Development of a new accurate lateral flow immunoassay for diagnosis of human leptospirosis.

PURPOSE: The current diagnostic methods for leptospirosis diagnosis are technically complex and expensive, with limited applicability to specialized laboratories. Furthermore, they lack diagnostic accuracy in the acute stage of the disease, which coincides with a period when antibiotics are highly effective. New simple and accurate tests are mandatory to decentralize and improve diagnosis. Here, we introduced a new lateral flow immunoassay (Lepto-LF) for human leptospirosis. METHODS: We conducted a double-blinded assay using 104 serum samples from patients with confirmed or discarded diagnosis for leptospirosis. The diagnostic performance of Lepto-LF was estimated across different ranges of days from onset of symptoms (dpo), considering the diagnostic algorithm as reference standard. Additionally, it was compared with the screening methods enzyme-linked immunosorbent assay (IgM-ELISA) and the slide agglutination test using temperature-resistant antigen (SATR). RESULTS: Lepto-LF exhibited perfect diagnostic performance with a Youden´s index J = 1 from 6 dpo in the acute phase. IgM-ELISA gave slightly lower accuracy with J = 0.91 and 95.5% of both sensitivity and specificity; while SATR showed a markedly inferior yield (J = 0.41, sensitivity = 95.5%, specificity = 45.5%). The performances remained consistent in the convalescence phase of the disease (> 10 dpo). CONCLUSION: Lepto-LF was found to be a reliable test for simple, rapid and early diagnosis of leptospirosis, resulting a promising tool for decentralizing leptospirosis diagnosis and enabling timely treatment of patients. In addition, Lepto-LF may be employed as confirmatory test, especially in remote areas and vulnerable contexts where the standard MAT is not available.

Leptospirosis and rickettsiosis, a diagnostic challenge for febrile syndrome in endemic areas / [Leptospirosis y rickettsiosis, reto diagnóstico para el síndrome febril en zonas endémicas].

This is the case of a 50-year-old male from the region of Urabá, Colombia, with a mixed infection by Rickettsia rickettsii and Leptospira interrogans serovar Copenhageni ST78 and negative test for malaria and dengue fever. The patient presented with febrile syndrome and was unresponsive to systemic antibiotic treatment, who finally died in the intensive care unit. We established the postmortem diagnosis through molecular typification of the two etiological agents. In the inspection at the patient's home, we found a Rattus rattus specimen infected with L. interrogans of the same serovar found in him. We found no ticks parasitizing the domestic animals cohabitating with the patient. This case of a mixed infection with progressive and fatal symptoms in a patient with occupational risk in a tropical disease endemic zone highlights the importance of considering the potential presentation of simultaneous etiologies in patients with multiple medical visits for unresolved febrile syndromes associated with risky exposure during agricultural activities.

Se presenta el caso de un hombre de 50 años de edad proveniente de la región de Urabá, Colombia, con una infección mixta por Rickettsia rickettsii y Leptospira interrogans serovar Copenhageni ST78, y pruebas negativas para malaria y dengue. El paciente presentó un síndrome febril que no mejoró con el tratamiento antibiótico sistémico y, finalmente, falleció en la unidad de cuidados intensivos. El diagnóstico post mortem se hizo mediante tipificación molecular de los dos agentes etiológicos. En la inspección del domicilio del paciente, se encontró un ejemplar de Rattus rattus infectado con L. interrogans del mismo serovar detectado en él. No se encontraron garrapatas en los animales domésticos que habitaban con el paciente. Se reporta una infección mixta con síntomas clínicos progresivos y fatales en un paciente con antecedentes laborales de riesgo en una zona endémica para enfermedades tropicales, lo que obliga a tener presente la posibilidad de infecciones simultáneas en personas procedentes de áreas endémicas que consulten reiteradamente por síndrome febril sin resolución y tengan riesgo laboral relacionado con actividades agrícolas.

Evaluation of three qPCR for the detection of pathogenic leptospires in domestic animals in Nicaragua / [Evaluación de tres PCR cuantitativas para la detección de leptospiras patógenas en animales domésticos en Nicaragua].

Introduction: Molecular biology diagnostic methods such as real-time PCR should be used in Nicaragua to improve the diagnosis of leptospirosis in humans and animals. Objective: To evaluate three qPCR methods for pathogenic Leptospira detection in domestic animals. Materials and methods: Real-time PCR primers were designed for the amplification of specific regions from the Lip 32 gene of Leptospira in SYBER Green (SYBER Green-A) and TaqMan, as well in SYBER Green-B as previously published. The sequences of 12 strains obtained from the database of the National Center for Biotechnology Information (NCBI) were aligned to select probes and primers. The analytical sensitivity was determined by calculating the detectable genomic equivalent while 18 pathogenic references strains and 28 negative controls were used to evaluate the sensitivity and specificity of each one of the three sets in 129 urine samples of domestic animals. Results: The detection limit of four genomic equivalents per reaction was obtained from SYBR Green-A. The specificities were 94.4% (95% CI: 81.1-100.0) for TaqMan, 77.8% (95% CI: 55.8-99.8) for SYBR Green-A, while for SYBR Green-B it was 61.1% (95% CI: 35.8-86.4). In the three tests, we obtained a specificity of 100% (95% CI: 98.2-100.0). In the field samples, 26.4% were positive with SYBR Green-A and 6.1% with SYBR Green-B. Conclusion: SYBR Green-A presented the lowest detection limit while the three techniques under evaluation showed high specificity while TaqMan was the most sensitive.

Introducción. En Nicaragua es necesario estandarizar pruebas moleculares como la PCR en tiempo real (quantitative Polymerase Chain Reaction, qPCR) que mejoren el diagnóstico de leptospirosis en humanos y animales. Objetivo. Evaluar tres qPCR para la detección de leptospiras patógenas en animales domésticos de Nicaragua. Materiales y métodos. Se diseñaron cebadores para la amplificación del gen LipL32 en SYBR Green (SYBR Green-A) y TaqMan, y en otros descritos previamente (SYBR Green-B). Las secuencias de 12 cepas obtenidas de la base de datos del National Center for Biotechnology Information (NCBI) se alinearon para la búsqueda de sondas y cebadores. La sensibilidad analítica se determinó calculando el equivalente genómico detectable, se utilizaron 18 cepas de referencia para la sensibilidad diagnóstica y 28 controles negativos para la especificidad. Los métodos se aplicaron en 129 muestras de orina de animales domésticos. Resultados. En SYBR Green-A se obtuvo un límite de detección de cuatro equivalentes genómicos; en TaqMan, la sensibilidad fue del 94,4 % (IC95% 81,1-100,0). Con SYBR Green-A, se obtuvo una sensibilidad del 77,8 % (IC95% 55,8-99,8), en tanto que con SYBR Green-B fue del 61,1 % (IC95% 35,8-86,4). En las tres pruebas se logró una especificidad del 100 % (IC95% 98,2-100,0). El 26,4 % de las muestras de animales domésticos fueron positivas con SYBR Green-A y el 6,2 % con SYBR Green-B. Conclusiones. El SYBR Green-A presentó un límite de detección bajo, en tanto que las tres técnicas evaluadas mostraron alta especificidad, en tanto que la TaqMan tuvo la mayor sensibilidad.

Clinical characterization of patients with severe leptospirosis in a tertiary hospital in Cali, Colombia, 2010-2016 / Caracterización clínica de la leptospirosis grave en un hospital de alta complejidad de Cali, Colombia, 2010-2016

Introduction: Leptospirosis is an endemic bacterial infection in Colombia. Its clinical course can be variable and occasionally fatal. There are few studies in the country about severe cases of leptospirosis. Objectives: To describe the demographic and clinical characteristics of patients with a diagnosis of leptospirosis, and their management in a high complexity hospital. Materials and methods: This was a descriptive retrospective study of patients with a serologic diagnosis of leptospirosis between 2010 and 2016. Results: We analyzed 87 patients, 74% of them were men, and 84% were older than 18 years; 35% had a comorbidity, the most common being arterial hypertension (16%) and diabetes mellitus (9%). The most frequent symptoms were fever, nausea, fatigue, myalgia, arthralgia, and abdominal pain. The majority of patients required hospitalization in general wards (61%), with a median stay of six days; 34% required management in the intensive care unit, with a median stay of five days. Mortality was 1.1% (n=1). All patients received treatment with either ceftriaxone or doxycycline. Conclusions: There is a risk of leptospira infections having a late diagnosis given their unspecific clinical presentation, which generates a high number of differential diagnoses. The early management in the intensive care unit could decrease the incidence of complications and the mortality of patients with leptospirosis.

Introducción. La leptospirosis es una infección bacteriana endémica en Colombia. Su curso clínico puede ser variable y, en ocasiones, fatal. Hay pocos estudios en el país sobre los casos graves de esta enfermedad. Objetivo. Describir las características demográficas y clínicas de los pacientes con diagnóstico de leptospirosis grave hospitalizados en salas generales o atendidos en la unidad de cuidados intensivos de un hospital de cuarto nivel. Materiales y métodos. Se llevó a cabo un estudio observacional descriptivo de los pacientes adultos y niños con diagnóstico serológico de leptospirosis entre el 2010 y el 2016. Resultados. Se analizaron las historias clínicas de 87 pacientes, 74 % de los cuales correspondía a hombres y, el 84 %, a mayores de 18 años. El 35 % tenía alguna comorbilidad y la hipertensión arterial sistémica (16 %) y la diabetes mellitus (9 %) fueron las más comunes. Los síntomas más frecuentes fueron: fiebre, náuseas, astenia, mialgias, artralgias y dolor abdominal. El 34 % requirió atención en la unidad de cuidados intensivos, con una mediana de estancia de 5 días. El 61 % requirió hospitalización en sala general, con una mediana de estancia de 6 días. Todos los casos recibieron tratamiento antibiótico con ceftriaxona o doxiciclina. La tasa de letalidad fue del 1,1 % (n=1). Conclusiones. La infección por Leptospira spp. tiene el riesgo de diagnosticarse de manera tardía por su presentación clínica inespecífica, lo que implica considerar un gran número de diagnósticos diferenciales. La atención temprana de los pacientes con cuadros graves de esta enfermedad en la unidad de cuidados intensivos, puede evitar una mayor incidencia de complicaciones y disminuir la mortalidad.

Caracterización clínica de la leptospirosis grave en un hospital de alta complejidad de Cali, Colombia, 2010-2016 / Clinical characterization of patients with severe leptospirosis in a tertiary hospital in Cali, Colombia, 2010-2016

Resumen Introducción. La leptospirosis es una infección bacteriana endémica en Colombia. Su curso clínico puede ser variable y, en ocasiones, fatal. Hay pocos estudios en el país sobre los casos graves de esta enfermedad. Objetivo. Describir las características demográficas y clínicas de los pacientes con diagnóstico de leptospirosis grave hospitalizados en salas generales o atendidos en la unidad de cuidados intensivos de un hospital de cuarto nivel. Materiales y métodos. Se llevó a cabo un estudio observacional descriptivo de los pacientes adultos y niños con diagnóstico serológico de leptospirosis entre el 2010 y el 2016. Resultados. Se analizaron las historias clínicas de 87 pacientes, 74 % de los cuales correspondía a hombres y, el 84 %, a mayores de 18 años. El 35 % tenía alguna comorbilidad y la hipertensión arterial sistémica (16 %) y la diabetes mellitus (9 %) fueron las más comunes. Los síntomas más frecuentes fueron: fiebre, náuseas, astenia, mialgias, artralgias y dolor abdominal. El 34 % requirió atención en la unidad de cuidados intensivos, con una mediana de estancia de 5 días. El 61 % requirió hospitalización en sala general, con una mediana de estancia de 6 días. Todos los casos recibieron tratamiento antibiótico con ceftriaxona o doxiciclina. La tasa de letalidad fue del 1,1 % (n=1). Conclusiones. La infección por Leptospira spp. tiene el riesgo de diagnosticarse de manera tardía por su presentación clínica inespecífica, lo que implica considerar un gran número de diagnósticos diferenciales. La atención temprana de los pacientes con cuadros graves de esta enfermedad en la unidad de cuidados intensivos, puede evitar una mayor incidencia de complicaciones y disminuir la mortalidad.

Abstract Introduction: Leptospirosis is an endemic bacterial infection in Colombia. Its clinical course can be variable and occasionally fatal. There are few studies in the country about severe cases of leptospirosis. Objectives: To describe the demographic and clinical characteristics of patients with a diagnosis of leptospirosis, and their management in a high complexity hospital. Materials and methods: This was a descriptive retrospective study of patients with a serologic diagnosis of leptospirosis between 2010 and 2016. Results: We analyzed 87 patients, 74% of them were men, and 84% were older than 18 years; 35% had a comorbidity, the most common being arterial hypertension (16%) and diabetes mellitus (9%). The most frequent symptoms were fever, nausea, fatigue, myalgia, arthralgia, and abdominal pain. The majority of patients required hospitalization in general wards (61%), with a median stay of six days; 34% required management in the intensive care unit, with a median stay of five days. Mortality was 1.1% (n=1). All patients received treatment with either ceftriaxone or doxycycline. Conclusions: There is a risk of leptospira infections having a late diagnosis given their unspecific clinical presentation, which generates a high number of differential diagnoses. The early management in the intensive care unit could decrease the incidence of complications and the mortality of patients with leptospirosis.

Rapid diagnosis of leptospirosis in patients with different clinical manifestations by 16S rRNA gene based nested PCR.

Leptospirosis, a zoonosis of global importance and it is underreported in India and more than 50,000 severe cases are reported each year. Here we present the evaluation of 16S rRNA based nested PCR assay for the rapid identification of human leptospires using serum and urine samples. The study includes 261 suspected cases for leptospirosis with different clinical manifestations. 16S rRNA based nested PCR assay was compared and evaluated against the conventional serological methods such as MAT and ELISA. The technique enabled amplification of a 289 bp product with notable percentage of positivity in all sample groups including 94.8 in pediatric cases, 93 in pregnant women, 94.2 in renal failure, 87.8 in jaundice and 94.6 in common febrile cases. The sensitivity and specificity was 94.4% and 100%, respectively. The technique proved to be prompt and effective for the diagnosis of leptospiral infection at the acute phase of the disease. PCR based approach detects leptospiral DNA from the clinical samples both at the acute and leptospiruria phase on comparison with its counter parts where detection is made possible only after 7 days or 7-30 days post-infection. In this regard PCR based diagnosis of leptospirosis should be made available for clinicians for the early diagnosis and prompt treatment of the disease.

IgM ELISA for leptospirosis diagnosis: a systematic review and meta-analysis / ELISA IgM para diagnóstico de leptospirose: revisão sistemática e meta-análise

Abstract A systematic review with meta-analysis was performed to estimate the accuracy of IgM ELISA for Leptospirosis diagnosis. A search of Medline, Lilacs, Embase, Cochrane Central Register of Controlled Trials and Grey literature (Google Scholar and British Library) was conducted. The medical subject headings (MeSHs) and the words "leptospirosis", "human leptospirosis" and "IgM ELISA" were used. Fifty-two studies were analyzed, which included 10,775 samples. The pooled sensitivity of all the studies was 86% (CI 95%, 85%-87%) and specificity was 90% (CI 95%, 89%-91%). In studies of the acute phase, the sensitivity and specificity were 84% (CI 95%, 82%-85%) and 91% (CI 95%, 90%-91%), respectively. In conclusion, IgM ELISA is sensitive for use as an initial screen for leptospiral infections.

Resumo O objetivo desta revisão sistemática e meta-análise foi avaliar a acurácia do ELISA IgM para o diagnóstico precoce da leptospirose em humanos. A busca foi realizada nas seguintes bases de dados: Medline, PubMed, LILACS, Embase e Cochrane Central Register of Controlled Trials e Grey literature (Google Scholar and British Library). As palavras-chaves usadas foram: "leptospirosis", "human leptospirosis" e "IgM ELI-SA". Foram analisados 52 estudos, que incluíram 10.775 amostras. A sensibilidade e especificidade combinada de todos os estudos foram 86% (CI 95%, 85%-87%) e 90% (CI 95%, 89%-91%), respectivamente. Nos estudos de fase aguda, a sensibilidade e especificidade foram, respectivamente, 84% (CI 95%, 82%-85%) e 91% (CI 95%, 90%-91%). Conclui-se que o ELISA IgM é um teste sensível para rastreamento inicial da leptospirose.