A Real-Time PCR Assay for the Diagnosis of Intestinal Schistosomiasis and Cure Assessment After the Treatment of Individuals With Low Parasite Burden.

The laboratorial diagnosis of the intestinal schistosomiasis is always performed using Kato-Katz technique. However, this technique presents low sensitivity for diagnosis of individuals with low parasite burden, which constitutes the majority in low endemicity Brazilian locations for the disease. The objective of this study was developed and to validate a real-time PCR assay (qPCR) targeting 121 bp sequence to detect Schistosoma spp. DNA for the diagnosis of intestinal schistosomiasis and a sequence of the human ß-actin gene as internal control. Firstly, the qPCR was standardized and next it was evaluated for diagnosis and cure assessment of intestinal schistosomiasis in the resident individuals in Tabuas and Estreito de Miralta, two locations in Brazil endemic for intestinal schistosomiasis. The qPCR assay results were compared with those of the Kato-Katz (KK) test, examining 2 or 24 slides, Saline Gradient (SG) and "reference test" (24 KK slides + SG). The cure assessment was measured by these diagnostic techniques at 30, 90, and 180 days post-treatment. In Tabuas, the positivity rates obtained by the qPCR was 30.4% (45/148) and by "reference test" was of 31.0% (46/148), with no statistical difference (p = 0.91). The presumed cure rates at 30, 90, and 180 days post-treatment were 100, 94.4, and 78.4% by the analysis of 24 KK slides, 100, 94.4, and 78.4% by the SG, and 100, 83.3, and 62.1% by the qPCR assay. In Estreito de Miralta, the positivity obtained by qPCR was 18.3% (26/142) and with "reference test" was 24.6% (35/142), with no statistical difference (p = 0.20). The presumed cure rates were 93.3, 96.9, and 96.5% by the KK, 93.3, 96.9, and 100% by the SG, and 93.3, 93.9, and 96.5% by the qPCR at 30, 90, and 180 days post-treatment, respectively. This study showed that the diagnostic techniques presented different performance in the populations from the two districts (Tabuas and Estreito de Miralta) and reinforces the need of combining techniques to improve diagnosis accuracy, increasing the detection of individuals with low parasite burden. This combination of techniques consists an important strategy for controlling the disease transmission.

Transmission control of schistosomiasis mansoni in a low endemicity area through a single intervention with rigorous prospection of infected cases treated with praziquantel: evaluation after 7 years of intervention.

A prospective cohort study with rigorous searching for schistosomiasis cases was conducted among residents of Pedra Preta, Montes Claros, Minas Gerais State, Brazil, seven years after an intervention. Kato-Katz (KK), Saline Gradient, Miracidia Hatch and Polymerase Chain Reaction (PCR) were used as the diagnostic methods in 2008. In the period of 2013-2016, 175 patients remaining in the area were examined using the diagnostic methods Kato-Katz (24 slides, 1 g of feces) and Saline Gradient (2 procedures, 1 g of feces). Sixty-eight out of the 69 infected and treated individuals in 2008 tested negative. The percentage of new cases was 2.29% (4/175), and the 4 infected individuals presented low parasitic load [1, 6, 7 and 19 eggs per gram (EPG)]. All the participants answered epidemiological questionnaires on risky behavior. All residences had pit latrines and domiciliary water supply. The primary transmission focus (lake) was dry for several months. Malacological surveys showed a few non-infected specimens of Biomphalaria glabrata . A clear dominance of Biomphalaria straminea was observed. It can be inferred that a significant decrease in the disease transmission occurred after a single action through an intense search for infected and treated cases under the ecoepidemiological conditions of this area.

Evaluation of diagnostic methods for the detection of intestinal schistosomiasis in endemic areas with low parasite loads: Saline gradient, Helmintex, Kato-Katz and rapid urine test.

BACKGROUND: In some tropical countries, such as Brazil, schistosomiasis control programs have led to a significant reduction in the prevalence and parasite burden of endemic populations. In this setting, the Kato-Katz technique, as the standard diagnostic method for the diagnosis of Schistosoma mansoni infections, which involves the analysis of two slides from one fecal sample, loses its sensitivity. As a result, a significant number of infected individuals are not detected. The objective of this study was to perform extensive parasitological testing of up to three fecal samples and include a rapid urine test (POC-CCA) in a moderate prevalence area in Northern Minas Gerais, Brazil, and evaluate the performance of each test separately and in combination. METHODS AND FINDINGS: A total of 254 individuals were examined with variants of the standard Kato-Katz technique (up to18 Kato-Katz slides prepared from three fecal samples), a modified Helmintex (30 g of feces), the saline gradient (500 mg of feces), and the POC-CCA methods. We established a reference standard taking into consideration all the positive results in any of the parasitological exams. Evaluation of the parasite burden by two Kato-Katz slides confirmed that most of the individuals harbored a light infection. When additional slides and different parasitological methods were included, the estimated prevalence rose 2.3 times, from 20.4% to 45.9%. The best sensitivity was obtained with the Helmintex method (84%). All parasitological methods readily detected a high or moderate intensity of infection; however, all lost their high sensitivity in the case of low or very low intensity infections. The overall sensitivity of POC-CCA (64.9%) was similar to the six Kato-Katz slides from three fecal samples. However, POC-CCA showed low concordance (κ = 0.34) when compared with the reference standard. CONCLUSIONS: The recommended Kato-Katz method largely underestimated the prevalence of S. mansoni infection. Because the best performance was achieved with a modified Helmintex method, this technique might serve as a more precise reference standard. An extended number of Kato-Katz slides in combination with other parasitological methods or with POC-CCA was able to detect more than 80% of egg-positive individuals; however, the rapid urine test (POC-CCA) produced a considerable percentage of false positive results.

Performance of POC-CCA® in diagnosis of schistosomiasis mansoni in individuals with low parasite burden.

INTRODUCTION: Schistosomiasis, caused by Schistosoma mansoni, is a public health concern in Brazil. However, the most popular diagnostic method, the Kato-Katz technique, exhibits low sensitivity in low-endemicity areas. We aimed to compare the performance of an immunological assay, the point-of-care circulating cathodic antigen (POC-CCA®) test, with that of two parasitological techniques in a low-endemicity population. METHODS: Our study included 141 individuals living in Estreito de Miralta, Minas Gerais, Brazil. Fecal samples were obtained from all participants and analyzed for schistosomiasis using two parasitological techniques: the Kato-Katz technique and the saline gradient technique. Additionally, POC-CCA® strips were utilized for testing urine samples. The results obtained by the different techniques were compared. RESULTS: Analysis of two or 24 slides using the Kato-Katz technique resulted in a positivity rate of 10.6% (15/141) or 19.1% (27/141), respectively. The saline gradient technique yielded a positivity rate of 17.0% (24/141). The prevalence according to both parasitological techniques was 24.1% (34/141). The POC-CCA® test yielded a positivity rate of 22.7% (32/141); however, the positivity rate was merely 2.1% if trace results were considered negative. The agreements observed between POC-CCA® and the parasitological techniques were good (Kappa indexes > 0.64). The POC-CCA® test was more sensitive than the two-slide Kato-Katz technique (p < 0.05) in detecting cases of S. mansoni infection when trace results were considered positive. CONCLUSIONS: These findings reinforce the importance of using multiple diagnostic techniques in low-endemicity areas for effective control of disease.

Performance of POC-CCA® in diagnosis of schistosomiasis mansoni in individuals with low parasite burden

Abstract: INTRODUCTION: Schistosomiasis, caused by Schistosoma mansoni, is a public health concern in Brazil. However, the most popular diagnostic method, the Kato-Katz technique, exhibits low sensitivity in low-endemicity areas. We aimed to compare the performance of an immunological assay, the point-of-care circulating cathodic antigen (POC-CCA®) test, with that of two parasitological techniques in a low-endemicity population. METHODS: Our study included 141 individuals living in Estreito de Miralta, Minas Gerais, Brazil. Fecal samples were obtained from all participants and analyzed for schistosomiasis using two parasitological techniques: the Kato-Katz technique and the saline gradient technique. Additionally, POC-CCA® strips were utilized for testing urine samples. The results obtained by the different techniques were compared. RESULTS: Analysis of two or 24 slides using the Kato-Katz technique resulted in a positivity rate of 10.6% (15/141) or 19.1% (27/141), respectively. The saline gradient technique yielded a positivity rate of 17.0% (24/141). The prevalence according to both parasitological techniques was 24.1% (34/141). The POC-CCA® test yielded a positivity rate of 22.7% (32/141); however, the positivity rate was merely 2.1% if trace results were considered negative. The agreements observed between POC-CCA® and the parasitological techniques were good (Kappa indexes > 0.64). The POC-CCA® test was more sensitive than the two-slide Kato-Katz technique (p < 0.05) in detecting cases of S. mansoni infection when trace results were considered positive. CONCLUSIONS: These findings reinforce the importance of using multiple diagnostic techniques in low-endemicity areas for effective control of disease.

Evaluation of parasitological and molecular techniques for the diagnosis and assessment of cure of schistosomiasis mansoni in a low transmission area.

This study evaluated parasitological and molecular techniques for the diagnosis and assessment of cure of schistosomiasis mansoni. A population-based study was performed in 201 inhabitants from a low transmission locality named Pedra Preta, municipality of Montes Claros, state of Minas Gerais, Brazil. Four stool samples were analysed using two techniques, the Kato-Katz® (KK) technique (18 slides) and the TF-Test®, to establish the infection rate. The positivity rate of 18 KK slides of four stool samples was 28.9% (58/201) and the combined parasitological techniques (KK+TF-Test®) produced a 35.8% positivity rate (72/201). Furthermore, a polymerase chain reaction (PCR)-ELISA assay produced a positivity rate of 23.4% (47/201) using the first sample. All 72 patients with positive parasitological exams were treated with a single dose of Praziquantel® and these patients were followed-up 30, 90 and 180 days after treatment to establish the cure rate. Cure rates obtained by the analysis of 12 KK slides were 100%, 100% and 98.4% at 30, 90 and 180 days after treatment, respectively. PCR-ELISA revealed cure rates of 98.5%, 95.5% and 96.5%, respectively. The diagnostic and assessment of cure for schistosomiasis may require an increased number of KK slides or a test with higher sensitivity, such as PCR-ELISA, in situations of very low parasite load, such as after therapeutic interventions.

Evaluation of parasitological and molecular techniques for the diagnosis and assessment of cure of schistosomiasis mansoni in a low transmission area

This study evaluated parasitological and molecular techniques for the diagnosis and assessment of cure of schistosomiasis mansoni. A population-based study was performed in 201 inhabitants from a low transmission locality named Pedra Preta, municipality of Montes Claros, state of Minas Gerais, Brazil. Four stool samples were analysed using two techniques, the Kato-Katz® (KK) technique (18 slides) and the TF-Test®, to establish the infection rate. The positivity rate of 18 KK slides of four stool samples was 28.9% (58/201) and the combined parasitological techniques (KK+TF-Test®) produced a 35.8% positivity rate (72/201). Furthermore, a polymerase chain reaction (PCR)-ELISA assay produced a positivity rate of 23.4% (47/201) using the first sample. All 72 patients with positive parasitological exams were treated with a single dose of Praziquantel® and these patients were followed-up 30, 90 and 180 days after treatment to establish the cure rate. Cure rates obtained by the analysis of 12 KK slides were 100%, 100% and 98.4% at 30, 90 and 180 days after treatment, respectively. PCR-ELISA revealed cure rates of 98.5%, 95.5% and 96.5%, respectively. The diagnostic and assessment of cure for schistosomiasis may require an increased number of KK slides or a test with higher sensitivity, such as PCR-ELISA, in situations of very low parasite load, such as after therapeutic interventions.

Acute schistosomiasis diagnosis: a new tool for the diagnosis of schistosomiasis in a group of travelers recently infected in a new focus of Schistosoma mansoni.

INTRODUCTION: The diagnosis of schistosomiasis mansoni on early stages of infection is important to prevent late morbidity. A simple, cheap, sensitive and specific assay for routine diagnosis of schistosome infection based on the detection of specific IgG for schistosomula tegument antigens (ELISA-SmTeg) was developed by our group. METHODS: We describe here an acute outbreak involving a travel group of 80 individuals from a non-endemic area of the State of Minas Gerais, Brazil. These individuals were in contact with a freshwater pool where Biomphalaria glabrata was found. Results obtained from our new methodology were compared to IgG antibody titers against soluble worm antigenic preparation (SWAP) by ELISA and, also to parasitological examination, nuclear magnetic resonance and clinical findings. RESULTS: ELISA-SmTeg was capable of detecting 64 positive cases among the 80 individuals participating at the survey with a positivity ratio of 80% and a higher sensitivity than ELISA-SWAP that was only sensitive for 56% of positive cases. Besides, a significant correlation was found for the severity of the infection and the specific IgG titers against SmTeg. CONCLUSIONS: Our data showed that ELISA-SmTeg might serve as the initial diagnostic tool for acute stages of the infection in community-based helminth control programs or for the surveillance of individuals from non-endemic areas.

Acute schistosomiasis diagnosis: a new tool for the diagnosis of schistosomiasis in a group of travelers recently infected in a new focus of Schistosoma mansoni

Introduction The diagnosis of schistosomiasis mansoni on early stages of infection is important to prevent late morbidity. A simple, cheap, sensitive and specific assay for routine diagnosis of schistosome infection based on the detection of specific IgG for schistosomula tegument antigens (ELISA-SmTeg) was developed by our group. Methods We describe here an acute outbreak involving a travel group of 80 individuals from a non-endemic area of the State of Minas Gerais, Brazil. These individuals were in contact with a freshwater pool where Biomphalaria glabrata was found. Results obtained from our new methodology were compared to IgG antibody titers against soluble worm antigenic preparation (SWAP) by ELISA and, also to parasitological examination, nuclear magnetic resonance and clinical findings. Results ELISA-SmTeg was capable of detecting 64 positive cases among the 80 individuals participating at the survey with a positivity ratio of 80% and a higher sensitivity than ELISA-SWAP that was only sensitive for 56% of positive cases. Besides, a significant correlation was found for the severity of the infection and the specific IgG titers against SmTeg. Conclusions Our data showed that ELISA-SmTeg might serve as the initial diagnostic tool for acute stages of the infection in community-based helminth control programs or for the surveillance of individuals from non-endemic areas. .

Evaluation of two coproscopic techniques for the diagnosis of schistosomiasis in a low-transmission area in the state of Minas Gerais, Brazil.

This population study, which evaluated two parasitological methods for the diagnosis of schistosomiasis mansoni, was performed in a low-transmission area in Pedra Preta, Montes Claros, Minas Gerais, Brazil. A total of 201 inhabitants of the rural area participated in this research. Four stool samples were obtained from all participants and analysed using the Kato-Katz method (18 slides) and a commercial test, the TF-Test®, which was performed quantitatively. The data were analysed to determine prevalence, the sensitivity of the diagnostic methods, the worm burden and the definition of the "gold standard", which was obtained by totalling the results of all samples examined using the Kato-Katz technique and the TF-Test®. The results showed that the prevalence obtained from the examination of one Kato-Katz slide (the methodology adopted by the Brazilian control programme) was 8% compared to 35.8% from the "gold standard", which was a 4.5-fold difference. This result indicates that the prevalence of schistosomiasis in so-called low-transmission areas is significantly underestimated.

Evaluation of two coproscopic techniques for the diagnosis of schistosomiasis in a low-transmission area in the state of Minas Gerais, Brazil

This population study, which evaluated two parasitological methods for the diagnosis of schistosomiasis mansoni, was performed in a low-transmission area in Pedra Preta, Montes Claros, Minas Gerais, Brazil. A total of 201 inhabitants of the rural area participated in this research. Four stool samples were obtained from all participants and analysed using the Kato-Katz method (18 slides) and a commercial test, the TF-Test®, which was performed quantitatively. The data were analysed to determine prevalence, the sensitivity of the diagnostic methods, the worm burden and the definition of the "gold standard", which was obtained by totalling the results of all samples examined using the Kato-Katz technique and the TF-Test®. The results showed that the prevalence obtained from the examination of one Kato-Katz slide (the methodology adopted by the Brazilian control programme) was 8 percent compared to 35.8 percent from the "gold standard", which was a 4.5-fold difference. This result indicates that the prevalence of schistosomiasis in so-called low-transmission areas is significantly underestimated.

Detection of IgG1 and IgG4 subtypes reactive against potato apyrase in schistosomiasis patients.

In this paper, we showed for the first time that the conserved domains within Schistosoma mansoni ATP diphosphohydrolase isoforms, shared with potato apyrase, possess epitopes for the IgG1 and IgG4 subtypes, as 24 (80%) of the 30 schistosomiasis patients were seropositive for this vegetable protein. The analyses for each patient cured (n = 14) after treatment (AT) with praziquantel revealed variable IgG1 and IgG4 reactivity against potato apyrase. Different antigenic epitopes shared between the vegetable and parasite proteins could be involved in susceptibility or resistance to S. mansoni AT with praziquantel and these possibilities should be explored.

Detection of IgG1 and IgG4 subtypes reactive against potato apyrase in schistosomiasis patients

In this paper, we showed for the first time that the conserved domains within Schistosoma mansoni ATP diphosphohydrolase isoforms, shared with potato apyrase, possess epitopes for the IgG1 and IgG4 subtypes, as 24 (80 percent) of the 30 schistosomiasis patients were seropositive for this vegetable protein. The analyses for each patient cured (n = 14) after treatment (AT) with praziquantel revealed variable IgG1 and IgG4 reactivity against potato apyrase. Different antigenic epitopes shared between the vegetable and parasite proteins could be involved in susceptibility or resistance to S. mansoni AT with praziquantel and these possibilities should be explored.

[Schistosoma mansoni: the action of lovastatin on the murine model]. / Schistosoma mansoni: ação da lovastatina no modelo murino.

Aimed to evaluate the lovastatin action on Schistosoma mansoni oviposition, infected mice with 100 plus minus 10 cercariae of the LE strain were used. Thirty days after infection the animals were treated with 100, 200 and 400mg/kg of lovastatin, per os, during five consecutive days and then sacrificed 7, 15, 30 or 60 days after treatment. We analyzed: distribution of worms in mesenteries and liver; mortality of worms in the liver; alteration of the oogram; eggs counting in the jejunum and liver; presence of intrauterine eggs and morphology of the worms from the treated and control groups (infected and not treated animals). Significant statistical differences were found between treated and control groups when the presence of intrauterine eggs was considered and also, alteration on the oogram, eggs at different stages of development in jejunum, liver and in the body length of males and females. The morphological study of the worms showed that the degenerative modifications occurred, mainly in the reproductive system, with reduction and alteration of the viteline follicles and the ovary of the females. Also modifications in the males testicles were observed. The results suggest that the drug under study reduces, considerably, the female S. mansoni, oviposition increases the worms' size, leads to alteration in the reproductive system of males and females. It also may induce death of a significant part of the worm populations at the dose of 400mg/kg.

Schistosoma mansoni: ação da lovastatina no modelo murino / Schistosoma mansoni: the action of lovastatin on the murine model

Aimed to evaluate the lovastatin action on Schistosoma mansoni oviposition, infected mice with 100 plus minus 10 cercariae of the LE strain were used. Thirty days after infection the animals were treated with 100, 200 and 400mg/kg of lovastatin, per os, during five consecutive days and then sacrificed 7, 15, 30 or 60 days after treatment. We analyzed: distribution of worms in mesenteries and liver; mortality of worms in the liver; alteration of the oogram; eggs counting in the jejunum and liver; presence of intrauterine eggs and morphology of the worms from the treated and control groups (infected and not treated animals). Significant statistical differences were found between treated and control groups when the presence of intrauterine eggs was considered and also, alteration on the oogram, eggs at different stages of development in jejunum, liver and in the body length of males and females. The morphological study of the worms showed that the degenerative modifications occurred, mainly in the reproductive system, with reduction and alteration of the viteline follicles and the ovary of the females. Also modifications in the males testicles were observed. The results suggest that the drug under study reduces, considerably, the female S. mansoni, oviposition increases the worms' size, leads to alteration in the reproductive system of males and females. It also may induce death of a significant part of the worm populations at the dose of 400mg/kg.