Morinda citrifolia L. (Noni) dietary supplementation ameliorates fluoride toxicity in the freshwater fish, Cyprinus carpio L

Fluoride is the organic and inorganic compound of the element fluorine, has detrimental effect to all animals including fish at higher concentration. Ground water with elevated concentration of fluoride is a major global problem including India. On the other hand, Morinda citrifolia (also called ‘Noni’ in Polynesia) is a medicinal plant, has antioxidative, anti-inflammatory and immunostimulatory properties. The ameliorating effect of M. citrifolia fruit extract dietary supplementation against fluoride induced toxicity on the growth performance, haematological parameters, and fluoride accumulation in the muscular tissue was investigated in the freshwater fish, Cyprinus carpio L. for 35 days in triplicate. One hundred and twenty acclimated C. carpio fingerlings were randomly allocated into five experimental groups (Groups I, II, III, IV and V) each with 24 fish divided into three replicates (eight fish/replicate). Group I (Control) and II (Toxic control or negative control) were provided with basal/control diet, whereas groups III, IV, and V were provided with 0.25% (2.5 g/kg), 0.50% (5.0 g/kg) and 0.75% (7.5 g/kg) M. citrifolia fruit extract supplemented diet respectively. All groups except for group I were exposed to 10% of the LC50 of fluoride. Selected growth and haematological parameters were estimated. The fluoride content in the muscular tissue of fish was also estimated following SPADNS method. Results revealed that exposure of sublethal concentration of fluoride significantly (P <0.05) reduced the growth performance and haematological parameters in Gr. II (toxic control) compared to the Gr. I (control). However, M. citrifolia dietary supplementation significantly (P <0.05) improved fluoride induced alteration of growth performance and haematological parameters of fish in the groups III, IV and V compared to Gr. II. Morinda citrifolia fruit extract dietary supplementation also significantly (P <0.05) decreased the accumulation fluoride in the muscular tissue of fish in groups III, IV and V compared to the Gr. II (toxic control). In conclusion, M. citrifolia dietary supplementation ameliorates the fluoride toxicity by preventing the accumulation of fluoride in the muscular tissue of the fish. The M. citrifolia dietary supplementation at 0.25% showed the best response, hence it is considered as the optimum dose.

Household Characteristics, Cost of Illness and Coping Mechanism of Families of Hospitalized Acute Encephalitis Syndrome Children: A Mix Method Study

Background: AES is responsible for causing high social and economic burden to the affected families. The study was conducted to know the household characteristics, cost of illness and coping strategy adopted by the family members of AES children admitted to a tertiary care facility in Assam, India. Methods: It was a sequential exploratory mixed method study, with a cross-sectional survey among AES chil-dren and their guardians, followed by In Depth interview. Results: Out of 51 cases 55% were male. The median age was 11 years. 53% of the families belonged to lower socioeconomic class. 96% of families reported to reside in rural areas. Commonly reported household assets were mobile phone 94%, bicycle 86%, television 31% and motorcycle 29%. Three major themes were emerged to contribute towards economic burden. The first major theme is “Direct cost” due to patient trans-portation cost (mean Rs 1161.00) and hospital costs: mostly due to medicinal cost (mean Rs1955.00), investi-gational cost (mean Rs 2920.00) and food cost (mean Rs 8375.00). The second theme “Indirect cost” is due to work days loss: 100% care providers had missed work days during hospital stay of their children and 84% had missed work days during post hospital care. The third theme is “Inherent cost spotted through coping mechanisms” which mostly 94% included borrowing money from the market and 31.4% selling household assets. Conclusion: Cost of illness is a huge burden to the AES afflicted families which demands reforms in health care financing and reimbursement in current context.

Acute toxicity of fluoride and aluminium on the freshwater fish, Cyprinus carpio L

Fluoride is a trace element which has beneficial effect at lower concentration but detrimental at higher concentration. The fluoride contamination in ground water is a major global problem. Similarly, aluminium is the most abundant metal of the earth, highly toxic to all organisms. In the present study, we investigated the acute toxicity of fluoride and aluminium on three month old fingerlings of Cyprinus carpio L. in hard water (Hardness 125 mg/L of CaCO3) under static renewal bioassay. The 96 h LC50 value of the fluoride and aluminium were 675.615 and 224.214 mg/L, respectively. There was a significant correlation (P <0.01) between mortality rate of C. carpio with all concentrations of fluoride as well as aluminium. The exposure time (24, 48, 72 and 96 h) and different concentration of fluoride (680, 684, 686 and 692 mg/L) and aluminium (228, 229, 231 and 232 mg/L) was also significantly correlated (P <0.05). The different abnormal behaviour displayed by the fluoride and aluminium exposed fishes were erratic swimming movements, rapid opercular activity and excessive secretion of mucous. The safe level of concentrations of fluoride and aluminium were 6.75 and 2.24 µg/L, respectively. The LC50 values of fluoride and aluminium of the present study may be useful in deriving water quality standards in West Bengal.

Early detection of multidrug resistant (MDR) Mycobacterium tuberculosis in a single tube with in-house designed fluorescence resonance energy transfer (FRET) probes using real-time PCR.

Rapid and correct diagnosis is crucial for the management of multidrug resistance (MDR) in Mycobacterium tuberculosis (MTB). The present study aims at rapid diagnosis for identification of multidrug resistance tuberculosis (MDR-TB) using real-time PCR. FRET hybridization probes targeting most prominent four selected codons for rpoB526 and 531 and for katG314 and 315 genes were designed and evaluated on 143 clinical MTB isolates and paired sputa for rapid detection of MDR-TB. The results of real-time PCR were compared with gold standard L-J proportion method and further validated by DNA sequencing. Of the 143 MTB positive cultures, 85 and 58 isolates were found to be ‘MDR’ and ‘pan susceptible’, respectively by proportion L-J method. The sensitivity of real-time PCR for the detection of rifampicin (RIF) and isoniazid (INH) were 85.88 and 94.11%, respectively, and the specificity of method was found to be 98.27%. DNA sequencing of 31 MTB isolates having distinct melting temperature (Tm) as compared to the standard drug susceptible H37Rv strain showed 100% concordance with real-time PCR results. DNA sequencing revealed the mutations at Ser531Leu, His526Asp of rpoB gene and Ser315Thr, Thr314Pro of katG gene in RIF and INH resistance cases. This real-time PCR assay that targets limited number of loci in a selected range ensures direct and rapid detection of MDR-TB in Indian settings. However, future studies for revalidation as well as refinement are required to break the limitations of MDR-TB detection.

Expression of CDX-2 and Ki-67 in different grades of colorectal adenocarcinomas.

Background: CDX2 is a caudal homeobox gene essential for intestinal differentiation and is specifi cally expressed in colorectal adenocarcinomas. Its role in colorectal carcinogenesis is not fully elucidated. Aims and Objectives: To study the expression pattern of CDX2 and Ki-67 in different grades of colorectal adenocarcinomas and to observe the relationship of their staining patterns in various tumor stages and to look for correlation if any, between Ki-67 labeling index (Ki-67 LI) and CDX2 expression. Materials and Methods: A total of 74 cases were enrolled. Detailed clinical profi le, peroperative fi ndings, histological grading and staging were noted. Immunohistochemistry for CDX2 and Ki-67 was done, and Ki-67 LI was calculated. CDX2 staining was graded semiquantitatively, and statistical analysis was done. Result: Age of presentation ranged from 20 to 75 years, and the male:female ratio was 1.83:1. There were 8, 47 and 13 cases of well, moderate and poorly differentiated adenocarcinomas, respectively. The mean Ki-67 LI of well, moderate and poorly differentiated adenocarcinomas were 14.25, 31.34 and 43.08 respectively, and their difference was statistically signifi cant, correlation was also noted with stage. CDX2 expression appeared to be stronger in poorly differentiated cases, but there was no signifi cant difference in its expression in the different grades and stages. There was no correlation between Ki-67 LI and CDX2 immunostaining pattern. The lymph node metastasis showed CDX2 positivity in all the cases. Conclusion: Expression of CDX2 does not signifi cantly change with the grade of colorectal adenocarcinomas. However, it is an important diagnostic marker in metastatic colonic lesions. The Ki-67 LI, on the other hand, showed a strong correlation with histopathological grades.

Central nervous system teratomas in infants: A report of two cases.

Intracranial teratomas are uncommon neoplasms with most of them being encountered in the pediatric age group. Teratomas are composed of derivatives of all the three germ cell layers and are classifi ed into mature, immature and teratoma with malignant transformation. Two cases of intracranial teratomas in infants are presented here with the idea of highlighting this relatively uncommon condition in an uncommon age.

Metastatic ganglioneuroma: A misnomer.

Neuroblastoma, ganglioneuroblastoma and ganglioneuroma atumors arising from the neural crest cells. Ganglioneuroma is considered as the most mature amongst the three and usually has no metastatic potential. Spontaneous maturation of neuroblastoma into ganglioneuroma is, however, quite well-known. Here, we present a case of an 8-year-old girl child with evidence of metastasis of ganglioneuroma into a lymph node.

Diffuse large B-cell lymphoma of the kidney: A rare neoplasm.

Primary renal lymphoma is a rare neoplasm, but it should be kept in mind in the differential diagnosis of renal neoplasms. A middle aged man presented with symptoms of weight loss, anorexia and fullness of the abdomen after meals. On clinical and radiological examination, a renal mass was revealed and operated upon. A diagnosis of primary high grade renal lymphoma was made on histopathological examination and immunohistochemically it was further classifi ed as diffuse large B-cell lymphoma. Unfortunately, the patient died after 5 months of diagnosis in spite of three cycles of chemotherapy following surgery. The pathological details of rare tumor are presented here.

Mycobacterium indicus pranii as stand-alone or adjunct immunotherapeutic in treatment of experimental animal tuberculosis.

Background & objectives: Mycobacterium w (M.w) is a saprophytic cultivable mycobacterium and shares several antigens with M. tuberculosis. It has shown good immunomodulation in leprosy patients. Hence in the present study, the efficacy of M.w immunotherapy, alone or in combination with multi drug chemotherapeutic regimens was investigated against drug sensitive M. tuberculosis H37Rv and three clinical isolates with variable degree of drug resistance in mice. Methods: BALB/c mice were infected with M. tuberculosis H37Rv (susceptible to all first and second line drugs) and three clinical isolates taken from the epository of the Institute. The dose of 200 bacilli was used for infection via respiratory route in an aerosol chamber. Chemotherapy (5 days/wk) was given one month after infection and the vaccinated group was given a dose of 1×107 bacilli by subcutaneous route. Bacterial load was measured at 4 and 6 wk after initiation of chemotherapy. Results: M.w when given along with chemotherapy (4 and 6 wk) led to a greater reduction in the bacterial load in lungs and other organs of TB infected animals compared to. However, the reduction was significantly (P<0.05) more in terms of colony forming units (cfu) in both organs (lungs and spleen). Conclusion: M.w (as immunomodulator) has beneficial therapeutic effect as an adjunct to chemotherapy.

Molecular typing of Mycobacterium leprae strains from northern India using short tandem repeats.

Background & objectives: Due to the inability to cultivate Mycobacterium leprae in vitro and most cases being paucibacillary, it has been difficult to apply classical genotyping methods to this organism. The objective of this study was therefore, to analyze the diversity among M. leprae strains from Uttar Pradesh, north India, by targeting ten short tandem repeats (STRs) as molecular markers. Methods: Ninety specimens including 20 biopsies and 70 slit scrappings were collected in TE buffer from leprosy patients, who attended the OPD of National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Tajganj, Agra, and from villages of Model Rural Health Research Unit (MRHRU) at Ghatampur, Kanpur, Uttar Pradesh. DNA was extracted from these specimens and ten STRs loci were amplified by using published and in-house designed primers. The copy numbers were determined by electrophoretic mobility as well as sequence analysis. Phylogenetic analysis was done on variable number of tandem repeats (VNTRs) data sets using start software. Results: Diversity was observed in the cross-sectional survey of isolates obtained from 90 patients. Allelic index for different loci was found to vary from 0.7 to 0.8 except for rpoT for which allelic index was 0.186. Similarity in fingerprinting profiles observed in specimens from the cases from same house or nearby locations indicated a possible common source of infection. Such analysis was also found to be useful in discriminating the relapse from possible reinfection. Interpretation & conclusions: This study led to identification of STRs eliciting polymorphism in north Indian strains of M. leprae. The data suggest that these STRs can be used to study the sources and transmission chain in leprosy, which could be very important in monitoring of the disease dynamics in high endemic foci.

Mycobacterium avium bacteremia and dual infection with mycobacterium avium and mycobacterium wolinskyi in the gut of an aids patient – First case report.

An AIDS patient was admitted to a tertiary care hospital in central India with fever, weight loss, breathlessness, night sweats, diarrhoea, BMI 14kg/m2, Hemoglobin 8gm% and CD4 counts 120 cells/cumm. His blood culture by BACTEC 460 TB system revealed Mycobacterium avium bacteremia and stool culture grew Mycobacterium avium and mycobacterium wolinskyi.

Comparative growth pattern of multi drug resistance versus susceptible isolates of Mycobacterium tuberculosis in mice lungs.

Background & objectives: Rise in prevalence of multi-drug resistance (MDR) in tubercle bacilli is a serious cause of concern. As mutations with two house keeping genes rpoB and katG are associated with resistance to two important anti-tubercular drugs rifampicin and isoniazid respectively, there is a need to understand the growth kinetics of organisms with such mutated genes in experimental animals. This study was undertaken to study the growth kinetics of susceptible as well multi-drug resistance Mycobacterium tuberculosis isolates in mice. Methods: Two MDR (having mutations in rpoB and catG) and two drug susceptible isolates of M. tuberculosis along with H37Rv were grown in mice after aerogenic infection. Results: The MDR isolates grew slowly up to 3 wk though the growth was significantly different from sensitive strains. However, after 3 wk, the growth in sensitive as well MDR strains was similar, suggesting that even the mutations in the MDR strains did not have any impact on the growth kinetics. Interpretation & conclusions: The effect of mutations in other parts of these genes need to be studied. Retention of property of MDR strains to establish infection after aerogenic infection has epidemiological significance in terms of the transmission of MDR tuberculosis.

Spectrum of Lactobacillus species present in healthy vagina of Indian women.

Background & objectives: Lactobacilli are depleted in vagina of women suffering from recurring episodes of bacterial vaginosis with vaginal pH >5. With the objective of making available probiotic lactobacilli for replenishment in such women, a study was undertaken to isolate and characterize the Lactobacilli present in women with eco-healthy vagina in Delhi. No information is so far available on the species of Lactobacilli resident in vagina of women in India. Methods: Vaginal swabs were taken from 80 women with informed consent after ethical approval and grown in MRS broth. Gram-positive, catalase-negative bacilli generating about 200 bp amplicon by PCR with Lactobacillus genus specifi c primers were further characterized by employing species specifi c primers followed by sequencing of 16S rDNA. Isolates of the same species were differentiated by random amplifi ed polymorphic DNA (RAPD) profi les. Results: The predominant species isolated were L. reuteri present in 26 (32.5%) women, L. fermentum in 20 (25%), and L. salivarius in 13 (16.25%) women. Sequencing of 16S rDNA of 20 isolates showed that except for two isolates of L. plantarum, sequences of the remaining agreed well with PCR identifi cation. None of the isolates had similar RAPD profi le. Interpretation & conclusions: Our fi ndings showed lactobacilli species present in healthy vagina of women in India differ from those reported from other countries. This information would be useful to development of probiotic tablets seeking to replenish the missing lactobacilli for reproductive health of women in India.

Identification of environmental mycobacteria isolated from Agra, north India by conventional & molecular approaches.

Background & objectives: Several environmental mycobacteria have been shown to be important human pathogens linked to immunomodulation especially in relation to effect on vaccination. Hence identification of mycobacteria to the species level is not only relevant to patient management but also to understand epidemiology of mycobacterial diseases and effect on vaccination. We undertook this study to assess the usefulness of various conventional and molecular methods in identification of environmental mycobacterial species from Agra, north India. Methods: One hundred nineteen isolates of environmental mycobacteria were grown from 291 (116 soil and 175 water) samples. These isolates were identified by standard biochemical tests, and a simple, rapid and cost-effective in-house developed gene amplification restriction analysis targeting 16S-23S rRNA spacer and flanking region and 16S rRNA sequencing. Results: Biochemical tests could clearly identify only 68.1 per cent (81/119) of isolates to species level. An in-house developed gene amplification - restriction analysis method could confirm the identity of 102 of 119 (85.7%) isolates and the remaining 17 isolates (14.3%) were confirmed by 16S rRNA sequencing also. These 119 environmental mycobacterial isolates, included several potentially pathogenic species such as M. fortuitum, M. chelonae, M. avium, M. marinum, M. manitobense, M. kansasii and others belonged to nonpathogenic species, M. terrae, M. smegmatis and M. flavescens. M. chelonae was isolated from water samples only whereas M. fortuitum was isolated from both water as well as soil samples. Interpretation & conclusion: The in-house developed gene amplification restriction analysis method though failed to accurately identify 14.3 per cent of isolates, facilitated rapid differentiation of most of environmental mycobacteria including potential pathogens from this area and thus would have diagnostic potential in cases with NTM infections. This combination strategy using PCR-RFLP and 16S rRNA sequencing may be useful for characterization of mycobacteria from similar environmental settings from other parts of world.

Nocardial mycetoma: diverse clinical presentations.

Nocardia spp are gram-positive, aerobic, acid-fast bacteria which exist as saprophytes in nature. Invasive disseminated infections are particularly common in immunocompromised or debilitated hosts. Superficial infections with Nocardia spp occur as a result of local trauma and contamination of the wound. Clinically, it presents as acute infection (abscesses or cellulitis), mycetoma, or sporotrichoid infection. Differential diagnosis includes eumycetoma, chromomycosis, blastomycosis, coccidioidomycosis, sporotrichosis, tuberculosis, botryomycosis, syphilis, yaws, and neoplasia. Its diagnosis is confirmed by demonstrating the causative organism in exudates (as granules), tissue specimens, or cultures. Early diagnosis will obviate need for drastic surgical measures as early institution of chemotherapy is effective in most patients. However, its diagnosis is often delayed due to diverse clinical presentations and for want of clinical suspicion, particularly in non-endemic areas. This paper presents 4 clinical forms of this not so uncommon disease, emphasizing the importance of high index of clinical suspicion, especially in non-endemic regions; and the significance of repeated examination of exudates for Nocardia granules for an early diagnosis.

Simultaneous ethambutol & isoniazid resistance in clinical isolates of Mycobacterium tuberculosis.

BACKGROUND & OBJECTIVE: There is a need to understand the nature of drug resistance patterns and predictors of emergence of drug resistance in Mycobacterium tuberculosis. There could be common factors/mechanisms for resistance to the drugs, isoniazid and ethambutol, both acting on cell wall. The present study was conducted to analyze the antimycobacterial susceptibility patterns of M. tuberculosis isolates to determine the minimum inhibitory concentrations (MICs) of ethambutol for M. tuberculosis; and to find out possible association of ethambutol resistance with isoniazid resistance. METHODS: A total of 380 M. tuberculosis isolates were tested for their susceptibilities to ethambutol at 2, 4, 6 microg/ml, isoniazid at 1 microg/ml and rifampicin at 64 microg/ml using MIC method. RESULTS: 44.21, 24.73 and 14.21 per cent isolates were resistant to ethambutol at concentrations of 2, 4 and 6 microg/ml respectively. At 6 microg/ml of ethambutol concentration, 85.18 per cent ethambutol resistant isolates were resistant to isoniazid also. At the same ethambutol concentration a fraction of 28.75 per cent isoniazid resistant isolates were ethambutol resistant. INTERPRETATION & CONCLUSION: Ethambutol resistance was accompanied with isoniazid resistance in a large percentage of isolates whereas ethambutol resistance was weakly linked with multidrug resistance. On the other hand, association between isoniazid and ethambutol resistance was weak showing one way linkage.