The importance of using a right test method in diagnosing leptospirosis

@#Leptospirosis is a common febrile illness in Malaysia. The disease is caused by pathogenic bacteria called leptospires that are transmitted directly or indirectly from animals to humans via contaminated water or soil. It is a potentially serious but treatable disease. Its symptoms may mimic those of other unrelated febrile illnesses such as dengue, influenza, meningitis, hepatitis or viral haemorrhagic fevers. The spectrum of the disease is extremely wide, ranging from subclinical infection to a severe syndrome of multiorgan infection with high mortality. The diagnosis requires high suspicion with history of exposure to water or environment possibly contaminated with infected animal urine. This is a case of a 13 year-oldgirl with no known medical illness, and a history of exposure to outdoor activities. However, paired sera for leptospirosis serology was not diagnostic. She then developed septic shock on day 14 of illness. But due to high suspicion of leptospirosis, antibiotic therapy was upgraded to ceftriaxone and samples were sent for further testing which revealed that leptospires were detected in the urine, using molecular technique. She improved after treated as leptospirosis.

Sclerotiorin is non-mutagenic and inhibits human PMNL 5-lipoxygenase and platelet aggregation.

Sclerotiorin, isolated from the fermented broth of Penicillium frequentans, exhibited potent inhibition against human polymorphonuclear leukocytes 5-lipoxygenase and human platelet aggregation with a half maximal value 36 µM and 250 µM, respectively. Further, the Ames test has demonstrated the sclerotiorin to be non-mutagenic.

Molecular genotyping of ABO blood groups in some population groups from India.

Background & objectives: Indian population is characterized by the presence of various castes and tribal groups. Various genetic polymorphisms have been used to differentiate among these groups. Amongst these, the ABO blood group system has been extensively studied. There is no information on molecular genotyping of ABO blood groups from India. Therefore, the main objective of this study was to characterize the common A, B and O alleles by molecular analysis in some Indian population groups. Methods: One hundred samples from the mixed population from Mumbai, 101 samples from the Dhodia tribe and 100 samples from the Parsi community were included in this study. Initially, the samples were phenotyped by standard serologic techniques. PCR followed by single strand conformational polymorphsim (SSCP) was used for molecular ABO genotyping. Samples showing atypical SSCP patterns were further analysed by DNA sequencing to characterize rare alleles. Results: Seven common ABO alleles with 19 different genotypes were found in the mixed population. The Dhodias showed 12 different ABO genotypes and the Parsis revealed 15 different ABO genotypes with six common ABO alleles identified in each of them. Two rare alleles were also identified. Interpretation & conclusions: This study reports the distribution of molecular genotypes of ABO alleles among some population groups from India. Considering the extremely heterogeneous nature of the Indian population, in terms of various genotype markers like blood groups, red cell enzymes, etc., many more ABO alleles are likely to be encountered.

Antioxidant, Cytotoxicity and Polyphenolic Content of Calotropis procera (Ait.) R. Br. Flowers.

Oxidative stress has been implicated with the pathology of many diseases such as inflammatory conditions, cancer, diabetes and aging. In view of that an attempt was made to evaluate free radical scavenging activity, cytotoxic activity and polyphenolic content of methanolic extract of Calotropis procera flowers.Free radical scavenging activity was estimated using in vitro models like 1,1,-diphenyl-2- picryl hydrazyl (DPPH), hydroxyl radical, hydrogen peroxide radical, reducing power and ferric thiocyanate method. Cytotoxicity was analysed following MTT assay using Hep2 and Vero cell lines and polyphenols were estimated using standard methods. Two way ANOVA was used for statistical analyses. The methanol extract of C. procera at 500 μg/ml showed better scavenging activity in ferric thiocyanade method (83.63 %) with the lowest IC50 of 100 μg/ml followed by hydrogen peroxide, hydroxyl radical scavenging and least activity was found to be present in DPPH assay (50.82 %). The extract had 100 % cytotoxicity on Hep2 cell lines. Flavonoids were found in greater amount than phenols and found to be had higher correlation with antioxidant activities. It was suggested that the flowers of C. procera possess in vitro antioxidant, cytotoxic activities and thus having much therapeutic value because of their polyphenolic content.

Murine monoclonal anti-H as typing reagent.

BACKGROUND & OBJECTIVE: The antigen H present on the surface of red cells in varying concentration, is maximum in O group red cells, but absent in Bombay phenotype individuals. This differentiation is generally detected by seed extracts of Ulex europaeus. The titre of such an extract is usually low and is subjected to batch variation. Hence, we carried out this study to raise potent murine monoclonal antibody against H antigen. METHODS: Spleen cells of female BALB/c mice immunized with O group red cells were fused in presence of polyethylene glycol (PEG) 1500 with a mouse myeloma cell line Sp2/0 Ag14 in hypoxanthine aminopterine thymidine (HAT) selective medium and incubated at 37 degrees, 5 per cent CO(2) and 95 per cent humidity for a week. RESULTS: The culture supernatants showing anti-H activity, were further subcloned and two clones 3E8A10 and 3E8A11 generated which showed a good potency, avidity and specificity. INTERPRETATION & CONCLUSION: The anti-H clones thus produced indigenously provided a potent reagent in distinguishing normal O group from Bombay phenotype individuals. The unlimited availability makes this reagent cost-effective to ensure a constant supply of hybrid clones with the similar specificities.

Varied distribution of RhD epitopes in the Indian population.

BACKGROUND: Inhabited by more than 4000 caste and tribal groups, India has an extremely heterogenous population. For thousands of years many tribal groups have practised endogamy and are practically genetically isolated. Traditionally, polyclonal anti-D reagent has been used for RhD typing; though monoclonal antibodies are increasingly being used. As a result, blood banks find it difficult to assign the RhD status to an increasing number of people. As monoclonal anti-D typing reagents may not detect all RhD antigen epitopes, we studied the RhD antigen epitope heterogeneity in different population groups in India. METHODS: Red cells of 5315 RhD-positive individuals belonging to different castes and tribes of India were tested with 30 different epitope-specific monoclonal anti-D antibodies. RESULTS: No single monoclonal antibody could detect all RhD-positive red cells detected by polyclonal antisera. The highest proportion of D antigen was detected by LHM 76/55 and BRAD-8 (98%) monoclonal antibodies. CONCLUSION: We need to determine the correct mix of monoclonal antibodies that will detect nearly all RhD antigens detected by polyclonal anti-D sera. Similarly, before accepting monoclonal anti-D for therapeutic use, it would be necessary to determine the appropriate ones for use in the Indian population.

Potential of commercial anti-D reagents in the identification of partial D variants in Indian population.

BACKGROUND & OBJECTIVE: As partial D variants are of clinical importance in transfusion medicine, the present study aims to determine the efficiency of commercial anti-D reagents to identify partial D variants. METHODS: Forty two samples of known partial D identified in the Indian population were tested with seven commercial monoclonal anti-D reagents. RESULTS: Most of the monoclonal anti-D reagents gave strong positive reactions (24 to 59%) to weak positive (28 to 47%) with partial D cells. Polyclonal anti-D detected all partial D variants as RhD positive, though reacting weakly with the majority (83%) of them. All the seven commercial monoclonal anti-D reagents detected some variants as D negative. Analysis of pairs of these reagents showed that the combinations of reagents 1 & 2 and 1 & 6 could detect all partial D variants as RhD positive and hence can be used for donor testing. INTERPRETATION & CONCLUSION: Findings of our study showed that none of the monoclonal reagents when used individually could detect all partial D variants. A combination of two suitable anti-D reagents are necessary to detect maximum number of partial D variants.

Production of murine monoclonal anti-B.

BACKGROUND & OBJECTIVE: Monoclonal antibodies against red blood cell antigens used in research and as diagnostics in India are commercially procured from western countries. Indigenously generated potent clones are not available in India. Hence, the objective of the present study was to raise potent murine monoclonal antibodies against A, B and H blood group antigens indigenously and establish a stable clone of anti-B secreting cells. METHODS: Spleen cells of female BALB/c mice immunized with B group red blood cells were fused in presence of polyethylene glycol (PEG) 1500 with a mouse myeloma cell line Sp 2/0 Ag. 14 in hypoxanthine aminopterine thymidine (HAT) selective medium and incubated at 37 degrees C, 5 per cent CO(2) and 95 per cent humidity for a week. RESULTS: The culture supernatant of the wells showing anti-B activity, were further subcloned and a clone 2C4D5F10 was generated which showed a good potency, avidity and specificity. INTERPRETATION & CONCLUSION: The anti-B clones thus produced indigenously provided a useful reagent in blood group typing. The unlimited availability unlike polyclonal antisera makes this reagent more cost-effective. It also ensures a regular supply with the similar specificity.

Intraocular gnathostomiasis.

We report a rare case of intraocular Gnathostomiasis, where a live worm, intracameral in location, was successfully removed. Its identity was confirmed by microscopy.

Acrodermatitis enteropathica--a case report.

Acrodermatitis enteropathica is a rare disease affecting infant girls. The skin in these cases develop rashes which start as vesicles and then dries to form erythematous squamous psoriasiform type of lesions. Dystrophy of nails and alopecia with loss of eye lashes and eye brows is also seen. Low serum zinc level is found in these patients and is thought to be the cause of this disease.