Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients.

An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease.

Trypanosoma cruzi necrotizing meningoencephalitis in a Venezuelan HIV⁺-AIDS patient: pathological diagnosis confirmed by PCR using formalin-fixed- and paraffin-embedded-tissues.

Coinfections with human immunodeficiency virus (HIV) and infectious agents have been recognized since the early 90s. In the central nervous system (CNS) of HIV(+) patients, parasitic protozoans like Toxoplasma gondii have been described as responsible for the space occupying lesions (SOL) developed. However, the involvement of Trypanosoma cruzi is also described but appears to be less frequent in acquired immunodeficiency syndrome (AIDS) and transplant recipients, associated with necrotizing myocarditis and neurological symptoms related to the occurrence of necrotizing pseudotumoral encephalitis (NPE) and meningoencephalitis (NME). The present work aims to present a Venezuelan case of NME associated with the coinfection of HIV and a T. cruzi-like trypanosomatid as well as its evolution and diagnosis by histopathological techniques, electron microscopy, and PCR methods using formalin-fixed- (FF-) and paraffin-embedded- (PE-) tissues. Postmortem cytological studies of leptomeninges imprints reveal the presence of trypomastigotes of Trypanosoma sp. Histopathological and electron microscopy studies allowed us to identify an amastigote stage and to reject the involvement of other opportunistic microorganisms as the etiological agent of the SOL. The definitive confirmation of T. cruzi as the etiological agent was achieved by PCR suggesting that the NME by T. cruzi was due to a reactivation of Chagas' disease.

Trypanosoma cruzi infection in Elche (Spain): comparison of the seroprevalence in immigrants from Paraguay and Bolivia.

Chagas disease is a global public health problem due to the recent emigration of people from Latin America to other regions, including Europe. The aim of this study is to determine the prevalence of Trypanosoma cruzi infection among Paraguayans and Bolivians living in Elche (Spain), a city located in the Mediterranean Coast of Spain. A cross-sectional study was conducted. Capillary blood samples were obtained through a finger prick, and collected on filter paper. An enzyme-linked immunosorbent assay and indirect immunofluorescence tests were performed to search for anti-T. cruzi IgG antibodies in the filter papers. Thirteen out of 201 participants were infected with T. cruzi in this study, seven out of 73 Bolivians and six out of 128 Paraguayans, corresponding to seroprevalences of 9·59% (95%CI, 4·72-18·5%) and 4·69% (95%CI, 2·17-9·85%), respectively. Palpitation, chest pain, and migration from rural endemic areas were the most common clinical and epidemiological risk factors associated with T. cruzi infection detected in the Paraguayan group. This study highlights that Chagas disease is no longer limited to the Bolivian population living in Spain. It is important to note this wider prevalence and, therefore, not discount Paraguayans in the screening for Chagas disease in Spain. Indeed, this should be considered for all immigrants from Latin America.

Serological screening of Chagas disease in an immigrant population in Asturias, Spain proceeding from Chagas-endemic areas.

Population movements from Chagas disease-endemic areas to non-endemic countries due to immigration make the occurrence of this disease in these latter areas possible. We describe the results of a screening programme conducted in an immigrant population from endemic areas, attending the Tropical Medicine Unit of the Hospital Central de Asturias between June 2006 and June 2008. The ID-Chagas antibody test (particle gel immunoassay (PaGIA); DiaMed-ID) was used as a screening assay. We analysed 64 patients, 9 of whom (14%) tested positive for Chagas disease antibodies, a diagnosis that was confirmed in all cases. Six patients came from Bolivia, 2 from Paraguay and 1 from Brazil. Chagas disease is of increasing importance, even in areas with low migratory flows; hence screening programmes for this population group are especially important.

Infección por virus Epstein Barr en niños VIH positivos: Relación con carga viral del VIH y contaje de linfocitos T CD4+ / Epstein Barr virus infection in HIV positive children: relationship with viral load and T CD4+ lymphocyte count

El objetivo de este trabajo fue estudiar la infección por Epstein Barr en niños VIH+ menores de 12 años, de ambos sexos, con TARAE y su relación con carga viral del VIH y contaje de linfocitos T CD4+. La detección de IgM/IgG anti CVA y anti EBNA1 se realizó por ELISA, la cuantificación de EBV en sangre periférica por PCR y el inmunofenotipaje por citometría de flujo. Estudio estadístico mediante el análisis de correlación de Spearman y la prueba de t de Student. Se evaluaron 21 niños con edades comprendidas entre 3 a 12 años, 18 de los cuales (85,7%) presentaron carga viral detectable para EBV. Dos de ellos tuvieron infección aguda temprana (IgM CVA e IgM EBNA1 +), 5 infección aguda tardía (IgM CVA - IgM EBNA +) con carga viral para EBV de 573 copias/ml, y 14 con infección pasada (IgG CVA e IgG EBNA positivos), 11 de los cuales tenían carga viral para EBV de 646 copias/ml (crónicos activos). No hubo diferencias estadísticamente significativas al comparar los promedios de las cargas virales de EBV en infecciones agudas y pasadas, ni entre esas cargas y las de VIH, pero si con los contajes de linfocitos T CD4+: cifras menores se correlacionaron con niveles altos de carga viral de EBV (p<0,05). Debe realizarse detección de los anticuerpos anti CVA y EBNA1 así como cuantificación de EBV por pruebas moleculares a todo niño VIH positivo, ya que a menor contaje de linfocitos T CD4+ mayor carga viral de EBV.

The purpose of this work was to study Epstein Barr infection in HIV+ children less than 12 years old of both sexes, with TARAE and its relationship with the HIV viral load and T CD4+ lymphocyte count. The detection of anti CVA and anti EBNA1 IgM/IgG was done by ELISA, the EBV quantification in peripheral blood by PCR, and the immunophenotype by flow cytometry. The statistical study was done through Spearman’s correlation analysis and Student’s t test. The evaluation included 21 children with ages between 3 and 12 years, 18 of which (85.7%) presented a detectable EBV viral load. Two of them had an acute early infection (IgM CVA and IgM EBNA1 +), 5 an acute late infection (IgM CVA - IgM EBNA+) with a EBV viral load equal to 573 copies/ml, and 14 with past infections ( IgG CVA and IgG EBNA positive), 11 of which had an EBV viral load equal to 646 copies/ml (active chronics). There were no statistical significant differences when comparing the mean EBV viral load in acute and past infections, nor between those loads and the HIV loads, but there were differences in the T CD4+ loads; lower numbers were correlated with high EBV viral loads (p<0.05). Detection of anti CVA and EBNA1 antibodies, as well as EBV quantification through molecular tests, should be done to all HIV positive children, since a lower T CD4+ lymphocyte count means a higher EBV viral load.

Despistaje de infección por citomegalovirus en niños VIH positivos mediante PCR: relación con serología y evolución clínica / PCR screening for cytomegalovirus infection in HIV-positive children: relationship between serology and clinical evolution

En pacientes VIH positivos es fundamental diagnósticar infección por CMV. La relación entre serología, detección viral y evolución clínica no está plenamente establecida. Detectar la presencia de CMV por PCR en sangre periférica en pacientes pediátricos, sin síntomas de infección, VIH positivos; relacionar estos resultados con la serología y la evolución clínica durante un año de seguimiento. Criterios de inclusión: Niños menores de 12 años, ambos sexos, infección diagnosticada por VIH, recibiendo terapia antirretroviral altamente efectiva (TARAE), consentimiento informado por escrito, firmado. La serología anti CMV se realizó mediante el método ELISA, la semicuantificación de CMV en sangre periférica, mediante PCR, y el inmunofenotipaje por citometría de flujo. Aquellos niños con carga viral inicial detectable para CMV fueron evaluados un año después (valoración cualitativa y oftalmológica). Correlación de Pearson, t Student. Se estudiaron 23 niños, 17 menores de 6 años; 21 de ellos (82.6 por ciento): lgG CMV +. Dos pacientes (8.7 por ciento): lgM CMV+ y promedio de carga viral: 11920 VID, dos IgM-IgG+, promedio de carga de 23129 VID; el resto negativos; todos con linfocitos CD4+ por encima del 25 por ciento. 50 por ciento de los niños con carga viral negativa para CMV, con contajes de CD4+ inferiores al 25 por ciento. El análisis de correlación de Pearson no mostró correlación entre la carga viral del VIH y los valores de VID para CMV (R²=0.13). Linfocitos CD8+: 32,3 ± 6,8 por ciento en los pacientes con carga para CMV, estadísticamente inferior al promedio del grupo sin cargas virales CMV: 49,1 ± 8,8. (t Student= 3,508; g.l: 17. P<0,003). Ningún niño presentó evidencia de enfermedad órgano específica (EOE), incluyendo retinitis. Independientemente de la presencia o no de IgM positiva para CMV, 4 pacientes tuvieron carga viral detectable. No hay correlación entre las cargas virales de ambos virus. Ningún niño con carga viral detectable para CMV...

In HIV-positive patients the diagnosis of CMV infection is essential. The relationship between serology, viral detection and clinical evolution has not been fully established. To detect the presence of CMV in peripheral blood by PCR testing in HIV positive pediatric patients with no infection symptoms, and to relate the results with serology and clinical evolution during a year follow up. Inclusion criteria: Children under twelve years of age, both genders, with a diagnosis of HIV infection, and receiving HAART therapy, written consent signed by parents. Anti CMV serology was performed by ELISA, semi-quantification of CMV in peripheral blood by PCR and immunophenotyping by flow-cytometry. Children with detectable initial viral load for CMV were submitted to aqualitative and ophtalmologic assessment one year later. Statistics: Pearson’s Correlation, Student’s t. 23 children, both genders, 17 under 6 years of age; 21 (82.6%): IgG CMV+. Two patients (8.7%): IgM CMV+ and viral load. 11920 IDV, two with IgM- IgG+, viral load average: 23129 IDV. The rest of the children were negative, all with lymphocytes CD4+ above 25%. 50% of the children had a negative viral load for CMV with CD4+ counts under 25%. There was no correlation between the HIV viral load and IDV values for CMV (r2=0.13). Lymphocytes CD8+32.3+ 6.8% in patients with viral load for CMV.This is statistically lower than the average for the group without viral loads CMV: 49.1 + 8.8 (Student’s t= 3.508; g.l: 17. p<0.003). No child showed evidence of specific organ disease (SOD), including retinitis. Regardless of serology for IgM, 4 patients had detectable viral loads. There was no correlation between the viral loads of the two viruses. No child with detectable viral load for CMV developed a specific organ disease, probably due to a highly efficient antiretroviral treatment. Viral load quantification for CMV in HIV + patients is recommended, regardless of specific IgM result.

Polymerase chain reaction detection and serologic follow-up after treatment with benznidazole in Bolivian children infected with a natural mixture of Trypanosoma cruzi I and II.

Thirty-five Bolivian children (5-10 years of age) seropositive for infection with T. cruzi underwent specific chemotherapy with benznidazole. Before treatment, 57.1% had a positive parasitologic diagnosis. Some patients presented an early conversion by polymerase chain reaction of blood samples, while others were still positive four and seven months after the end of the treatment, which indicated an absence of parasite clearance. Strain typing showed that most patients were infected by a mixture of clones I and II of T. cruzi. Serologic conversion in conventional tests and antibodies to shed acute-phase antigen were observed in two and four patients, respectively. For the other patients, the average rate of antibody decay was half the initial rate. The parasitologic and serologic data indicated that chemotherapy acts throughout the course of infection in a long-lasting process in which the decrease of specific antibody production is related to the reduction of the live parasite load.

Comparación de pruebas para detección de microalbuminuria en pacientes con diabetes mellitus tipo 2 / Comparative study to detect microalbuminuria in diabetic patients

Introducción: La prevalencia mundial de nefropatía en pacientes con diabetes mellitus es de 35 por ciento, y la de microalbuminuria de 20 a 25 por ciento. Cuando la excreción de la última oscila entre 15 a 30 ?g/minuto, avanza rápidamente a insuficiencia renal. Objetivo: Validar el método con ácido sulfosalicílico como prueba para identificar microalbuminuria en pacientes con diabetes mellitus. Material y métodos: Se incluyeron 71 pacientes con diabetes mellitus tipo 2, adscritos a unidades del primer nivel de atención médica del Instituto Mexicano del Seguro Social. Las muestras de orina fueron sometidas a prueba con ácido sulfosalicílico. Resultados: La edad promedio fue de 55 años y la evolución de la diabetes osciló entre dos meses y 14 años. La prueba analizada tuvo sensibilidad de 70 por ciento y especificidad de 93 por ciento al compararla con el radioinmunoanálisis (estándar de oro). Conclusiones: La técnica con ácido sulfosalicílico demostró ser una prueba tamiz confiable, de bajo costo y fácil realización, ideal para la identificación temprana de microalbuminuria en pacientes con diabetes mellitus.