Trypanosoma cruzi loop-mediated isothermal amplification (Trypanosoma cruzi Loopamp) kit for detection of congenital, acute and Chagas disease reactivation.

A Trypanosoma cruzi Loopamp kit was recently developed as a ready-to-use diagnostic method requiring minimal laboratory facilities. We evaluated its diagnostic accuracy for detection of acute Chagas disease (CD) in different epidemiological and clinical scenarios. In this retrospective study, a convenience series of clinical samples (venous blood treated with EDTA or different stabilizer agents, heel-prick blood in filter paper or cerebrospinal fluid samples (CSF)) from 30 infants born to seropositive mothers (13 with congenital CD and 17 noninfected), four recipients of organs from CD donors, six orally-infected cases after consumption of contaminated guava juice and six CD patients coinfected with HIV at risk of CD reactivation (N = 46 patients, 46 blood samples and 1 CSF sample) were tested by T. cruzi Loopamp kit (Tc LAMP) and standardized quantitative real-time PCR (qPCR). T. cruzi Loopamp accuracy was estimated using the case definition in the different groups as a reference. Cohen's kappa coefficient (κ) was applied to measure the agreement between Tc LAMP (index test) and qPCR (reference test). Sensitivity and specificity of T. cruzi Loopamp kit in blood samples from the pooled clinical groups was 93% (95% CI: 77-99) and 100% (95% CI: 80-100) respectively. The agreement between Tc LAMP and qPCR was almost perfect (κ = 0.92, 95% CI: 0.62-1.00). The T. cruzi Loopamp kit was sensitive and specific for detection of T. cruzi infection. It was carried out from DNA extracted from peripheral blood samples (via frozen EDTA blood, guanidine hydrochloride-EDTA blood, DNAgard blood and dried blood spots), as well as in CSF specimens infected with TcI or TcII/V/VI parasite populations. The T. cruzi Loopamp kit appears potentially useful for rapid detection of T. cruzi infection in congenital, acute and CD reactivation due to HIV infection.

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples.

BACKGROUND: Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI-TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). METHODS/PRINCIPAL FINDINGS: The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. CONCLUSIONS/SIGNIFICANCE: Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.

Human papilloma virus risk factors for infection and genotype distribution in aboriginal women from Northern Argentina.

The mortality rate for cervical cancer (CC) in Northern Argentina is three times higher than the average for the country (7.8 deaths/100,000 women). We determined the prevalence and genotype distribution of human papillomavirus (HPV) in 227 sexually active women of the native Pilagá community in Formosa, Argentina. We also conducted an HPV-16 variant analysis and studied several community factors that might play a role in viral entry and infection. Endo and exocervical samples were tested for HPV DNA with MY09/11-PCR or with GP5+/6+-PCR. HPV was detected in 46.7% of the samples and 21 different types were found; the most frequent being HPV-16 (19.4%), -6 and -18 (5.3%), -58 (3.5%) and -31 and -33 (3.1%). In relation to HPV-16 variants, 68.2% were European and 31.8% Asian-American. Among the cofactors analyzed only disposal of hu man excreta to the open air (P=0.01) was significantly associated with HPV infection. Our prevalence estimates clearly show that Pilagá women are highly exposed to or infected with high risk HPV types and therefore are at a high risk of developing precancerous lesions and eventually CC at the population level.

Human papillomavirus risk factors for infection and genotype distribution in aboriginal women from Northern Argentina / Virus papiloma humano (hpv) en mujeres de la etnia Pilagá del nordeste argentino: Factores de riesgo de infección y distribución de tipos virales

The mortality rate for cervical cancer (CC) in Northern Argentina is three times higher than the average for the country (7.8 deaths/100 000 women). We determined the prevalence and genotype distribution of human papillomavirus (HPV) in 227 sexually active women of the native Pilagá community in Formosa, Argentina. We also conducted an HPV-16 variant analysis and studied several community factors that might play a role in viral entry and infection. Endo- and exocervical samples were tested for HPV DNA with MY09/11-PCR or with GP5+/6+-PCR. HPV was detected in 46.7% of the samples and 21 different types were found; the most frequent being HPV-16 (19.4%), -6 and -18 (5.3%), -58 (3.5%) and -31 and -33 (3.1%). In relation to HPV-16 variants, 68.2% were European and 31.8% Asian-American. Among the cofactors analyzed only disposal of human excreta to the open air (P=0.01) was significantly associated with HPV infection. Our prevalence estimates clearly show that Pilagá women are highly exposed to or infected with high risk HPV types and therefore are at a high risk of developing precancerous lesions and eventually CC at the population level.

La tasa de mortalidad por cáncer cervical (CC) en la región norte de la Argentina es tres veces más alta que la media del país (7.8 muertes/100 000 mujeres). En el presente trabajo se determinó la prevalencia de infección por virus papiloma humano (VPH) y la distribución y frecuencia de los genotipos en 227 mujeres sexualmente activas de la etnia aborigen Pilagá (Formosa, Argentina). También se realizó un análisis de las variantes intratípicas de VPH-16 presentes en la comunidad y se analizaron diversos factores socioculturales que podrían tener algún rol destacado en la transmisión de la infección viral. Se estudiaron muestras de células endo-exocervicales mediante PCR basadas en los cebadores MY09/11 y GP5+/6+ con posterior restricción enzimática y/o hibridación dot-blot. La infección por VPH fue detectada en el 46.7% de las mujeres analizadas. Fueron identificados 21 genotipos, de los cuales los más frecuentes fueron HPV-16 (19.4%), -6 y -18 (5.3%), -58 (3.5%) y -31 y -33 (3.1%). Respecto al HPV-16, se encontraron 68.2% de variantes europeas y 31.8% de asiático-americanas. Entre los cofactores analizados, solo la disposición de excretas al aire libre estuvo significativamente asociada con la infección por VPH (P = 0.01). Los datos obtenidos reflejan que la comunidad Pilagá está altamente expuesta a las infecciones por genotipos de alto riesgo de VPH, lo cual puede estar asociado a una alta incidencia de lesiones cervicales preneoplásicas y neoplásicas.

Human papillomavirus risk factors for infection and genotype distribution in aboriginal women from Northern Argentina / Virus papiloma humano (hpv) en mujeres de la etnia Pilagá del nordeste argentino: Factores de riesgo de infección y distribución de tipos virales

The mortality rate for cervical cancer (CC) in Northern Argentina is three times higher than the average for the country (7.8 deaths/100 000 women). We determined the prevalence and genotype distribution of human papillomavirus (HPV) in 227 sexually active women of the native Pilagá community in Formosa, Argentina. We also conducted an HPV-16 variant analysis and studied several community factors that might play a role in viral entry and infection. Endo- and exocervical samples were tested for HPV DNA with MY09/11-PCR or with GP5+/6+-PCR. HPV was detected in 46.7% of the samples and 21 different types were found; the most frequent being HPV-16 (19.4%), -6 and -18 (5.3%), -58 (3.5%) and -31 and -33 (3.1%). In relation to HPV-16 variants, 68.2% were European and 31.8% Asian-American. Among the cofactors analyzed only disposal of human excreta to the open air (P=0.01) was significantly associated with HPV infection. Our prevalence estimates clearly show that Pilagá women are highly exposed to or infected with high risk HPV types and therefore are at a high risk of developing precancerous lesions and eventually CC at the population level.(AU)

La tasa de mortalidad por cáncer cervical (CC) en la región norte de la Argentina es tres veces más alta que la media del país (7.8 muertes/100 000 mujeres). En el presente trabajo se determinó la prevalencia de infección por virus papiloma humano (VPH) y la distribución y frecuencia de los genotipos en 227 mujeres sexualmente activas de la etnia aborigen Pilagá (Formosa, Argentina). También se realizó un análisis de las variantes intratípicas de VPH-16 presentes en la comunidad y se analizaron diversos factores socioculturales que podrían tener algún rol destacado en la transmisión de la infección viral. Se estudiaron muestras de células endo-exocervicales mediante PCR basadas en los cebadores MY09/11 y GP5+/6+ con posterior restricción enzimática y/o hibridación dot-blot. La infección por VPH fue detectada en el 46.7% de las mujeres analizadas. Fueron identificados 21 genotipos, de los cuales los más frecuentes fueron HPV-16 (19.4%), -6 y -18 (5.3%), -58 (3.5%) y -31 y -33 (3.1%). Respecto al HPV-16, se encontraron 68.2% de variantes europeas y 31.8% de asiático-americanas. Entre los cofactores analizados, solo la disposición de excretas al aire libre estuvo significativamente asociada con la infección por VPH (P = 0.01). Los datos obtenidos reflejan que la comunidad Pilagá está altamente expuesta a las infecciones por genotipos de alto riesgo de VPH, lo cual puede estar asociado a una alta incidencia de lesiones cervicales preneoplásicas y neoplásicas.(AU)

La Enfermedad de Chagas en poblaciones aborígenes del Noreste de Argentina / No disponible

Objetivos: Se estudió la infección por T. cruzi en aborígenes pertenecientes a tres etnias del noreste de Argentina. Métodos: Se estudiaron 455 individuos (2-62 años de edad), de los cuales 145 pertenecen a la etnia Quomo Toba residentes en dos localidades de la provincia de Chaco (112 en Pampa del Indio y 33 en Quitilipi), 199a la etnia wichi, también residentes en dos localidades de Chaco (120 en Misión Nueva Pompeya y 79 en Sauzalito) y 111 a la etnia Pilagá residentes en la localidad de Estanislao del Campo, provincia de Formosa. Mediante la colaboración de asistentes sociales bilingües se convocó a los individuos a los Centros sanitarios próximos a sus comunidades para explicarles los objetivos del estudio, solicitarles su consentimiento para participar del mismo y tomarles muestras de sangre para investigar anticuerpos anti T. cruzi mediante los tests de hemoaglutinación, ELISA e inmunoflorescencia. Resultados: La seroprevalencia total de la población analizada fue 54,3% (247/455); 46,9% para los individuos de Formosa y 57,3% para los de Chaco, con aumento con la edad pero con altos valores entre los menores de 15 años. No hubo diferencias significativas en relación con el género. La población Quom presentó una seroprevalencia del 54,5%, la Wichi del 59,3% y la Pilagá del 46,8%.Conclusiones: Los datos obtenidos muestran la magnitud de la infección chagásica aún presente entre los aborígenes de ciertas áreas de la región del Gran Chaco americano. Esta región presenta ambientes ecoepidemiológicos que reducen el impacto de las acciones de control con probabilidades de reinfestación, por lo que se requieren intervenciones con una mirada de región y no de áreas con límites políticos-administrativos (AU)

Objectives: To evaluate T. cruzi human infection in indigenous people belonging to three ethnic groups livingin northeast Argentina. Methods: We studied 455 individuals (2-62 years old), 145 belonging to Quom or Toba ethnic group inhabiting two towns of province of Chaco (112 from Pampa del Indio and 33 from Quitilipi), 199 belonging to the Wichiethnic group, also inhabiting in two towns of Chaco (120 from Misión Nueva Pompeya and 79 from El Sauzalito) and 111 belonging to the Pilagá ethnic group from Estanislao del Campo, in the province of Formosa. Bilingual social workers were resorted to convene the subjects to the closest health care center in order to explain thepurposes of the study, to obtain their consent to be recruited and to take blood samples for the detection ofantibodies against Chagas disease by indirect hemoagglutination, ELISA and indirect immunoflorescence tests. Results: Total seroprevalence in the population studied was 54.3% (247/455); 46.9% in the individuals from the province of Formosa and 57.3% in those from Chaco, increasing with age but with high values even in children under 15 years of age. No significant differences regarding gender were found. Quom population showed 54.5%of seroprevalence, the Wichi population showed 59.3% and the Pilagá group had a seroprevalence of 46.8%.Conclusions: Data found in this work are showing the magnitude of chagasic infection still present amongindigenous people living in some areas of the Gran Chaco region. As this region presents eco-epidemiological environments which reduce the impact of the control actions with probabilities of re-infestation, future interventions should take into account the region as a whole and not as areas with political-administrative limits (AU)

Detección de Leishmania (Viannia) braziliensis mediante la Reacción enCadena de la Polimerasa (PCR) en hamster dorado (Mesocricetusauratus) experimentalmente infectado / No disponible

El diagnóstico inmunológico y parasitológico de la leishmaniasis tegumentaria (LT) y visceral (LV)continúa siendo un desafío ya que una limitante es la demostración etiológica mediante extendidos coloreados, histopatología, cultivos, inoculación en animales de laboratorio, xenodiagnósticos. El procedimiento de PCR incrementa la sensibilidad del diagnóstico microscópico directo considerándola actualmente una herramienta muy valorable en la identificación y caracterización de las diferentes especies de Leishmania. En el presente trabajo se evalúan dos métodos de extracción de ADN en diferentes tejidos de hámster (Mesocricetus auratus) experimentalmente infectado con Leishmania (Viannia) braziliensis con material de un enfermo con LT con confirmación parasitológica. Los ADN obtenidos fueron sometidos a una técnica de PCR específica para Le. braziliensis. Se consideró positiva a toda muestra que evidenciara un amplicón de 126 pb en electroferograma de agarosa al 2%.Se realizaron otros métodos como el xenodiagnóstico y técnicas microscópicas directas para comparar la sensibilidad con la PCR. Todas las muestras del roedor infectado evidenciaron el amplicón correspondiente a Le. braziliensis. En este trabajo se demuestra que el detergente CTAB es adecuado para obtener una ADN óptimo para una amplificación específica y que PCR es el método más sensible ensayado (AU)

The etiologic diagnostic of Tegumentary Leishmaniasis (TL) and Visceral Leishmaniasis (VL) is performed by microscopic examination of stained smears of skin lesions, by histopathology, by culture, inoculation in animals or by xenodiagnostic. PCR procedure is currently considered as one promisory diagnostic tool due to its sensitivity, being usually used in identification and characteristization of Leishmania genus.In this work two DNA extraction methods using different golden hamster’s (Mesocricetus auratus)tissues experimentaly infected with Leishmania (Viannia) braziliensis from a patient with parasitologic confirmation of TL are evaluated. DNA obtained were put through specific L. braziliensis PCR, considering positive every sample showing an amplicon of 126 pb in the electroferogram of agarose 2%.PCR sensitivity was compared with xenodiagnostic and direct microscopic examination. All infected mice’s samples showed the corresponding amplicon to L. braziliensis, demonstrating that CTAB use is adecuated to obtain a top quality DNA and that, in these conditions, PCR is the highest sensitivity method (AU)

Enfermedad de Chagas congénito en Hospitales de la ciudad de Corrientes-Argentina / No disponible

Entre Enero de 2005 y Junio de 2006 se estudiaron 2075 embarazadas de los Hospitales “A. I. de Llano” y “J. R. Vidal” de la Ciudad de Corrientes. Se les efectuó Hemoaglutinación Indirecta (HAI) y test de ELISA. En los recién nacidos de aquellas pacientes que resultaron ser reactivas se les practicaron métodos parasitológicos directos (MPD) de Microstrout y Gota Gruesa, y amplificación por PCR y se consideró positiva toda muestra que presentara la banda específica de 330pb del ADN kinetoplastídico. Del total de embarazadas, 130 resultaron seropositivas (6,26%). Se estudiaron 104 recién nacidos, detectándose 8 positivos por los MPD (7,7%) y 12 positivos por PCR (11,5%).Los casos confirmados fueron tratados con Beznidazol durante 60 días. La prevalencia de la infección chagásica encontrada en embarazadas de la Provincia. de Corrientes está dentro de la esperada para áreas endémicas de Latinoamérica (entre 2% y 51%), mientras que la prevalencia de infección connatal hallada del 11,5% es significativamente mayor que la registrada en otras áreas endémicas de Argentina. Nuestros resultados demuestran, como otros reportados anteriormente en la literatura, que un protocolo de PCR optimizado como el usado aquí fue más sensible en la detección de Trypanosoma cruzien pacientes con Chagas congénito (AU)

Between January 2005 and June 2006, 2075 pregnant women from “A.I. de Llano “ and “J. R. Vidal” Hospitals from Corrientes were studied. Indirect Hemaglutination (HAI) and ELISA test were performed. In seropositive patients’ newborn direct parasitologic methods (DPM) of Microstrout and bulk drop, and PCR amplification were made. Every sample with the specific band of 330pb of the kinetoplastide DNA was considered positive. Confirmed cases were treated during 60 days with Beznidazole. From total of pregnant women, 130 were seropositive (6,26%).From 104 newborn studied, 8 were positives by the DPM (7,7%) and 12 were positives by PCR (11,5%). From all diagnosed patients, only7 cases received treatment due to the impossibility to locate them. The prevalence of Chagas infection found in pregnant women of the Province of Corrientes is the usual for endemic areas of Latin America(between 2% and 51%), whereas the prevalence of congenital infection (11.5%) is significantly greater than the recorded one in other endemic areas of Argentina. Our results as well as others reported in the literature, show that the optimized PCR protocol used here was more sensitive in detecting the presence of Trypanosoma cruzi in Congenital Chagas’ Disease (AU)