Quantitative PCR as a marker for preemptive therapy and its role in therapeutic control in Trypanosoma cruzi/HIV coinfection.

BACKGROUND: Trypanosoma cruzi and HIV coinfection can evolve with depression of cellular immunity and increased parasitemia. We applied quantitative PCR (qPCR) as a marker for preemptive antiparasitic treatment to avoid fatal Chagas disease reactivation and analyzed the outcome of treated cases. METHODOLOGY: This mixed cross-sectional and longitudinal study included 171 Chagas disease patients, 60 coinfected with HIV. Of these 60 patients, ten showed Chagas disease reactivation, confirmed by parasites identified in the blood, cerebrospinal fluid, or tissues, 12 exhibited high parasitemia without reactivation, and 38 had low parasitemia and no reactivation. RESULTS: We showed, for the first time, the success of the timely introduction of benznidazole in the non-reactivated group with high levels of parasitemia detected by qPCR and the absence of parasites in reactivated cases with at least 58 days of benznidazole. All HIV+ patients with or without reactivation had a 4.0-5.1 higher chance of having parasitemia than HIV seronegative cases. A positive correlation was found between parasites and viral loads. Remarkably, treated T. cruzi/HIV-coinfected patients had 77.3% conversion from positive to negative parasitemia compared to 19.1% of untreated patients. Additionally, untreated patients showed ~13.6 times higher Odds Ratio of having positive parasitemia in the follow-up period compared with treated patients. Treated and untreated patients showed no differences regarding the evolution of Chagas disease. The main factors associated with all-cause mortality were higher parasitemia, lower CD4 counts/µL, higher viral load, and absence of antiretroviral therapy. CONCLUSION: We recommend qPCR prospective monitoring of T. cruzi parasitemia in HIV+ coinfected patients and point out the value of pre-emptive therapy for those with high parasitemia. In parallel, early antiretroviral therapy introduction is advisable, aiming at viral load control, immune response restoration, and increasing survival. We also suggest an early antiparasitic treatment for all coinfected patients, followed by effectiveness analysis alongside antiretroviral therapy.

Assessment of biomarkers and clinical parameters as predictors of survival in patients with chagasic heart failure.

BACKGROUND: Chagas disease, endemic in Latin America and spreading globally due to emigration, has a significant health burden, particularly in relation to chagasic heart failure (HF). Chagasic cardiomyopathy (CCM) is characterized by chronic inflammatory myocardial disease. This study aimed to identify inflammatory parameters and biomarkers that could aid in the management of patients with chagasic HF. METHODS AND FINDINGS: A cohort study was conducted at a tertiary cardiology single-center over a mean follow-up period of 2.4 years. The study included patients with HF secondary to CCM enrolled between October 2013 and July 2017. Various clinical parameters, echocardiography findings, parasitemia status, brain natriuretic peptide (BNP) and troponin T (TnT) levels, and inflammatory biomarkers (IL-6, IL-10, IL-12p70, IL-17A, adiponectin, and IFN-γ) were assessed. The study encompassed a cohort of 103 patients, with a median age of 53 years and 70% being male. The left ventricular ejection fraction (LVEF) was 28%, with 40% of patients classified as NYHA II functional class. The median BNP level was 291 pg/ml. The observed mortality rate during the study period was 38.8%. Predictors of lower survival were identified as elevated levels of BNP, TnT, reduced LVEF, and increased adiponectin (thresholds: BNP > 309 pg/ml, TnT > 27.5 ng/ml, LVEF < 25.5%, adiponectin > 38 µg/mL). Notably, there was no evidence indicating a relationship between parasitemia and the inflammatory parameters with lower survival in these patients, including INF-γ, IL-6, IL-10, IL12-(p70), and IL17a. CONCLUSION: Despite the presence of a chronic inflammatory process, the evaluated inflammatory biomarkers in this cohort were not predictive of survival in patients with chagasic HF with reduced ejection fraction (HFrEF). However, reduced LVEF, elevated BNP, adiponectin levels, and troponin T were identified as predictors of lower survival in these patients.

Genetic diversity of Trypanosoma cruzi strains isolated from chronic chagasic patients and non-human hosts in the state of São Paulo, Brazil.

BACKGROUND: Trypanosoma cruzi shows an exuberant genetic diversity. Currently, seven phylogenetic lineages, called discrete typing units (DTUs), are recognised: TcI-TcVI and Tcbat. Despite advances in studies on T. cruzi and its populations, there is no consensus regarding its heterogeneity. OBJECTIVES: This study aimed to perform molecular characterisation of T. cruzi strains, isolated in the state of São Paulo, to identify the DTUs involved and evaluate their genetic diversity. METHODS: T. cruzi strains were isolated from biological samples of chronic chagasic patients, marsupials and triatomines through culture techniques and subjected to molecular characterisation using the fluorescent fragment length barcoding (FFLB) technique. Subsequently, the results were correlated with complementary information to enable better discrimination between the identified DTUs. FINDINGS: It was possible to identify TcI in two humans and two triatomines; TcII/VI in 19 humans, two marsupials and one triatomine; and TcIII in one human host, an individual that also presented a result for TcI, which indicated the possibility of a mixed infection. Regarding the strains characterised by the TcII/VI profile, the correlation with complementary information allowed to suggest that, in general, these parasite populations indeed correspond to the TcII genotype. MAIN CONCLUSIONS: The TcII/VI profile, associated with domestic cycles and patients with chronic Chagas disease, was the most prevalent among the identified DTUs. Furthermore, the correlation of the study results with complementary information made it possible to suggest that TcII is the predominant lineage of this work.

Genetic diversity of Trypanosoma cruzi strains isolated from chronic chagasic patients and non-human hosts in the state of São Paulo, Brazil

BACKGROUND Trypanosoma cruzi shows an exuberant genetic diversity. Currently, seven phylogenetic lineages, called discrete typing units (DTUs), are recognised: TcI-TcVI and Tcbat. Despite advances in studies on T. cruzi and its populations, there is no consensus regarding its heterogeneity. OBJECTIVES This study aimed to perform molecular characterisation of T. cruzi strains, isolated in the state of São Paulo, to identify the DTUs involved and evaluate their genetic diversity. METHODS T. cruzi strains were isolated from biological samples of chronic chagasic patients, marsupials and triatomines through culture techniques and subjected to molecular characterisation using the fluorescent fragment length barcoding (FFLB) technique. Subsequently, the results were correlated with complementary information to enable better discrimination between the identified DTUs. FINDINGS It was possible to identify TcI in two humans and two triatomines; TcII/VI in 19 humans, two marsupials and one triatomine; and TcIII in one human host, an individual that also presented a result for TcI, which indicated the possibility of a mixed infection. Regarding the strains characterised by the TcII/VI profile, the correlation with complementary information allowed to suggest that, in general, these parasite populations indeed correspond to the TcII genotype. MAIN CONCLUSIONS The TcII/VI profile, associated with domestic cycles and patients with chronic Chagas disease, was the most prevalent among the identified DTUs. Furthermore, the correlation of the study results with complementary information made it possible to suggest that TcII is the predominant lineage of this work.

Valores normais da atividade da colinesterase plasmática em anseriformes, com destaque para Alopochen aegyptiacus / Normal values of plasma cholinesterase activity in anseriformes, with emphasis on Alopochen aegyptiacus

A inibição da enzima colinesterase plasmática (BChE) pode ser utilizada como biomarcador para os efeitos da intoxicação por organofosforados e carbamatos. Nas aves, esta inibição ocorre de forma mais acentuada que nos mamíferos, porém poucos são os trabalhos publicados nestas espécies. O objetivo do estudo fo a dosagem da BChE em gansos-egípcios (Alopochen aegyptiacus) e nos anseriformes domésticos: gansos-domésticos (Anser anser domesticus) e marrecos (Anas platyrhynchos domesticus), para o estabelecimento de valores de referência normais. O trabalho possui ineditismo com relação à determinação desta enzima nos gansos-egípcios e domésticos. Os gansos e marrecos são mantidos em confinamento com fornecimento de alimentos e água ad libitum e em espaço adequado à sua manutenção no Instituto Adolfo Lutz (IAL), com a finalidade de fornecimento de sangue para a alimentação de triatomídeos do insetário de criação no Núcleo de Parasitoses Sistêmicas. Nos Alopochen aegyptiacus a média e o desvio padrão da BChE foram de 1.868 + 263,6 U/L, nos Anser anser domesticus 2.311 + 673,2 U/L e nos Anas platyrhynchos domesticus 4.290 + 86,11 U/L. (AU)

The inhibition of the plasma cholinesterase enzyme (BChE) can be used as a biomarker for the effects of intoxication by organophosphates and carbamates. In birds, this inhibition is more pronounced than in mammals, however there are few specific studies were conducted in this field. The aim of this study was to measure BChE in Egyptian geese (Alopochen aegyptiacus) and domestic anseriforms: domestic geese (Anser anser domesticus) and mallards (Anas platyrhynchos domesticus), not exposed to pesticides, for the establishment of normal values. The work is unprecedented regarding the determination of this enzyme in egyptian geese and domestic geese. Geese and mallards are kept in confinement with ad liditum food and water supply and in adequate space for their maintenance at the Adolfo Lutz Institute (IAL), for the purpose of supplying blood for the feeding of triatomines from the insectary of the Nucleus of Systemic Parasitoses. In Alopochen aegyptiacus the mean and standard deviation of BChE were 1,868 + 263,6 U/L, in Anser anser domesticus 2,311 + 673,2 U/L and in Anas platyrhynchos domesticus 4,290 + 86.11 U/L. (AU)

Aspectos epidemiológicos e históricos do controle da doença de Chagas no continente americano / Epidemiological and historical aspects of Chagas disease control in the american continent

A doença de Chagas é um dos maiores problemas de saúde pública da AméricaLatina. Após 103 anos de sua descoberta, continua negligenciada e ainda representa a maior causa de óbitos entre as doenças parasitárias. Asustentabilidade dos programas para o controle da transmissão da infecçãochagásica, instituídos em países como os do Cone Sul, deve-se a iniciativasintergovernamentais e políticas que resultaram na melhoria dos serviços deatenção à saúde, condições sanitárias e no desenvolvimento sócio-econômico.No entanto, mesmo com a disponibilidade de tecnologia para o controle da transmissão domiciliar, vários países ainda apresentam áreas infestadas por triatomíneos e incidência variável da infecção chagásica, demonstrando que o subdesenvolvimento, a pobreza e a falta de prioridade política são fatoresdeterminantes para a sua existência, emergência e re-emergência. Nas últimasdécadas, a transmissão de Trypanosoma cruzi transfusional, vetorial passiva ouoral e congênita assumiram maior importância na manutenção da endemia e, devido à imigração, a doença de Chagas passou a ser um problema de saúde pública também em outros continentes.

Real-time PCR in HIV/Trypanosoma cruzi coinfection with and without Chagas disease reactivation: association with HIV viral load and CD4 level.

BACKGROUND: Reactivation of chronic Chagas disease, which occurs in approximately 20% of patients coinfected with HIV/Trypanosoma cruzi (T. cruzi), is commonly characterized by severe meningoencephalitis and myocarditis. The use of quantitative molecular tests to monitor Chagas disease reactivation was analyzed. METHODOLOGY: Polymerase chain reaction (PCR) of kDNA sequences, competitive (C-) PCR and real-time quantitative (q) PCR were compared with blood cultures and xenodiagnosis in samples from 91 patients (57 patients with chronic Chagas disease and 34 with HIV/T. cruzi coinfection), of whom 5 had reactivation of Chagas disease and 29 did not. PRINCIPAL FINDINGS: qRT-PCR showed significant differences between groups; the highest parasitemia was observed in patients infected with HIV/T. cruzi with Chagas disease reactivation (median 1428.90 T. cruzi/mL), followed by patients with HIV/T. cruzi infection without reactivation (median 1.57 T. cruzi/mL) and patients with Chagas disease without HIV (median 0.00 T. cruzi/mL). Spearman's correlation coefficient showed that xenodiagnosis was correlated with blood culture, C-PCR and qRT-PCR. A stronger Spearman correlation index was found between C-PCR and qRT-PCR, the number of parasites and the HIV viral load, expressed as the number of CD4(+) cells or the CD4(+)/CD8(+) ratio. CONCLUSIONS: qRT-PCR distinguished the groups of HIV/T. cruzi coinfected patients with and without reactivation. Therefore, this new method of qRT-PCR is proposed as a tool for prospective studies to analyze the importance of parasitemia (persistent and/or increased) as a criterion for recommending pre-emptive therapy in patients with chronic Chagas disease with HIV infection or immunosuppression. As seen in this study, an increase in HIV viral load and decreases in the number of CD4(+) cells/mm(3) and the CD4(+)/CD8(+) ratio were identified as cofactors for increased parasitemia that can be used to target the introduction of early, pre-emptive therapy.

Estimativa do número mínimo de promastigotas de Leishmania (Viannia) braziliensis necessário para a proliferação em sistema de cultivo em microplacas / Estimated minimum number of promastigotes of Leishmania (Viannia) braziliensis necessary for proliferation in a culture system in microplates

O diagnóstico de certeza da leishmaniose cutânea depende do encontro doagente etiológico. A técnica tradicional de cultura (TTC) realizada emtubosdemanda prolongada incubação, grande volume de meio de cultivo edeamostra. A técnica de microcultura (TMC) realizada em placas de 96poçosutiliza pequeno volume de meio de cultivo e de amostra, possibilitandorealizar diversos inóculos por amostra examinada. Ocrescimento de L. (V.) braziliensis (MHOM/84/LTB300) foi realizado nasduas técnicasobjetivando determinar o número mínimo de parasitasnecessários para aproliferação. A partir de diluições seriadas de suspensãocom L. (V.) braziliensis, foram obtidos inóculos em concentrações de 1x100 até1x103 para o cultivo. Na concentração de 1x103 foi observadaproliferação0arasitária a partir de 48 horas do cultivo. Um único parasita(1x100) foisuficiente para permitir a proliferação nas duas técnicas. Noinóculo de18concentração 1x101 a TTC atingiu 1x108 parasitas, porém aproliferação foi3observada primeiramente na TMC. Na concentração de 1x103 a TMC atingiu 2x108 parasitas. Concluiu-se que a TMC é uma estratégiaque permitiu ocultivo a partir de uma única forma flagelada. Novos estudosestão sendorealizados para avaliar a viabilidade da TMC na utilização comométododiagnóstico