Deep sequencing of the Trypanosoma cruzi GP63 surface proteases reveals diversity and diversifying selection among chronic and congenital Chagas disease patients.

BACKGROUND: Chagas disease results from infection with the diploid protozoan parasite Trypanosoma cruzi. T. cruzi is highly genetically diverse, and multiclonal infections in individual hosts are common, but little studied. In this study, we explore T. cruzi infection multiclonality in the context of age, sex and clinical profile among a cohort of chronic patients, as well as paired congenital cases from Cochabamba, Bolivia and Goias, Brazil using amplicon deep sequencing technology. METHODOLOGY/ PRINCIPAL FINDINGS: A 450bp fragment of the trypomastigote TcGP63I surface protease gene was amplified and sequenced across 70 chronic and 22 congenital cases on the Illumina MiSeq platform. In addition, a second, mitochondrial target--ND5--was sequenced across the same cohort of cases. Several million reads were generated, and sequencing read depths were normalized within patient cohorts (Goias chronic, n = 43, Goias congenital n = 2, Bolivia chronic, n = 27; Bolivia congenital, n = 20), Among chronic cases, analyses of variance indicated no clear correlation between intra-host sequence diversity and age, sex or symptoms, while principal coordinate analyses showed no clustering by symptoms between patients. Between congenital pairs, we found evidence for the transmission of multiple sequence types from mother to infant, as well as widespread instances of novel genotypes in infants. Finally, non-synonymous to synonymous (dn:ds) nucleotide substitution ratios among sequences of TcGP63Ia and TcGP63Ib subfamilies within each cohort provided powerful evidence of strong diversifying selection at this locus. CONCLUSIONS/SIGNIFICANCE: Our results shed light on the diversity of parasite DTUs within each patient, as well as the extent to which parasite strains pass between mother and foetus in congenital cases. Although we were unable to find any evidence that parasite diversity accumulates with age in our study cohorts, putative diversifying selection within members of the TcGP63I gene family suggests a link between genetic diversity within this gene family and survival in the mammalian host.

The Trypanosoma cruzi satellite DNA OligoC-TesT and Trypanosoma cruzi kinetoplast DNA OligoC-TesT for diagnosis of Chagas disease: a multi-cohort comparative evaluation study.

BACKGROUND: The Trypanosoma cruzi satellite DNA (satDNA) OligoC-TesT is a standardised PCR format for diagnosis of Chagas disease. The sensitivity of the test is lower for discrete typing unit (DTU) TcI than for TcII-VI and the test has not been evaluated in chronic Chagas disease patients. METHODOLOGY/PRINCIPAL FINDINGS: We developed a new prototype of the OligoC-TesT based on kinetoplast DNA (kDNA) detection. We evaluated the satDNA and kDNA OligoC-TesTs in a multi-cohort study with 187 chronic Chagas patients and 88 healthy endemic controls recruited in Argentina, Chile and Spain and 26 diseased non-endemic controls from D.R. Congo and Sudan. All specimens were tested in duplicate. The overall specificity in the controls was 99.1% (95% CI 95.2%-99.8%) for the satDNA OligoC-TesT and 97.4% (95% CI 92.6%-99.1%) for the kDNA OligoC-TesT. The overall sensitivity in the patients was 67.9% (95% CI 60.9%-74.2%) for the satDNA OligoC-TesT and 79.1% (95% CI 72.8%-84.4%) for the kDNA OligoC-Test. CONCLUSIONS/SIGNIFICANCE: Specificities of the two T. cruzi OligoC-TesT prototypes are high on non-endemic and endemic controls. Sensitivities are moderate but significantly (p = 0.0004) higher for the kDNA OligoC-TesT compared to the satDNA OligoC-TesT.

Comparison of the performance of rapid prescreening, 10% random review, and clinical risk criteria as methods of internal quality control in cervical cytopathology.

BACKGROUND: Rapid prescreening (RPS) is an internal quality-control (IQC) method that is used both to reduce errors in the laboratory and to measure the sensitivity of routine screening (RS). Little direct comparison data are available comparing RPS with other more widely used IQC methods. METHODS: The authors compared the performance of RPS, 10% random review of negative smears (R-10%), and directed rescreening of negative smears based on clinical risk criteria (RCRC) over 1 year in a community clinic setting. RESULTS: In total, 6,135 smears were evaluated. The sensitivity of RS alone was 71.3%. RPS detected significantly more (132 cases) false-negative (FN) cases than either R-10% (7 cases) or RCRC (32 cases). RPS significantly improved the overall sensitivity of the laboratory (71.3-92.2%; P = .001); neither R-10% nor RCRC significantly changed the sensitivity of RS. RPS was not as specific as the other methods, although nearly 68% of all abnormalities detected by RPS were verified as real. RPS of 100% of smears required the same amount of time as RCRC but required twice as much time as R-10%. CONCLUSIONS: The current results demonstrated that RPS is a much more effective IQC method than either R-10% or RCRC. RPS detects significantly more errors and can improve the overall sensitivity of a laboratory with either a modest increase or no increase in overall time spent on IQC. R-10% is an insensitive IQC method, and neither R-10% nor RCRC can significantly improve the overall sensitivity of a laboratory.

Controle externo da qualidade dos diagnósticos citológicos no rastreamento do câncer cervical: estudo piloto / External quality control in the cytologic diagnosis in screening cervical cancer: piloty study

Todos os sistemas de avaliação do desempenho dos Laboratórios de Citopatologia exigem um programa de controle interno e externo da qualidade. Este estudo tem por objetivo avaliar a concordância dos diagnósticos citopatológicos do Laboratório Rômulo Rocha com os diagnósticos da Unidade de Monitoramento Externo da Qualidade (UMEQ), bem como, avaliar a freqüência de casos discordantes, falso-positivos e falso-negativos. Foram enviados a UMEQ 537 esfregaços selecionados pelo sistema SISCOLO durante o ano de 2003, incluindo todos os casos positivos, todos os insatisfatórios e no mínimo 5% dos exames negativos. Apenas seis casos foram considerados discordantes pela UMEQ, dos quais três casos foram considerados negativos pelo Rômulo Rocha, destes, dois foram reavaliados como ASC e um como ASC/AG, um caso negativo foi reavaliado como insatisfatório, um caso de NIC 2/HPV foi reavaliado como ASC e um caso de NIC 1/HPV reavaliado como NIC 2/HPV pela UMEQ. A concordância diagnóstica entre o Rômulo Rocha e a UMEQ foi de 0,99 de acordo com o coeficiente Kappa. Este estudo mostrou estrito controle interno da qualidade desenvolvido no Laboratório Rômulo Rocha. Sua prática pode ser útil para orientar diversas medidas de educação continuada.

Systems of laboratory development assessment in cytopathology require an intern and external quality control. The aim of the present study was to assess concordance of cytopathologic diagnosis in the Laboratory Rômulo Rocha with cytopathologic diagnosis carried out in the External Quality Control Unit (UMEQ), as well as to evaluate frequency of discordant cases, false-positive and false-negative. For this, 537 smears were randomly selected by SISCOLO throughout the year 2003, including all positive and unsatisfactory cases and at least 5% of the negative ones. Only six discordant cases were detected. Three of these cases were considered negative by Rômulo Rocha, two were rescreened as ASC and one case was rescreened ASC/AG, one negative case was rescreened as unsatisfactory, one case NIC 2/HPV was rescreened as ASC and another case NIC 1/HPV was rescreened as NIC 2/HPV by UMEQ. The diagnostic agreement between Rômulo Rocha and UMEQ was 0.99 according to kappa coefficient. Results of the study reveal that the Rômulo Rocha uses a very strict quality control, which can be helpful in guiding students in continued education programs.

[Congenital transmission of Trypanosoma cruzi in Brazil: estimation of prevalence based on preliminary data of national serological surveys in children under 5 years old and other sources]. / Transmisión congénita del Trypanosoma cruzi en Brasil: estimativa de prevalencia baseada en resultados preliminares de la encuesta nacional serológica en niños menores de cinco años, así como otras fuentes.

A prevalence estimation of congenital transmission in Brazil is performed, based on several sources of recent data. From a serological survey conducted now in Brazil, with children below 5 years old, preliminary data from the state of Minas Gerais only 19/9,556 children did have antibodies against Trypanosoma cruzi. All 19 mothers were infected, but only one child persisted with antibodies on a second blood collection, hence diagnosed as congenital. The other were just passive transference of maternal antibodies. From a recent publication, 278 children born from 145 infected mothers were studied. Two cases (0.7%) were congenital. In other source, from 1,348 blood donors, 35 were born in non endemic areas. When 10 of them were called, 8 were born from infected mothers and five may be congenital. Finally, no infection was detected in 93 children born from 78 infected mothers. The reasons for this low prevalence are discussed, are lower than in other countries of the South Cone, that harbor also T. cruzi 2, but are unrecognized up to now.