Ana Maria de Castro

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.

Immunological imbalance between IFN-³ and IL-10 levels in the sera of patients with the cardiac form of Chagas disease

The immune response is crucial for protection against disease; however, immunological imbalances can lead to heart and digestive tract lesions in chagasic patients. Several studies have evaluated the cellular and humoral immune responses in chagasic patients in an attempt to correlate immunological findings with clinical forms of Chagas disease. Moreover, immunoglobulins and cytokines are important for parasitic control and are involved in lesion genesis. Here, cytokine and IgG isotype production were studied, using total epimastigote antigen on sera of chagasic patients with indeterminate (IND, n = 27) and cardiac (CARD, n = 16) forms of the disease. Samples from normal,uninfected individuals (NI, n = 30) were use as controls. The results showed that sera from both IND and CARD patients contained higher levels of Trypanosoma cruzi-specific IgG1 (IgG1) antibodies than sera from NI. No difference in IgG2 production levels was observed between NI, IND and CARD patients, nor was a difference in IL-10 and IFN-gamma production detected in the sera of IND, CARD and NI patients. However, IND patients displayed a positive correlation between IL-10 and IFN-gamma levels in serum, while CARD patients showed no such correlation, indicating an uncontrolled inflammatory response in CARD patients. These findings support the hypothesis that a lack of efficient regulation between IFN-gamma and IL-10 productions in CARD patients may lead to cardiac immunopathology.

Chagas Disease: Increased Parasitemia during Pregnancy Detected by Hemoculture

One hundred fifty-two Trypanosoma cruzi seropositive women were submitted to a single hemoculture; 101 were pregnant, and 51 were not pregnant. Seven tubes from each individual were harvested with liver infusion tryptose (LIT) medium and observed monthly until the fifth month. Hemocultures were positive in 50% (76 of 152) of the women. Results showed that the positivity was 29.4% (15 of 51) among non-pregnant women and 60.4% (61 of 101) in pregnant women (P < 0.05). In relation to gestational age, there were significant differences in positivity, with a higher proportion of women with positive hemocultures (20 of 25) before 21 weeks and lower after 30 weeks (10 of 21; P = 0.02). We conclude that pregnancy enhances the parasitemia in Chagas disease, with a higher effect early in pregnancy.

Prevalence of toxoplasmosis in pregnant women and vertical transmission of Toxoplasma gondii in patients from basic units of health from Gurupi, Tocantins, Brazil, from 2012 to 2014

Introduction Toxoplasmosis is a parasitary disease that presents high rates of gestational and congenital infection worldwide being therefore considered a public health problem and a neglected disease. Objective To determine the prevalence of toxoplasmosis amongst pregnant women and vertical transmission of Toxoplasma gondii in their newborns attended in the Basic Units of Health (BUH) from the city of Gurupi, state of Tocantins, Brazil. Methods A prevalence study was performed, including 487 pregnant women and their newborns attended in the BUH of the urban zone of the city of Gurupi, state of Tocantins, Brazil, during the period from February 2012 to February 2014. The selection of the pregnant women occurred by convenience. In the antenatal admission they were invited to participate in this study. Three samples of peripheral blood were collected for the detection of specific anti-T. gondii IgG, IgM and IgA through ELISA, for the polimerase chain reaction (PCR) and IgG avidity during pregnancy. When IgM antibodies were detected the fetal and newborn infection investigation took place. The newborn was investigated right after birth and after one year of age through serology and PCR to confirm/exclude the vertical transmission. The analyses were performed in the Studies of the Host-Parasite Relationship Laboratory (LAERPH, IPTSP-UFG), Goiania, state of Goias, Brazil. The results were inserted in a data bank in Epi-Info 3.3.2 statistic software in which the analysis was performed with p≤5%. Results The toxoplasmosis infection was detected in 68.37% (333/487, CI95%: 64.62–72.86). The toxoplasmosis chronic infection prevalence was of 63.03% (307/487, CI95%: 58.74–67.32). The prevalence of maternal acute infection was of 5.33% (26/487; CI95%: 3.3–7.3) suspected by IgM antibodies detection in the peripheral blood. The prevalence of confirmed vertical transmission was of 28% (7/25; CI95%: 10.4–45.6). Conclusions These results show an elevated prevalence of toxoplasmosis in pregnant women and vertical transmission of T. gondii in the city of Gurupi, state of Tocantins, Brazil.

Congenital toxoplasmosis: evaluation of serological methods for the detection of anti-Toxoplasma gondii IgM and IgA antibodies.

A study was carried out to evaluate the presence of serological markers for the immunodiagnosis of the vertical transmission of toxoplasmosis. We tested the sensitivity, specificity and predictive values (positive and negative) of different serological methods for the early diagnosis of congenital toxoplasmosis. In a prospective longitudinal study, 50 infants with suspected congenital toxoplasmosis were followed up in the ambulatory care centre of Congenital Infections at University Hospital in Goiânia, Goiás, Brazil, from 1 January 2004-30 September 2005. Microparticle Enzyme Immunoassay (MEIA), Enzyme-Linked Fluorescent Assay (ELFA) and Immune-Fluorescent Antibody Technique (IFAT) were used to detect specific IgM anti-Toxoplasma gondii antibodies and a capture ELISA was used to detect specific IgA antibodies. The results showed that 28/50 infants were infected. During the neonatal period, IgM was detected in 39.3% (11/28) of those infected infants and IgA was detected in 21.4% (6/28). The sensitivity, specificity and predictive values (positive and negative) of each assay were, respectively: MEIA and ELFA: 60.9%, 100%, 100%, 55.0%; IFAT: 59.6%, 91.7%, 93.3%, 53.7%; IgA capture ELISA: 57.1%, 100%, 100%, 51.2%. The presence of specific IgM and IgA antibodies during the neonatal period was not frequent, although it was correlated with the most severe cases of congenital transmission. The results indicate that the absence of congenital disease markers (IgM and IgA) in newborns, even after confirming the absence with several techniques, does not constitute an exclusion criterion for toxoplasmosis.

Assessment of laboratory methods used in the diagnosis of congenital toxoplasmosis after maternal treatment with spiramycin in pregnancy

BackgroundThe different laboratory methods used in the diagnosis of congenital toxoplasmosis have variable sensitivity and specificity. There is no evidence to prove that maternal treatment reduces the risk of fetal infection. The purpose of this study was to assess methods for the confirmation of congenital toxoplasmosis after maternal treatment with spiramycin during pregnancy, and to evaluate the effect of this treatment on clinical manifestations of the disease in newborns (NB).MethodsThis was a community-based, cross-sectional study of acute toxoplasmosis in newborns at risk of acquiring congenital infection. Participating newborns were born in the Clinical Hospital Maternity Ward of the Federal University of Goiás. Eligible participants were divided into 2 groups: group 1 consisted of 44 newborns born to mothers treated with spiramycin during pregnancy and group 2 consisted of 24 newborns born to mothers not treated with spiramycin during pregnancy because the diagnosis of toxoplasmosis was not performed. The sensitivity and specifity of PCR for T. gondii DNA in peripheral blood and serological testing for specific anti-T. gondii IgM and IgA, and the effects of maternal spiramycin treatment on these parameters, were determined by associating test results with clinical manifestations of disease.ResultsThe sensitivity of the markers (T. gondii DNA detected by PCR, and the presence of specific anti-T. gondii IgM and IgA) for congenital toxoplasmosis was higher in group 2 than in group 1 (31.6, 68.4, 36.8% and 3.7, 25.9, 11.1% respectively). Even with a low PCR sensitivity, the group 2 results indicate the importance of developing new techniques for the diagnosis of congenital toxoplasmosis in newborns. Within group 1, 70.4% of the infected newborns were asymptomatic and, in group 2, 68.4% showed clinical manifestations of congenital toxoplasmosis.ConclusionsThe higher proportion of infants without clinical symptoms in group 1 (70.4%) suggests the maternal treatment with spiramycin delays fetal infection, reducing the clinical sequelae of the disease in newborns. Given the low sensitivity of the tests used, when there is suspicion of congenital transmission several serological and parasitological tests are required in order to confirm or exclude congenital toxoplasmosis in newborns.

Epidemiological factors associated with seropositivity for toxoplasmosis in pregnant women from Gurupi, State of Tocantins, Brazil

INTRODUCTION Knowledge of the prevalence and risk factors for Toxoplasma gondii dissemination among pregnant women is relevant because the parasite can be spread from mother to infant. The objective of this study was to assess the epidemiology and risk factors of toxoplasmosis in pregnant women from Gurupi, State of Tocantins, Brazil, from February 2012 to June 2013. METHODS The study population included 487 pregnant women. Sociodemographic, dietary and cultural data were collected using a standardized and validated form. Peripheral blood was collected for serologic testing using the ELISA test (IgM/IgG antibodies). The data were analyzed by comparing seropositivity with risk factors using crude and adjusted odds ratios. RESULTS The prevalence rate for IgG and IgM antibodies was 68.7% and 5.7%, respectively. Sociodemographic characteristics associated with toxoplasmosis risk included the following: education level ≤ 8 years (OR: 6.612; CI: 1.450-30.144), age ≥ 30 years (OR: 5.273; CI: 1.166-23.844), working outside the home (OR: 1.604; CI: 1.015-2.536), and family income of two minimum wages or lower (OR: 2.700; CI: 1.891-8.182). Regarding dietary habits, there was a significant association of seropositivity with meat intake (OR: 1.78; CI: 1.149-4.080), cutting vegetables without washing the cutting board beforehand (OR: 2.051; CI: 1.165-3.614), frequent intake of vegetables (OR: 2.051; CI: 1.368-3.006) and in natura milk intake (OR: 2.422; CI: 1.014-5.785). CONCLUSIONS The high prevalence rates of toxoplasmosis in Gurupi are related to age, raw meat and in natura milk intake, as well as education level, working outside the home, and poor hygienic habits during meal preparation.

Infeccoes de transmissao vertical em material abortivo e sangue com enfase em Toxoplasmose gondii

PURPOSE To analyze the serological, anatomopathological and parasitological results obtained from abortive material in order to detect infections with the risk of vertical transmission, with emphasis on toxoplasmosis. METHODS A cross-sectional cohort study was conducted in order to determine the prevalence of infectoparasitic diseases. A total of 105 women who suffered spontaneous complete or incomplete abortion participated in the study. The women were interviewed, answered a questionnaire and had their blood and abortive material collected. Immunological tests were carried out in order to detect toxoplasmosis, Chagas disease, rubeola, cytomegalovirus and syphilis, and anatomopathological analysis of the ovular remains was performed. RESULTS 55% of the women studied were 20 to 30 years old. Most of them (68%) presented a gestational age between the 7th and 14th week. 54.3% of the women had complete or incomplete high school education. Serological analysis showed cytomegalovirus (CMV) as the most common vertically transmitted infection with 97.1% positivity, followed by rubeola with 95.2%. Toxoplasmosis showed 54.3% positivity, Chagas disease 1.9% and syphilis 0.95%. Anatomopathological analysis showed inflammation in 63.1% of the cases and absence of inflammation in 34%. The results of the serological, anatomopathological and parasitological analysis of the 105 participants showed that 57 women were T. gondii positive. However, none showed positivity in the polymerase chain reaction (PCR) or in mouse inoculation. CONCLUSIONS The prevalence of diseases with the risk of vertical transmission is important in women with spontaneous abortion, indicating the need for more research in order to investigate the etiology of abortion.

Prevalência de toxoplasmose em gestantes atendidas em dois centros de referência em uma cidade do Nordeste, Brasil

PURPOSE: To determine the prevalence of toxoplasmosis and to identify the main factors associated with seroreactivity in pregnant women cared for at two reference centers in a city in Northeast Brazil.METHODS: A cross-sectional study was conducted on 561 pregnant women at two high-risk prenatal reference centers in a city in Northeast Brazil. All women were interviewed using an epidemiological questionnaire and had their blood samples collected for the following serological tests: anti-Toxoplasma gondii IgG and IgM (ELISA), IgG avidity test, and polymerase chain reaction (PCR). Statistical analysis was carried out using SPSS version 18.0 for Windows, calculating odds ratio, confidence interval of 95% and with the level of significance set at 5%.RESULTS: Seroreactivity for toxoplasmosis was detected in 437 women (77.0%), susceptibility in 124 (22.1%) and active infection in 5 (0.9%). There was no significant association between seroreactivity for toxoplasmosis and age, location, income, education, availability of sewage, number of pregnancies or gestational age. The variables significantly associated (p≤0.05) with seroreactivity were multiparity (p=0.03) and living with stray dogs (p=0.01).CONCLUSIONS: This study identified high seroreactivity for toxoplasmosis among patients seen during prenatal care, as well as factors associated with seroreactivity. Appropriate guidelines about primary preventive measures should be emphasized and quarterly serological monitoring is recommended for pregnant women in this city and elsewhere in the Northeast of Brazil.

Seroprevalence of Triatoma virus (Dicistroviridae: Cripaviridae) antibodies in Chagas disease patients

BackgroundChagas disease is caused by Trypanosoma cruzi, and humans acquire the parasite by exposure to contaminated feces from hematophagous insect vectors known as triatomines. Triatoma virus (TrV) is the sole viral pathogen of triatomines, and is transmitted among insects through the fecal-oral route and, as it happens with T. cruzi, the infected insects release the virus when defecating during or after blood uptake.MethodsIn this work, we analysed the occurrence of anti-TrV antibodies in human sera from Chagas disease endemic and non-endemic countries, and developed a mathematical model to estimate the transmission probability of TrV from insects to man, which ranged between 0.00053 and 0.0015.ResultsOur results confirm that people with Chagas disease living in Bolivia, Argentina and Mexico have been exposed to TrV, and that TrV is unable to replicate in human hosts.ConclusionsWe presented the first experimental evidence of antibodies against TrV structural proteins in human sera.