Environmental hepatitis E virus detection supported by serological evidence in the northwest of Argentina.

Background: Hepatitis E virus (HEV) is an emergent cause of acute hepatitis worldwide. Water contamination is a possible source of viral infection. In South America, particularly in Argentina, little is known about environmental HEV circulation, including recreational water. The aim of this work was to provide evidence of current environmental and human circulation of HEV in northern Argentina. Methods: Molecular detection of HEV in water samples from the Arias-Arenales River in the city of Salta by nested polymerase chain reaction (ORF2 region) and anti-HEV immunoglobulin G (IgG) and IgM detection in the general population by enzyme-linked immunosorbent assay was carried out. Results: HEV RNA was detected in 1.6% (3/189) of the environmental samples. All sequences belonged to HEV genotype 3 and were very similar to those previously detected in the country. The prevalence of IgG anti-HEV was 9% (13/143) and three samples were positive for specific IgM. Conclusions: Circulation of HEV in the northwest of Argentina was demonstrated for the first time, showing viral presence in environmental samples and infections in people who attended health care centres for routine control. These findings show that recreational waters are a possible source of virus and highlight the need to carry out HEV detection when a case of hepatitis occurs.

Detección de Trypanosoma cruzi en tejido y sangre murina por PCR convencional y en tiempo real / Detection of Trypanosoma cruzi in murine blood and tissue by conventional and real time PCR / Detecção de Trypanosoma cruzi em tecido e sangue murinos por PCR convencional e em tempo real

El objetivo del presente trabajo fue comparar la detección de ADN de Trypanosoma cruzi mediante PCR en tiempo real (qPCR) y PCR convencional en sangre periférica (n=25) y músculo esquelético (n=20) de ratones tratados con drogas tripanomicidas luego de 6 meses post-tratamiento. En las muestras de sangre se detectaron un total de 7 positivas por qPCR, mientras que por PCR convencional sólo se detectaron 2. En músculo esquelético, 15 muestras fueron positivas por qPCR y 3 por PCR convencional. Los resultados obtenidos demuestran que la fuerza de concordancia es débil entre las técnicas de PCR utilizadas para la detección de ADN de T. cruzi (k=0,37; 49% positivas por qPCR vs. 11% por PCR convencional, p=0,0001). En las muestras de sangre, los valores diagnósticos de qPCR con respecto a la PCR convencional fueron: 100% sensibilidad; 78% especificidad; 30% VPP; 100% VPN; 4,6 RVP; 0 RVN. Para las muestras de músculo esquelético se obtuvieron los siguientes valores diagnósticos de qPCR: 100% sensibilidad; 29% especificidad; 20% VPP; 100% VPN; 1,4 RVP; 0 RVN. Ambas técnicas fueron igualmente sensibles en el rango de mediana-alta concentración, pero qPCR fue más efectiva para detectar bajas cargas parasitarias, en particular en las muestras de tejido.

The aim of this work was to compare detection of Trypanosoma cruzi DNA by real time (qPCR) and conventional PCR in peripheral blood (n=25), and skeletal muscle (n=20) of mice treated with trypanocidal compounds after 6 months post-treatment. A total of 7 blood samples were positive by qPCR; whereas, by conventional PCR only 2 were detected. In skeletal muscle, 15 samples were regarded positive by qPCR and 3 by conventional PCR. These results showed a weak concordance strength among PCR techniques employed to detect T. cruzi DNA in the studied samples (k=0.37; 49% positives by qPCR vs. 11% by conventional PCR, p=0.0001). In blood samples, qPCR diagnostic values in comparison with conventional PCR were: 100% sensibility; 78% specificity; 30% PPV; 100% NPV; 4.6 PVR; 0 NVR. For skeletal muscle samples, qPCR diagnostic values were: 100% sensibility; 29% specificity; 20% PPV; 100% NPV; 1.4 PVR; 0 NVR. Both techniques were equally sensitive in the medium-high concentration range, but qPCR was more effective to detect low parasitic burden, particularly in skeletal muscle samples.

O objetivo deste estudo foi comparar a detecção de DNA de Trypanosoma cruzi por PCR em tempo real (qPCR) e PCR convencional no sangue periférico (N=25) e músculo esquelético (N= 20) de camundongos tratados com medicamentos tripanomicidas depois de 6 meses de pós-tratamento. Nas amostras de sangue foi detectado um total de sete positivas por qPCR; enquanto que apenas foram encontradas 2 por PCR convencional. No músculo esquelético, 15 amostras foram positivas por qPCR e 3 por PCR convencional. Os resultados mostram que a força de concordância é fraca entre as técnicas de PCR utilizadas para a detecção de DNA de T. cruzi (k=0,37, 49% positivas por qPCR vs. 11% para a PCR convencional, p=0,0001). Nas amostras de sangue, os valores diagnósticos de qPCR em relação a PCR convencional foram de 100% sensibilidade; 78% de especificidade; 30% de VPP; 100% VPN; 4,6 RVP; 0 RVN. Para as amostras de músculo esquelético, os seguintes valores diagnósticos de qPCR foram obtidos: 100% sensibilidade; 29% de especificidade; 20% de VPP; 100% VPN; 1,4 RVP; 0 RVN. Ambas as técnicas são igualmente sensíveis na faixa de concentração média-alta, mas qPCR foi mais eficaz na detecção de baixas cargas parasitárias, especialmente em amostras de tecido.

Detección de Trypanosoma cruzi en tejido y sangre murina por PCR convencional y en tiempo real / Detection of Trypanosoma cruzi in murine blood and tissue by conventional and real time PCR / DetecþÒo de Trypanosoma cruzi em tecido e sangue murinos por PCR convencional e em tempo real

El objetivo del presente trabajo fue comparar la detección de ADN de Trypanosoma cruzi mediante PCR en tiempo real (qPCR) y PCR convencional en sangre periférica (n=25) y músculo esquelético (n=20) de ratones tratados con drogas tripanomicidas luego de 6 meses post-tratamiento. En las muestras de sangre se detectaron un total de 7 positivas por qPCR, mientras que por PCR convencional sólo se detectaron 2. En músculo esquelético, 15 muestras fueron positivas por qPCR y 3 por PCR convencional. Los resultados obtenidos demuestran que la fuerza de concordancia es débil entre las técnicas de PCR utilizadas para la detección de ADN de T. cruzi (k=0,37; 49% positivas por qPCR vs. 11% por PCR convencional, p=0,0001). En las muestras de sangre, los valores diagnósticos de qPCR con respecto a la PCR convencional fueron: 100% sensibilidad; 78% especificidad; 30% VPP; 100% VPN; 4,6 RVP; 0 RVN. Para las muestras de músculo esquelético se obtuvieron los siguientes valores diagnósticos de qPCR: 100% sensibilidad; 29% especificidad; 20% VPP; 100% VPN; 1,4 RVP; 0 RVN. Ambas técnicas fueron igualmente sensibles en el rango de mediana-alta concentración, pero qPCR fue más efectiva para detectar bajas cargas parasitarias, en particular en las muestras de tejido.(AU)

The aim of this work was to compare detection of Trypanosoma cruzi DNA by real time (qPCR) and conventional PCR in peripheral blood (n=25), and skeletal muscle (n=20) of mice treated with trypanocidal compounds after 6 months post-treatment. A total of 7 blood samples were positive by qPCR; whereas, by conventional PCR only 2 were detected. In skeletal muscle, 15 samples were regarded positive by qPCR and 3 by conventional PCR. These results showed a weak concordance strength among PCR techniques employed to detect T. cruzi DNA in the studied samples (k=0.37; 49% positives by qPCR vs. 11% by conventional PCR, p=0.0001). In blood samples, qPCR diagnostic values in comparison with conventional PCR were: 100% sensibility; 78% specificity; 30% PPV; 100% NPV; 4.6 PVR; 0 NVR. For skeletal muscle samples, qPCR diagnostic values were: 100% sensibility; 29% specificity; 20% PPV; 100% NPV; 1.4 PVR; 0 NVR. Both techniques were equally sensitive in the medium-high concentration range, but qPCR was more effective to detect low parasitic burden, particularly in skeletal muscle samples.(AU)

O objetivo deste estudo foi comparar a detecþÒo de DNA de Trypanosoma cruzi por PCR em tempo real (qPCR) e PCR convencional no sangue periférico (N=25) e músculo esquelético (N= 20) de camundongos tratados com medicamentos tripanomicidas depois de 6 meses de pós-tratamento. Nas amostras de sangue foi detectado um total de sete positivas por qPCR; enquanto que apenas foram encontradas 2 por PCR convencional. No músculo esquelético, 15 amostras foram positivas por qPCR e 3 por PCR convencional. Os resultados mostram que a forþa de concordÔncia é fraca entre as técnicas de PCR utilizadas para a detecþÒo de DNA de T. cruzi (k=0,37, 49% positivas por qPCR vs. 11% para a PCR convencional, p=0,0001). Nas amostras de sangue, os valores diagnósticos de qPCR em relaþÒo a PCR convencional foram de 100% sensibilidade; 78% de especificidade; 30% de VPP; 100% VPN; 4,6 RVP; 0 RVN. Para as amostras de músculo esquelético, os seguintes valores diagnósticos de qPCR foram obtidos: 100% sensibilidade; 29% de especificidade; 20% de VPP; 100% VPN; 1,4 RVP; 0 RVN. Ambas as técnicas sÒo igualmente sensíveis na faixa de concentraþÒo média-alta, mas qPCR foi mais eficaz na detecþÒo de baixas cargas parasitárias, especialmente em amostras de tecido.(AU)

Performance characteristics of qPCR assays targeting human- and ruminant-associated bacteroidetes for microbial source tracking across sixteen countries on six continents.

Numerous quantitative PCR assays for microbial fecal source tracking (MST) have been developed and evaluated in recent years. Widespread application has been hindered by a lack of knowledge regarding the geographical stability and hence applicability of such methods beyond the regional level. This study assessed the performance of five previously reported quantitative PCR assays targeting human-, cattle-, or ruminant-associated Bacteroidetes populations on 280 human and animal fecal samples from 16 countries across six continents. The tested cattle-associated markers were shown to be ruminant-associated. The quantitative distributions of marker concentrations in target and nontarget samples proved to be essential for the assessment of assay performance and were used to establish a new metric for quantitative source-specificity. In general, this study demonstrates that stable target populations required for marker-based MST occur around the globe. Ruminant-associated marker concentrations were strongly correlated with total intestinal Bacteroidetes populations and with each other, indicating that the detected ruminant-associated populations seem to be part of the intestinal core microbiome of ruminants worldwide. Consequently tested ruminant-targeted assays appear to be suitable quantitative MST tools beyond the regional level while the targeted human-associated populations seem to be less prevalent and stable, suggesting potential for improvements in human-targeted methods.

Desarrollo de una PCR en tiempo real para la cuantificación de leishmania SP en muestras de pacientes con leishmaniasis tegumentaria americana / Development of a real time PCR assay for the quantification of leishmania SP in samples from patients with american tegumentary leishmaniasis

INTRODUCCION: La leishmaniasis tegumentaria americana (LTA) es una enfermedad endémica reemergente en la provincia de Salta. Los recursos terapéuticos disponibles presentan serias limitaciones. Se han detectado numerosos casos de fallas terapéuticas debido a que el criterio de curación se basa en la evolución clínica de las lesiones.OBJETIVO: A fin de contar con un sistema adecuado para monitorear la enfermedad, se propuso optimizar una PCR en tiempo real (RT-PCR) basada en el uso de un agente intercalante y oligonucleótidos que amplifican secuencias del ADN del kinetoplasto (KADN), directamente de muestras de raspados de lesiones (en pacientes con LTA) contenidas en buffer TE (Tris-EDTA), sin extracción de ADN.METODOS: Para obtener una curva estándar (CE) se realizaron diluciones seriadas de parásitos (p) de cepas de referencia de especies que circulan en la zona. También se compararon 3 métodos de procesamiento de muestras (PM): Buffer de lisis, Insta-GeneTM Matrix (BIO-RAD) y TE. Luego se analizaron negativos para evaluar la especificidad y sensibilidad del sistema. Estas muestras provenían de raspados de lesiones cutáneas o mucocutáneas, tomados con palillos de madera en 300 μl TE.RESULTADOS: El límite de detección fue de 0,001 p/300 μl TE. La CE construida a partir de una cepa de Leishmania Viannia braziliensis mostró una pendiente de -3,40, eficiencia de amplificación de 96,66%, coeficiente de Pearson (R2) de 0,997 e intersección en la ordenada de 44,079. Al comparar los PM, el método de buffer TE fue el más eficiente. La RT-PCR desarrollada mostró un 100% de especificidad frente a muestras controles negativos y un 100% de sensibilidad frente a controles positivos.CONCLUSIONES: El sistema analizado es altamente sensible y permite detectar parásitos de Leishmania sp directamente de muestras clínicas provenientes de raspados de lesiones.

INTRODUCTION: The american tegumentary leishmaniasis (ATL) is an endemic, re-emergent disease in the Province of Salta. The therapeutic resources which are available have serious limitations. Numerous treatment failures have been detected due to the fact that the evaluation of chemotherapy is based on the clinical outcome of lesionsOBJECTIVE: In order to find a system for the correct follow-up of the disease, the study aimed at optimized the real time PCR (RT-PCR) based on intercalating agent and primers that amplify kinetoplastic DNA sequences (KDNA), directly on samples from skin lesion scrappings (from patients with ATL) contained in buffer TE, without DNA purification..METHODS: To obtain a stardard curve (SC) serial dilutions of parasites (p) were made (from reference strains of species that circulate in the region). 3 different methods of sample processing (SP) were compared: Lysis buffer, Insta-GeneTM Matrix (BIO-RAD) and TE (Tris-EDTA). The study evaluated the specificity and sensibility of the system by analyzing positive (by conventional PCR) and negative clinical samples. These samples were from cutaneous or mucocutaneous lesion scrapings, taken with toothpicks in 300 μl TE.RESULTS: The detection limit was 0.001 p/300 μl TE. The SC built with a Leishmania Viannia braziliensis strain showed a slope of -3.40, amplification efficiency of 96.66%, Pearson coefficient (R2) of 0.997 and 44.079 x-intersection. When comparing the SP, the buffer TE method was the most efficient one. With respect to positive and negative control samples, this RT-PCR showed a sensitivity and specificity of 100% respectively.CONCLUSIONS: This system is highly sensitive and allows to detect Leishmania sp parasites directly from clinical samples of lesion scrapings.

Desarrollo de una PCR en tiempo real para la cuantificación de leishmania SP en muestras de pacientes con leishmaniasis tegumentaria americana / Development of a real time PCR assay for the quantification of leishmania SP in samples from patients with american tegumentary leishmaniasis

INTRODUCCION: La leishmaniasis tegumentaria americana (LTA) es una enfermedad endémica reemergente en la provincia de Salta. Los recursos terapéuticos disponibles presentan serias limitaciones. Se han detectado numerosos casos de fallas terapéuticas debido a que el criterio de curación se basa en la evolución clínica de las lesiones.OBJETIVO: A fin de contar con un sistema adecuado para monitorear la enfermedad, se propuso optimizar una PCR en tiempo real (RT-PCR) basada en el uso de un agente intercalante y oligonucleótidos que amplifican secuencias del ADN del kinetoplasto (KADN), directamente de muestras de raspados de lesiones (en pacientes con LTA) contenidas en buffer TE (Tris-EDTA), sin extracción de ADN.METODOS: Para obtener una curva estándar (CE) se realizaron diluciones seriadas de parásitos (p) de cepas de referencia de especies que circulan en la zona. También se compararon 3 métodos de procesamiento de muestras (PM): Buffer de lisis, Insta-GeneTM Matrix (BIO-RAD) y TE. Luego se analizaron negativos para evaluar la especificidad y sensibilidad del sistema. Estas muestras provenían de raspados de lesiones cutáneas o mucocutáneas, tomados con palillos de madera en 300 μl TE.RESULTADOS: El límite de detección fue de 0,001 p/300 μl TE. La CE construida a partir de una cepa de Leishmania Viannia braziliensis mostró una pendiente de -3,40, eficiencia de amplificación de 96,66%, coeficiente de Pearson (R2) de 0,997 e intersección en la ordenada de 44,079. Al comparar los PM, el método de buffer TE fue el más eficiente. La RT-PCR desarrollada mostró un 100% de especificidad frente a muestras controles negativos y un 100% de sensibilidad frente a controles positivos.CONCLUSIONES: El sistema analizado es altamente sensible y permite detectar parásitos de Leishmania sp directamente de muestras clínicas provenientes de raspados de lesiones.

INTRODUCTION: The american tegumentary leishmaniasis (ATL) is an endemic, re-emergent disease in the Province of Salta. The therapeutic resources which are available have serious limitations. Numerous treatment failures have been detected due to the fact that the evaluation of chemotherapy is based on the clinical outcome of lesionsOBJECTIVE: In order to find a system for the correct follow-up of the disease, the study aimed at optimized the real time PCR (RT-PCR) based on intercalating agent and primers that amplify kinetoplastic DNA sequences (KDNA), directly on samples from skin lesion scrappings (from patients with ATL) contained in buffer TE, without DNA purification..METHODS: To obtain a stardard curve (SC) serial dilutions of parasites (p) were made (from reference strains of species that circulate in the region). 3 different methods of sample processing (SP) were compared: Lysis buffer, Insta-GeneTM Matrix (BIO-RAD) and TE (Tris-EDTA). The study evaluated the specificity and sensibility of the system by analyzing positive (by conventional PCR) and negative clinical samples. These samples were from cutaneous or mucocutaneous lesion scrapings, taken with toothpicks in 300 μl TE.RESULTS: The detection limit was 0.001 p/300 μl TE. The SC built with a Leishmania Viannia braziliensis strain showed a slope of -3.40, amplification efficiency of 96.66%, Pearson coefficient (R2) of 0.997 and 44.079 x-intersection. When comparing the SP, the buffer TE method was the most efficient one. With respect to positive and negative control samples, this RT-PCR showed a sensitivity and specificity of 100% respectively.CONCLUSIONS: This system is highly sensitive and allows to detect Leishmania sp parasites directly from clinical samples of lesion scrapings.