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.

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.

Multiple myeloma and Chagas disease: qPCR as a marker for preemptive antiparasitic therapy: a case reports series and review.

Multiple myeloma (MM) associated with Chagas disease is rarely described. This disease and its therapy suppress T cell and macrophage functions and increase regulatory T cell function, allowing the increase of parasitemia and the risk of Chagas Disease Reactivation (CDR). We aimed to analyze the role of conventional (cPCR) and quantitative Polymerase Chain Reaction (qPCR) for prospective monitoring of T. cruzi parasitemia, searching for markers of preemptive antiparasitic therapy in MM patients with Chagas disease. Moreover, we investigated the incidence and management of hematological diseases and CDR both inside and outside the transplant setting in the MEDLINE database. We found 293 studies and included 31 of them. Around 1.9-2.0% of patients with Chagas disease were reported in patients undergoing Stem Cell Transplantation. One case of CDR was described in eight cases of MM and Chagas disease. We monitored nine MM and Chagas disease patients, seven under Autologous Stem Cell Transplantation (ASCT), during 44.56±32.10 months (mean±SD) using parasitological methods, cPCR, and qPCR. From these patients, three had parasitemia. In the first, up to 256 par Eq/mL were detected, starting from 28 months after ASCT. The second patient dropped out and died soon after the detection of 161.0 par Eq/mL. The third patient had a positive blood culture. Benznidazole induced fast negativity in two cases; followed by notably lower levels in one of them. Increased T. cruzi parasitemia was related to the severity of the underlying disease. We recommend parasitemia monitoring by qPCR for early introduction of preemptive antiparasitic therapy to avoid CDR.

Multiple myeloma and Chagas disease: qPCR as a marker for preemptive antiparasitic therapy: a case reports series and review

ABSTRACT Multiple myeloma (MM) associated with Chagas disease is rarely described. This disease and its therapy suppress T cell and macrophage functions and increase regulatory T cell function, allowing the increase of parasitemia and the risk of Chagas Disease Reactivation (CDR). We aimed to analyze the role of conventional (cPCR) and quantitative Polymerase Chain Reaction (qPCR) for prospective monitoring of T. cruzi parasitemia, searching for markers of preemptive antiparasitic therapy in MM patients with Chagas disease. Moreover, we investigated the incidence and management of hematological diseases and CDR both inside and outside the transplant setting in the MEDLINE database. We found 293 studies and included 31 of them. Around 1.9-2.0% of patients with Chagas disease were reported in patients undergoing Stem Cell Transplantation. One case of CDR was described in eight cases of MM and Chagas disease. We monitored nine MM and Chagas disease patients, seven under Autologous Stem Cell Transplantation (ASCT), during 44.56±32.10 months (mean±SD) using parasitological methods, cPCR, and qPCR. From these patients, three had parasitemia. In the first, up to 256 par Eq/mL were detected, starting from 28 months after ASCT. The second patient dropped out and died soon after the detection of 161.0 par Eq/mL. The third patient had a positive blood culture. Benznidazole induced fast negativity in two cases; followed by notably lower levels in one of them. Increased T. cruzi parasitemia was related to the severity of the underlying disease. We recommend parasitemia monitoring by qPCR for early introduction of preemptive antiparasitic therapy to avoid CDR.

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.

Aplastic Anemia and Chagas Disease: T. cruzi Parasitemia Monitoring by Quantitative PCR and Preemptive Antiparasitic Therapy.

Background: Aplastic anemia is a rare and life-threatening condition, seldomly witnessed concomitantly with Chagas disease. We aim to discuss the management of these patients under risk of chronic Chagas disease reactivation (CDR), a severe condition with a high morbimortality that occurs in chronic Chagas disease patients under immunosuppression. Case reports: Trypanosoma cruzi (T. cruzi) parasitemia was monitored in three patients for 4−58 months by conventional PCR (cPCR), quantitative PCR (qPCR), microhematocrit/buffy coat, blood culture, and/or xenodiagnosis. One patient received antiparasitic treatment (benznidazole) and the other received allopurinol. Although parasitemia was controlled during and after benznidazole treatment at 300 mg/d for 51 days, in one patient, hematologic parameters worsened continuously before, during, and after treatment. Allopurinol led only to the temporary suppression of T. cruzi parasitemia in the second patient, but after danazol and hematological improvement, parasitemia became undetectable until the end of monitoring. Discussion and Conclusion: Unexpected undetectable or low parasitemia by cPCR/qPCR was reported. We show that the monitoring of parasitemia by qPCR and the use of preemptive therapy when the parasitemia was positive proved to be beneficial to our patients. As a result of the toxicity of more effective antiparasitics, shorter regimens of benznidazole or less toxic drugs in preemptive therapy are options that deserve future studies.

Análise de técnicas parasitológicas utilizadas no seguimento de pacientes com doença de chagas e tratados por meio do transplante cardíaco / Analysis of parasitological techniques in the monitoring of patients with Chagas disease and treated by heart transplantation

Este estudo teve como objetivo analisar a função da hemocultura, do xenodiagnóstico e do creme leucocitário no acompanhamento e diagnóstico de possível reativação em pacientescrônicos tratados por meio do transplante de coração. Foram examinadas 70 amostras das quais15,7por cento (11/70) se mostraram positivas, sendo 8,5por cento (6/70) na hemocultura e 12,8por cento (9/70) noxenodiagnóstico. Apresentaram concordância nos dois métodos quatro amostras (36,36por cento). Nãohouve positividade no creme leucocitário. Os achados corroboram informações sobre a superiorsensibilidade do xenodiagnóstico em relação à hemocultura. A amostra do paciente 20, positiva noxenodiagnóstico (décimo quinto dia) e que apresentou miocardite com ninhos de amastigotas 15 dias antes dadetecção laboratorial, sinaliza a importância da leitura precoce dos exames parasitológicos comopreditivos de possível reativação da doença.

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.

Trypanosoma cruzi, hemocultura: uma abordagem prática / Trypanosoma cruzi, hemoculture: a practical approach

JUSTIFICATIVA E OBJETIVOS: A hemocultura permanece útil para diagnosticar a doença de Chagas crônica. O objetivo deste estudo foi comparar a técnica "clássica" e a "modificada" mais usada para essa finalidade no Brasil. MÉTODO: A inoculação de "creme leucocitário" com sedimento de sangue centrifugado caracteriza ambas as técnicas. Amostras provenientes de dois grupos de 137 pacientes chagásicos crônicos foram examinadas. A "clássica" utiliza 10 mL de sangue; a "modificada", 30 mL. RESULTADOS: A técnica "modificada" mostrou-se mais sensível do que a "clássica". CONCLUSÃO: Sugere-se o uso rotineiro da técnica "modificada".

BACKGROUND AND OBJECTIVES: Hemoculture remains a valuable tool for the diagnosis of chronic Chagas disease. We tested the two techniques routinely used in Brazil. METHOD: The "modified" technique includes the inoculation of "buffy coat" and centrifuged blood sediment from a 30 mL sample, the serum being washed out after centrifugation and replaced by equal volume of culture medium; the "classic" one requires the inoculation of centrifuged blood sediment plus "buffy coat" from a 10 mL sample. RESULTS: A higher sensitivity of the "modified" technique was observed when the results of two samples of 137 chronic Chagas disease patients were compared. CONCLUSION: The routine use of the "modified" technique should be preferred.

[An evaluation of the efficacy of the CSF method for diagnosing intestinal helminthiases]. / Uma avaliação sobre a eficácia do método CSF no diagnóstico de helmintíases intestinais.

The recently proposed CSF method for diagnosing intestinal helminthiases was compared with the other methods (direct; Faust et al.; spontaneous sedimentation in water; and Kato-Katz) that are routinely for this purpose. The CSF method performed satisfactorily, thus showing that this technique can be adopted for use in diagnoses or epidemiological analyses.

[Observations on Blastocystis hominis and Cyclospora cayetanensis in routine parasitological examinations]. / Observações sobre Blastocystis hominis e Cyclospora cayetanensis em exames parasitológicos efetuados rotineiramente.

We report some observations made from routine parasitological examinations on feces. The methods of Faust et al. and of spontaneous sedimentation in water are not enough to identify Blastocystis hominis. Significant percentage presence of this protozoan was found, especially when staining with iron hematoxylin was performed. Cyclospora cayetanensis was found in 0.7% of the cases, which suggests that this parasite should also routinely be investigated by appropriate techniques.

Uma avaliação sobre a eficácia do método CSF no diagnóstico de helmintíases intestinais / An evaluation of the efficacy of the CSF method for diagnosing intestinal helminthiases

O método CSF, recentemente proposto para diagnóstico de helmintíases intestinais foi comparado com outros (direto; Faust e cols; sedimentação espontânea em água; Kato-Katz) habitualmente usados com essa finalidade. Houve desempenho satisfatório, revelando que tal técnica pode ser adotada em tarefas para diagnóstico ou análise epidemiológicas.

The recently proposed CSF method for diagnosing intestinal helminthiases was compared with the other methods (direct; Faust et al.; spontaneous sedimentation in water; and Kato-Katz) that are routinely for this purpose. The CSF method performed satisfactorily, thus showing that this technique can be adopted for use in diagnoses or epidemiological analyses.

Observações sobre Blastocystis hominis e Cyclospora cayetanensis em exames parasitológicos efetuados rotineiramente / Observations on Blastocystis hominis and Cyclospora cayetanensis in routine parasitological examinations

Relatamos algumas observações, efetuadas com exames parasitológicos de fezes, em atividades rotineiras: os métodos de Faust e cols e de sedimentação espontânea em água não servem para evidenciação de Blastocystis hominis; foram encontradas expressivas porcentagens de presença desse protozoário, sobretudo quando realizada coloração pela hematoxilina férrica; houve 0,7 por cento de registro de positividade para Cyclospora cayetanensis, sugerindo inclusão habitual de pesquisa, por técnicas apropriadas, de tal parasita.

We report some observations made from routine parasitological examinations on feces. The methods of Faust et al. and of spontaneous sedimentation in water are not enough to identify Blastocystis hominis. Significant percentage presence of this protozoan was found, especially when staining with iron hematoxylin was performed. Cyclospora cayetanensis was found in 0.7 percent of the cases, which suggests that this parasite should also routinely be investigated by appropriate techniques.

[Blastocystosis: a high proportion of cases found in schoolchildren of São Paulo, State of São Paulo, Brazil]. / Elevada porcentagem de blastocistose em escolares de São Paulo, SP.

As a part of medical assistance activities, parasitological examination of fecal samples from 227 school children from a public institution of São Paulo (SP) revealed a rather high proportion of results positive for Blastocystis hominis. Other protozoan and worm species were markedly scarcer, a peculiar situation according to our judgement. It is acknowledged that blastocystosis is still largely an indefinite and controversial subject, which deserves adequate analysis to avoid drawbacks in the sphere of action of public health and general medical assistance.

Elevada porcentagem de blastocistose em escolares de São Paulo, SP / Blastocystosis: a high proportion of cases found in schoolchildren of São Paulo, State of São Paulo, Brazil

Em exame parasitológico de fezes de 227 alunos de escola pública de São Paulo (SP), encontramos 87 (38,3 por cento) positivas para Blastocystis hominis. A blastocistose ainda suscita controvérsias e indefinições, merecedoras de esclarecimentos sobretudo para evitar contratempos no âmbito da saúde pública e das atenções médico-assistenciais.

Blastocistose: controvérsias e indefinições / Blastocystosis: controversy and ill-defined

A blastocistose, enterite devida ao Blastocystis hominis, ainda causa controvérsias. É comum verificar que expressivo número de médicos, patologistas e profissionaisque exercem atividades no âmbito da saúde pública conhecem ainda pouco sobre aspectos nosológicos desse protozoário. Essa situação faz com que, por vezes, sejam adotadas condutas inadequadas na clínica...

[Blastocystosis: controversy and indefinedness]. / Blastocistose: controvérsias e indefinições.

Blastocystosis is the infection caused by Blastocystis hominis. It is associated with frequent and unquestionably very important controversy and lack of definition, above all due to its implications for general assistance and medical care. In that connection, there is considerable disagreement on the subject of the pathogenicity of this protozoan, which should be categorically defined. Other aspects besides the above, require clarification through results from well conducted studies aiming at attributing Blastocystis hominis a proper role within the context of public health. Another matter worthy of attention is the diagnostic value of the parasitological stool examination, with the proviso that it is adequate, as are fecal smears suspended in saline solution or permanent mounts stained with iron hematoxylin or thionine. The use of inadequate techniques tends to produce false negative results, thereby impeding investigation into the real importance of this microorganism.

Blastocistose: controvérsias e indefinições / Blastocystosis: controversy and indefinedness

Blastocistose é a infecçäo causada pelo Blastocystis hominis. Está relacionada com várias controvérsias e indefinições, sem dúvida muito importantes sobretudo pelas implicações médico-assistenciais que suscitam. A propósito, pendência expressiva diz respeito à patogenicidade do referido protozoário, que precisa ser categoricamente definida. Ao lado dessa particularidade outros assuntos exigem elucidações através de pesquisas bem conduzidas, para que a blastocistose fique devidamente situada no contexto da saúde pública. Aspecto também digno de atençäo é o diagnóstico pelo exame parasitológico de fezes, necessariamente executável por meio de métodos adequados, como o direto e os permanentes, exemplificados pelos que usam a a hematoxilina férrica ou a tionina. O emprego de técnicas impróprias propicia resultados falsos-negativos, conturbando o aconselhável bom conhecimento da real participaçäo do microrganismo em questäo