RESUMO
The population of Brazil is currently characterised by many individuals harbouring low-intensity Schistosoma mansoni infections. The Kato-Katz technique is the diagnostic method recommended by the World Health Organization (WHO) to assess these infections, but this method is not sensitive enough in the context of low egg excretion. In this regard, potential alternatives are being employed to overcome the limits of the Kato-Katz technique. In the present review, we evaluated the performance of parasitological and immunological approaches adopted in Brazilian areas. Currently, the diagnostic choices involve a combination of strategies, including the utilisation of antibody methods to screen individuals and then subsequent confirmation of positive cases by intensive parasitological investigations.
Assuntos
Anticorpos Anti-Helmínticos/análise , Antígenos de Helmintos/análise , Técnicas de Laboratório Clínico/métodos , Fezes/parasitologia , Schistosoma mansoni , Esquistossomose mansoni/diagnóstico , Animais , Brasil/epidemiologia , Doenças Endêmicas , Humanos , Técnicas Imunoenzimáticas , Contagem de Ovos de Parasitas , Schistosoma mansoni/imunologia , Schistosoma mansoni/isolamento & purificação , Esquistossomose mansoni/epidemiologia , Sensibilidade e Especificidade , Índice de Gravidade de DoençaRESUMO
BACKGROUND: Despite decades of use of control programs, schistosomiasis remains a global public health problem. To further reduce prevalence and intensity of infection, or to achieve the goal of elimination in low-endemic areas, there needs to be better diagnostic tools to detect low-intensity infections in low-endemic areas in Brazil. The rationale for development of new diagnostic tools is that the current standard test Kato-Katz (KK) is not sensitive enough to detect low-intensity infections in low-endemic areas. In order to develop new diagnostic tools, we employed a proteomics approach to identify biomarkers associated with schistosome-specific immune responses in hopes of developing sensitive and specific new methods for immunodiagnosis. METHODS AND FINDINGS: Immunoproteomic analyses were performed on egg extracts of Schistosoma mansoni using pooled sera from infected or non-infected individuals from a low-endemic area of Brazil. Cross reactivity with other soil-transmitted helminths (STH) was determined using pooled sera from individuals uniquely infected with different helminths. Using this approach, we identified 23 targets recognized by schistosome acute and chronic sera samples. To identify immunoreactive targets that were likely glycan epitopes, we compared these targets to the immunoreactivity of spots treated with sodium metaperiodate oxidation of egg extract. This treatment yielded 12/23 spots maintaining immunoreactivity, suggesting that they were protein epitopes. From these 12 spots, 11 spots cross-reacted with sera from individuals infected with other STH and 10 spots cross-reacted with the negative control group. Spot number 5 was exclusively immunoreactive with sera from S. mansoni-infected groups in native and deglycosylated conditions and corresponds to Major Egg Antigen (MEA). We expressed MEA as a recombinant protein and showed a similar recognition pattern to that of the native protein via western blot. IgG-ELISA gave a sensitivity of 87.10% and specificity of 89.09% represented by area under the ROC curve of 0.95. IgG-ELISA performed better than the conventional KK (2 slides), identifying 56/64 cases harboring 1-10 eggs per gram of feces that were undiagnosed by KK parasitological technique. CONCLUSIONS: The serological proteome approach was able to identify a new diagnostic candidate. The recombinant egg antigen provided good performance in IgG-ELISA to detect individuals with extreme low-intensity infections (1 egg per gram of feces). Therefore, the IgG-ELISA using this newly identified recombinant MEA can be a useful tool combined with other techniques in low-endemic areas to determine the true prevalence of schistosome infection that is underestimated by the KK method. Further, to overcome the complexity of ELISA in the field, a second generation of antibody-based rapid diagnostic tests (RDT) can be developed.
Assuntos
Antígenos de Helmintos/sangue , Proteínas de Helminto/sangue , Proteoma/metabolismo , Schistosoma mansoni/imunologia , Esquistossomose mansoni/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antígenos de Helmintos/imunologia , Biomarcadores/sangue , Brasil , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Fezes/parasitologia , Feminino , Proteínas de Helminto/imunologia , Humanos , Imunoglobulina G/sangue , Lactente , Masculino , Pessoa de Meia-Idade , Óvulo/imunologia , Contagem de Ovos de Parasitas , Proteoma/imunologia , Proteômica , Proteínas Recombinantes/imunologia , Esquistossomose mansoni/sangue , Sensibilidade e Especificidade , Testes Sorológicos/métodosRESUMO
The population of Brazil is currently characterised by many individuals harbouring low-intensity Schistosoma mansoni infections. The Kato-Katz technique is the diagnostic method recommended by the World Health Organization (WHO) to assess these infections, but this method is not sensitive enough in the context of low egg excretion. In this regard, potential alternatives are being employed to overcome the limits of the Kato-Katz technique. In the present review, we evaluated the performance of parasitological and immunological approaches adopted in Brazilian areas. Currently, the diagnostic choices involve a combination of strategies, including the utilisation of antibody methods to screen individuals and then subsequent confirmation of positive cases by intensive parasitological investigations.
Assuntos
Humanos , Schistosoma mansoni , ImunoensaioRESUMO
Background: Prior to eliminating schistosomiasis, efforts must address accurate and fast individual diagnosis. Diagnosis is still inaccurate by parasitological and point-of-care circulating cathodic antigen (POC-CCA) in areas of low endemicity. Methods: Our group has optimized POC-CCA with a 30 min urine concentration step with no need for specialized technicians or equipment and with high accuracy. We evaluated this new method, called POC-CCA filter (FLT), in two Brazilian endemic areas with distinct profiles. Results: At baseline, POC-CCA had a poor performance with several false results and undefined trace readings, revealing a prevalence rate of 10% against a rate of 23% for POC-CCA FLT, which was similar to the parasitological rates. Accuracy increased from as low as 0.36 to 0.96 after urine concentration in one area. POC-CCA properly diagnosed only half of the cases at three post-treatment time points, while POC-CCA FLT was able to diagnose 96, 83 and 100%, respectively. Conclusions: The improvement of conventional POC methodology by a fast and simple urine concentration step provided not only an increase in its accuracy before and after praziquantel treatment, but also preserved its applicability in low-prevalence endemic areas, allowing the definition of trace readings as negative cases.
Assuntos
Antígenos de Helmintos/urina , Sistemas Automatizados de Assistência Junto ao Leito , Schistosoma mansoni/imunologia , Esquistossomose mansoni/diagnóstico , Esquistossomose mansoni/urina , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anti-Helmínticos/uso terapêutico , Brasil/epidemiologia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Praziquantel/uso terapêutico , Estudos Prospectivos , Reprodutibilidade dos Testes , Esquistossomose mansoni/tratamento farmacológico , Esquistossomose mansoni/epidemiologia , Sensibilidade e Especificidade , Adulto JovemRESUMO
Schistosomiasis is still a public health problem in Brazil. The Kato-Katz test is the most frequently used diagnostic method for Schistosoma mansoni infection. However, it lacks sensitivity in areas of low prevalence. We have assessed the positivity rate of S. mansoni infection in Bananeiras, a village on Capistrano, Ceara, Brazil by performing a point-of-care test in urine to determine the circulating cathodic antigens (POC-CCA), and we compared the findings with those of the Kato-Katz technique for egg detection in stool and an enzyme-linked immunosorbent assay for specific antibodies against adult worms (SWAP-ELISA) in serum before treatment (baseline). Additionally, the POC-CCA and Kato-Katz test results were compared at one and two years post-treatment, and only POC-CCA strips were utilised for follow-up testing on urine samples at 3-6 weeks. Only one sample of stool and urine was collected per event. Overall, 258 individuals were investigated at the baseline. The POC-CCA test detected 10 (3.9%) positive cases; however, this amount increased to 30 (11.6%) when considering trace readings as positive (tâ¯+â¯), whereas the Kato-Katz method found only 4 (1.6%) positive cases and the SWAP-ELISA detected 105 (40.7%) positive cases. The consistency observed between a single POC-CCA (tâ¯+â¯) or (t-) and the Kato-Katz (three slides) was poor (Kappa indexes <0.20). The highest positivity rate as determined by CCA and Kato-Katz was found in adults. At the baseline, a praziquantel treatment was administered to all individuals regardless of their infection status. According to the POC-CCA test, 93% of the previous positive cases became negative by the third week after the treatment; this rate reached 100% at the sixth week assessment. The follow-up showed that of the 175 individuals evaluated at one year post-treatment, only one (0.6%) showed 'trace' results, and all the individuals were negative for eggs in the stool. At two years, all 185 examined individuals were negative by the Kato-Katz method, and 11 (5.9%) presented traces by POC-CCA. Our results indicate that a single POC-CCA test reveals a significantly higher number of positive cases than the Kato-Katz technique for diagnosing S. mansoni in a low endemic setting, when trace results are considered as positive cases. Nevertheless, the true significance of the trace is not clear. These findings reinforce the need to associate different tools for improved schistosomiasis diagnosis in individuals with low parasite burdens.
Assuntos
Anticorpos Anti-Helmínticos/análise , Antígenos de Helmintos/análise , Sistemas Automatizados de Assistência Junto ao Leito , Schistosoma mansoni/imunologia , Esquistossomose mansoni/diagnóstico , Testes Sorológicos/métodos , Adulto , Animais , Anti-Helmínticos/uso terapêutico , Brasil , Criança , Ensaio de Imunoadsorção Enzimática , Fezes/parasitologia , Feminino , Humanos , Masculino , Praziquantel/uso terapêutico , Esquistossomose mansoni/tratamento farmacológico , Sensibilidade e EspecificidadeRESUMO
Current schistosomiasis control strategies are largely based on chemotherapeutic agents and a limited number of drugs are available today. Praziquantel (PZQ) is the only drug currently used in schistosomiasis control programs. Unfortunately, this drug shows poor efficacy in patients during the earliest infection phases. The effects of PZQ appear to operate on the voltage-operated Ca2+ channels, which are located on the external Schistosoma mansoni membrane. Because some Ca2+ channels have dihydropyridine drug class (a class that includes nifedipine) sensitivity, an in vitro analysis using a calcium channel antagonist (clinically used for cardiovascular hypertension) was performed to determine the antischistosomal effects of nifedipine on schistosomula and adult worm cultures. Nifedipine demonstrated antischistosomal activity against schistosomula and significantly reduced viability at all of the concentrations used alone or in combination with PZQ. In contrast, PZQ did not show significant efficacy when used alone. Adult worms were also affected by nifedipine after a 24 h incubation and exhibited impaired motility, several lesions on the tegument and intense contractility. These data support the idea of Ca2+ channels subunits as drug targets and favour alternative therapeutic schemes when drug resistance has been reported. In this paper, strong arguments encouraging drug research are presented, with a focus on exploring schistosomal Ca2+ channels.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Nifedipino/farmacologia , Praziquantel/farmacologia , Schistosoma mansoni/efeitos dos fármacos , Esquistossomicidas/farmacologia , Animais , Camundongos , Testes de Sensibilidade ParasitáriaRESUMO
Current schistosomiasis control strategies are largely based on chemotherapeutic agents and a limited number of drugs are available today. Praziquantel (PZQ) is the only drug currently used in schistosomiasis control programs. Unfortunately, this drug shows poor efficacy in patients during the earliest infection phases. The effects of PZQ appear to operate on the voltage-operated Ca2+channels, which are located on the external Schistosoma mansoni membrane. Because some Ca2+channels have dihydropyridine drug class (a class that includes nifedipine) sensitivity, an in vitro analysis using a calcium channel antagonist (clinically used for cardiovascular hypertension) was performed to determine the antischistosomal effects of nifedipine on schistosomula and adult worm cultures. Nifedipine demonstrated antischistosomal activity against schistosomula and significantly reduced viability at all of the concentrations used alone or in combination with PZQ. In contrast, PZQ did not show significant efficacy when used alone. Adult worms were also affected by nifedipine after a 24 h incubation and exhibited impaired motility, several lesions on the tegument and intense contractility. These data support the idea of Ca2+channels subunits as drug targets and favour alternative therapeutic schemes when drug resistance has been reported. In this paper, strong arguments encouraging drug research are presented, with a focus on exploring schistosomal Ca2+channels.
Assuntos
Animais , Camundongos , Bloqueadores dos Canais de Cálcio/farmacologia , Nifedipino/farmacologia , Praziquantel/farmacologia , Schistosoma mansoni/efeitos dos fármacos , Esquistossomicidas/farmacologia , Testes de Sensibilidade ParasitáriaRESUMO
Schistosomiasis diagnosis is based on the detection of eggs in the faeces, which is laborious and lacks sensitivity, especially for patients with a low parasite burden. Immunological assays for specific antibody detection are available, but they usually demonstrate low sensitivity and/or specificity. In this study, two simple immunological assays were evaluated for the detection of soluble Schistosoma mansoni adult worm preparation (SWAP) and egg-specific IgGs. These studies have not yet been evaluated for patients with low parasite burdens. Residents of an endemic area in Brazil donated sera and faecal samples for our study. The patients were initially diagnosed by a rigorous Kato-Katz analysis of 18 thick smears from four different stool samples. The ELISA-SWAP was successful for human diagnosis with 90% sensitivity and specificity, confirming the Kato-Katz diagnosis with nearly perfect agreement, as seen by the Kappa index (0.85). Although the ELISA-soluble S. mansoni egg antigen was 85% sensitive, it exhibited low specificity (80%; Kappa index: 0.75) and was more susceptible to cross-reactivity. We believe that immunological assays should be used in conjunction with Kato-Katz analysis as a supplementary tool for the diagnosis of schistosomiasis for patients with low infection burdens, which are usually hard to detect.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Schistosoma mansoni/imunologia , Esquistossomose mansoni/diagnóstico , Adulto , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/imunologia , Fezes/parasitologia , Feminino , Humanos , Masculino , Esquistossomose mansoni/epidemiologia , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos , Surtos de Doenças , Imunoglobulina G , Schistosoma mansoni/imunologia , Esquistossomose mansoni/diagnóstico , Viagem , Doença Aguda , Animais , Brasil/epidemiologia , Ensaio de Imunoadsorção Enzimática , Fezes/parasitologia , Humanos , Espectroscopia de Ressonância Magnética , Contagem de Ovos de Parasitas , Esquistossomose mansoni/epidemiologia , Sensibilidade e EspecificidadeRESUMO
Schistosomiasis diagnosis is based on the detection of eggs in the faeces, which is laborious and lacks sensitivity, especially for patients with a low parasite burden. Immunological assays for specific antibody detection are available, but they usually demonstrate low sensitivity and/or specificity. In this study, two simple immunological assays were evaluated for the detection of soluble Schistosoma mansoni adult worm preparation (SWAP) and egg-specific IgGs. These studies have not yet been evaluated for patients with low parasite burdens. Residents of an endemic area in Brazil donated sera and faecal samples for our study. The patients were initially diagnosed by a rigorous Kato-Katz analysis of 18 thick smears from four different stool samples. The ELISA-SWAP was successful for human diagnosis with 90% sensitivity and specificity, confirming the Kato-Katz diagnosis with nearly perfect agreement, as seen by the Kappa index (0.85). Although the ELISA-soluble S. mansoni egg antigen was 85% sensitive, it exhibited low specificity (80%; Kappa index: 0.75) and was more susceptible to cross-reactivity. We believe that immunological assays should be used in conjunction with Kato-Katz analysis as a supplementary tool for the diagnosis of schistosomiasis for patients with low infection burdens, which are usually hard to detect.