Graciela Russomando

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.

A SNP in the ABCC11 gene is the determinant of human earwax type

Human earwax consists of wet and dry types. Dry earwax is frequent in East Asians, whereas wet earwax is common in other populations. Here we show that a SNP, 538G → A (rs17822931), in the ABCC11 gene is responsible for determination of earwax type. The AA genotype corresponds to dry earwax, and GA and GG to wet type. A 27-bp deletion in ABCC11 exon 29 was also found in a few individuals of Asian ancestry. A functional assay demonstrated that cells with allele A show a lower excretory activity for cGMP than those with allele G. The allele A frequency shows a north-south and east-west downward geographical gradient; worldwide, it is highest in Chinese and Koreans, and a common dry-type haplotype is retained among various ethnic populations. These suggest that the allele A arose in northeast Asia and thereafter spread through the world. The 538G → A SNP is the first example of DNA polymorphism determining a visible genetic trait.

Congenital Chagas Disease: Recommendations for Diagnosis, Treatment and Control of Newborns, Siblings and Pregnant Women

In May 2010, the sixty-third World Health Assembly adopted resolution WHA63.20 on the control and elimination of Chagas disease, highlighting the need “to promote the development of public health measures in disease-endemic and disease non-endemic countries, with special focus on endemic areas, for the early diagnosis of congenital transmission and management of cases” [1]. This article summarizes the recommendations of the Technical Group IVa on “Prevention and Control of Congenital Transmission and Case Management of Congenital Infections” of the World Health Organizations Programme on Control of Chagas disease (infection with Trypanosoma cruzi). The present recommendations derive from those obtained in the meetings listed in Box 1. Box 1. Meetings from Which the Present Recommendations Derive Meeting ULB (Belgium)/UMSS (Bolivia)), Cochabamba, Bolivia, November 6–8, 2002: “Congenital Infection with Trypanosoma cruzi: From Mechanisms of Transmission to Strategies for Diagnosis And Control”, Carlier Y and Torico F, Revista da Sociedade Brasileira de Medicina Tropical 2003, 6: 767–771. Meeting PAHO/CLAP/ULB (Belgium)/IRD (France), Montevideo, Uruguay, June 24–25, 2004: “Congenital Chagas Disease: Its Epidemiology and Management”, http://www.paho.org/English/AD/DPC/CD/dch-chagas-congenita-2004.htm Meeting PAHO/CLAP/ULB (Belgium), Montevideo, Uruguay, May 17–18, 2007: “Information, Education and Communication in Congenital Chagas Disease”, http://www.paho.org/English/AD/DPC/CD/dch-congenita-iec-07.doc Meeting WHO, Geneva, Switzerland, July 4–6, 2007: “Revisiting Chagas Disease: From a Latin American Health Perspective to a Global Health Perspective” Meeting of the WHO TG IVa (congenital and paediatric Chagas disease), New Orleans, Louisiana, United States, December 11, 2008, satellite meeting to the ASTMH 57th annual meeting Meeting of the 6th European Congress of Tropical Medicine and International Health, Verona, Italy, September 6–10, 2009: “Chagas Disease in Europe” Meeting of WHO-HQ and the WHO regional office for Europe, Geneva, Switzerland, December 17–18, 2009: “Consultation on Chagas Disease in Europe”

Trypanosoma cruzi I genotypes in different geographical regions and transmission cycles based on a microsatellite motif of the intergenic spacer of spliced-leader genes.

The intergenic region of spliced-leader (SL-IR) genes from 105 Trypanosoma cruzi I (Tc I) infected biological samples, culture isolates and stocks from 11 endemic countries, from Argentina to the USA were characterised, allowing identification of 76 genotypes with 54 polymorphic sites from 123 aligned sequences. On the basis of the microsatellite motif proposed by Herrera et al. (2007) to define four haplotypes in Colombia, we could classify these genotypes into four distinct Tc I SL-IR groups, three corresponding to the former haplotypes Ia (11 genotypes), Ib (11 genotypes) and Id (35 genotypes); and one novel group, Ie (19 genotypes). Genotypes harbouring the Tc Ic motif were not detected in our study. Tc Ia was associated with domestic cycles in southern and northern South America and sylvatic cycles in Central and North America. Tc Ib was found in all transmission cycles from Colombia. Tc Id was identified in all transmission cycles from Argentina and Colombia, including Chagas cardiomyopathy patients, sylvatic Brazilian samples and human cases from French Guiana, Panama and Venezuela. Tc Ie gathered five samples from domestic Triatoma infestans from northern Argentina, nine samples from wild Mepraia spinolai and Mepraia gajardoi and two chagasic patients from Chile and one from a Bolivian patient with chagasic reactivation. Mixed infections by Tc Ia+Tc Id, Tc Ia+Tc Ie and Tc Id+Tc Ie were detected in vector faeces and isolates from human and vector samples. In addition, Tc Ia and Tc Id were identified in different tissues from a heart transplanted Chagas cardiomyopathy patient with reactivation, denoting histotropism. Trypanosoma cruzi I SL-IR genotypes from parasites infecting Triatoma gerstaeckeri and Didelphis virginiana from USA, T. infestans from Paraguay, Rhodnius nasutus and Rhodnius neglectus from Brazil and M. spinolai and M. gajardoi from Chile are to our knowledge described for the first time.

Immunological identification of Trypanosoma cruzi lineages in human infection along the endemic area.

Genotyping studies show a polarized geographic distribution of Trypanosoma cruzi lineages in humans. Here, we assessed their distribution along Latin America through an immunological approach we designated Western blot (WB) assay with Trypomastigote small-surface antigen (TSSA) I and TSSA II (TSSA-WB). These antigens are expressed by T. cruzi I (TCI; now TcI) and T. cruzi II (TCII; reclassified as TcII to TcVI) parasites. TSSA-WB showed good concordance with genotyping tests. An unexpected frequency of TSSA II recognition was observed in Colombia, Venezuela, and Mexico (northern region of Latin America). In Argentina and Paraguay (southern region), immunophenotyping confirmed the already reported TCII (TcII to TcVI) dominance. The lineage distribution between these regions showed significant difference but not among countries within them (except for Colombia and Venezuela). TSSA-WB shows TCII emergence in the northern region where TCI was reported as dominant or even as the unique T. cruzi lineage infecting humans.

Trypanosoma cruzi Lineages Detected in Congenitally Infected Infants and Triatoma infestans from the Same Disease-Endemic Region under Entomologic Surveillance in Paraguay

Trypanosoma cruzi II is associated with Chagas disease in the southern part of South America. We analyzed T. cruzi variants in field-collected triatomines and congenitally infected infants living in the same disease-endemic region in Paraguay. Results of polymerase chain reactions for T. cruzi kinetoplast DNA and satellite DNA were positive in 83 triatomine feces samples and 58 infant blood samples. However, lineages were detected in 33 and 38 samples, respectively. Trypanosoma cruzi genotypes were determined in 56 (97%) blood samples after hybridization by using specific probes. The Tc I genotype was not detected. The prevalent sublineage was Tc IId in triatomines (27 of 33) and infant blood (36 of 58) as assessed by amplification of the 24Salpha ribosomal RNA and the mini-exon region genes. The Tc IIc genotype was detected in 20 infant blood samples and in 1 triatomine. This study shows T. cruzi II is the predominant lineage circulating in triatomines and humans in endemic areas of eastern region of Paraguay.

Whole-genome analyses reveals the animal origin of a rotavirus G4P[6] detected in a child with severe diarrhea

Group A rotaviruses are a major cause of severe gastroenteritis in children worldwide. Currently, two rotavirus vaccines are being used in vaccination programs, and one of the factors involved in lower vaccine efficacy is the mismatch among the circulating strains and the vaccine strains. Thus, the emergence of animal strains in the human population could affect the efficacy of vaccination programs. Here we report the presence of a G4P[6] strain in a Paraguayan child presenting acute gastroenteritis in 2009. Genomic analyses revealed that the strain presents a porcine-like genome (G4-P[6]-I1-R1-C1-M1-A8-N1-T7-E1-H1), suggesting a direct animal-to-human transmission. Continuous surveillance of rotaviruses in humans and animals will help us to better understand rotavirus epidemiology and evolution.

Molecular epidemiology of norovirus strains in Paraguayan children during 2004-2005: description of a possible new GII.4 cluster.

BACKGROUND Noroviruses (NoV) have been shown to be an important cause of morbidity and mortality in children worldwide, only second after Group A rotaviruses (RVA). In Paraguay, acute gastroenteritis (AGE) is the third cause of mortality in children ≤5 years old. OBJECTIVES To analyze the presence and diversity of NoV in Paraguayan children ≤5 years old presenting AGE. STUDY DESIGN Three hundred seventy eight fecal samples, negative for pathogenic bacteria and RVA, were collected from children admitted as ambulatory and hospitalized patients in a large private hospital from Asuncion, Paraguay from 2004 to 2005. The presence and diversity of NoV was determined by two different RT-PCR strategies and nucleotide sequencing. RESULTS One hundred and sixty one samples were positive for NoV by partial amplification of the viral polymerase gene (RdRp). No seasonality or differences in the viral prevalence for the different age-groups were detected. GII and GI NoVs were associated to 58% and 42% of the infections, respectively. The genotype was determined in 18% (29/161) NoV-positive samples. The genotypes detected were: GII.4 (18%), GII.17 (18%), GII.6 (14%), GII.7 (14%), GII.3 (10%), GII.5 (3%), GII.8 (3%), GII.16 (3%), GI.3 (14%) and GI.8 (3%). Amplification of the ORF2 from the GII.4 strains showed the presence of a new GII.4 variant. CONCLUSIONS The results showed a continuous circulation of NoV in children throughout the two years of study and an extensive diversity of genotypes co-circulating, highlighting the need for better surveillance of NoV in Paraguayan children.

Viral load of human bocavirus-1 in stools from children with viral diarrhoea in Paraguay

Since their discovery, four species of human bocavirus (HBoV) have been described in patients with respiratory and gastrointestinal diseases. However, a clear causal association between HBoV-1 and gastroenteritis has not been demonstrated. In this study, we describe the detection and quantification of HBoV-1 in stools from children with acute non-bacterial gastroenteritis using quantitative polymerase chain reaction. HBoV-1 genome was detected in 10.6% of stools with frequent association with rotavirus and norovirus. The median of HBoV-1 viral load was 1.88 × 104 genome/ml, lower than previously shown in secretions of patients with respiratory infections, without any obvious association between high viral load and presence of HBoV as single agent. Thus, although HBoV-1 was frequently detected in these patients, there is no clear causal association of this agent with diarrhoea. Indeed, HBoV-1 DNA in stools of patients with gastroenteritis without respiratory symptoms may be a remnant of previous infections or associated with prolonged shedding of virus in the respiratory or digestive tracts.

Microorganisms Associated With Pneumonia in Children <5 Years of Age in Developing and Emerging Countries: The GABRIEL Pneumonia Multicenter, Prospective, Case-Control Study

Summary In a multicenter, prospective case-control study involving 1758 children aged <5 years in developing and emerging countries, the main microorganisms associated with pneumonia were Streptococcus pneumoniae, human metapneumovirus, rhinovirus, and respiratory syncytial virus.