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hsp65 PCR-restriction analysis (PRA) with capillary electrophoresis in comparison to three other methods for identification of Mycobacterium species
No full textWe developed a scheme for rapid identification of Mycobacterium species using an automated fluorescence capillary electrophoresis instrument. A 441-bp region of the hsp65 gene was examined using PCR-restriction analysis (PRA). The assay was initially evaluated on 38 reference strains. The observed sizes of restriction fragments were consistently smaller than the real sizes for each of the species as deduced from the sequence analysis (mean variance=7bp). Nevertheless, the obtained PRA patterns were highly reproducible and resulted in correct species identifications. A blind test was then successfully performed on 64 test isolates previously characterized by conventional biochemical methods, a commercial INNO-LiPA Mycobacteria assay and/or sequence determination of the 5' end of 16S rRNA gene. A total of 14 of 64 isolates were erroneously identified by conventional methods (78% accuracy). In contrast, PRA performed very well in comparison with the LiPA (89% concordance) and especially with DNA sequencing (93.3% of concordant results). Also, PRA identified seven isolates representing five previously unreported hsp65 alleles. We conclude that hsp65 PRA based on automated capillary electrophoresis is a rapid, simple and reliable method for identification of mycobacteria. Copyright 2010 Elsevier B.V. All rights reserved
Costs of hospital-based methadone maintenance treatment in HIV/AIDS control among injecting drug users in Indonesia
No full textOBJECTIVE: To assess the cost of hospital-based methadone maintenance treatment (MMT) for injecting drug users (IDUs) in Bandung, Indonesia; to address concerns of financial sustainability at the hospital level and financial accessibility and economic attractiveness at the health care policy level. METHODS: In a 1 year observation period in 2006-2007, MMT service delivery costs were estimated on the basis of a micro-costing approach. Patient costs were estimated on the basis of a survey among 48 methadone clients. RESULTS: A total number of 129 clients attended the MMT clinic, resulting in a total of 16,335 client visits. Total annual societal costs of running the MMT clinic equalled Rp 1130 mln (US7.57) per client visit. Of total costs, patient costs established the largest share (65%), followed by that of central government (20%), and the hospital (15%). Present consultation tariffs already cover hospital costs and the patient costs of accessing MMT services constitute almost 70% of their income. CONCLUSION: Under current circumstances, MMT services are financially sustainable to the hospital. MMT services are subsidized by the central government, and this is warranted considering the important role of the program in HIV/AIDS among IDUs. Still, the present user fee seems a barrier to utilization, and a higher level of subsidy might be justified to reduce the cost to the patient. Copyright 2009 Elsevier Ireland Ltd. All rights reserved
Cold tolerance of the harlequin ladybird Harmonia axyridis in Europe
No full textAs an essential aspect of its invasive character in Europe, this study examined the cold hardiness of the harlequin ladybird Harmonia axyridis. This was done for field-collected populations in Belgium overwintering either in an unheated indoor or an outdoor hibernaculum. The supercooling point, lower lethal temperature and lower lethal time at 0 and -5 degrees C were determined. Possible seasonal changes were taken into account by monitoring the populations during each winter month. The supercooling point and lower lethal temperature remained relatively constant for the overwintering populations in the outdoor hibernaculum, ranging from -17.5 to -16.5 degrees C and -17.1 to -16.3 degrees C, respectively. In contrast, the supercooling point and lower lethal temperature of the population overwintering indoors clearly increased as the winter progressed, from -18.5 to -13.2 degrees C and -16.7 to -14.1 degrees C, respectively. A proportion of the individuals overwintering indoors could thus encounter problems surviving the winter due to premature activation at times when food is not available. The lower lethal time of field populations at 0 and -5 degrees C varied from 18 to 24 weeks and from 12 to 22 weeks, respectively. Morph type and sex had no influence on the cold hardiness of the overwintering adults. In addition, all cold tolerance parameters differed greatly between the laboratory population and field populations, implying that cold tolerance research based solely on laboratory populations may not be representative of field situations. We conclude from this study that the strong cold hardiness of H. axyridis in Europe may enable the species to establish in large parts of the continent. Copyright (c) 2009 Elsevier Ltd. All rights reserved
Flow cytometry-based methods for assessing soluble scFv activities and detecting antigens in solution
No full textNovel methods are reported for evaluating and utilizing single chain fragment variable (scFv) antibodies derived from yeast-display libraries. Yeast-display was used to select scFv specific to invariant surface glycoproteins (ISG) of Trypanosoma brucei. A limiting step in the isolation of scFv from non-immune libraries is the conversion of highly active yeast-displayed scFv into soluble antibodies that can be used in standard immunoassays. Challenges include limited solubility or activity following secretion and purification of scFv. For this reason, few scFv derived from yeast-display platforms have moved into development and implementation as diagnostic reagents. To address this problem, assays were developed that employ both yeast-displayed and -secreted scFv as analytical reagents. The first is a competitive inhibition flow cytometry (CIFC) assay that detects secreted scFv by virtue of their ability to competitively inhibit the binding of biotinylated antigen to yeast-displayed scFv. The second is an epitope binning assay that uses secreted scFv to identify additional yeast-displayed scFv that bind non-overlapping or non-competing epitopes on an antigen. The epitope binning assay was used not only to identify sandwich assay pairs with yeast-displayed scFv, but also to identify active soluble scFv present in low concentration in a crude expression extract. Finally, a CIFC assay was developed that bypasses entirely the need for soluble scFv expression, by using yeast-displayed scFv to detect unlabeled antigen in samples. These methods will facilitate the continued development and practical implementation of scFv derived from yeast-display libraries. (c) 2009 Wiley Periodicals, Inc
High prevalence of anti-Trichinella IgG in domestic pigs of the Son La province, Vietnam
No full textAlthough several outbreaks of Trichinella infection among humans have occurred in northwestern Vietnam, no information is available on the circulation of Trichinella among domestic pigs. The objective of the present study was to estimate the seroprevalence of anti-Trichinella IgG in free-roaming pigs (Sus scrofa) in the Son La province of northwestern Vietnam, where a human outbreak of trichinellosis occurred in June 2008. Serum samples were collected from free-roaming pigs of four communes of the Bac Yen district (Son La province) and tested for Trichinella antibodies with a commercial ELISA kit using excretory/secretory antigens. Of 1035 pigs from which serum samples were collected, 206 were positive (19.9%). There was a significant difference in the prevalence among communes (chi(2)=22.87, 3 d.f., p<0.0001). Muscle samples from 76 serologically positive pigs were tested by artificial digestion. Trichinella larvae were detected in 11 (14.5%) of them. The larvae were identified by multiplex PCR as Trichinella spiralis. This study provides the first data on the circulation of T. spiralis in domestic pigs reared in Vietnam, and the results are useful for evaluating the risk of infection for humans. The results indicate that pigs act as a reservoir and play an important role in the maintenance of the domestic cycle of T. spiralis in northwestern Vietnam
Elimination of Taenia solium transmission to pigs in a field trial of the TSOL18 vaccine in Cameroon
No full textA pilot field trial of the TSOL18 vaccine was undertaken in Cameroon. Two hundred and forty, 2-3 month-old piglets were distributed to 114 individual households in pairs. Vaccinated animals received three immunisations with 200 microg TSOL18 plus 5 mg Quil A and 30 mg/kg oxfendazole at the time of the second immunisation. Necropsies were undertaken when the pigs were approximately 12 months of age. Viable Taenia solium cysticerci were identified in 20 control pigs (prevalence 19.6%); no cysticerci were found in any of the vaccinated animals (P<0.0001). Combined application of TSOL18 vaccination and a single oxfendazole treatment in pigs may be a relatively simple and sustainable procedure that has the potential to control T. solium transmission in endemic areas and, indirectly, reduce the number of new cases of neurocysticercosis in humans. 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved
Fitness of Mycobacterium tuberculosis strains of the W-Beijing and Non-W-Beijing genotype
No full textBACKGROUND: Multidrug resistant tuberculosis (MDR-TB) is a major threat for global tuberculosis control. The W-Beijing Mycobacterium tuberculosis genotype has been associated with drug resistance. Elucidation of the mechanisms underlying this epidemiological finding may have an important role in the control of MDR-TB. The aim of this study was to evaluate the fitness of drug-susceptible and MDR M. tuberculosis strains of the W-Beijing genotype compared with that of Non-W-Beijing strains. METHODOLOGY/PRINCIPAL FINDINGS: Fitness of M. tuberculosis strains was determined by evaluating the difference in the growth curves obtained in the MGIT960 automated system and assessing the competitive growth capacity between W-Beijing and non-W-Beijing strains. The W-Beijing MDR strains had a significant longer lag phase duration compared to the other strains but did not present a significant fitness cost. When grown in competition they had an advantage only in medium containing 0.1% Tween 80. CONCLUSIONS/SIGNIFICANCE: It was not possible to confirm a selective advantage of W-Beijing strains to grow, except for differences in their resistance to Tween 80. Further studies are needed to elucidate the putative advantage of W-Beijing strains compared to other genotypes
A changing environment and the epidemiology of tsetse-transmitted livestock trypanosomiasis
No full textThe distribution, prevalence and impact of vector-borne diseases are often affected by anthropogenic environmental changes that alter the interactions between the host, the parasite and the vector. In the case of tsetse-transmitted livestock trypanosomiasis these changes are a result of the encroachment of people and their livestock into tsetse-infected wild areas. This has created a sequence of new epidemiological settings that is changing the relative importance of the domestic or sylvatic trypanosome transmission cycles and is causing concomitant changes in the impact of the disease on livestock. These changes in the dynamics of the epidemiology have an important impact on the factors that need to be considered when developing area-specific strategies for the future management of tsetse-transmitted livestock trypanosomiasis