Polymorphism of gp15/400 allergen gene of W. bancrofti from different regions of India endemic for lymphatic filariasis.

Nematode polyprotein allergens (NPA) are lipid binding/transport molecules that elicit elevated levels of IgE response in the infected host, leading to Th2 type of immune response. They also transport arachidonic acid and its metabolites that are known to be involved in the action of antifilarial drug, Diethylcarbamazine and hence are of great significance for the control of lymphatic filariasis. We investigated the polymorphism of gp15/400 polyprotein of 35 isolates of lymphatic filarial parasite Wuchereria bancrofti collected from different geographic locations of India. The repeat sub-unit of the gene was found to be highly conserved in all the isolates with only two nucleotide synonymous changes at positions 286 (A-G) and 337 (C-T). Since this molecule is highly conserved and has multifarious roles in the survival and pathogenesis of the parasite it has good potential as a target for drug, immunodulation tool and immunotherapy development.     

A method for detecting microfilaraemia, filarial specific antigens and antibodies and typing of parasites for drug resistance and genotypes using finger prick blood sample

Monitoring and evaluation of programme to eliminate lymphatic filariasis (LF) depends on epidemiological assessment using appropriate indicators. Minimum efforts using reliable tests are necessary to guide the programme managers in decision-making. Impact of Mass Drug Administration (MDA) towards filariasis elimination can be assessed by the detection of microfilariae (mf) or parasite DNA (infective), filarial antigens (infected) and antibodies (exposure). It is also important to monitor drug resistance and variation in genetic structure of parasite populations using molecular markers. We developed a method to carry out parasitological, molecular, immunological and genetic analysis from a minimum volume of blood sample (about 150 μl) drawn from finger tip of an individual residing in LF endemic area. The method involves separation of sera for immunological assays and isolation of mf of Wuchereria bancrofti from the blood clots for counting, which were then used for W. bancrofti specific PCR, screening for albendazole sensitivity/resistance alleles by AS-PCR, RAPD profiling and ITS 2 PCR for genotyping. A protocol is also suggested for the separation of sera for assays to detect antigen and antibodies and isolation of mf from clots for genetic analysis. The protocol developed has shown potential application in monitoring several immunological, parasitological and molecular parameters from a limited amount of blood sample collected by finger prick, in large-scale operations.     

A simple and rapid DNA extraction method for the detection of Wuchereria bancrofti infection in the vector mosquito,Culex quinquefasciatus by Ssp I PCR assay

A simple, rapid and inexpensive method for the extraction of DNA from filarial vector, Culex quinquefasciatus, useful in Ssp I PCR assay for xenomonitoring of infection with Wuchereria bancrofti is presented. The DNA extracted by this method was found suitable for PCR detection of W. bancrofti infection in pools of 10-30 mosquitoes. The PCR assay employing the simplified DNA extraction method was evaluated for its sensitivity on field caught Cx. quinquefasciatus, in comparison with the conventional dissection and microscopy technique. When assayed on dissection washings of vector mosquitoes the PCR assay detected 45 pools out of 49 dissection positive pools as positive for infection and hence found to be less sensitive than the conventional technique. The reason for detecting four dissection positive pools as negatives by the PCR assay may be due to the loss of a few numbers of parasites (1-3) present in these pools during the transfer of washings of dissected mosquitoes. The PCR assay detected ten out of 72 dissection negative pools as positives, while it did not detect any of the 62 known negative (laboratory reared, uninfected) mosquito pools as positives. When 38 pools (10 mosquitoes/pool) of intact mosquitoes were assessed for infection by each method, the infection rates obtained by the two methods were almost similar (3.35 and 3.01%, respectively, for conventional method and PCR assay). The results thus show that the DNA extraction method, which is simple, rapid, safe and inexpensive, is efficient to generate DNA from vector mosquitoes useful in PCR assay and hence has potential application in xenomonitoring.     

Oxidative stress in severe dengue viral infection: association of thrombocytopenia with lipid peroxidation

Oxidative stress in viral infections has been suggested. The study was carried out to assess the oxidative stress in the different clinical spectrums of dengue infection and to evaluate if thrombocytopenia is associated with lipid and protein oxidative injury. Twenty-seven dengue fever (DF), 32 dengue hemorrhagic fever (DHF) and 21 dengue shock syndrome (DSS) cases were studied at 3, 5 and 7 days of illness. Sixty-three healthy subjects were selected as controls. Serum protein carbonyls (PCOs), malendialdehyde (MDA) and total antioxidant status (TAS) were estimated in blood. Dengue infected individuals had significantly high levels of PCOs and MDA on the three days tested in comparison to controls. In DF cases, no significant changes in the levels of MDA and PCOs were found in course of time. However, among DHF and DSS, significant increase in MDA levels was found in the fifth and seventh day samples in comparison to their respective third day sample (P < 0.05). Using one way ANOVA, high PCOs levels were found in DSS in comparison to DF and DHF cases on all the three days tested (P < 0.001). TAS levels were found to be low among DSS on days 5 and 7 and day 7 in DHF when compared with DF cases. Correlation analysis between MDA and hematocrit revealed a significant positive association between them in DHF and DSS on day 5 (DHF r = 0.372; p = 0.024 and DSS r = 0.535; p = 0.0-01) and day 7 (DHF r = 0.412; p = 0.003 and DSS r = 0.765; p < 0.0001). There was an important negative correlation between platelet count and plasma lipid peroxidation levels among DHF and DSS on all three days tested [day 3 (DHF r = -0.392; p = 0.012 and DSS r = -0.453; p = 0.004), day 5 (DHF r = -0.592; p < 0.001 and DSS r = -0.581; p < 0.001) and day 7 (DHF r = -0.418; p = 0.001 and DSS r = -0.515; p = 0.002)]. This study concludes that an increase in oxidative stress was found in dengue viral infection. The level of oxidative stress was maximal in DSS followed by DHF and its severity was minimal in DF. The thrombocytopenia of dengue infection was associated with the extent of lipid peroxidation. Future studies might be carried out to find the role of oxidative damage in the ethiopathogenesis of thrombocytopenia and vascular leakage in dengue infection.     

Operative Risks of Domino Liver Transplantation for the Familial Amyloid Polyneuropathy Liver Donor and Recipient: A Double Analysis

Although domino liver transplantation (LT) is an established procedure, data about the operative risks are limited. This study aimed at evaluating the operative risks of domino LT. Two retrospective analyses were conducted (comparison of familial amyloid polyneuropathy [FAP] liver donors [61 patients] vs. FAP nondonors [39 patients] and FAP liver recipients [61 patients] vs. deceased donor liver recipients [61 patients]). First analysis showed a 60‐day mortality of 6.6% for FAP donors and 7.7% for FAP nondonors (p = 1.0). No patient developed primary graft nonfunction. Acute rejection was higher in FAP nondonors compared to FAP donors (38.5% vs. 13.1%). Both groups had similar vascular and biliary complication rates. ICU stay was similar, whereas total hospitalization was longer for FAP nondonors. Both groups had similar 1‐ and 5‐year patient and graft survival rates (83.4% vs. 87.2%, and 79.8% vs. 71.8%, p = 0.7) and (83.3% vs. 87.2%, and 79.1% vs.71.8%, p = 0.7). The second analysis showed a 1.6% mortality for FAP liver recipients vs. 3.2% of the control group (p = 1). Both groups had similar morbidity and technical complication rates (18.0% vs. 13.1%, p = 0.45) and (0.18 vs. 0.15, p = 0.65). The domino procedure does not add any risk to FAP donor or recipient. It increases the organ pool allowing transplantation of marginal recipients who otherwise are denied deceased donor liver transplantation.     

Family-based clusters of cognitive test performance in familial schizophrenia

Background  

Cognitive traits derived from neuropsychological test data are considered to be potential endophenotypes of schizophrenia. Previously, these traits have been found to form a valid basis for clustering samples of schizophrenia patients into homogeneous subgroups. We set out to identify such clusters, but apart from previous studies, we included both schizophrenia patients and family members into the cluster analysis. The aim of the study was to detect family clusters with similar cognitive test performance.     

Development of rapid assessment procedures for the delimitation of lymphatic filariasis-endemic areas

Lymphatic filariasis caused by Wuchereria bancrofti is a major public health problem in 73 tropical and subtropical countries including India. Delimitation of endemic areas is essential to plan control operations. The current method of night blood survey (NBS) for delimitation is cumbersome, time-consuming and expensive. Therefore, there is a need to develop assessment procedures which can rapidly delimit endemic areas. For this purpose we evaluated three procedures: direct interviewing of key informants using structured questionnaires, an indirect method of a self-administered questionnaires to key informants and physical examination by health workers for the presence of chronic filarial disease. Thirty rural communities in a filariasis-endemic region in Cuddalore district in Tamil Nadu State in southern India constituted the study population. The determination of filariasis endemicity in the village communities assessed by the above procedures was compared in terms of rapidity, specificity, sensitivity and cost with the microfilaria rate and disease rate obtained by night blood sample survey and clinical examination by physicians. Prevalence score, control preference score and weighted mean number of cases with filarial disease per village were calculated using the key informant questionnaire techniques. While the prevalence and control preference score showed low sensitivity and moderate specificity, weighted mean number of cases showed high sensitivity and moderate specificity in identifying endemic villages. The prevalence of disease as determined by the physical examination of a sample population by health workers was highly sensitive in identifying communities endemic for filariasis. The degree of association between the disease rates estimated by physician and trained health workers was significant (r = 0.56; P < 0.05). These observations suggest that the weighted mean number of cases per village obtained through key informant techniques may be considered at a primary level to crudely identify endemic areas, followed by physical examination by health workers for filariasis, since it is relatively cheap and rapid.