli was purified to generate anti-TCPA antibodies in rabbit. Immunoblot and G(M1)-ELISA verified the synthesis and assembly of pentameric chimeric proteins in fruit tissue of transgenic tomato plants. The chimeric protein CTB-P4 and CTB-P6 accumulated up to 0.17 and 0.096% of total soluble protein (TSP), respectively, in tomato fruits. Whereas expression of TCPA, CTB-P4 and CTB-P6 in E. coli can be utilized for development of conventional vaccine, expression of these antigens which can provide both anti-toxin as well as anti-colonization immunity, has been demonstrated in plants, in a form which is potentially capable of inducing immune response against cholera infection.

Class I integrons and SXT elements in El Tor strains isolated before and after 1992 Vibrio cholerae O139 outbreak, Calcutta, India.

We examined the distribution of class I integrons and SXT elements in Vibrio cholerae O1 El Tor strains, isolated in Calcutta, India, before and after the V. cholerae O139 outbreak in 1992. Class I integrons, with aadA1 gene cassette, were detected primarily in the pre-O139 strains; the SXT element was found mainly in the post-O139 strains.

Occurrence of antibiotic resistance gene cassettes aac(6')-Ib, dfrA5, dfrA12, and ereA2 in class I integrons in non-O1, non-O139 Vibrio cholerae strains in India.

Molecular mechanisms of multidrug resistance in Vibrio cholerae belonging to non-O1, non-O139 serogroups isolated during 1997 to 1998 in Calcutta, India, were investigated. Out of the 94 strains examined, 22 strains were found to have class I integrons. The gene cassettes identified were dfrA1, dfrA15, dfrA5, and dfrA12 for trimethoprim; aac(6')-Ib for amikacin and tobramycin; aadA1 and aadA2 for streptomycin and spectinomycin; and ereA2 for erythromycin resistance. To our knowledge, this is the first report of the presence of dfrA5, dfrA12, aac(6')-Ib, and ereA2 cassettes in class I integrons of V. cholerae. Forty-three of 94 strains also had plasmids, and out of these, 14 contained both class I integrons and plasmids. Pulsed-field gel electrophoresis followed by Southern hybridization revealed that in the 14 plasmid-bearing strains, class I integrons resided either on chromosomes, on plasmids, or on both. Our results indicated that besides class I integrons and plasmids, a conjugative transposon element, SXT, possibly contributed to the multiple antibiotic resistance.

Molecular analysis of non-O1, non-O139 Vibrio cholerae associated with an unusual upsurge in the incidence of cholera-like disease in Calcutta, India.

There was an inexplicable upsurge in the incidence of non-O1, non-O139 Vibrio cholerae among hospitalized patients admitted to the Infectious Diseases Hospital, Calcutta, India, between February and March 1996. Of the 18 strains of V. cholerae isolated during this period, 15 belonged to the non-O1, non-O139 serogroups (4 belonged to O144, 3 belonged to O11, 1 each belonged to O6, O8, O12, O19, O39, and O58, and 2 strains could not be typed), 2 belonged to the O139 serogroup, and 1 belonged to the O1 serogroup. Cell-free culture supernatants of 13 representative non-O1, non-O139 V. cholerae strains evoked a distinct cytotoxic effect on CHO and HeLa cells, and the strains examined produced the nonmembrane-damaging cytotoxin. By several PCR assays, it was determined that none of the non-O1, non-O139 strains were positive for the ctxA, zot, ace, and tcpA genes and for the genes representing the heat-labile toxin, heat-stable toxin, and verotoxin of Escherichia coli and the various variants of these genes. Studies on the clonality of non-O1, non-O139 V. cholerae strains by restriction fragment length polymorphism (RFLP) analysis of rRNA genes and of other genes (hlyA, hlyU, hlx, toxR, and attRS1) and by pulsed-field gel electrophoresis (PFGE) collectively indicate that the upsurge which occurred in February and March 1996 was caused by strains belonging to different clones. Overall, there was an excellent correlation between the results of ribotyping, RFLP analysis of various genes, and PFGE, with strains belonging to a particular serogroup showing nearly identical restriction patterns and PFGE profiles. It is clear from this study that some serogroups of V. cholerae can cause diarrhea by a mechanism quite different from that of toxigenic V. cholerae O1 and O139, and we have proposed the nomenclature of enteropathogenic V. cholerae to include these serogroups.