Validation of a simple resazurin-based promastigote assay for the routine monitoring of miltefosine susceptibility in clinical isolates of Leishmania donovani

Simple, cost-effective approach for routine surveillance of parasite susceptibility to antileishmanial drug miltefosine (MIL) is highly desirable for controlling emergence of drug resistance in visceral leishmaniasis (VL). We validated a simple resazurin-based fluorimetric assay using promastigotes to track natural MIL tolerance in Leishmania donovani parasites from VL cases (n = 17) against standard amastigote assay, in two different labs in India. The inter-stage MIL susceptibility correlated strongly (r = 0.70, p = 0.0018) using J774.A.1 macrophage cell line-based amastigote assay and fluorescence-based resazurin assay for promastigotes. Investigation of inter-stage MIL susceptibility for the same set of clinical isolates in another lab also showed a strong correlation (r = 0.72, p = 0.0012) using mouse peritoneal macrophages for amastigote assay and resazurin-based alamar blue assay for promastigotes. Additionally, parasites from post-kala-azar dermal leishmaniasis (PKDL) lesions (n = 7, r = 0.78, p = 0.046) and MIL-induced parasites (r = 0.92, p = 0.0001; n = 3) also exhibited a strongly correlated inter-stage miltefosine susceptibility. Thus, our results support the utility of resazurin assay as a simplified biological tool for MIL susceptibility monitoring in clinical isolates from MIL-treated VL/PKDL patients

Seroepidemiological study of caprine toxoplasmosis in East and West Shewa Zones, Oromia Regional State, Central Ethiopia

Toxoplasmosis is a global zoonosis caused by Toxoplasma gondii, an intracellular apicomplexan parasite. The objectives of this study were to estimate the animal and flock level seroprevalence and risk factors associated with toxoplasmosis in goats of Central Ethiopia. In Ethiopia, goats are economically important animals used for meat and milk production. The study was cross-sectional and 927 blood samples from 187 goat flocks were collected to examine T. gondii specific IgG antibodies by enzyme linked immunosorbent assay (ELISA). A questionnaire was used to collect data on the potential risk factors. The study revealed flock and animal level seroprevalence of 58.3% (109/187; 95% confidence interval [CI]: 51.16, 65.42) and 19.7% (183/927; 95% CI: 17.17, 22.31), respectively. The likelihood of acquiring T. gondii infection was higher in semi-intensively managed goats (Odds ratio [OR]=2.48, 95% confidence interval [CI]: 1.14, 5.37; P=0.022) than in extensively managed goats, in females than in males (OR=1.84, 95% CI: 1.16, 2.92; P=0.010), in adults than in young animals (OR=2.00, 95% CI: 1.21, 3.28; P=0.006), in small than in large flocks (OR=2.03, 95% CI: 1.03, 3.98; P=0.040), in goats kept under sedentary (OR=2.81, 95% CI: 1.41, 5.59; P=0.003) and agropastoral farming system (OR=3.62, 95% CI: 1.83, 7.18; P<0.001) than pastoral farming system and in goats allowed to drink water from the tap than those drinking from river and stagnant water bodies (OR=9.25, 95% CI: 3.04, 28.15; P<0.001). Our study indicates that exposure of goats to oocysts of T. gondii is widespread. We recommend further studies to determine the genotype of the parasite, public health and economic impacts of toxoplasmosis and the role of raw goat meat and milk as a source of infection for consumers

Effects of managed care mechanisms on access to healthcare: results from a qualitative study in Colombia

BACKGROUND: Managed competition has underpinned most health sector reforms aimed at improving access and efficiency, in Latin America and other countries. The aim of the paper is to analyse barriers to healthcare that emerge from the introduction of managed care mechanisms in Colombia. METHODS: Qualitative, exploratory, and descriptive-interpretative research was carried out on the basis of case studies of four healthcare networks, comprised of insurers and their providers. Individual semi-structured interviews were conducted with a theoretical sample of informants (managers, professionals, and users), between 24 and 61 per network. The final sample size was reached by saturation of information. An inductive thematic content analysis was conducted. The study areas were two municipalities of Colombia, in which most of the population live in poverty. RESULTS: A number of managed care mechanisms that act as barriers to access were identified by all informants, regardless of area and type of insurance regime. These mechanisms act directly on the patient (authorizations, fragmented insurance) or on the providers (purchasing mechanisms or limits to medical practice). The predominant mechanism appears to be related to the type of agreement established between insurers and providers. The reason for these barriers, according to informants, is insurers' search for profitability. As a consequence, there is delay in or no access to adequate treatment. This is particularly evident in secondary care. CONCLUSION: A variety of managed care strategies that effectively hinder access to healthcare have been introduced by insurers, casting doubt on the usefulness of their application in low-income countries and profit-making contexts. Copyright (c) 2012 John Wiley & Sons, Ltd

M48U1 CD4 mimetic has a sustained inhibitory effect on cell-associated HIV-1 by attenuating virion infectivity through gp120 shedding

BACKGROUND: HIV-1 infected cells can establish new infections by crossing the vaginal epithelia and subsequently producing virus in a milieu that avoids the high microbicide concentrations of the vaginal lumen. FINDINGS: To address this problem, here, we report that pretreatment of HIV-infected peripheral blood mononuclear cells (PBMCs) with a 27 amino acid CD4-mimetic, M48U1, causes dramatic and prolonged reduction of infectious virus output, due to its induction of gp120 shedding. CONCLUSIONS: M48U1 may, therefore, be valuable for prophylaxis of mucosal HIV-1 transmission

HIV-1 protease inhibitors for treatment of visceral leishmaniasis in HIV-co-infected individuals

The global prevalence of HIV is a major challenge for control of visceral leishmaniasis, a disseminated protozoan infection. In some east African regions, up to 40% of patients with visceral leishmaniasis are co-infected with HIV. Management of visceral leishmaniasis in such patients is complicated by treatment failures and relapses, even while patients are receiving standard antiretroviral therapy. In-vitro studies have consistently documented an inhibitory effect of specific HIV-1 protease inhibitors on leishmania parasites, and the underlying mechanism is partly explained. With the global scaling up of HIV treatment, HIV-1 protease inhibitors are increasingly becoming available for second-line HIV treatment in regions where visceral leishmaniasis and HIV are endemic. However, additional research is needed before HIV-1 protease inhibitors can be taken forward for clinical use against visceral leishmaniasis in HIV-infected patients. Since the effect of protease inhibitors against Leishmania species was generally observed at high drug concentrations, efficacy and dose-response relationships should be studied in animals before these drugs are used in clinical trials. More extensive studies of all available HIV protease inhibitors are needed, including investigation of drug interactions and emergence of drug-resistant parasites. In addition to exploring the full potential of current HIV-1 protease inhibitors against visceral leishmaniasis, leishmania-specific protease inhibitors should be developed

A cluster-randomized trial of insecticide-treated curtains for dengue vector control in Thailand

The efficacy of insecticide-treated window curtains (ITCs) for dengue vector control was evaluated in Thailand in a cluster-randomized controlled trial. A total of 2,037 houses in 26 clusters was randomized to receive the intervention or act as control (no treatment). Entomological surveys measured Aedes infestations (Breteau index, house index, container index, and pupae per person index) and oviposition indices (mean numbers of eggs laid in oviposition traps) immediately before and after intervention, and at 3-month intervals over 12 months. There were no consistent statistically significant differences in entomological indices between intervention and control clusters, although oviposition indices were lower (P < 0.01) in ITC clusters during the wet season. It is possible that the open housing structures in the study reduced the likelihood of mosquitoes making contact with ITCs. ITCs deployed in a region where this house design is common may be unsuitable for dengue vector control

Comparison of visceral leishmaniasis diagnostic antigens in African and Asian Leishmania donovani reveals extensive diversity and region-specific polymorphisms

BACKGROUND: Visceral leishmaniasis (VL), caused by infection with complex, remains a major public health problem in endemic regions of South Asia, East Africa, and Brazil. If untreated, symptomatic VL is usually fatal. Rapid field diagnosis relies principally on demonstration of anti- antibodies in clinically suspect cases. The rK39 immunochromatographic rapid diagnostic test (RDT) is based on rK39, encoded by a fragment of a kinesin-related gene derived from a Brazilian , now recognised as , originating from Europe. Despite its reliability in South Asia, the rK39 test is reported to have lower sensitivity in East Africa. A reason for this differential response may reside in the molecular diversity of the rK39 homologous sequences among East African strains. METHODOLOGYPRINCIPAL FINDINGS: Coding sequences of rK39 homologues from East African strains were amplified from genomic DNA, analysed for diversity from the rK39 sequence, and compared to South Asian sequences. East African sequences were revealed to display significant diversity from rK39. Most coding changes in the 5' half of repeats were non-conservative, with multiple substitutions involving charge changes, whereas amino acid substitutions in the 3' half of repeats were conservative. Specific polymorphisms were found between South Asian and East African strains. Diversity of HASPB1 and HASPB2 gene repeat sequences, used to flank sequences of a kinesin homologue in the synthetic antigen rK28 designed to reduce variable RDT performance, was also investigated. Non-canonical combination repeat arrangements were revealed for HASPB1 and HASPB2 gene products in strains producing unpredicted size amplicons. CONCLUSIONSSIGNIFICANCE: We demonstrate that there is extensive kinesin genetic diversity among strains in East Africa and between East Africa and South Asia, with ample scope for influencing performance of rK39 diagnostic assays. We also show the importance of targeted comparative genomics in guiding optimisation of recombinant/synthetic diagnostic antigens