Applicability of an optimized non-conventional flow cytometry method to detect anti-Trypanosoma cruzi immunoglobulin G for the serological diagnosis and cure assessment following chemotherapeutic treatment of Chagas disease.

One of the challenges on immunodiagnostic of Chagas disease in endemic areas has been the search for more practical and safe antigenic preparation that provides tests with higher sensitivity and specificity, with low cross-reactivity. A new approach using fixed Trypanosoma cruzi epimastigotes to detect IgG reactivity was investigated previously. In order to continue this investigation, this study aimed at optimizing the flow cytometry-based method to the diagnosis of Chagas disease patients after specific chemotherapy. To achieve our goal, serum samples from 93 subjects - 52 adults chronically infected by T. cruzi, and 41 uninfected controls were tested by flow cytometry. Secondly, serum samples from patients Treated Cured and Treated Uncured from Chagas disease were also tested to evaluate the potential of the method on assessing cure. After establishing the ideal serum dilution and cut off, 121 serum samples from patients with other endemic infections were tested to check cross-reactivity. The results showed that parasite staining with Evan's blue dye eliminated debris, allowing trustworthy analysis of anti-fixed epimastigote IgG reactivity. The applicability of the method to diagnose Chagas disease was confirmed by the high sensitivity (98.1%) and specificity (100%) found. This method also contributed for post-therapeutic assessment of cure, identifying 94.1% of Treated Uncured and 83.3% of Treated Cured patients. Cross-reactivity was observed in a very low number (6.7%). On the whole, these data highly recommend the use of anti-fixed T. cruzi epimastigote IgG reactivity by flow cytometry to the diagnosis and cure monitoring of Chagas disease in endemic areas.

Anti-Leishmania chagasi immunoglobulin G3 detected by flow cytometry for early cure assessment in American visceral leishmaniasis.

We have previously reported a novel flow cytometric based methodology to access the reactivity of seric anti-live (FC-ALPA) and fixed (FC-AFPA) L. chagasi IgG antibodies applicable for cure assessment after specific therapy of VL. Both, FC-ALPA-IgG and FC-AFPA-IgG are promising targets to be used for early cure assessment. However, our finding suggested that further refinements were still required to improve the performance of FC-AFPA IgG for early cure assessment in VL. In the present investigation, we have established and evaluated the performance of FC-AFPA-IgG1/IgG2/IgG3/IgG4 aiming to increase the performance index of the previously reported for FC-AFPA-IgG. The data was expressed as percentage of fluorescent positive parasites after incubation of pre-fixed L. chagasi promastigotes with the test sera samples and addition of second-step FITC-labeled anti-human IgG subclasses conjugates. The analysis of anti-L. chagasi IgG reactivity in polled sera samples from VL patients demonstrated that, before the etiological treatment, the IgG subclass profile was characterized by IgG1>IgG3 with the absence of IgG2 and IgG4 at the specific sera dilution tested. Following the establishment of specific PPFP cut-off-edges to segregate negative and positive results (PPFP of 50% for FC-AFPA-IgG1 and PPFP of 40% for FC-AFPA-IgG3), the analysis of IgG1 and IgG3 reactivity demonstrated good performance for early cure assessment in VL. The analysis of individual samples indicated that despite at 2 mAT, most treated VL patients (81%) still displayed positive results in FC-AFPA-IGg1 analysis, an increased fraction of treated patients (76%) presented negative in FC-AFPA-IgG1 analysis at 6 mAT. Interestingly, the data from FC-AFPA-IgG3 demonstrated an outstanding performance of this method to early cure assessment in VL with increased frequency of treated patients displaying negative results at 2 mAT (90.5%) as well as at 6 mAT (95.2%). The analysis of likelihood ratio (LR) further confirmed the remarkable performance of FC-AFPA-IgG3 as an early complementary biomarker useful to monitor the post-therapeutic cure in human VL.

Anti-fixed Leishmania chagasi promastigotes IgG antibodies detected by flow cytometry (FC-AFPA-IgG) as a tool for serodiagnosis and for post-therapeutic cure assessment in American visceral leishmaniasis.

Visceral leishmaniasis (VL) is a systemic infection, caused by an intracellular protozoan parasite belonging to the Leishmania donovani complex. The diagnosis of VL is complex because most clinical features are shared with other commonly occurring febrile hepatosplenic diseases that can be endemic along with VL. A number of serological devices are available but still require improvement mainly due to residual post-therapeutic serology and the cross-reactivity with other Trypanosomatidae protozooses. This study intended to describe and evaluate the performance of an indirect immunofluorescence assay referred as flow cytometry anti-fixed Leishmania chagasi promastigote IgG antibodies (FC-AFPA-IgG) for serodiagnosis of VL and assessment of post-therapeutic cure. The sera reactivity is reported as the percentage of positive fluorescent parasite (PPFP) along the titration curve. The analysis of sera titration curve indicated the sera dilution 1/32,000 and the PPFP=25% as the cut-off to segregate positive and negative results. Using these parameters, the FC-AFPA-IgG displayed outstanding sensitivity and specificity for diagnosis and post-therapeutic cure assessment purposes. The inter-test reproducibility of FC-AFPA-IgG was also verified, considering two independent Analysts and validated the results obtained by FC-AFPA-IgG. Moreover, the comparison between FC-AFPA-IgG and the conventional serologic test (ELISA) showed that besides the statistically analogous results with strong positive correlation the FC-AFPA-IgG displayed higher performance indexes. Further analysis demonstrated that while cross-reactivity was observed in 8% of samples tested by ELISA, no cross-reactivity was detected by FC-AFPA-IgG. Together, the findings presented in this study showed the potential of FC-AFPA-IgG in both diagnosis and post-therapeutic cure assessment of VL.

Upgrading the flow-cytometric analysis of anti-Leishmania immunoglobulins for the diagnosis of American tegumentary leishmaniasis.

We have previously described a flow cytometry-based assay to detect anti-live Leishmania (Viania) braziliensis promastigote antibodies (FC-ALPA) with prominent performance of FC-ALPA to diagnosis American tegumentary leishmaniasis (ATL). However, the laboriousness to work with live parasites represented the major drawback for using FC-ALPA in routine clinical laboratory. Herein, we have presented an upgraded technology using fixed Leishmania (Leishmania) amazonensis promastigotes as antigen (FC-AFPA). Our data demonstrated that FC-AFPA-IgG displays outstanding performance for ATL diagnosis with high sensitivity (99%) and specificity (100%). Moreover, Likelihood Ratio indicated that positive results (LR+) has an infinite times more chance to come from ATL than from non-infected individuals (NI). Despite the high frequency of cross-reactivity with putative ATL co-endemic diseases, including visceral leishmaniasis, Chagas disease and malaria, FC-AFPA-IgG showed remarkable potential for differential diagnosis with other dermatological illnesses such as leprosy and sporotrichosis. FC-AFPA-IgG subclasses analysis revealed that LTA is characterized by IgG1>IgG3>IgG2 = IgG4 anti-L. amazonensis profiling, electing FC-AFPA-IgG1 and IgG3 with better performances to diagnosis ATL diagnosis. Additionally, FC-AFPA-IgG3 showed to be a better diagnostic tool in endemic areas for malarial disease. Despite the substantial advance to work with fixed promastigotes that contributes to its higher sensitivity, the lower specificity of FC-AFPA represented the major flaws as compared to FC-ALPA, suggesting that further improvement is still required to minimize the cross-reactivity with trypanosomatidae infections. Perspectives for using a flow cytometry multiplex based methodology to simultaneously assess anti-L. amazonensis, anti-L. chagasi and anti-Trypanosoma cruzi IgG reactivity is currently under investigation.

Detection of anti-leishmania (Leishmania) chagasi immunoglobulin G by flow cytometry for cure assessment following chemotherapeutic treatment of American visceral leishmaniasis.

The residual serological reactivity observed in patients cured of visceral leishmaniasis (VL) represents the major factor underlying the low efficiency of most anti-Leishmania serological approaches to assess posttherapeutic cure in VL. Herein, we have described a detuned flow cytometry-based methodology to detect anti-live (FC-ALPA-immunoglobulin G [IgG]) and anti-fixed (FC-AFPA-IgG) L. chagasi promastigote IgG, along the titration curve (1:2,000 to 1:128,000), as a tool to assess late (12 months after treatment [12 mAT]) and early (2 and 6 mAT) posttherapeutic cure of pediatric American visceral leishmaniasis. Reactivities were reported as the percentage of positive fluorescent parasite (PPFP), using a PPFP of 50% as a cutoff to segregate positive and negative results. Our data demonstrated that both FC-ALPA-IgG at 1:4,000 and FC-ALPA-IgG at 1:32,000 are useful for late cure assessment in VL, with 100% specificity and outstanding likelihood ratio indices. Cure assessment at 6 mAT also showed promising performance indices, identifying 81% and 71.4% of the treated patients with negative results. However, new interpretation parameters were necessary to monitor cure at 2 mAT. We then introduced the differential PPFP (DeltaPPFP) of 25% as a new cutoff for early cure assessment at specific serum dilutions to analyze IgG reactivity by FC-ALPA-IgG and FC-AFPA-IgG. Our data demonstrated that at 2 mAT, DeltaPPFP was >25% in 60% and 57.1% of treated patients, whereas at 6 mAT, a DeltaPPFP of >25% was observed in 100% and 95.2% of samples assayed by FC-ALPA-IgG and FC-AFPA-IgG, respectively. Together, our findings showed the potential of both FC-ALPA-IgG and FC-AFPA-IgG regarding their applicability to detect differential serological reactivity and further contribution to posttherapeutic cure assessment in VL.

Desempenho da pesquisa de anticorpos anti-Leishmania (Leishmania) chagasi, por citometria de fluxo, na monitoração de cura na Leishmaniose Visceral Humana

A busca de marcadores de doença ativa e a validação de critérios de cura pós-terapêutica têm sido amplamente investigados na LV. Entretanto, o estabelecimento de um critério de cura sorológico ainda permanece inconclusivo. Os critérios adotados atualmente para estabelecer uma eficácia terapêutica dependem da avaliação clínica 12 meses pós-tratamento. O objetivo desse trabalho, foi avaliar a aplicabilidade da citometria de fluxo na pesquisa de anticorpos anti-promastigotas fixadas de L. (L.) chagasi (AAPF) na avaliação 12 meses após o tratamento e encontrar um indicador laboratorial de cura 2 meses e 6 meses pós-tratamento. Foi estabelecida uma relação entre os níveis de anticorpos anti L. (L.) chagasi e a eficácia do tratamento na LV, através da AAPF. Neste estudo foram avaliados soros de 21 pacientes com diagnóstico parasitológico positivo para LV, coletados antes e após o tratamento. As amostras de soro diluídas (1:1.000 a 1:128.000) foram incubadas com promastigotas fixadas, posteriormente incubadas com anti-IgG humana marcadas com FITC e IgG subclasses marcadas com SAPE na diluição de 1.000 a 128.000 para a pesquisa de IgG1 e 1:40 a 1:5.120 na pesquisa de IgG2, IgG3 e IgG4 e analisadas no citômetro de fluxo. Os resultados foram expressos como o percentual de parasitas fluorescentes positivos (PPFP) para cada amostra individual, estabelecendo 50 porcento como ponto de pacientes avaliados 12 meses após o tratamento (71,4 porcento). Entretanto, 2 meses após o tratamento foi encontrado valores de PPFP<50 porcento em menos de 50 porcento dos pacientes. Em relação a subclasses de IgG foi encontrado valores reagentes em IgG1 e IgG3. Na pesquisa da AAPF para IgG1 pacientes apresentou melhor desempenho na monitoração terapêutica. Observamos 90,5 porcento de queda nos níveis de PPFP 6 meses e 71,4 porcento 2 meses pós-tratamento. Nossos resultados mostraram a aplicabilidade da citometria de fluxo para detecção de anticorpos anti-L. (L.) chagasi e na monitoração de ...

Desempenho da pesquisa de anticorpos anti-Leishmania (Leishmania) chagasi, por citometria de fluxo, na monitoração de cura na Leishmaniose Visceral Humana / Performance of the anti-Leishmania research of antibodies (Leishmania) chagasi, for citometry of flow, in the monitor of cure in the Visceral Leishmaniasis Human being

A busca de marcadores de doença ativa e a validação de critérios de cura pós-terapêutica têm sido amplamente investigados na LV. Entretanto, o estabelecimento de um critério de cura sorológico ainda permanece inconclusivo. Os critérios adotados atualmente para estabelecer uma eficácia terapêutica dependem da avaliação clínica 12 meses pós-tratamento. O objetivo desse trabalho, foi avaliar a aplicabilidade da citometria de fluxo na pesquisa de anticorpos anti-promastigotas fixadas de L. (L.) chagasi (AAPF) na avaliação 12 meses após o tratamento e encontrar um indicador laboratorial de cura 2 meses e 6 meses pós-tratamento. Foi estabelecida uma relação entre os níveis de anticorpos anti L. (L.) chagasi e a eficácia do tratamento na LV, através da AAPF. Neste estudo foram avaliados soros de 21 pacientes com diagnóstico parasitológico positivo para LV, coletados antes e após o tratamento. As amostras de soro diluídas (1:1.000 a 1:128.000) foram incubadas com promastigotas fixadas, posteriormente incubadas com anti-IgG humana marcadas com FITC e IgG subclasses marcadas com SAPE na diluição de 1.000 a 128.000 para a pesquisa de IgG1 e 1:40 a 1:5.120 na pesquisa de IgG2, IgG3 e IgG4 e analisadas no citômetro de fluxo. Os resultados foram expressos como o percentual de parasitas fluorescentes positivos (PPFP) para cada amostra individual, estabelecendo 50 por cento como ponto de pacientes avaliados 12 meses após o tratamento (71,4 por cento). Entretanto, 2 meses após o tratamento foi encontrado valores de PPFP<50 por cento em menos de 50 por cento dos pacientes. Em relação a subclasses de IgG foi encontrado valores reagentes em IgG1 e IgG3. Na pesquisa da AAPF para IgG1 pacientes apresentou melhor desempenho na monitoração terapêutica. Observamos 90,5 por cento de queda nos níveis de PPFP 6 meses e 71,4 por cento 2 meses pós-tratamento. Nossos resultados mostraram a aplicabilidade da citometria de fluxo para detecção de anticorpos anti-L. (L.) chagasi...

Desempenho da pesquisa de anticorpos anti-Leishmania (Leishmania) chagasi, por citometria de fluxo, na monitoração de cura na Leishmaniose Visceral Humana / Performance of the anti-Leishmania research of antibodies (Leishmania) chagasi, for citometry of flow, in the monitor of cure in the Visceral Leishmaniasis Human being

A busca de marcadores de doença ativa e a validação de critérios de cura pós-terapêutica têm sido amplamente investigados na LV. Entretanto, o estabelecimento de um critério de cura sorológico ainda permanece inconclusivo. Os critérios adotados atualmente para estabelecer uma eficácia terapêutica dependem da avaliação clínica 12 meses pós-tratamento. O objetivo desse trabalho, foi avaliar a aplicabilidade da citometria de fluxo na pesquisa de anticorpos anti-promastigotas fixadas de L. (L.) chagasi (AAPF) na avaliação 12 meses após o tratamento e encontrar um indicador laboratorial de cura 2 meses e 6 meses pós-tratamento. Foi estabelecida uma relação entre os níveis de anticorpos anti L. (L.) chagasi e a eficácia do tratamento na LV, através da AAPF. Neste estudo foram avaliados soros de 21 pacientes com diagnóstico parasitológico positivo para LV, coletados antes e após o tratamento. As amostras de soro diluídas (1:1.000 a 1:128.000) foram incubadas com promastigotas fixadas, posteriormente incubadas com anti-IgG humana marcadas com FITC e IgG subclasses marcadas com SAPE na diluição de 1.000 a 128.000 para a pesquisa de IgG1 e 1:40 a 1:5.120 na pesquisa de IgG2, IgG3 e IgG4 e analisadas no citômetro de fluxo. Os resultados foram expressos como o percentual de parasitas fluorescentes positivos (PPFP) para cada amostra individual, estabelecendo 50 porcento como ponto de pacientes avaliados 12 meses após o tratamento (71,4 porcento). Entretanto, 2 meses após o tratamento foi encontrado valores de PPFP<50 porcento em menos de 50 porcento dos pacientes. Em relação a subclasses de IgG foi encontrado valores reagentes em IgG1 e IgG3. Na pesquisa da AAPF para IgG1 pacientes apresentou melhor desempenho na monitoração terapêutica. Observamos 90,5 porcento de queda nos níveis de PPFP 6 meses e 71,4 porcento 2 meses pós-tratamento. Nossos resultados mostraram a aplicabilidade da citometria de fluxo para detecção de anticorpos anti-L. (L.) chagasi e na monitoração de...