Rubén O. Cimino

Diagnostic Accuracy of Five Serologic Tests for Strongyloides stercoralis Infection

Background The diagnosis of Strongyloides stercoralis (S. stercoralis) infection is hampered by the suboptimal sensitivity of fecal-based tests. Serological methods are believed to be more sensitive, although assessing their accuracy is difficult because of the lack of sensitivity of a fecal-based reference (“gold”) standard. Methods The sensitivity and specificity of 5 serologic tests for S. stercoralis (in-house IFAT, NIE-ELISA and NIE-LIPS and the commercially available Bordier-ELISA and IVD-ELISA) were assessed on 399 cryopreserved serum samples. Accuracy was measured using fecal results as the primary reference standard, but also using a composite reference standard (based on a combination of tests). Results According to the latter standard, the most sensitive test was IFAT, with 94.6% sensitivity (91.2–96.9), followed by IVD-ELISA (92.3%, 87.7–96.9). The most specific test was NIE-LIPS, with specificity 99.6% (98.9–100), followed by IVD-ELISA (97.4%, 95.5–99.3). NIE-LIPS did not cross-react with any of the specimens from subjects with other parasitic infections. NIE-LIPS and the two commercial ELISAs approach 100% specificity at a cut off level that maintains ≥70% sensitivity. Conclusions NIE-LIPS is the most accurate serologic test for the diagnosis of S. stercoralis infection. IFAT and each of the ELISA tests are sufficiently accurate, above a given cut off, for diagnosis, prevalence studies and inclusion in clinical trials.

A Public Health Response against Strongyloides stercoralis: Time to Look at Soil-Transmitted Helminthiasis in Full

Strongyloides stercoralis infections have a worldwide distribution with a global burden in terms of prevalence and morbidity that is largely ignored. A public health response against soil-transmitted helminth (STH) infections should broaden the strategy to include S. stercoralis and overcome the epidemiological, diagnostic, and therapeutic challenges that this parasite poses in comparison to Ascaris lumbricoides, Trichuris trichiura, and hookworms. The relatively poor sensitivity of single stool evaluations, which is further lowered when quantitative techniques aimed at detecting eggs are used, also complicates morbidity evaluations and adequate drug efficacy measurements, since S. stercoralis is eliminated in stools in a larval stage. Specific stool techniques for the detection of larvae of S. stercoralis, like Baermanns and Kogas agar plate, despite superiority over direct techniques are still suboptimal. New serologies using recombinant antigens and molecular-based techniques offer new hopes in those areas. The use of ivermectin rather than benzimidazoles for its treatment and the need to have curative regimens rather than lowering the parasite burden are also unique for S. stercoralis in comparison to the other STH due to its life cycle, which allows reproduction and amplification of the worm burden within the human host. The potential impact on STH of the benzimidazoles/ivermectin combinations, already used for control/elimination of lymphatic filariasis, should be further evaluated in public health settings. While waiting for more effective single-dose drug regimens and new sensitive diagnostics, the evidence and the tools already available warrant the planning of a common platform for STH and S. stercoralis control.

Accuracy of Five Serologic Tests for the Follow up of Strongyloides stercoralis Infection

Background Traditional faecal-based methods have poor sensitivity for the detection of S. stercoralis, therefore are inadequate for post-treatment evaluation of infected patients who should be carefully monitored to exclude the persistence of the infection. In a previous study, we demonstrated high accuracy of five serology tests for the screening and diagnosis of strongyloidiasis. Aim of this study is to evaluate the performance of the same five tests for the follow up of patients infected with S. stercoralis. Methods Retrospective study on anonymized, cryo-preserved samples available at the Centre for Tropical Diseases (Negrar, Verona, Italy). Samples were collected before and from 3 to 12 months after treatment. The samples were tested with two commercially-available ELISA tests (IVD, Bordier), two techniques based on a recombinant antigen (NIE-ELISA and NIE-LIPS) and one in-house IFAT. The results of each test were evaluated both in relation to the results of fecal examination and to those of a composite reference standard (classifying as positive a sample with positive stools and/or at least three positive serology tests). The associations between the independent variables age and time and the dependent variable value of serological test (for all five tests), were analyzed by linear mixed-effects regression model. Results A high proportion of samples demonstrated for each test a seroreversion or a relevant decline (optical density/relative light units halved or decrease of at least two titers for IFAT) at follow up, results confirmed by the linear mixed effects model that showed a trend to seroreversion over time for all tests. In particular, IVD-ELISA (almost 90% samples demonstrated relevant decline) and IFAT (almost 87%) had the best performance. Considering only samples with a complete negativization, NIE-ELISA showed the best performance (72.5% seroreversion). Conclusions Serology is useful for the follow up of patients infected with S. stercoralis and determining test of cure.

Trypanosoma cruzi diversity in the Gran Chaco: mixed infections and differential host distribution of TcV and TcVI.

The transmission cycles of Trypanosoma cruzi in the Gran Chaco are complex networks involving domestic and wild components, whose interrelationships are not well understood. Knowing the circuit of transmission of the different Discrete Typing Units (DTUs) of T. cruzi in the complex environment of the Chaco region is relevant to understanding how the different components (reservoirs, vectors, ecotopes) interact. In the present study we identified the DTUs infecting humans and dogs in two rural areas of the Gran Chaco in Argentina, using molecular methods which avoid parasite culture. Blood samples of humans and dogs were typified by PCR-DNA blotting and hybridization assays with five specific DNA probes (TcI, TcII, TcIII, TcV and TcVI). PCR analyses were performed on seropositive human and dog samples and showed the presence of T. cruzi DNA in 41.7% (98/235) and 53% (35/66) samples, respectively. The identification of infective DTUs was determined in 83.6% (82/98) and 91.4% (32/35) in human and dog samples, respectively. Single infections (36.7% - 36/98) and a previously not detected high proportion of mixed infections (47.9% - 47/98) were found. In a 15.3% (15/98) of samples the infecting DTU was not identified. Among the single infections TcV was the most prevalent DTU (30.6% - 30/98) in human samples; while TcVI (42.8% - 15/35) showed the highest prevalence in dog samples. TcV/TcVI was the most prevalent mixed infection in humans (32.6% - 32/98); and TcI/TcVI (14.3% - 5/35) in dogs. Significant associations between TcV with humans and TcVI with dogs were detected. For the first time, the presence of TcIII was detected in humans from this region. The occurrence of one human infected whit TcIII (a principally wild DTU) could be suggested the emergence of this, in domestic cycles in the Gran Chaco.

Urban Transmission of American Cutaneous Leishmaniasis in Argentina: Spatial Analysis Study

We used kernel density and scan statistics to examine the spatial distribution of cases of pediatric and adult American cutaneous leishmaniasis in an urban disease-endemic area in Salta Province, Argentina. Spatial analysis was used for the whole population and stratified by women > 14 years of age (n = 159), men > 14 years of age (n = 667), and children < 15 years of age (n = 213). Although kernel density for adults encompassed nearly the entire city, distribution in children was most prevalent in the peripheral areas of the city. Scan statistic analysis for adult males, adult females, and children found 11, 2, and 8 clusters, respectively. Clusters for children had the highest odds ratios (P < 0.05) and were located in proximity of plantations and secondary vegetation. The data from this study provide further evidence of the potential urban transmission of American cutaneous leishmaniasis in northern Argentina.

Effect of Poor Access to Water and Sanitation As Risk Factors for Soil-Transmitted Helminth Infection: Selectiveness by the Infective Route.

Background Soil-transmitted helminth (STH) infections are a public health problem in resource-limited settings worldwide. Chronic STH infection impairs optimum learning and productivity, contributing to the perpetuation of the poverty-disease cycle. Regular massive drug administration (MDA) is the cardinal recommendation for its control; along with water, sanitation and hygiene (WASH) interventions. The impact of joint WASH interventions on STH infections has been reported; studies on the independent effect of WASH components are needed to contribute with the improvement of current recommendations for the control of STH. The aim of this study is to assess the association of lacking access to water and sanitation with STH infections, taking into account the differences in route of infection among species and the availability of adequate water and sanitation at home. Methods and Findings Cross-sectional study, conducted in Salta province, Argentina. During a deworming program that enrolled 6957 individuals; 771 were randomly selected for stool/serum sampling for parasitological and serological diagnosis of STH. Bivariate stratified analysis was performed to explore significant correlations between risk factors and STH infections grouped by mechanism of entry as skin-penetrators (hookworms and Strongyloides stercoralis) vs. orally-ingested (Ascaris lumbricoides and Trichuris trichiura). After controlling for potential confounders, unimproved sanitation was significantly associated with increased odds of infection of skin-penetrators (adjusted odds ratio [aOR] = 3.9; 95% CI: 2.6–5.9). Unimproved drinking water was significantly associated with increased odds of infection of orally-ingested (aOR = 2.2; 95% CI: 1.3–3.7). Conclusions Lack of safe water and proper sanitation pose a risk of STH infections that is distinct according to the route of entry to the human host used by each of the STH species. Interventions aimed to improve water and sanitation access should be highlighted in the recommendations for the control of STH.

Performance of different Trypanosoma cruzi antigens in the diagnosis of Chagas disease in patients with American cutaneous leishmaniasis from a co-endemic region in Argentina.

To determine the ability of recombinant antigens to detect cases of infection with Trypanosoma cruzi among cases of infection with Leishmania spp. by serological methods.

Biological behavior of different Trypanosoma cruzi isolates circulating in an endemic area for Chagas disease in the Gran Chaco region of Argentina

The biological behavior of the different Trypanosoma cruzi strains is still unclear and the importance of exploring the relevance of these differences in natural isolates is of great significance. Herein we describe the biological behavior of four T. cruzi isolates circulating sympatrically in a restricted geographic area in Argentina endemic for Chagas Disease. These isolates were characterized as belonging to the Discrete Typing Units (DTUs) TcI, TcIII, TcV and TcVI as shown by Multilocus Enzyme Electrophoresis and Multilocus Sequence Typing. In order to study the natural behavior of the different isolates and to preserve their natural properties, we developed a vector transmission model that allows their maintenance in the laboratory. The model consisted of serial passages of these parasites between insect vectors and mice. Vector-derived parasite forms were then inoculated in C57BL/6J mice and number of parasite in peripheral blood, serological response and histological damage in acute and chronic phases of the infection were measured. Parasites from DTUs TcI, TcIII and TcVI were detected by direct fresh blood examination, while TcV parasites could only be detected by Polimerase Chain Reaction. No significant difference in the anti-T. cruzi antibody response was found during the chronic phase of infection, except for mice infected with TcV parasites where no antibodies could be detected. Histological sections showed that TcI isolate produced more damage in skeletal muscle while TcVI induced more inflammation in the heart. This work shows differential biological behavior among different parasite isolates obtained from the same cycle of transmission, permitting the opportunity to formulate future hypotheses of clinical and epidemiological importance.

Immuno-enzymatic evaluation of the recombinant TSSA-II protein of Trypanosoma cruzi in dogs and human sera: a tool for epidemiological studies

The rTSSA-II (recombinant Trypomastigote Small Surface II) antigen was evaluated by ELISA to detect anti-Trypanosoma cruzi antibodies in sera from naturally infected dogs and humans. For this evaluation ELISA-rTSSA-II was standardized and groups were classified according to the results obtained through xenodiagnosis, ELISA and PCR. Sensitivity (Se), Specificity (Sp), Kappa index (KI) and area under curve (AUC) were determined. The Se was determined by using 14 sera from dogs infected with T. cruzi VI (TcVI) whereas Sp was determined by using 95 non-chagasic sera by xenodiagnosis, ELISA-Homogenate and PCR. The performance of ELISA-rTSSA-II in dog sera was high (AUC=0·93 and KI=0·91). The Se was 92·85% (1 false negative) and Sp was 100%. Two sera from dogs infected with TcI and 1 with TcIII were negative. For patients infected with T. cruzi, reactivity was 87·8% (36/41), there was only 1 indeterminate, and Sp was 100%. Fifty-four sera from non-chagasic and 68 sera from patients with cutaneous leishmaniasis did not react with rTSS-II. ELISA-rTSSA-II showed a high performance when studying sera from naturally infected dogs and it also presented 100% Sp. This assay could be an important tool to carry out sero-epidemiological surveys on the prevalence of T. cruzi circulating lineages in the region.

Benznidazole treatment in chronic children infected with Trypanosoma cruzi: serological and molecular follow-up of patients and identification of Discrete Typing Units.

A total of 221 children from two rural settlements in Northeast Argentina were examined for T. cruzi infection. Blood samples were taken for serology tests and PCR assays. In addition, T. cruzi Discrete Typing Units (DTUs) were determined by hybridization with specific DNA probes of the minicircle hypervariable regions (mHVR). Serological results indicated that 26% (57/215) were reactive against T. cruzi antigens. PCR analyses were performed on seropositive samples showing presence of parasite DNA in 31 out of 53 samples (58.5%). All seropositive children underwent specific chemotherapy with Benznidazole (5mg/kg/day) for a period of two months and were monitored two and five years after treatment. Overall the treatment was well tolerated and low side effects were observed. Serological conversion was observed at two years post -treatment in one child form Pampa Ávila and at five years in two children from Tres Estacas. However, at the end of the follow-up period, T. cruzi DNA could not be detected by PCR in samples from treated children, except in two cases. In addition, the results of hybridizations with specific DNA probes showed that DTU TcV was detected in 68% (21/31), TcVI in 7% (2/31) and TcV/VI in 3% (1/31) of the samples. Altogether, results of the follow-up of treated children showed a low rate of seroconversion; however trend toward seroconversion was evident at five years post-treatment. On the other hand, detection of T. cruzi DNA by PCR significantly decreased after Benznidazole treatment. The existence of data regarding serological and molecular follow-ups from controlled studies in the Chaco Region will be important for future treatment efforts against T. cruzi infection in this region. The results obtained in the present study represent a contribution in this regard.