Prospective multicenter evaluation of real time PCR Kit prototype for early diagnosis of congenital Chagas disease.

BACKGROUND: Current algorithm for Congenital Chagas Disease (cCD) diagnosis is unsatisfactory due to low sensitivity of the parasitological methods. Moreover, loss to follow-up precludes final serodiagnosis after nine months of life in many cases. A duplex TaqMan qPCR kit for Trypanosoma cruzi DNA amplification was prospectively evaluated in umbilical cord (UCB) and peripheral venous blood (PVB) of infants born to CD mothers at endemic and non-endemic sites of Argentina. METHODS: We enrolled and followed-up 370 infants; qPCR was compared to gold-standard cCD diagnosis following studies of diagnostic accuracy guidelines. FINDINGS: Fourteen infants (3·78%) had cCD. The qPCR sensitivity and specificity were higher in PVB (72·73%, 99·15% respectively) than in UCB (66·67%, 96·3%). Positive and negative predictive values were 80 and 98·73% and 50 and 98·11% for PVB and UCB, respectively. The Areas under the Curve (AUC) of ROC analysis for qPCR and micromethod (MM) were 0·81 and 0·67 in UCB and 0·86 and 0·68 in PVB, respectively. Parasitic loads ranged from 37·5 to 23,709 parasite equivalents/mL. Discrete typing Unit Tc V was identified in five cCD patients and in six other cCD cases no distinction among Tc II, Tc V or Tc VI was achieved. INTERPRETATION: This first prospective field study demonstrated that qPCR was more sensitive than MM for early cCD detection and more accurate in PVB than in UCB. Its use, as an auxiliary diagnostic tool to MM will provide more accurate records on cCD incidence. FUNDING: FITS SALUD 001-CHAGAS (FONARSEC, MINCyT, Argentina) to the Public-Private Consortium (INGEBI-CONICET, INP-ANLIS MALBRAN and Wiener Laboratories); ERANET-LAC-HD 328 to AGS and PICT 2015-0074 (FONCYT, MinCyT) to AGS and FA.

Detection and identification of Kinetoplastids of zoonotic interest by HRM-qPCR analysis in Canis lupus familiaris from Argentinean Mesopotamia.

This work aimed to conduct a first PCR-based approach for differential diagnosis of kinetoplastidean infections in dogs. Diagnosis of Kinetoplastid infections in domestic animals is difficult, since parasitemia is intermittent and signs are nonspecific; it is mainly based on parasitological smears or concentration techniques, which lack sensitivity and depend on operator` expertise. Dogs are relevant reservoirs in transmission of Kinetoplastids; they function as sentinels to detect active transmission cycles before they involve humans. Trypanosoma cruzi, Trypanosoma evansi, and various species of Leishmania genus are multi-host parasites, capable of parasitizing dogs among a vast number of reservoirs. An algorithm based on sequential Real-Time PCR-High Resolution Melting (HRM) (qPCR-HRM) assays directed at 24S alpha ribosomal DNA, ITS1 and Hsp70 designed to distinguish among T. cruzi, T. rangeli, T. evansi and Leishmania spp. was tested in fourteen dogs with suspicion of kinetoplastid diseases. A qPCR control of DNA integrity in the tested sample, targeted to the mammalian interphotoreceptor retinoid-binding protein (IRBP) gene fragment was incorporated to the algorithm. T. evansi was detected in four dogs and L. infantum in one. Two of five qPCR positive cases were smear negative. Smear and T. evansi qPCR positive cases corresponded to animals that died despite being treated, indicating the association of parasitemia with disease severity. This laboratory tool increases the possibility of confirming outbreaks of kinetoplastid diseases with zoonotic potential and identify the etiological agents involved.

Real-time polymerase chain reaction based algorithm for differential diagnosis of Kinetoplastidean species of zoonotic relevance.

Kinetoplastids are a group of flagellated protozoa that infect a vast repertoire of mammals and insect vectors. From a zoonotic point of view, domestic animals are critical reservoirs for transmission of Kinetoplastidean parasites. Due to their proximity to humans, they assume substantial epidemiological importance in the context of these zoonoses and consequently in public health. Their reliable identification is relevant to understand their eco-epidemiological involvement in transmission cycles. This work aimed to develop an algorithm based on sequential Real-Time PCR (qPCR) assays targeted to different loci (24S alpha rDNA, ITS1 and Hsp70) allowing distinction among Trypanosoma cruzi, Trypanosoma rangeli, Trypanosoma evansi and Leishmania species in biological samples collected from mammalian reservoirs and triatomine vectors. The algorithm includes a first qPCR test targeted to endogenous genes conserved within mammals and within triatomine vectors as internal controls of DNA sample integrity and/or qPCR inhibition. This algorithm was evaluated in biological samples from domestic cattle (N = 14), dogs (N = 19) and triatomines (N = 19). Analytical sensitivity of 24S alpha rDNA for detection of T. rangeli was 10 fg of DNA, with a linear range between 10 fg and 10 ng. For T. cruzi it varied depending on the Discrete typing unit. The ITS1 qPCR showed an analytical sensitivity of 100 pg/reaction and 100 fg/reaction of Leishmania spp. and T. evansi DNAs. In mammal field samples, four T. cruzi 24S alpha rDNA sequences and fourteen ITS1 amplicons specific for T. evansi were detected. qPCR-HRM analysis directed to the Hsp70 gene diagnosed two dogs with Leishmania infantum infection. Among 19 triatomine field samples, T. cruzi was detected in five; T. rangeli in eight and one specimen showed a mixed infection. This diagnostic algorithm can provide more accurate records of kinetoplastidean infection burden in vectors and reservoirs, relevant to update current eco-epidemiological maps in co-endemic regions.

Chlamydia trachomatis as a probable cofactor in human papillomavirus infection in aboriginal women from northeastern Argentina

Objectives: High-risk types of human papillomavirus (HPV) are strongly associated with cervical cancer (CC), and Chlamydia trachomatis (CT), the most frequent sexually transmitted bacterial infection (STBI) worldwide, seems to be a risk factor for HPV infection and for CC. It is also known that both agents are more prevalent in vulnerable communities where lack of adequate primary health care is a cause for concern. The aim of this work was to determine the impact of CT and HPV infections in women belonging to an isolated aboriginal population (Pilaga community) from a poor region in Northern Argentina (province of Formosa). For this purpose, a cross-sectional study was performed in all sexually active Pilaga women, who attended a local community-based gynecological health screening project. The polymerase chain reaction (PCR) method on a cervical brush specimen was used to detect both agents. Results: A total of 227 women (20 percent of the total female population of the Pilaga community) were studied and the overall prevalence was 26.4 percent for CT, 46.7 percent for HPV and 16.3 percent for concurrent infection. CT infection was higher in HPV DNA positive (34.2 percent) than in HPV DNA negative women (19 percent; OR: 2.22/95 percent CI = 1.16-4.28 / p = 0.009) and the most prevalent HPV types were HPV-16 (19.4 percent), 6 and 18 (5.3 percent), 58 (3.5 percent) and 33 (3.1 percent). Conclusions: The prevalence of CT and HPV observed in Pilaga women are among the worst registered in Latin America. Also, data collected suggest that chlamydial infection may play an important role in the natural history of HPV infection. On this respect, we propose that the association between these two agents seems to be more related to a mutual potentiation than to the fact that they share a common route of transmission.

International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients.

BACKGROUND: A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation. METHODOLOGY/FINDINGS: An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05-0.5 parasites/mL whereas specific kDNA tests detected 5.10(-3) par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3-94.4%, specificity of 85-95%, accuracy of 86.8-89.5% and kappa index of 0.7-0.8 compared to consensus PCR reports of the 16 good performing tests and 63-69%, 100%, 71.4-76.2% and 0.4-0.5, respectively compared to serodiagnosis. Method LbD2 used solvent extraction followed by Sybr-Green based Real time PCR targeted to Sat-DNA; method LbD3 used solvent DNA extraction followed by conventional PCR targeted to Sat-DNA. The third method (LbF1) used glass fiber column based DNA extraction followed by TaqMan Real Time PCR targeted to Sat-DNA (cruzi 1/cruzi 2 and cruzi 3 TaqMan probe) and the fourth method (LbQ) used solvent DNA extraction followed by conventional hot-start PCR targeted to kDNA (primer pairs 121/122). These four methods were further evaluated at the coordinating laboratory in a subset of human blood samples, confirming the performance obtained by the participating laboratories. CONCLUSION/SIGNIFICANCE: This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.

Chlamydia trachomatis as a probable cofactor in human papillomavirus infection in aboriginal women from northeastern Argentina.

OBJECTIVES: High-risk types of human papillomavirus (HPV) are strongly associated with cervical cancer (CC), and Chlamydia trachomatis (CT), the most frequent sexually transmitted bacterial infection (STBI) worldwide, seems to be a risk factor for HPV infection and for CC. It is also known that both agents are more prevalent in vulnerable communities where lack of adequate primary health care is a cause for concern. The aim of this work was to determine the impact of CT and HPV infections in women belonging to an isolated aboriginal population (Pilaga community) from a poor region in Northern Argentina (province of Formosa). For this purpose, a cross-sectional study was performed in all sexually active Pilaga women, who attended a local community-based gynecological health screening project. The polymerase chain reaction (PCR) method on a cervical brush specimen was used to detect both agents. RESULTS: A total of 227 women (20% of the total female population of the Pilaga community) were studied and the overall prevalence was 26.4% for CT, 46.7% for HPV and 16.3% for concurrent infection. CT infection was higher in HPV DNA positive (34.2%) than in HPV DNA negative women (19%; OR: 2.22/95% CI = 1.16-4.28 / p = 0.009) and the most prevalent HPV types were HPV-16 (19.4%), 6 and 18 (5.3%), 58 (3.5%) and 33 (3.1%). CONCLUSIONS: The prevalence of CT and HPV observed in Pilaga women are among the worst registered in Latin America. Also, data collected suggest that chlamydial infection may play an important role in the natural history of HPV infection. On this respect, we propose that the association between these two agents seems to be more related to a mutual potentiation than to the fact that they share a common route of transmission.

Molecular identification of Trypanosoma cruzi discrete typing units in end-stage chronic Chagas heart disease and reactivation after heart transplantation.

BACKGROUND: One hundred years after the discovery of Chagas disease, it remains a major neglected tropical disease. Chronic Chagas heart disease (cChHD) is the most severe manifestation. Heart transplantation is the proper treatment for end-stage heart failure, although reactivation of disease may result after receipt of immunosuppressive therapy. T. cruzi strains cluster into 6 discrete typing units (DTUs; I-VI) associated with different geographical distribution, transmission cycles and varying disease symptoms. In the southern cone of South America, T. cruzi II, V, and VI populations appear to be associated with Chagas disease and T. cruzi I with sylvatic cycles. METHODS: Molecular characterization of DTUs, T. cruzi I genotypes (on the basis of spliced-leader gene polymorphisms), and minicircle signatures was conducted using cardiac explant specimens and blood samples obtained from a cohort of 16 Argentinean patients with cChHD who underwent heart transplantation and from lesion samples obtained from 6 of these patients who presented with clinical reactivation of Chagas disease. RESULTS: Parasite persistence was associated with myocarditis progression, revealing T. cruzi I (genotype Id) in 3 explant samples and T. cruzi II, V, or VI in 5 explant samples. Post-heart transplantation follow-up examination of bloodstream DTUs identified T. cruzi I in 5 patients (genotypes Ia or Id) and T. cruzi II, V, or VI in 7 patients. T. cruzi I, V, and VI were detected in skin chagoma specimens, and T. cruzi V and VI were detected in samples obtained from patients with myocarditis reactivations. Multiple DTUs or genotypes at diverse body sites and polymorphic minicircle signatures at different cardiac regions revealed parasite histotropism. T. cruzi I infections clustered in northern Argentina (latitude, 23 degrees S-27 degrees S), whereas T. cruzi II, V, or VI DTUs were more ubiquitous. CONCLUSIONS: Multiple DTUs coexist in patients with Chagas disease. The frequent finding of T. cruzi I associated with cardiac damage was astounding, revealing its pathogenic role in cChHD at the southern cone.

Human papillomavirus genotypes in women with cervical cytological abnormalities from an area with high incidence of cervical cancer.

It has been well demonstrated the relationship between the infection with high-risk human papillomavirus (HPVs) genotypes and cervical cancer. In Northeastern Argentina a high incidence of this pathology has been described and therefore a high prevalence of HPV infection is expected. In order to identify HPV genotypes associated with malignant and pre-malignant cervical lesions present in the area, 53 ecto-endo cervical cell specimens obtained from women with cytohistological alterations were studied by a PCR-RFLP technique. Out of 53 patients, 34 (64.2%) were positive for HPV infection, being HPV-16 (32.3%) the most frequently found genotype, followed by HPV-58 (14.7%), -6, -18 and -45 (5.9%), -33, -52, -53, -54, -56, -66, -MM4 and -LVX100 (2.9%). Also 5 cases of infection caused by multiple genotypes were found, which corresponded to 14.7% of the positive cases. Results indicate that besides HPV-16 and -18, the most prevalent high-risk HPV genotypes worldwide, others like -45 and -58 as well as co-infection cases are frequent between women of Northeastern Argentina, and a particular attention should be paid to this circumstance because it could be an epidemiological feature of regional importance and a useful information for a future vaccination program.

Human papillomavirus genotypes in women with cervical cytological abnormalities from an area with high incidence of cervical cancer

La relación entre la infección por los virus papiloma humanos (HPVs) de alto riesgo y el cáncer de cuello de útero ha sido bien demostrada. En el Nordeste de Argentina se observa una alta incidencia de esta patología y en consecuencia se estima una alta prevalencia de infección por HPV. A fin de identificar los genotipos de HPV presentes en el área, asociados a casos de lesiones malignas y premalignas de cuello de útero, se estudiaron 53 muestras ecto-endo cervicales de mujeres con alteraciones citohistológicas residentes permanentes de las ciudades de Resistencia y Corrientes. De las 53 pacientes estudiadas, 34 resultaron positivas para HPV (64.2 por ciento), correspondiendo la mayor frecuencia a HPV-16 (32.3 por ciento), seguido por HPV-58 (14.7 por ciento), HPV-6, -18 y -45 (5.9 por ciento), -33, -52, -53, -54, -56, -66, -MM4 y -LVX100 (2.9 por ciento). Además, se encontraron 5 casos de infecciones mixtas causadas por mas de un genotipo, lo que resulta de importancia ya que representan el 14.7 por ciento del total de los casos positivos. Los resultados demuestran que, además de HPV-16 y -18 que son los genotipos de alto riesgo de mayor prevalencia a nivel mundial, otros como el HPV-45 y -58 y los casos de infecciones múltiples son frecuentes en mujeres del nordeste argentino, lo que podría constituir un rasgo epidemiológico de importancia regional y ser de utilidad en el futuro en los programas de vacunación.