Desafios da implantação de testes rápidos para o diagnóstico da leishmaniose visceral em serviços de saúde de município endêmico para a doença / Challenges of the implantation of rapid tests for the diagnosis of visceral leishmaniasis in health services of a municipality endemic to the disease

O objetivo deste estudo foi descrever e discutir fatores facilitadores e desafios enfrentados durante o processo de incorporação de um teste rápido para o diagnóstico da leishmaniose visceral (LV) humana em serviços de saúde de município endêmico para a doença no Brasil. Este estudo foi dividido em quatro eixos de análise, seguindo as etapas de execução do estudo: 1) Descrição do sistema de saúde local e da tecnologia a ser implantada; 2) Contexto e atividades preparatórias; 3) Resultados da avaliação da implantação, da aceitação e do desempenho do algoritmo diagnóstico; 4) Conclusões, considerações e recomendações. O estudo foi conduzido em Ribeirão das Neves, no estado de Minas Gerais; o teste rápido implantado, o IT LEISH®, executado a partir de sangue capilar. Impasses e desafios estiveram relacionados à recusa de profissionais de saúde em realizar o IT LEISH® durante as capacitações, como dificuldade no processo de compra do teste rápido e atraso na entrega, dificuldades para coleta do sangue capilar relatada por pacientes e profissionais de saúde e falta de clareza entre os profissionais sobre suas atribuições e responsabilidades nas unidades de saúde, além de evasão de pacientes para cidade de maior porte. Este estudo apontou para a viabilidade da implantação de um teste rápido que descentralizasse e favorecesse o acesso ao diagnóstico laboratorial da LV. No entanto, no período do estudo, a maioria dos casos de LV residentes em Ribeirão das Neves foi diagnosticada em outro município, Belo Horizonte. Tal constatação aponta para desarticulação interna envolvendo os serviços de saúde do município, seja no acolhimento e na identificação dos suspeitos de LV, seja na efetiva utilização dos recursos disponíveis. Mesmo assim, identificamos dois aspectos determinantes para a realização da implantação: o engajamento de profissionais lotados em setores estratégicos da gestão municipal e a existência de financiamento. Estes resultados demonstram a complexidade do processo de implantação de uma nova tecnologia e apontam para a necessidade de trabalho integrado. Do contrário, a disponibilidade de testes rápidos para LV não será suficiente para garantir acesso e redução da letalidade pela doença.

This study aims to describe and discuss facilitating factors and challenges faced during the process of incorporating a rapid test for the diagnosis of human visceral leishmaniasis (VL) in health services of a municipality endemic to the disease in Brazil. This study was divided in four axis of analysis, following the study stages of execution: 1) Description of the local health system and the technology to be implemented; 2) Context and preparatory activities; 3) Results of evaluation of the implementation, acceptance and performance of the diagnostic algorithm; 4) Conclusions, considerations and recommendations. The study was conducted in Ribeirão das Neves, Minas Gerais and the rapid test implanted was IT LEISH®, performed from capillary blood. Impasses and challenges were related to the refusal of health professionals to perform IT LEISH® during training, difficulty in purchasing the rapid test and its delayed delivery, difficulties in capillary blood collection reported by patients and health professionals, lack of clarity among the professionals regarding their duties and responsibilities in health units, as well as avoidance of patients to a larger city. This study pointed to the feasibility of the implantation of a rapid test that decentralizes and favors the access to laboratory diagnosis of VL. However, in the study period, the majority of VL cases residing in Ribeirão das Neves were diagnosed in another municipality, Belo Horizonte. This finding points to internal disarticulation involving the health services of the municipality, either in the reception and identification of suspected LV, or in the effective use of available resources. Even so, we identified two determinant aspects for the implementation: the engagement of crowded professionals in strategic sectors of municipal management and the existence of financing. These results demonstrate the complexity of the process of implementing a new technology and point to the necessity of integrated work. Otherwise, the availability of rapid tests for LV will not be enough to guarantee access and reduction of disease lethality.

Study of implementation and direct cost estimates for diagnostic tests for human visceral leishmaniasis in an urban area in Brazil / Estudo da implantação e estimativas de custo direto de testes diagnósticos para leishmaniose visceral humana em uma área urbana no Brasil / Estudio de la implementación y el costo directo estimado de pruebas de diagnóstico de la leishmaniasis visceral humana en una zona urbana en Brasil

Abstract This work reports the process and costs of comprehensively implementing two tests to decentralize the diagnosis of visceral leishmaniasis (VL) in an endemic city in Brazil: a rapid test (IT LEISH) and a direct agglutination test (DAT-LPC). The implementation began by training health professionals to perform the tests. Estimation of the training costs considered the proportional remuneration of all professionals involved and the direct costs of the tests used for training. The study was conducted between November 2011 and November 2013. During that time, 17 training sessions were held, and 175 professionals were trained. The training cost for each professional was US$ 7.13 for the IT LEISH and US$ 9.93 for the DAT-LPC. The direct costs of the IT LEISH and DAT-LPC were estimated to be US$ 6.62 and US$ 5.44, respectively. This first evaluation of the implementation of these diagnostic tests indicates the feasibility of decentralizing both methods to extend access to VL diagnosis in Brazil.

Resumo Este trabalho relata o processo e os custos da implantação de dois testes para descentralizar o diagnóstico da leishmaniose visceral (LV) em um município endêmico no Brasil: um teste rápido (IT LEISH) e um teste de aglutinação direta (DAT-LPC). A implantação iniciou com o treinamento dos profissionais de saúde do município na realização dos testes diagnósticos. Os itens incluídos nas estimativas de custo das capacitações foram a remuneração proporcional de todos os profissionais envolvidos e os custos diretos dos testes usados. O estudo foi conduzido entre novembro de 2011 e novembro de 2013. Durante esse período, 17 capacitações foram realizadas e 175 profissionais treinados. O custo relacionado a cada profissional de saúde capacitado na realização do IT LEISH foi de US$ 7,13 e na realização do DAT-LPC, de US$ 9,93. O custo direto do IT LEISH e do DAT-LPC foi estimado em US$ 6,62 e US$ 5,44, respectivamente. Esta primeira avaliação da implantação desses dois testes aponta para a viabilidade da descentralização de ambos os métodos, que aumentam o acesso ao diagnóstico da LV no Brasil.

Resumen Este trabajo relata la puesta en funcionamiento y los costos de pruebas de diagnóstico de VL en un municipio endémico en Brasil: el test rápido (IT LEISH) y la prueba de aglutinación directa (DAT-LPC). Esta puesta en marcha comenzó por capacitar al personal sanitario del municipio para la realización de las pruebas. Para estimar los costos de la capacitación, se consideró la remuneración proporcional de todo el personal involucrado y los costos directos derivados de la aplicación de las pruebas. El estudio fue realizado entre noviembre de 2011 y noviembre de 2013. En ese periodo se realizaron 17 capacitaciones y se formaron 175 profesionales. Se calcula que el costo derivado de capacitar cada profesional para realizar el IT LEISH fue de 7.13 US$ y 9.93 US$ para el DAT-LPC. Los costos directos del IT LEISH y del DAT-LPC se estimaron en 6,62 US$ y 5,44 US$ respectivamente. La primera evaluación de la puesta en funcionamiento de las dos pruebas en este municipio señala que es viable descentralizar su realización, lo que amplía el acceso al diagnóstico de la VL en Brasil.

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.

Parasite load and risk factors for poor outcome among children with visceral leishmaniasis. A cohort study in Belo Horizonte, Brazil, 2010-2011

Clinical and laboratory risk factors for death from visceral leishmaniasis (VL) are relatively known, but quantitative real-time polymerase chain reaction (qPCR) might assess the role of parasite load in determining clinical outcome. The aim of this study was to identify risk factors, including parasite load in peripheral blood, for VL poor outcome among children. This prospective cohort study evaluated children aged ≤ 12 years old with VL diagnosis at three times: pre-treatment (T0), during treatment (T1) and post-treatment (T2). Forty-eight patients were included and 16 (33.3%) met the criteria for poor outcome. Age ≤ 12 months [relative risk (RR) 3.51; 95% confidence interval (CI) 1.89-6.52], tachydyspnoea (RR 3.46; 95% CI 2.19-5.47), bacterial infection (RR 3.08; 95% CI 1.27-7.48), liver enlargement (RR 3.00; 95% CI 1.44-6.23) and low serum albumin (RR 7.00; 95% CI 1.80-27.24) were identified as risk factors. qPCR was positive in all patients at T0 and the parasite DNA was undetectable in 76.1% of them at T1 and in 90.7% at T2. There was no statistical association between parasite load at T0 and poor outcome.

Diagnosing schistosomiasis: where are we?

In light of the World Health Organization's initiative to extend schistosomiasis morbidity and mortality control programs by including a disease elimination strategy in low endemic settings, this paper reviews diagnostic tools described during the last decades and provide an overview of ongoing efforts in making an efficient diagnostic tool available worldwide. A literature search on PubMed using the search criteria schistosomiasis and diagnosis within the period from 1978 to 2013 was carried out. Articles with abstract in English and that used laboratory techniques specifically developed for the detection of schistosomiasis in humans were included. Publications were categorized according to the methodology applied (parasitological, immunological, or molecular) and stage of development (in house development, limited field, or large scale field testing). The initial research generated 4,535 publications, of which only 643 met the inclusion criteria. The vast majority (537) of the publications focused on immunological techniques; 81 focused on parasitological diagnosis, and 25 focused on molecular diagnostic methods. Regarding the stage of development, 307 papers referred to in-house development, 202 referred to limited field tests, and 134 referred to large scale field testing. The data obtained show that promising new diagnostic tools, especially for Schistosoma antigen and deoxyribonucleic acid (DNA) detection, which are characterized by high sensitivity and specificity, are being developed. In combination with international funding initiatives these tools may result in a significant step forward in successful disease elimination and surveillance, which is to make efficient tests accessible and its large use self-sustainable for control programs in endemic countries.

Polymerase chain reaction for the evaluation of Schistosoma mansoni infection in two low endemicity areas of Minas Gerais, Brazil

This study aimed to evaluate the occurrence of schistosomiasis in areas with low endemicity using polymerase chain reaction (PCR) as a diagnostic method. We analysed faecal samples from 219 individuals residing in Piau and Coronel Pacheco, state of Minas Gerais, Brazil, using a single faecal sample from each individual and two slides of the Kato-Katz technique as a gold standard. Fifteen out of the 219 samples were positive with both methods of diagnosis. One sample was diagnosed as positive by the Kato-Katz technique only and 61 were diagnosed only by PCR. The positivity rates were 7.3% with the Kato-Katz method and 34.7% with PCR. When both techniques were assumed to have 100% specificity and positive individuals were identified by both methods, the sensitivity of the Kato-Katz method was 20.8% and the PCR sensitivity was 98.7%. The Kappa index between the two techniques was 0.234, suggesting weak agreement. The assessment of a single faecal sample by PCR detected more cases of infection than the analysis of one sample with two slides using the Kato-Katz technique, suggesting that PCR can be a useful diagnostic tool, particularly in areas with low endemicity.

Análise do perfil de expressão gênica por cDNA MICRORRAY em amostras de mucosa normal, metaplasia intestinal e adenocarcicoma do esôfago e estomago / Analysis of gene expression profile by cDNA Microarray of esophagus and stomach samples representing normal mucosa, intestinal metaplasia and adenocarcinoma

Esse estudo comparou pela metodologia do cDNA microarray o perfil de expressão gênica de 71 amostras do esôfago e estômago~ representando mucosa normal~ metaplasia intestinal e carcinomas. A análise fatorial e a técnica dos componentes principais foram empregadas para gerar fatores que melhor representassem o conjunto inicial de 4600 genes. Quatro fatores foram selecionados e divididos para formarem oito escores. As amostras de esôfago normal~ esofagite~ esôfago de Barrett e adenocarcinomas do esôfago e junção esofagogástrica foram comparadas com base na média da somatória da intensidade de expressão dos genes que compõem cada escore. Dois perfis foram obtidos: para alguns escores individuais ou determinadas combinações entre eles foi possível diferenciar apenas o conjunto de amostras de esôfago normal e esofagite do cotliunto de amostras de Barrett e adenocarcinomas~ já para outros escores e combinações entre eles foi possível diferenciar o conjunto de amostras de esôfago normal e esofagite das amostras de Barrett e adenocarcinoma separadamente, como também entre essas duas últimas amostras. Uma outra abordagem de análise foi empregada para comparar o perfil de expressão gênica entre amostras de esôfago e estômago. A ativação ou inibição de módulos funcionais que consistiam em conjuntos de genes relacionados a processos biológicos de acordo com o banco de dados KEGG (K yoto Encyclopeedia of Genes and Genomes) foram avaliadas nas diferentes amostras. Os módulos de interação ligante-receptor para citocinas e metabolismo de glicerolípídeos foram destacados pelo número e tipo de tecidos em que se mostraram ativos ou inibidos. A análise de agrupamento pelo algoritmo K-médias para esses módulos gerou três grupos. Um grupo foi constituído por amostras de tecido escamoso, outro por amostras de tecido co lunar maligno (carcinomas do esôfago e estômago) e um outro com todas as amostras de tecido colunar não maligno (mucosa gástrica normal e metaplasia intestinal do esôfago e estômago). Para esses módulos foram identificados os genes que contribuíram para o perfil de ativação ou inibição nas diferentes amostras. Esses genes foram os mesmos genes responsáveis pelos tipos de agrupamentos observados para as diferentes amostras. Os genes ILIR2, CCL20, CCL18, INHBA, IL4R e IFNA2 foram consistentes com o módulo de interação ligante-receptor para citocinas e os genes AKRIBIO, ALDH3A2, ADHIB e CDSI foram consistentes com o módulo de metabolismo de glicerolipídeos. Futuros estudos poderão melhor identificar o papel dos genes dos escores formados e das vias de metabolismo de glicerolipídeos e sinalização por citocinas na compreensão do desenvolvimento da metaplasia intestinal do esôfago e estômago e bem como, do seu risco de progressão para o adenocarcinoma (AU)

The expression profile of 71 samples of esophagus and stomach, representing normal mucosa, intestinal metaplasia and carcinoma tissues were determined using cDNA microarrays technology. The factor analysis and the principal component analysis (PCA) were used to extract factors or components that better represent the group of 4,600 initial genes. Four factors were extracted and partitioned forming eight scores. Samples of normal esophageal mucosa, esophagitis, Barrett' s mucosa and adenocarcinomas o f the esophagus and esophagogastric junction were compared by the mean of the sum of expression intensity of the genes representing each score. Two profiles were acquired. Some individual scores and some combinations among them permitted the differentiation of the group composed of normal esophageal and esophagitis samples from the group composed of Barrett' s and adenocarcinomas samples. Additionally, different individual scores and combinations among them permitted the differentiation of the group composed of normal esophageal and esophagitis samples from the Barrett and adenocarcinoma samples, independently. These two last tissues were also di:fferentiated between them Another analysis was performed to compare the expression profile of esophagus and stomach samples. The activation and repression of functional modules related to biological processes according to the KEGG base data (Kyoto Encyclopdia o f Genes and Genomes) were assessed among the samples. The cytokine-cytokine receptor interaction and the glycerolipid metabolism modules showed interesting profile considering the number and kind of tissue that they were active or repressed. The cluster analysis by K-means algorithm was performed and the best result was obtained with K=3 and, for both modules, a clear separation between squamous tissue samples (normal esophagus mucosa and esophagitis mucosa); malignant columnar samples (gastric and esophagic carcinomas) and non-malignant columnar samples (intestinal metaplasia of the esophagus and stomach and normal gastric mucosa) was observed. The genes which contribute to the significant expression of the modules among samples were identified. The consistent genes found for the cytokine-cytokine receptor interaction module were ILIR2, CCL20, CCL18, INHB~ IL4R and IFNAR2, and for the glycerolipid metabolism module were AKRIBIO, ALDH3A2, ADHIB and CDSI. Futher investigation concerning the group of genes formed and a better understanding of the role o f glycerolipid metabolism and cytokine signaling could contribute to elucidate the development of intestinal metaplasia as well as the progression to adenocarcinoma (AU)