by Furaha Mramba, Francis Oloo, Mechtilda Byamungu, Thomas Kröber, Andrew McMullin, Steve Mihok, Patrick M. Guerin

Background

Here we set out to standardize long-lasting, visually-attractive devices for Glossina swynnertoni, a vector of both human and animal trypanosomiasis in open savannah in Tanzania and Kenya, and in neighbouring conservation areas used by pastoralists. The goal was to determine the most practical device/material that would induce the strongest landing response in G. swynnertoni for use in area-wide population suppression of this fly with insecticide-impregnated devices.

Methods and Findings

Trials were conducted in wet and dry seasons in the Serengeti and Maasai Mara to measure the performance of traps and targets of different sizes and colours, with and without chemical baits, at different population densities and under different environmental conditions. Adhesive film was used as a simple enumerator at these remote locations to compare trapping efficiencies of devices. Independent of season or presence of chemical baits, targets in phthalogen blue or turquoise blue cloth with adhesive film were the best devices for capturing G. swynnertoni in all situations, catching up to 19 times more flies than pyramidal traps. Baiting with chemicals did not affect the relative performance of devices. Fly landings were two times higher on 1 m2 blue-black targets as on pyramidal traps when equivalent areas of both were covered with adhesive film. Landings on 1 m2 blue-black targets were compared to those on smaller phthalogen blue 0.5 m2 all-blue or blue-black-blue cloth targets, and to landings on all-blue plastic 0.32–0.47 m2 leg panels painted in phthalogen blue. These smaller targets and leg panels captured equivalent numbers of G. swynnertoni per unit area as bigger targets.

Conclusions

Leg panels and 0.5 m2 cloth targets show promise as cost effective devices for management of G. swynnertoni as they can be used for both control (insecticide-impregnated cloth) and for sampling (rigid plastic with insect glue or adhesive film) of populations.

by Niall Conroy, Susan Vlack, Julian M. Williams, John J. Patten, Robert L. Horvath, Stephen B. Lambert

Background

Australia uses a protocol combining human rabies immunoglobulin (HRIG) and rabies vaccine for post-exposure prophylaxis (PEP) of rabies and Australian bat lyssavirus (ABLV), with the aim of achieving an antibody titre of ≥0.5 IU/ml, as per World Health Organization (WHO) guidelines, as soon as possible.

Methodology/Principal Findings

We present the course of PEP administration and serological testing for four men with complex requirements. Following dog bites in Thailand, two men (62 years old, 25 years old) received no HRIG and had delayed vaccine courses: 23 days between dose two and three, and 18 days between dose one and two, respectively. Both seroconverted following dose four. Another 62-year-old male, who was HIV-positive (normal CD4 count), also suffered a dog bite and had delayed care receiving IM rabies vaccine on days six and nine in Thailand. Back in Australia, he received three single and one double dose IM vaccines followed by another double dose of vaccine, delivered intradermally and subcutaneously, before seroconverting. A 23-year-old male with a history of allergies received simultaneous HRIG and vaccine following potential ABLV exposure, and developed rash, facial oedema and throat tingling, which was treated with a parenteral antihistamine and tapering dose of steroids. Serology showed he seroconverted following dose four.

Conclusions/Significance

These cases show that PEP can be complicated by exposures in tourist settings where reliable prophylaxis may not be available, where treatment is delayed or deviates from World Health Organization recommendations. Due to the potentially short incubation time of rabies/ABLV, timely prophylaxis after a potential exposure is needed to ensure a prompt and adequate immune response, particularly in patients who are immune-suppressed or who have not received HRIG. Serology should be used to confirm an adequate response to PEP when treatment is delayed or where a concurrent immunosuppressing medical condition or therapy exists.

by Tapan Bhattacharyya, Marleen Boelaert, Michael A. Miles

Background

Visceral leishmaniasis (VL), caused by infection with Leishmania donovani 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-Leishmania 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 L. chagasi, now recognised as L. infantum, 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 L. donovani strains.

Methodology/Principal Findings

Coding sequences of rK39 homologues from East African L. donovani 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.

Conclusions/Significance

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.

by Peter J. Hotez, T. Dorina Papageorgiou

by Deu John M. Cruz, Andrea Cristine Koishi, Juliana Bosso Taniguchi, Xiaolan Li, Rafaela Milan Bonotto, Joo Hwan No, Keum Hyun Kim, Sungmin Baek, Hee Young Kim, Marc Peter Windisch, Ana Luiza Pamplona Mosimann, Luana de Borba, Michel Liuzzi, Michael Adsetts Edberg Hansen, Claudia Nunes Duarte dos Santos, Lucio Holanda Freitas-Junior

Dengue virus is a mosquito-borne flavivirus that has a large impact in global health. It is considered as one of the medically important arboviruses, and developing a preventive or therapeutic solution remains a top priority in the medical and scientific community. Drug discovery programs for potential dengue antivirals have increased dramatically over the last decade, largely in part to the introduction of high-throughput assays. In this study, we have developed an image-based dengue high-throughput/high-content assay (HT/HCA) using an innovative computer vision approach to screen a kinase-focused library for anti-dengue compounds. Using this dengue HT/HCA, we identified a group of compounds with a 4-(1-aminoethyl)-N-methylthiazol-2-amine as a common core structure that inhibits dengue viral infection in a human liver-derived cell line (Huh-7.5 cells). Compounds CND1201, CND1203 and CND1243 exhibited strong antiviral activities against all four dengue serotypes. Plaque reduction and time-of-addition assays suggests that these compounds interfere with the late stage of viral infection cycle. These findings demonstrate that our image-based dengue HT/HCA is a reliable tool that can be used to screen various chemical libraries for potential dengue antiviral candidates.

by Direk Limmathurotsakul, Manas Kanoksil, Vanaporn Wuthiekanun, Rungrueng Kitphati, Bianca deStavola, Nicholas P. J. Day, Sharon J. Peacock

Background

Melioidosis is a serious infectious disease caused by the Category B select agent and environmental saprophyte, Burkholderia pseudomallei. Most cases of naturally acquired infection are assumed to result from skin inoculation after exposure to soil or water. The aim of this study was to provide evidence for inoculation, inhalation and ingestion as routes of infection, and develop preventive guidelines based on this evidence.

Methods/Principal Findings

A prospective hospital-based 1∶2 matched case-control study was conducted in Northeast Thailand. Cases were patients with culture-confirmed melioidosis, and controls were patients admitted with non-infectious conditions during the same period, matched for gender, age, and diabetes mellitus. Activities of daily living were recorded for the 30-day period before onset of symptoms, and home visits were performed to obtain drinking water and culture this for B. pseudomallei. Multivariable conditional logistic regression analysis based on 286 cases and 512 controls showed that activities associated with a risk of melioidosis included working in a rice field (conditional odds ratio [cOR] = 2.1; 95% confidence interval [CI] 1.4–3.3), other activities associated with exposure to soil or water (cOR = 1.4; 95%CI 0.8–2.6), an open wound (cOR = 2.0; 95%CI 1.2–3.3), eating food contaminated with soil or dust (cOR = 1.5; 95%CI 1.0–2.2), drinking untreated water (cOR = 1.7; 95%CI 1.1–2.6), outdoor exposure to rain (cOR = 2.1; 95%CI 1.4–3.2), water inhalation (cOR = 2.4; 95%CI 1.5–3.9), current smoking (cOR = 1.5; 95%CI 1.0–2.3) and steroid intake (cOR = 3.1; 95%CI 1.4–6.9). B. pseudomallei was detected in water source(s) consumed by 7% of cases and 3% of controls (cOR = 2.2; 95%CI 0.8–5.8).

Conclusions/Significance

We used these findings to develop the first evidence-based guidelines for the prevention of melioidosis. These are suitable for people in melioidosis-endemic areas, travelers and military personnel. Public health campaigns based on our recommendations are under development in Thailand.

by Carli M. Halpenny, Claire Paller, Kristine G. Koski, Victoria E. Valdés, Marilyn E. Scott

Background

Few studies have investigated the relative influence of individual susceptibility versus household exposure factors versus regional clustering of infection on soil transmitted helminth (STH) transmission. The present study examined reinfection dynamics and spatial clustering of Ascaris lumbricoides, Trichuris trichiura and hookworm in an extremely impoverished indigenous setting in rural Panamá over a 16 month period that included two treatment and reinfection cycles in preschool children.

Methodology/Principle Findings

Spatial cluster analyses were used to identify high prevalence clusters for each nematode. Multivariate models were then used (1) to identify factors that differentiated households within and outside the cluster, and (2) to examine the relative contribution of regional (presence in a high prevalence cluster), household (household density, asset-based household wealth, household crowding, maternal education) and individual (age, sex, pre-treatment eggs per gram (epg) feces, height-for-age, latrine use) factors on preschool child reinfection epgs for each STH. High prevalence spatial clusters were detected for Trichuris and hookworm but not for Ascaris. These clusters were characterized by low household density and low household wealth indices (HWI). Reinfection epg of both hookworm and Ascaris was positively associated with pre-treatment epg and was higher in stunted children. Additional individual (latrine use) as well as household variables (HWI, maternal education) entered the reinfection models for Ascaris but not for hookworm.

Conclusions/Significance

Even within the context of extreme poverty in this remote rural setting, the distinct transmission patterns for hookworm, Trichuris and Ascaris highlight the need for multi-pronged intervention strategies. In addition to poverty reduction, improved sanitation and attention to chronic malnutrition will be key to reducing Ascaris and hookworm transmission.

by Wuelton Marcelo Monteiro, Ana Paula Margioto Teston, Ana Paula Gruendling, Daniele dos Reis, Mônica Lúcia Gomes, Silvana Marques de Araújo, Maria Terezinha Bahia, Laylah Kelre Costa Magalhães, Jorge Augusto de Oliveira Guerra, Henrique Silveira, Max Jean de Ornelas Toledo, Maria das Graças Vale Barbosa

Background

In the Brazilian Amazon, clinical and epidemiological frameworks of Chagas disease are very dissimilar in relation to the endemic classical areas of transmission, possibly due to genetic and biological characteristics of the circulating Trypanosoma cruzi stocks. Twenty six T. cruzi stocks from Western Amazon Region attributed to the TcI and TcIV DTUs were comparatively studied in Swiss mice to test the hypothesis that T. cruzi clonal structure has a major impact on its biological and medical properties.

Methodology/Principal Findings

Seventeen parameters were assayed in mice infected with 14 T. cruzi strains belonging to DTU TcI and 11 strains typed as TcIV. In comparison with TcI, TcIV stocks promoted a significantly shorter pre-patent period (p<0.001), a longer patent period (p<0.001), higher values of mean daily parasitemia (p = 0.009) and maximum of parasitemia (p = 0.015), earlier days of maximum parasitemia (p<0.001) and mortality (p = 0.018), higher mortality rates in the acute phase (p = 0.047), higher infectivity rates (p = 0.002), higher positivity in the fresh blood examination (p<0.001), higher positivity in the ELISA at the early chronic phase (p = 0.022), and a higher positivity in the ELISA at the late chronic phase (p = 0.003). On the other hand TcI showed higher values of mortality rates in the early chronic phase (p = 0.014), higher frequency of mice with inflammatory process in any organ (p = 0.005), higher frequency of mice with tissue parasitism in any organ (p = 0.027) and a higher susceptibility to benznidazole (p = 0.002) than TcIV. Survival analysis showing the time elapsed from the day of inoculation to the beginning of the patent period was significantly shorter for TcIV strains and the death episodes triggered following the infection with TcI occurred significantly later in relation to TcIV. The notable exceptions come from positivity in the hemocultures and PCR, for which the results were similar.

Conclusion/Significance

T. cruzi stocks belonging to TcI and TcIV DTUs from Brazilian Amazon are divergent in terms of biological and medical properties in mice.

by Eduardo A. Undurraga, Yara A. Halasa, Donald S. Shepard

Background

Dengue virus infection is the most common arthropod-borne disease of humans and its geographical range and infection rates are increasing. Health policy decisions require information about the disease burden, but surveillance systems usually underreport the total number of cases. These may be estimated by multiplying reported cases by an expansion factor (EF).

Methods and Findings

As a key step to estimate the economic and disease burden of dengue in Southeast Asia (SEA), we projected dengue cases from 2001 through 2010 using EFs. We conducted a systematic literature review (1995–2011) and identified 11 published articles reporting original, empirically derived EFs or the necessary data, and 11 additional relevant studies. To estimate EFs for total cases in countries where no empirical studies were available, we extrapolated data based on the statistically significant inverse relationship between an index of a country's health system quality and its observed reporting rate. We compiled an average 386,000 dengue episodes reported annually to surveillance systems in the region, and projected about 2.92 million dengue episodes. We conducted a probabilistic sensitivity analysis, simultaneously varying the most important parameters in 20,000 Monte Carlo simulations, and derived 95% certainty level of 2.73–3.38 million dengue episodes. We estimated an overall EF in SEA of 7.6 (95% certainty level: 7.0–8.8) dengue cases for every case reported, with an EF range of 3.8 for Malaysia to 19.0 in East Timor.

Conclusion

Studies that make no adjustment for underreporting would seriously understate the burden and cost of dengue in SEA and elsewhere. As the sites of the empirical studies we identified were not randomly chosen, the exact extent of underreporting remains uncertain. Nevertheless, the results reported here, based on a systematic analysis of the available literature, show general consistency and provide a reasonable empirical basis to adjust for underreporting.

by Roy Faiman, Ibrahim Abbasi, Charles Jaffe, Yoav Motro, Abdelmagid Nasereddin, Lionel F. Schnur, Moshe Torem, Francine Pratlong, Jean-Pierre Dedet, Alon Warburg

In 2006/7, 18 cases of cutaneous leishmaniasis (CL) were reported for the first time from Sde Eliyahu (pop. 650), a village in the Beit She'an valley of Israel. Between 2007–2011, a further 88 CL cases were diagnosed bringing the total to 106 (16.3% of the population of Sde Eliyahu). The majority of cases resided in the south-western part of the village along the perimeter fence. The causative parasite was identified as Leishmania major Yakimoff & Schokhor, 1914 (Kinetoplastida: Trypanosomatidae). Phlebotomus papatasi (Scopoli), 1786 (Diptera: Psychodidae) was found to be the most abundant phlebotomine species comprising 97% of the sand flies trapped inside the village, and an average of 7.9% of the females were positive for Leishmania ITS1 DNA. Parasite isolates from CL cases and a sand fly were characterized using several methods and shown to be L. major. During a comprehensive survey of rodents 164 Levant voles Microtus guentheri Danford & Alston, 1880 (Rodentia: Cricetidae) were captured in alfalfa fields bordering the village. Of these 27 (16.5%) tested positive for Leishmania ITS1 DNA and shown to be L. major by reverse line blotting. A very high percentage (58.3% - 21/36) of Tristram's jirds Meriones tristrami Thomas, 1892 (Rodentia: Muridae), found further away from the village also tested positive for ITS1 by PCR. Isolates of L. major were successfully cultured from the ear of a wild jird found positive by ITS1 PCR. Although none of the wild PCR-positive voles exhibited external pathology, laboratory-reared voles that were infected by intradermal L. major inoculation, developed patent lesions and sand flies became infected by feeding on the ears of these laboratory-infected voles. This is the first report implicating M. guentheri and M. tristrami as reservoirs of Leishmania. The widespread co-distribution of M. guentheri and P. papatasi, suggests a significant threat from the spread of CL caused by L. major in the Middle East, central Asia and southern Europe.

by Donald S. Shepard, Eduardo A. Undurraga, Yara A. Halasa

Background

Dengue poses a substantial economic and disease burden in Southeast Asia (SEA). Quantifying this burden is critical to set policy priorities and disease-control strategies.

Methods and Findings

We estimated the economic and disease burden of dengue in 12 countries in SEA: Bhutan, Brunei, Cambodia, East-Timor, Indonesia, Laos, Malaysia, Myanmar, Philippines, Singapore, Thailand, and Viet Nam. We obtained reported cases from multiple sources—surveillance data, World Health Organization (WHO), and published studies—and adjusted for underreporting using expansion factors from previous literature. We obtained unit costs per episode through a systematic literature review, and completed missing data using linear regressions. We excluded costs such as prevention and vector control, and long-term sequelae of dengue. Over the decade of 2001–2010, we obtained an annual average of 2.9 million (m) dengue episodes and 5,906 deaths. The annual economic burden (with 95% certainty levels) was US$950m (US$610m–US$1,384m) or about US$1.65 (US$1.06–US$2.41) per capita. The annual number of disability-adjusted life years (DALYs), based on the original 1994 definition, was 214,000 (120,000–299,000), which is equivalent to 372 (210–520) DALYs per million inhabitants.

Conclusion

Dengue poses a substantial economic and disease burden in SEA with a DALY burden per million inhabitants in the region. This burden is higher than that of 17 other conditions, including Japanese encephalitis, upper respiratory infections, and hepatitis B.

by Juan David Ramírez, Marleny Montilla, Zulma M. Cucunubá, Astrid Carolina Floréz, Pilar Zambrano, Felipe Guhl

Background

Trypanosoma cruzi, the causative agent of Chagas disease, displays significant genetic variability revealed by six Discrete Typing Units (TcI-TcVI). In this pathology, oral transmission represents an emerging epidemiological scenario where different outbreaks associated to food/beverages consumption have been reported in Argentina, Bolivia, Brazil, Ecuador and Venezuela. In Colombia, six human oral outbreaks have been reported corroborating the importance of this transmission route. Molecular epidemiology of oral outbreaks is barely known observing the incrimination of TcI, TcII, TcIV and TcV genotypes.

Methodology and Principal Findings

High-throughput molecular characterization was conducted performing MLMT (Multilocus Microsatellite Typing) and mtMLST (mitochondrial Multilocus Sequence Typing) strategies on 50 clones from ten isolates. Results allowed observing the occurrence of TcI, TcIV and mixed infection of distinct TcI genotypes. Thus, a majority of specific mitochondrial haplotypes and allelic multilocus genotypes associated to the sylvatic cycle of transmission were detected in the dataset with the foreseen presence of mitochondrial haplotypes and allelic multilocus genotypes associated to the domestic cycle of transmission.

Conclusions

These findings suggest the incrimination of sylvatic genotypes in the oral outbreaks occurred in Colombia. We observed patterns of super-infection and/or co-infection with a tailored association with the severe forms of myocarditis in the acute phase of the disease. The transmission dynamics of this infection route based on molecular epidemiology evidence was unraveled and the clinical and biological implications are discussed.

by Juliane Schroeder, H. Adrienne McGachy, Stuart Woods, Robin Plevin, James Alexander

We have recently demonstrated that MAP kinase phosphatase 2 (MKP-2) deficient C57BL/6 mice, unlike their wild-type counterparts, are unable to control infection with the protozoan parasite Leishmania mexicana. Increased susceptibility was associated with elevated Arginase-1 levels and reduced iNOS activity in macrophages as well as a diminished TH1 response. By contrast, in the present study footpad infection of MKP-2−/− mice with L. major resulted in a healing response as measured by lesion size and parasite numbers similar to infected MKP-2+/+ mice. Analysis of immune responses following infection demonstrated a reduced TH1 response in MKP-2−/− mice with lower parasite specific serum IgG2b levels, a lower frequency of IFN-γ and TNF-α producing CD4+ and CD8+ T cells and lower antigen stimulated spleen cell IFN-γ production than their wild-type counterparts. However, infected MKP-2−/− mice also had similarly reduced levels of antigen induced spleen and lymph node cell IL-4 production compared with MKP-2+/+ mice as well as reduced levels of parasite-specific IgG1 in the serum, indicating a general T cell hypo-responsiveness. Consequently the overall TH1/TH2 balance was unaltered in MKP-2−/− compared with wild-type mice. Although non-stimulated MKP-2−/− macrophages were more permissive to L. major growth than macrophages from MKP-2+/+ mice, reflecting their reduced iNOS and increased Arginase-1 expression, LPS/IFN-γ activation was equally effective at controlling parasite growth in MKP-2−/− and MKP-2+/+ macrophages. Consequently, in the absence of any switch in the TH1/TH2 balance in MKP-2−/− mice, no significant change in disease phenotype was observed.

by Javier Carrión, M. Angeles Abengozar, María Fernández-Reyes, Carlos Sánchez-Martín, Eduardo Rial, Gustavo Domínguez-Bernal, M. Mar González-Barroso

Background

Uncoupling protein 2 (UCP2) is a mitochondrial transporter that has been shown to lower the production of reactive oxygen species (ROS). Intracellular pathogens such as Leishmania upregulate UCP2 and thereby suppress ROS production in infected host tissues, allowing the multiplication of parasites within murine phagocytes. This makes host UCP2 and ROS production potential targets in the development of antileishmanial therapies. Here we explore how UCP2 affects the outcome of cutaneous leishmaniosis (CL) and visceral leishmaniosis (VL) in wild-type (WT) C57BL/6 mice and in C57BL/6 mice lacking the UCP2 gene (UCP2KO).

Methodology and Findings

To investigate the effects of host UCP2 deficiency on Leishmania infection, we evaluated parasite loads and cytokine production in target organs. Parasite loads were significantly lower in infected UCP2KO mice than in infected WT mice. We also found that UCP2KO mice produced significantly more interferon-γ (IFN-γ), IL-17 and IL-13 than WT mice (P<0.05), suggesting that UCP2KO mice are resistant to Leishmania infection.

Conclusions

In this way, UCP2KO mice were better able than their WT counterparts to overcome L. major and L. infantum infections. These findings suggest that upregulating host ROS levels, perhaps by inhibiting UPC2, may be an effective approach to preventing leishmaniosis.

by Sonja Hartnack, Christine M. Budke, Philip S. Craig, Qiu Jiamin, Belgees Boufana, Maiza Campos-Ponce, Paul R. Torgerson

Background

The diagnosis of canine echinococcosis can be a challenge in surveillance studies because there is no perfect gold standard that can be used routinely. However, unknown test specificities and sensitivities can be overcome using latent-class analysis with appropriate data.

Methodology

We utilised a set of faecal and purge samples used previously to explore the epidemiology of canine echinococcosis on the Tibetan plateau. Previously only the purge results were reported and analysed in a largely deterministic way. In the present study, additional diagnostic tests of copro-PCR and copro-antigen ELISA were undertaken on the faecal samples. This enabled a Bayesian analysis in a latent-class model to examine the diagnostic performance of a genus specific copro-antigen ELISA, species-specific copro-PCR and arecoline purgation. Potential covariates including co-infection with Taenia, age and sex of the dog were also explored. The dependence structure of these diagnostic tests could also be analysed.

Principle findings

The most parsimonious result, indicated by deviance-information criteria, suggested that co-infection with Taenia spp. was a significant covariate with the Echinococcus infection. The copro-PCRs had estimated sensitivities of 89% and 84% respectively for the diagnoses of Echinococcus multilocularis and E. granulosus. The specificities for the copro-PCR were estimated at 93 and 83% respectively. Copro-antigen ELISA had sensitivities of 55 and 57% for the diagnosis of E. multilocularis and E. granulosus and specificities of 71 and 69% respectively. Arecoline purgation with an assumed specificity of 100% had estimated sensitivities of 76% and 85% respectively.

Significance

This study also shows that incorporating diagnostic uncertainty, in other words assuming no perfect gold standard, and including potential covariates like sex or Taenia co-infection into the epidemiological analysis may give different results than if the diagnosis of infection status is assumed to be deterministic and this approach should therefore be used whenever possible.

by Inés Zulantay, Werner Apt, Daniel Ramos, Lorena Godoy, Claudio Valencia, Matías Molina, Eduardo Sepúlveda, Patricio Thieme, Gabriela Martínez, Gabriela Corral

by Andrés E. Ciocchini, Diego A. Rey Serantes, Luciano J. Melli, Jeremy A. Iwashkiw, Bettina Deodato, Jorge Wallach, Mario F. Feldman, Juan E. Ugalde, Diego J. Comerci

Brucellosis is a highly contagious zoonosis and still a major human health problem in endemic areas of the world. Although several diagnostic tools are available, most of them are difficult to implement especially in developing countries where complex health facilities are limited. Taking advantage of the identical structure and composition of the Brucella spp. and Yersinia enterocolitica O:9 O-polysaccharide, we explored the application of a recombinant Y. enterocolitica O:9-polysaccharide-protein conjugate (OAg-AcrA) as a novel antigen for diagnosis of human brucellosis. We have developed and validated an indirect immunoassay using OAg-AcrA coupled to magnetic beads. OAg-AcrA was produced and purified with high yields in Y. enterocolitica O:9 cells co-expressing the oligosaccharyltransferase PglB and the protein acceptor AcrA of Campylobacter jejuni without the need for culturing Brucella. Expression of PglB and AcrA in Y. enterocolitica resulted in the transfer of the host O-polysaccharide from its lipid carrier to AcrA. To validate the assay and determine the cutoff values, a receiver-operating characteristic analysis was performed using a panel of characterized serum samples obtained from healthy individuals and patients of different clinical groups. Our results indicate that, using this assay, it is possible to detect infection caused by the three main human brucellosis agents (B. abortus, B. melitensis and B. suis) and select different cutoff points to adjust sensitivity and specificity levels as needed. A cutoff value of 13.20% gave a sensitivity of 100% and a specificity of 98.57%, and a cutoff value of 16.15% resulted in a test sensitivity and specificity of 93.48% and 100%, respectively. The high diagnostic accuracy, low cost, reduced assay time and simplicity of this new glycoconjugate-magnetic beads assay makes it an attractive diagnostic tool for using not only in clinics and brucellosis reference laboratories but also in locations with limited laboratory infrastructure and/or minimally trained community health workers.

by Michel A. Marin, Cristiane C. Thompson, Fernanda S. Freitas, Erica L. Fonseca, A. Oladipo Aboderin, Sambo B. Zailani, Naa Kwarley E. Quartey, Iruka N. Okeke, Ana Carolina P. Vicente

Background

The current millennium has seen a steep rise in the number, size and case-fatalities of cholera outbreaks in many African countries. Over 40,000 cases of cholera were reported from Nigeria in 2010. Variants of Vibrio cholerae O1 El Tor biotype have emerged but very little is known about strains causing cholera outbreaks in West Africa, which is crucial for the implementation of interventions to control epidemic cholera.

Methodology/Principal Findings

V. cholerae isolates from outbreaks of acute watery diarrhea in Nigeria from December, 2009 to October, 2010 were identified by standard culture methods. Fifteen O1 and five non-O1/non-O139 strains were analyzed; PCR and sequencing targeted regions associated with virulence, resistance and biotype were performed. We also studied genetic interrelatedness among the strains by multilocus sequence analysis and pulsed-field gel electrophoresis. The antibiotic susceptibility was tested by the disk diffusion method and E-test. We found that multidrug resistant atypical El Tor strains, with reduced susceptibility to ciprofloxacin and chloramphenicol, characterized by the presence of the SXT element, and gyrASer83Ile/parCSer85Leu alleles as well CTX phage and TCP cluster characterized by rstRElTor, ctxB-7 and tcpACIRS alleles, respectively, were largely responsible for cholera outbreaks in 2009 and 2010. We also identified and characterized a V. cholerae non-O1/non-O139 lineage from cholera-like diarrhea cases in Nigeria.

Conclusions/Significance

The recent Nigeria outbreaks have been determined by multidrug resistant atypical El Tor and non-O1/non-O139 V. cholerae strains, and it seems that the typical El Tor, from the beginning of seventh cholera pandemic, is no longer epidemic/endemic in this country. This scenario is similar to the East Africa, Asia and Caribbean countries. The detection of a highly virulent, antimicrobial resistant lineage in Nigeria is worrisome and points to a need for vaccine-based control of the disease. This study has also revealed the putative importance of non-O1/non-O139 V. cholerae in diarrheal disease in Nigeria.

by Vladimir A. Timoshevskiy, David W. Severson, Becky S. deBruyn, William C. Black, Igor V. Sharakhov, Maria V. Sharakhova

Background

Aedes aegypti, the yellow fever mosquito, is an efficient vector of arboviruses and a convenient model system for laboratory research. Extensive linkage mapping of morphological and molecular markers localized a number of quantitative trait loci (QTLs) related to the mosquito's ability to transmit various pathogens. However, linking the QTLs to Ae. aegypti chromosomes and genomic sequences has been challenging because of the poor quality of polytene chromosomes and the highly fragmented genome assembly for this species.

Methodology/Principal Findings

Based on the approach developed in our previous study, we constructed idiograms for mitotic chromosomes of Ae. aegypti based on their banding patterns at early metaphase. These idiograms represent the first cytogenetic map developed for mitotic chromosomes of Ae. aegypti. One hundred bacterial artificial chromosome clones carrying major genetic markers were hybridized to the chromosomes using fluorescent in situ hybridization. As a result, QTLs related to the transmission of the filarioid nematode Brugia malayi, the avian malaria parasite Plasmodium gallinaceum, and the dengue virus, as well as sex determination locus and 183 Mbp of genomic sequences were anchored to the exact positions on Ae. aegypti chromosomes. A linear regression analysis demonstrated a good correlation between positions of the markers on the physical and linkage maps. As a result of the recombination rate variation along the chromosomes, 12 QTLs on the linkage map were combined into five major clusters of QTLs on the chromosome map.

Conclusion

This study developed an integrated linkage, chromosome, and genome map—iMap—for the yellow fever mosquito. Our discovery of the localization of multiple QTLs in a few major chromosome clusters suggests a possibility that the transmission of various pathogens is controlled by the same genomic loci. Thus, the iMap will facilitate the identification of genomic determinants of traits responsible for susceptibility or refractoriness of the mosquito to diverse pathogens.

by Epco Hasker, Sangeeta Kansal, Paritosh Malaviya, Kamlesh Gidwani, Albert Picado, Rudra Pratap Singh, Ankita Chourasia, Abhishek Kumar Singh, Ravi Shankar, Joris Menten, Mary Elizabeth Wilson, Marleen Boelaert, Shyam Sundar

Introduction

Asymptomatic persons infected with the parasites causing visceral leishmaniasis (VL) usually outnumber clinically apparent cases by a ratio of 4–10 to 1. We describe patterns of markers of Leishmania donovani infection and clinical VL in relation to age in Bihar, India.

Methods

We selected eleven villages highly endemic for Leishmania donovani. During a 1-year interval we conducted two house to house surveys during which we collected blood samples on filter paper from all consenting individuals aged 2 years and above. Samples were tested for anti-leishmania serology by Direct Agglutination Test (DAT) and rK39 ELISA. Data collected during the surveys included information on episodes of clinical VL among study participants.

Results

We enrolled 13,163 persons; 6.2% were reactive to DAT and 5.9% to rK39. Agreement between the tests was weak (kappa = 0.30). Among those who were negative on both tests at baseline, 3.6% had converted to sero-positive on either of the two tests one year later. Proportions of sero-positives and sero-converters increased steadily with age. Clinical VL occurred mainly among children and young adults (median age 19 years).

Discussion

Although infection with L. donovani is assumed to be permanent, serological markers revert to negative. Most VL cases occur at younger ages, yet we observed a steady increase with age in the frequency of sero-positivity and sero-conversion. Our findings can be explained by a boosting effect upon repeated exposure to the parasite or by intermittent release of parasites in infected subjects from safe target cells. A certain proportion of sero-negative subjects could have been infected but below the threshold of antibody abundance for our serologic testing.