A phase 2 randomized controlled trial of oral resistant starch supplements in the prevention of acute radiation proctitis in patients treated for cervical cancer

Background: Radiation induced proctitis is frequently encountered during the radiation therapy of cervical and prostate cancers that causes pain and occasionally with bleeding and may affect the continuity of radiation therapy. Aims and Objectives: The purpose of the study is to look at the benefit of administration of an oral prebiotic amylase resistant starch in reducing the incidence of acute radiation proctitis, a distressing symptom in patients receiving radiation therapy for cancer of the cervix. Material and Methods: The study was conducted between 2011 and 2014 in 104 patients receiving radical chemo-radiotherapy for carcinoma cervix. Patients were randomized in to two arms, one receiving 30 gm of resistant starch and the other digestible starch on a daily basis throughout the course of the external radiotherapy. All patients received standard 4-field box radiation portals, 50 Gy in 25 fractions with 4 cycles of weekly concurrent Cisplatin. At completion of external beam radiotherapy, all patients underwent LDR/HDR brachytherapy. The study was double blinded and allocation was concealed from the investigators. The investigator recorded the radiotherapy related toxicity of the patients according to CTC V 3.0. The incidence and severity of grade 2-4 diarrhoea and proctitis were documented on a weekly basis and compared across the two groups and analyzed. Stool short chain fatty acid concentrations were measured at baseline at 2nd and 4th week and after 6 weeks of completion of radiotherapy in both study placebo arms and reported. The pattern of microbiota in the stool were also estimated in all patients at 4 time points. Two patients who progressed during therapy were not included in the analyses and two patients discontinued the intervention. A per protocol analyses was done. Results: At analysis there were 50 patients in each arm. The severity of clinical proctitis was found to be similar in both groups of patients with 12.2 % of patients experiencing toxicity of grade 2 and above in digestible starch group versus 14.6% in the resistant starch group. Functional proctitis was similarly graded and it was found that 16.3 % patients in digestible starch group experienced toxicity against 10.2 % patients in the resistant starch group. This difference was seen at 4th week and continued in the subsequent weeks till the end of radiation. Both groups had similar reported toxicity at 6 weeks post intervention and similar incidence of grade 2 and above diarrhea. The resistant starch group was found to have 8% incidence as compared to 2% in the other group at the 5th and 6th week. The short chain fatty acid concentrations were not significantly different in the groups at any point. Conclusion: The study did not demonstrate a significant benefit in administering resistant starch over and above normal diet to patients receiving pelvic radiotherapy. The reasons may be attributed to concurrent use of chemotherapy and decrease in intestinal probiotics. The use of digestible starch in the control arm may have contributed to lower incidence of the toxicity endpoints as well

A Cross Sectional Study of Drug Utilization Pattern in Indoor Patients of Tertiary Care Teaching Hospitals in Central India

OBJECTIVE: To evaluate the pattern of drug utilization with special focus on the use of antimicrobials. METHODS: Single day hospital-wide point prevalence survey was conducted to assess drug utilization pattern.  Information regarding age, gender, occupation, income group, diagnosis, patient’s present/past medical history, treatment, any adverse drug reactions, and investigations were recorded in a proforma. Drug utilization pattern was evaluated using quality indicators of drug use recommended by WHO. RESULTS: A total of 77 patients were included, 62% male and 38% female. Maximum number of patients was admitted for infectious diseases (34%). Pantoprazole was most commonly prescribed drug & ceftriaxone was most commonly prescribed antibiotic. The average number of drugs prescribed per encounter was 4.87.  Oral route was the most preferred route. Percentage of drugs prescribed by generic name was 42.44%. Percentage of drugs prescribed from essential medical list was 49.33%. Multivitamins & non steroidal anti-inflammatory drugs (NSAIDS) were most commonly prescribed fixed dose combination in our study.  CONCLUSION: Majority of drugs were prescribed by oral route. Essential medical list and updated guidelines were available in the hospital. Poly-pharmacy, over use of proton-pump inhibitors & multivitamins was areas of concern. Prolonged and irrational use of antibiotics for surgical prophylaxis was noted in surgical indoor patients. There was underutilization of microbiological facilities.

Alterations of mucosal microbiota in the colon of patients with inflammatory bowel disease revealed by real time polymerase chain reaction amplification of 16S ribosomal ribonucleic acid.

Background & objectives: Alterations in microbial communities closely associated with the intestinal mucosa are likely to be important in the pathogenesis of inflammatory bowel disease (IBD). We examined the abundance of specific microbial populations in colonic mucosa of patients with ulcerative colitis (UC), Crohn’s disease (CD) and controls using reverse transcription quantitative polymerase chain reaction (RT-qPCR) amplification of 16S ribosomal ribonucleic acid (16S rRNA). Methods: RNA was extracted from colonic mucosal biopsies of patients with UC (32), CD (28) and patients undergoing screening colonoscopy (controls), and subjected to RT-qPCR using primers targeted at 16S rRNA sequences specific to selected microbial populations. Results: Bacteroides-Prevotella-Porphyromonas group and Enterobacteriaceae were the most abundant mucosal microbiota. Bacteroides and Lactobacillus abundance was greater in UC patients compared with controls or CD. Escherichia coli abundance was increased in UC compared with controls. Clostridium coccoides group and C. leptum group abundances were reduced in CD compared with controls. Microbial population did not differ between diseased and adjacent normal mucosa, or between untreated patients and those already on medical treatment. The Firmicutes to Bacteroidetes ratio was significantly decreased in both UC and CD compared with controls, indicative of a dysbiosis in both conditions. Interpretation & conclusions: Dysbiosis appears to be a primary feature in both CD and UC. Microbiome-directed interventions are likely to be appropriate in therapy of IBD.

Probiotic potential of lactic acid bacteria present in home made curd in southern India.

Background & objectives: The human gut microbiota play a significant role in nutritional processes. The concept of probiotics has led to widespread consumption of food preparations containing probiotic microbes such as curd and yogurt. Curd prepared at home is consumed every day in most homes in southern India. In this study the home-made curd was evaluated for lactic acid bacteria (LAB) with probiotic potential. Methods: Fifteen LAB (12 lactobacilli, 1 lactococcus, 2 Lleuconostoc) and one yeast isolated from home-made curd were evaluated for resistance to acid, pepsin, pancreatin and bile salts; antimicrobial resistance; intrinsic antimicrobial activity; adherence to Caco-2 epithelial cells; ability to block pathogen adherence to Caco-2 cells; ability to inhibit interleukin (IL)-8 secretion from HT-29 epithelial cells in response to Vibrio cholerae; and ability to induce anti-inflammatory cytokine expression in THP-1 monocyte cells. Results: Llactobacillus abundance in fermenting curd peaked sharply at 12 h. Nine of the strains survived exposure to acid (pH 3.0) for at least one hour, and all strains survived in the presence of pancreatin or bile salts for 3 h. None showed haemolytic activity. All were resistant to most antimicrobials tested, but were sensitive to imipenem. Most strains inhibited the growth of Salmonella Typhimurium while five inhibited growth of V. cholerae O139. Seven strains showed adherence to Caco-2 cells ranging from 20-104 per cent of adherence of an adherent strain of Escherichia coli, but all inhibited V. cholerae adherence to Caco-2 cells by 20-100 per cent. They inhibited interleukin-8 secretion from HT-29 cells, in response to V. cholerae, by 50-80 per cent. Two strains induced IL-10 and IL-12 messenger ribonucleic acid (mRNA) expression in THP-1 cells. Interpretation & conclusions: LAB in curd had properties consistent with probiotic potential, but these were not consistent across species. LAB abundance in curd increased rapidly at 12 h of fermentation at room temperature and declined thereafter.

Adherent Escherichia coli in colorectal mucosal biopsies: A histological and ultrastructural evaluation.

Background: Colorectal mucosal biopsies occasionally demonstrate the presence of bacteria adherent to the epithelium. This study evaluated the histological and ultrastructural correlates of such bacterial adherence. Materials and Methods: Rectal mucosal biopsies from eight patients in whom histopathological examination of biopsies had earlier demonstrated adherent bacteria were examined by electron microscopy and by bacterial culture. Colorectal biopsies of 69 patients with adherent bacteria detected histologically were retrospectively evaluated for histological changes at sites proximal and distant to adherent bacteria. Results: Escherichia coli of different serogroups were isolated from 7 of 8 rectal biopsies demonstrating bacterial adherence. All isolates showed diffuse or focal adherence to HEp-2 cell monolayers. Ultrastructural changes noted included microvillus damage, pedestal formation, actin web condensation, and protrusions of the apical cytoplasm of epithelial cells into the lumen towards the bacteria. Histological changes noted at light microscopy included reduction in epithelial cell height, focal epithelial cell degeneration, cryptitis and neutrophil infiltration at sites of bacterial adherence whereas these were usually absent at sites distant to adherent bacteria. Bacterial adherence was noted more often in biopsies from Crohn's disease patients than in patients without this diagnosis (P < 0.001). Conclusion: Adherent Escherichia coli in colorectal biopsies were associated with focal epithelial damage and showed an association with Crohn's disease.

Environmental factors associated with Crohn’s disease in India.

Background The frequency of diagnosis of Crohn’s disease (CD) in India is increasing. This case-control study was designed to detect associations of environmental and dietary factors with the diagnosis of CD. Methods In 200 consecutive patients with CD and 200 control subjects without gastrointestinal disease, environmental hygiene exposures in childhood and in the past one year, and dietary preferences were recorded using a questionnaire. Univariate and multivariate analyses were done. Results In univariate analysis, CD showed positive association with urban residence (at birth and current), availability of protected drinking water (childhood and current), availability of piped water in the house (childhood and current), and strict vegetarian dietary habit, and negative association with regular fish consumption and presence of cattle in the house compound. Multivariate analysis showed that regular fish consumption (OR 0.52, 95% CI 0.33–0.80, p=0.003), and presence of cattle in the house compound currently (OR 0.57, 95% CI 0.35–0.92, p=0.023) were significant protective associations, whereas use of safe drinking water was positively associated (OR 1.59, 95% CI 1.02–2.47, p=0.042) with the disease. Conclusion Occurrence of CD was associated with dietary and environmental exposures, which indicate that diet and hygiene may influence the development of this disease.

Faecal bifidobacteria in Indian neonates & the effect of asymptomatic rotavirus infection during the first month of life.

Background & Objectives: Bifidobacteria colonize the gut after the first week of life and remain an important component of the gut microbiota in infancy. This study was carried out to characterize the diversity and number of bifidobacteria colonizing the gut in Indian neonates and to investigate whether asymptomatic infection with rotavirus in the first month of life affected gut colonization by bidifobacteria. Methods: DNA was isolated from faeces of 14 term-born neonates who were under surveillance for rotavirus infection. Bacterial and bifidobacterial diversity was evaluated by temporal temperature gradient electrophoresis (TTGE) of 16S rDNA amplified using total bacteria and bifidobacteria-specific primers. Real time PCR, targeting 16S rDNA, was used to quantitate faecal bifidobacteria and enterobacteria. Results: TTGE of conserved bacterial 16S rDNA showed 3 dominant bands of which Escherichia coli (family Enterobacteriaceae) and Bifidobacterium (family Bifidobacteriaceae) were constant. TTGE of Bifidobacterium genus-specific DNA showed a single band in all neonates identified by sequencing as Bifidobacterium longum subsp. infantis. Faecal bifidobacterial counts (log10 cfu/g faeces) ranged from 6.1 to 9.3 and enterobacterial counts from 6.3 to 9.5. Neonates without and with rotavirus infection in the first week of life did not show significant differences in the median count of bifidobacteria (log10 count 7.48 vs. 7.41) or enterobacteria (log10 count 8.79 vs. 7.92). Interpretation & Conclusions: B. longum subsp. infantis was the sole bifidobacterial species colonizing the gut of Indian neonates. Asymptomatic rotavirus infection in the first month of life was not associated with alteration in faecal bifidobacteria or enterobacteria.

Fecal polymerase chain reaction for Mycobacterium tuberculosis IS6110 to distinguish Crohn’s disease from intestinal tuberculosis.

Background We have previously shown that amplification of Mycobacterium tuberculosis specific DNA (TB PCR) from feces reliably diagnosed intestinal tuberculosis. This study was undertaken to determine how well this test would distinguish intestinal tuberculosis from Crohn’s disease in a country endemic for tuberculosis. Methods Consecutive patients with diagnoses of Crohn’s disease and intestinal tuberculosis were enrolled, and the diagnoses confirmed by follow up. DNA was extracted from fecal samples and subjected to polymerase chain reaction TB PCR for IS6110 sequence which is specific for M. tuberculosis. Results Twenty one of 24 patients with intestinal tuberculosis and 5 of 44 patients with Crohn’s disease tested positive by TB PCR. The sensitivity, specificity, positive predictive and negative predictive values for TB PCR in distinguishing tuberculosis from Crohn’s disease were 0.79 (95% confidence interval 0.57–0.92), 0.88 (0.75–0.96), 0.79 (0.57–0.92) and 0.88 (0.75–0.96), respectively. A combination of fecal TB PCR with mycobacterial culture of mucosal biopsy specimens identified 23 of 24 (96.2%) of patients with intestinal TB, with sensitivity, specificity, positive predictive and negative predictive values (95% CI) of 0.95 (0.78–0.99), 0.88 (0.75–0.96), 0.82 (0.63–0.93) and 0.97 (0.86–0.99), respectively. Conclusion Fecal TB PCR is a good screening test to distinguish intestinal tuberculosis from Crohn’s disease.

Protective effect of Withaferin-A on tumour formation in 7,12-dimethylbenz[a]anthracene induced oral carcinogenesis in hamsters.

With an aim to investigate the protective effect of Withaferin-A on 7,12-dimethylbenz[a]anthracene (DMBA) induced oral carcinogenesis in Syrian golden hamsters, tumour incidence, tumour volume and tumour burden and status of detoxication agents, lipid peroxidation and antioxidants in DMBA administered (3 times/week for 14 weeks) hamsters were assessed. Hundred percent tumour formation in DMBA alone administered animal was observed. Oral administration of Withaferin-A (20 mg/kg body weight) to DMBA administered animals for 14 weeks completely prevented the tumour incidence, tumour volume and tumour burden. Also, Withaferin-A showed significant anti-lipid peroxidative and antioxidant properties and maintained the status of phase-I and phase-II detoxication agents during DMBA-induced oral carcinogenesis. The results thus indicate that the protective effect of Withaferin-A is probably due to its anti-lipid peroxidative and antioxidant functions as well as modulating effect on carcinogen detoxication during DMBA-induced oral carcinogenesis.

Common NOD2 mutations are absent in patients with Crohn's disease in India.

BACKGROUND: Crohn's disease is being increasingly diagnosed in the Indian subcontinent. Three apparently common mutations in the NOD2 gene are found in up to 30% of sporadic patients with Crohn's disease in western countries. We examined whether such mutations are also found in Indian patients with Crohn's disease. METHODS: Venous blood was collected from 82 patients (age range: 7-65 years, 53 men) with Crohn's disease and 149 control subjects; DNA was extracted and subjected to polymerase chain reaction using specific primers. The amplified fragments of size 185, 163 and 151 bp for R702W, G908R and 1007fs, respectively, were digested with MspI, HhaI and ApaI, and the restriction pattern noted after electrophoresis. RESULTS: Twenty-eight patients had ileocolonic disease, 26 ileal disease, 20 colonic disease and 8 had disease limited to proximal small bowel or stomach. None of the 82 patients showed any of the three NOD2 mutations. The control subjects (93 men) had a variety of chronic gastrointestinal disorders (ulcerative colitis 52, irritable bowel syndrome 30, intestinal tuberculosis 20, colon cancer 7, miscellaneous 37). None of the control subjects showed a mutation in any of the three NOD2 mutation analyses. CONCLUSION: The three NOD2 gene mutations described above are uncommon in Indian patients with Crohn's disease. This study complements information provided by recent studies on NOD2 mutations in Indians.

Estimation of faecal carriage of Clostridium difficile in patients with ulcerative colitis using real time polymerase chain reaction.

BACKGROUND & OBJECTIVE: Ulcerative colitis (UC) is a disease of unknown aetiology in which exacerbations are sometimes linked to intestinal colonization by toxin-producing Clostridium difficile. We undertook this study to detect and quantitatively assess C. difficile in the stool of patients with UC using real time polymerase chain reaction (RT-PCR), and to compare it with healthy individuals. METHODS: A total of 37 consecutive patients with UC (26 male, mean age 41.3 yr) and 36 healthy adult volunteers (20 male, mean age 36.4), none of whom had received antibiotics within two months prior to faecal collection, were included in the study. Faecal DNA was extracted, quantitative PCR (qPCR) carried out using primers to amplify species-specific segments of 16S rDNA of C. difficile, and expressed as relative fold difference against amplification of highly conserved (universal) segments. Toxins A and B were assayed by ELISA. RESULTS: Quantitative PCR detected C. difficile sensitively, and spiking with increasing numbers of the organism resulted in linear increase in amplification (R(2)=0.974). C. difficile was detected by qPCR in faeces of 20 of 36 healthy volunteers and 34 of 37 patients with UC. Relatively greater amplification of C. difficile (fold difference) was noted in UC compared to controls (P<0.0001). There was no significant difference in C. difficile amplification between patients with proctitis, left sided colitis and pancolitis, or between active and quiescent colitis. Toxin was detected in the faeces of 8 of 37 patients with UC compared to 2 of 36 healthy volunteers. INTERPRETATION & CONCLUSION: Findings of this study showed overgrowth of C. difficile in the stool of Indian patients with UC. However, its relevance to disease pathogenesis and severity in a tropical country like India needs to be investigated further.

Antigenotoxic Effect of Ferulic Acid in 7;12-Dimethyl Beza (A) - Anthracene (DMBA) Induced Genotoxicity

The antigenotoxic effect of ferulic acid was carried out by evaluating the cytogenetic markers; the micronuclei frequency and chromosomal aberrations; in the bone marrow of hamsters in 7;12dimethylbenz(a)anthracene (DMBA) induced genotoxicity. Genotoxicity was induced in experimental hamsters by single intraperitoneal injection of DMBA (30mg kg-1 b.w). Pretreatment of ferulic acid orally at a dose of 40mg kg-1 b.w for five days significantly reduced the frequency of micronucleated polychromatic erythrocytes (MnPCEs) and the percentage of chromosomal aberrations in hamster's bone marrow. Our results thus suggest that ferulic acid has potent antigenotoxic effect in DMBA induced genotoxicity in golden Syrian hamsters

Response of liver antioxidant system to taurine in rats fed high fructose diet.

Fructose-fed rats were more susceptible to peroxidative damage as measured by thiobarbituric acid reactive species. The concentrations of lipid peroxides, diene conjugates, lipofuscin and hydroperoxides were significantly higher. The levels of enzymic antioxidants such as vitamin C, vitamin E and glutathione and activities of antioxidant enzymes were significantly lower in fructose-fed rats. When these rats received taurine in drinking water, peroxidative damage was minimal in both plasma and liver. Taurine was effective in inducing the antioxidant potential in fructose-fed rats. Increased peroxidative damage in liver is likely to be associated with fructose dependent pathology, which could be reduced by taurine by enhancing the antioxidant potential.

Role of nitric oxide on insulin induced seizures in mice.

The role of nitric oxide (NO) on acute hypoglycemia-induced seizures in mice was investigated using insulin as the hypoglycemic agent. The NO precursor L-arginine in the doses of 150, 500 and 750 mg/kg exhibited a dose-dependent protective effect against seizures induced by 8 mu/kg insulin. The NO synthase inhibitor (L-NMMA) at the doses of 50 and 100 mg/kg potentiated the subconvulsive doses of insulin (2 mu/kg). The onset, duration, number of seizures and the mortality were noted in a 2 hr study period. The results of this study suggest than NO plays an important protective role in acute hypoglycemia induced seizures which are known to occur through the activation of NMDA receptors.

Cognitive dysfunction induced by phenytoin and valproate in rats: effect of nitric oxide.

Phenytoin (PHT) and Valproate (VPA) are known to induce cognitive dysfunction, in terms of long term memory loss. Nitric oxide (NO) on the other hand is said to help in long term potentiation and hence enhance memory. The effects of nitric oxide donor L-arginine (L-Arg) and nitric oxide synthase inhibitor N-W-L-Nitroarginine (L-NOARG) were studied on the cognitive dysfunction, induced by PHT and VPA in normal healthy rats, using the step-through passive avoidance test (PAT). It was observed that combining L-Arg with PHT significantly enhanced long term memory while, combining PHT with L-NOARG decreased it, as compared to PHT alone. When combined with VPA, L-Arg and L-NOARG increased the retention latency as compared to PVA alone but this was not statistically significant. We conclude that the No donor L-Arg is able to increase the difference in LTE in acquisition and retention trials with both PHT and VPA, but with VPA the increase is not statistically significant.

Anticonvulsant profile of nimodipine and nitrendipine against pentylenetetrazole induced seizures in rats.

The activity of nimodipine and nitrendipine against pentylenetetrazole (PTZ) induced seizures in Albino rats was studied alone and in combination with valproate. The median effective dose [ED50] of valproate, nimodipine and nitrendipine were initially determined. All the 3 drugs were injected i.p. 30 min before the induction of seizures. Seizures were induced by PTZ 85 mg/kg i.p., and subsequently the effect of combining ED50 doses of nimodipine and nitrendipine with ED50 dose of valproate was evaluated. ED50 of valproate and nitrendipine were 129 and 2.5 mg/kg respectively. ED50 of nimodipine could not be established since a dose-response relationship was not obtained. Hence, for the purpose of combination studies, 4 mg/kg of nimodipine was used. Both nimodipine (4 mg/kg) and nitrendipine (2.5 mg/kg) decreased the ED50 of valproate from 129 to 40 mg/kg. Both nimodipine and nitrendipine potentiate the activity of valproate against PTZ induced seizures and can be considered as potential adjuvant anticonvulsants which merit further study.

Effect of training on lipid peroxidation, thiol status and antioxidant enzymes in tissues of rats.

The effect of training on lipid peroxidation, thiol-status and certain antioxidant enzymes of glutathione system was studied in blood and tissues such as liver and skeletal muscle of rats. Exercise training was given by subjecting the rats to treadmill running. Training for a period of 6 weeks resulted in increased lipid peroxidation (P < 0.001) as indicated by thiobarbituric acid test and conjugated diene measurement in liver and muscle. Thiol levels (total and non-protein) were reduced. However, glutathione level in blood was increased and blood lipid peroxides were unaltered as a result of training compared to sedentary controls. Adaptive increases in three antioxidant enzyme activities were observed. The study indicates that training induces adaptation in the glutathione system.

Effect of nimodipine on the efficacy of commonly used antiepileptic drugs in rats.

Effect of nimodipine was studied alone and in combination with phenytoin and valproate in maximal electroshock seizures in rats. The test drug was injected i.p. and seizures elicited by a 60 Hz alternating current of 150 mA intensity for 0.25 sec duration through corneal electrodes. The median effective dose (ED50) of phenytoin, valproate and nimodipine were found to be 13, 255 and 4 mg/kg respectively. Addition of ED50 of nimodipine to ED50 of phenytoin and valproate produced an additive effect. Addition of ED25 of nimodipine to ED25 of phenytoin and valproate produced asynergistic effect. Our results show that addition of nimodipine significantly potentiates the anticonvulsant efficacy of phenytoin and valproate.

Histological and immunological correlates of suspected leprosy lesions.

Thirty-two subjects with suspected leprosy lesions were investigated to assess various modalities of sensibility and sweat function and these were correlated with immunological and histological parameters. It was found that pain and temperature, mediated by small unmyelinated fibres were impaired in the early lesions. Impairment of sweat function was seen only when one of the modalities of sensibility was also affected. Antibodies specific to a protein (35 kDa) antigen and phenolic glycolipid 1 of Mycobacterium leprae were positive in nine and 12 cases respectively, while 15 of the 31 biopsies revealed the presence of mycobacterial antigens in these lesions. The implications of these findings are discussed.