FC-TRIPLEX Chagas/Leish IgG1: a multiplexed flow cytometry method for differential serological diagnosis of chagas disease and leishmaniasis.

Differential serological diagnosis of Chagas disease and leishmaniasis is difficult owing to cross-reactivity resulting from the fact that the parasites that cause these pathologies share antigenic epitopes. Even with optimized serological assays that use parasite-specific recombinant antigens, inconclusive test results continue to be a problem. Therefore, new serological tests with high sensitivity and specificity are needed. In the present work, we developed and evaluated the performance of a new flow cytometric serological method, referred to as FC-TRIPLEX Chagas/Leish IgG1, for the all-in-one classification of inconclusive tests. The method uses antigens for the detection of visceral leishmaniasis, localized cutaneous leishmaniasis, and Chagas disease and is based on an inverted detuned algorithm for analysis of anti-Trypanosomatidae IgG1 reactivity. First, parasites were label with fluorescein isothiocyanate or Alexa Fluor 647 at various concentrations. Then serum samples were serially diluted, the dilutions were incubated with suspensions of mixed labeled parasites, and flow cytometric measurements were performed to determine percentages of positive fluorescent parasites. Using the new method, we obtained correct results for 76 of 80 analyzed serum samples (95% overall performance), underscoring the outstanding performance of the method. Moreover, we found that the fluorescently labeled parasite suspensions were stable during storage at room temperature, 4 °C, and -20 °C for 1 year. In addition, two different lots of parasite suspensions showed equivalent antigen recognition; that is, the two lots showed equivalent categorical segregation of anti-Trypanosomatidae IgG1 reactivity at selected serum dilutions. In conclusion, we have developed a sensitive and selective method for differential diagnosis of Chagas disease, visceral leishmaniasis, and localized cutaneous leishmaniasis.

Evaluation of anti-lived and anti-fixed Leishmania (Viannia) braziliensis promastigote IgG antibodies detected by flow cytometry for diagnosis and post-therapeutic cure assessment in localized cutaneous leishmaniasis.

This study aims to investigate a flow cytometry performance-based methodology to detect anti-live (FC-ALPA-IgG) and anti-fixed (FC-AFPA-IgG) Leishmania (Viannia) braziliensis promastigote IgG as a means to monitor post-therapeutic cure of patients with localized cutaneous leishmaniasis (LCL). Serum samples from 30 LCL patients infected with L. (V.) braziliensis were assayed, comparing the IgG reactivity before and after specific treatment with pentavalent antimonial. Reactivities were reported as the percentage of positive fluorescent parasites (PPFP), using a PPFP of 60% as a cut-off value. In the serum dilution of 1:1024, the positive percentage of LCL serum sample for FC-ALPA-IgG and FC-AFPA-IgG was 86% and 90%, respectively, before treatment. Analysis of ∆PPFP that represents the difference between PPFP after and before treatment appeared as a new approach to monitor post-therapeutic IgG reactivity in LCL. Our data support the perspective of using FC-ALPA and FC-AFPA as a useful serologic tool for diagnosis and for post-therapeutic follow-up of LCL patients.

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.

[Evaluation of the performance of immunological parameters as indicators for clinical progression of chronic HTLV-1 infection]. / Avaliação do desempenho de parâmetros imunológicos como indicadores de progressão clínica da infecção crônica pelo HTLV-1.

This study evaluated the performance of single and combined laboratory parameters, B-lymphocyte percentages (%LB), T/B cell ratio and %CD8+HLA-DR+/CD8+, to differentiate asymptomatic cases (AS) from HAM/TSP patients (HT) within a population of HTLV-1 seropositive cases. Percentage indices demonstrated that each parameter alone presented moderate performance, with co-negativity of 83 and 91% for %LB and T/B cell ratio, respectively, and co-positivity of 78% for %CD8+HLA-DR+/CD8+. Combined analysis (%CD8+HLA-DR+/CD8+ and T/B cell ratio) did not show any substantial performance enhancement (co-positivity = 75% and co-negativity = 74%). Likelihood ratio analysis using different value ranges for the separate parameters revealed that HTLV-1 seropositive cases with %LB<7%, T/B cell ratio>11 and %CD8+HLA-DR+/CD8+>70% would have, respectively, 11, 19 and 10 times greater chance of belonging to the HT group. Therefore, use of these phenotypic indicators as complementary laboratory methods for monitoring the clinical progression of chronic HTLV-1 infection is recommended.

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.

Clinical value of anti-Leishmania (Leishmania) chagasi IgG titers detected by flow cytometry to distinguish infected from vaccinated dogs.

Leishmune vaccination covers a broader number of endemic areas of canine visceral leishmaniasis (CVL) and therefore the development of new serological devices able to discriminate CVL from Leishmune vaccinees becomes an urgent need considering the post-vaccine seroconversion detected throughout conventional methodologies. Herein, we have described the establishment of a flow cytometry based methodology to detect anti-fixed L. (L.) chagasi promastigotes antibodies (FC-AFPA-IgG, FC-AFPA-IgG1 and FC-AFPA-IgG2) in sera samples from Leishmania (Leishmania) chagasi infected dogs and Leishmune vaccinees. The results of FC-AFPA were reported along the sera titration curve (1:128-1:524,288), as percentage-of-positive-fluorescent-parasite (PPFP). The use of PPFP=20% as a cut-off edge to segregate negative and positive results at sera dilution 1:2048 revealed outstanding performance indexes that elect FC-AFPA-IgG and IgG2 (both detected by polyclonal FITC-labeled second step reagent) applicable to the serological diagnosis of CVL, with 100% of specificity for both IgG and IgG2 and 97 and 93% of sensitivity, respectively. Moreover, FC-AFPA-IgG, applied at sera dilution 1:2048, also appeared as a useful tool to discriminate L. chagasi infected dogs from Leishmune vaccinees, with 76% of specificity. Outstanding likelihood indexes further support the performance of FC-AFPA-IgG for exclusion diagnosis of CVL in Leishmune vaccinees. Analysis of FC-AFPA-IgG at sera dilution 1:8192 revealed the most outstanding indexes, demonstrating that besides the ability of PPFP

Avaliação do desempenho de parâmetros imunológicos como indicadores de progressão clínica da infecção crônica pelo HTLV-1 / Evaluation of the performance of immunological parameters as indicators for clinical progression of chronic HTLV-1 infection

Neste estudo, foi avaliado o desempenho isolado e combinado de parâmetros laboratoriais, percentual de linfócitos B ( por centoLB), a razão entre células T/B e o por centoCD8+HLA-DR+/CD8+, na identificação de indivíduos assintomáticos-AS ou portadores de HAM/TSP-HT numa população de casos soropositivos para HTLV-1. índices expressos em porcentagem demonstram que cada parâmetro, isoladamente, apresenta desempenho moderado, com co-negatividade=83 por cento e 91 por cento para por centoLB e razão entre células T/B, respectivamente e co-positividade=78 por cento para por centoCD8+HLA-DR+/CD8+. A análise combinada ( por centoCD8+HLA-DR+/CD8+ e razão T/B) não revelou ganho significativo no desempenho (co-positividade=75 por cento, co-negatividade=74 por cento). A análise das razões de verossimilhança em diferentes faixas de valores, para os parâmetros isolados, revelou que um indivíduo soropositivo para HTLV-1 com por centoLB<7 por cento, razão entre células T/B>11 e por centoCD8+HLA-DR+/CD8+>70 por cento possui, respectivamente, 11, 19 e quase 10 vezes mais chances de pertencer ao grupo HT. Portanto, recomenda-se o uso desses indicadores fenótipos na propedêutica laboratorial complementar de monitoração da progressão clínica da infecção crônica pelo HTLV-1.

This study evaluated the performance of single and combined laboratory parameters, B-lymphocyte percentages ( percentLB), T/B cell ratio and percentCD8+HLA-DR+/CD8+, to differentiate asymptomatic cases (AS) from HAM/TSP patients (HT) within a population of HTLV-1 seropositive cases. Percentage indices demonstrated that each parameter alone presented moderate performance, with co-negativity of 83 and 91 percent for percentLB and T/B cell ratio, respectively, and co-positivity of 78 percent for percentCD8+HLA-DR+/CD8+. Combined analysis ( percentCD8+HLA-DR+/CD8+ and T/B cell ratio) did not show any substantial performance enhancement (co-positivity = 75 percent and co-negativity = 74 percent). Likelihood ratio analysis using different value ranges for the separate parameters revealed that HTLV-1 seropositive cases with percentLB<7 percent, T/B cell ratio>11 and percentCD8+HLA-DR+/CD8+>70 percent would have, respectively, 11, 19 and 10 times greater chance of belonging to the HT group. Therefore, use of these phenotypic indicators as complementary laboratory methods for monitoring the clinical progression of chronic HTLV-1 infection is recommended.

Non-conventional flow cytometry approaches to detect anti-Trypanosoma cruzi immunoglobulin G in the clinical laboratory.

We have recently developed a flow cytometric approach to detect anti-live trypomastigote and anti-fixed epimastigote IgG antibodies (FC-ALTA and FC-AFEA) in sera from individuals infected by Trypanosoma cruzi. Here, we present the first evaluation of the applicability of FC-AFEA-IgG as a diagnostic tool for Chagas disease. Performance analysis demonstrated that FC-AFEA-IgG has a sensitivity of 82% and a specificity of 100%. The assessment for prognosis performed by FC-ALTA-IgG1 and FC-AFEA-IgG, after classification of chagasic patients as belonging to indeterminate (IND), cardiac (CARD) or digestive (DIG) clinical forms, showed that most of IND have higher amounts of IgG than individuals' carrying CARD or DIG Chagas disease. FC-AFEA-IgG was also evaluated as a method to monitor chemotherapy efficacy in individuals classified into three distinct categories: not treated (NT), treated but not cured (TNC), and treated and cured (TC). Performance analysis demonstrated that FC-AFEA-IgG has an extraordinary capacity as a serological criterion to assess cure after therapeutic intervention in Chagas disease. These results represent a great advance in the application of serological techniques for clinical investigations on Chagas disease, and they clearly define new directions and perspectives. We intend to continue this field research focusing our attention on the influence of the degree of clinical damage on the FC-ALTA-IgG1 and FC-AFEA-IgG reactivity.

Aplicações da pesquisa de anticorpos IgG anti-formas promastigotas vivas de Leishmania (V. ) braziliensis, por citometria de fluxo, em estudos clínicos da Leishmaniose cutânea localizada / Applications of the research of a flow cytometry to detected anti-live Leishmania (V. ) braziliensis promastigotes IgG antibodies (FC-ALPA-IgG) in clinical studies of the localized cutaneous leishmaniasis

Este estudo se refere ao desenvolvimento de uma nova metodologia,fundamentada na pesquisa de anticorpos IgG anti-formas promastigotas vivas de Leishmania(V.) braziliensis,por citometria de fluxo-AAPV-IgG para diagnóstico e monitoramento de cura da leishmaniose cutânea localizada-LCL.Em uma primeira etapa de padronização do método,ficou estabelecida a nova metodologia que permite discriminar a reatividade de IgG do soro de pacientes portadores de LCL(L+)daquela observada para indivíduos sem manifestação clínica de LCL(L-),empregando-se em cada teste:a)500.000 promastigotas vivas de 10 dias de cultivo in vitro;b)soro teste na diluição 1:1.024;c)anticorpo secundário anti-IgG humano-FITC na diluição 1:400;d)ambas incubações a 37ºC por 30 minutos e)o valor do percentual de parasito fluorescentes positivos-PPFP de 60por cento como ponto de corte entre resultados positivos e negativos.Uma segunda etapa,foi feita a avaliação comparativa do desempenho do método proposto e de um método tradicional,a Reação de Imunofluorescência Indireta-RIFI.A análise dos índices de desempenho...Posteriormente...desempenho da pesquisa AAPV-IgG aplicada ao diagnóstico da LCL,uma vez que podem apresentar resultados falso-negativos e falso-positivos,respectivamente.Considerando a queda no desempenho da AAPV-IgG na identificação de LCL numa amostragem populacional que inclui LV e DC,foi proposta a avaliação do desempenho da quantificação das subclasses de IgG no esclarecimento de situações positivas no teste original.A análise combinada da AAPV-IgG1/IgG2 mostrou-se útil no esclarecimento de situações positivas no teste original, quando aplicado numa amostragem populacional que inclui LV e DC.O estabelecimento do ponto de corte de 60por cento de PPFP para AAPV-IgG1 e de 50por cento de PPFP para AAPV-IgG2 permitiu o esclarecimento de 93por cento das situações AAPV-IgG positivas que não apresentavam LCL e confirmar 100por cento dos casos de LCL em atividade clínica.A análise das RVs demonstrou que um resultado positivo na análise combinada praticamente confirma a presença de LCL e que um resultado negativo praticamente confirma a ausência de LCL.Finalmente,foi demonstrada a utilidade da AAPV-IgG no monitoramento precoce e tardio da cura pós-terapêutica específica da LCL.Observou-se que,71por cento dos pacientes sem recidivas após 2 anos do tratamento foram identificados precocemente(30d-PT), com queda na reatividade de PPFP,superior a 10por cento, na diluição do soro 1:4.096.

Aplicação da pesquisa de anticorpos IgG anti-formas promastigotas vivas de Leishmania (V. ) braziliensis, por citometria de fluxo, em estudos clínicos da Leishmaniose cutânea localizada

Este estudo se refere ao desenvolvimento de uma nova metodologia, fundamentada na pesquisa de anticorpos IgG anti-formas promastigotas vivas de Leishmania (V.) braziliensis, por citometria de fluxo-AAPV-IgG para diagnóstico e monitoramento de cura da leishmaniose cutânea localizada-LCL. Em uma primeira etapa de padronização do método, ficou estabelecida a nova metodologia que permite discriminar a reatividade de IgG do soro de pacientes portadores de LCL(L+) daquela observada para indivíduos sem manifestação clínica de LCL(L-), empregando-se em cada teste: a) 500.000 promastigotas vivas de 10 dias de cultivo; b) soro teste na diluição 1:1.024; c) anticorpo secundário anti-IgG humano-FITC na diluição 1:400; d) ambas incubações a 37ºC por 30 minutos e e) o valor do percentual de parasito fluorescentes positivos-PPFP de 60 porcento como ponto de corte entre resultados positivos e negativos

Em uma segunda etapa, foi feita a avaliação comparativa do desempenho do método proposto e de um método tradicional, a Reação de Imunofluorescência Indireta-RIFI. A análise dos índices de desempenho expressos em percentual (sensibilidade, especificidade, valores preditivos, probabilidade de doença pós-teste negativo, taxa de falso-positivo, acurácia e Índice J de Youden) e a Razão de Verossimilhança-RV demonstrou a melhor performance da AAPV-IgG em relação à RIFI, na identificação de casos de LCL em atividade clínica. A análise das RVs demonstrou que os títulos da RIFI foram desprovidos de valor diagnóstico enquanto que na AAPV-IgG, valores de PPFP>60 porcento contribuem para a confirmação do diagnóstico da LCL e valores de PPFP£60 porcento praticamente excluem o diagnóstico de LCL

Posteriormente, foram feitas simulações com situações clínicas que podem concorrer para falso-positividade ou falso-negatividade do teste em estudo. O tempo de duração da lesão na LCL, casos de leishmaniose visceral-LV e doença de Chagas-DC foram identificados como situações clínicas capazes de interferir no desempenho da pesquisa AAPV-IgG aplicada ao diagnóstico da LCL, uma vez que podem apresentar resultados falso-negativos e falso-positivos, respectivamente. Considerando a queda no desempenho da AAPV-IgG na identificação de LCL numa amostragem populacional que inclui LV e DC, foi proposta a avaliação do desempenho da quantificação das subclasses de IgG no esclarecimento de situações positivas no teste original. A análise combinada da AAPV-IgG1/IgG2 mostrou-se útil no esclarecimento de situações positivas no teste original, quando aplicado numa amostragem populacional que inclui LV e DC.. O estabelecimento do ponto de corte de 60 porcento de PPFP para AAPV-IgG1 e de 50 porcento de PPFP para AAPV-IgG2 permitiu o esclarecimento de 93 porcento das situações AAPV-IgG positivas que não apresentavam LCL e confirmar 100 porcento dos casos de LCL em atividade clínica. A análise das RVs demonstrou que um resultado positivo na análise combinada praticamente confirma a presença de LCL e que um resultado negativo praticamente confirma a ausência de LCL. Finalmente, foi demonstrada a utilidade da AAPV-IgG no monitoramento precoce e tardio da cura pós-terapêutica específica da LCL. Observou-se que, que 71porcento dos pacientes sem recidivas após 2 anos do tratamento foram identificados precocemente (30d-PT), com queda na reatividade de PPFP, superior a 10 porcento, na diluição do soro 1:4.096

Aplicação da pesquisa de anticorpos IgG anti-formas promastigotas vivas de Leishmania (V. ) braziliensis, por citometria de fluxo, em estudos clínicos da Leishmaniose cutânea localizada / Application of antibodies IgG anti-living promastigotes of Leishmania (V. ) braziliensis by flow cytometry in clinical studies of localized cutaneous leishmaniasis

Este estudo se refere ao desenvolvimento de uma nova metodologia, fundamentada na pesquisa de anticorpos IgG anti-formas promastigotas vivas de Leishmania (V.) braziliensis, por citometria de fluxo-AAPV-IgG para diagnóstico e monitoramento de cura da leishmaniose cutânea localizada-LCL. Em uma primeira etapa de padronização do método, ficou estabelecida a nova metodologia que permite discriminar a reatividade de IgG do soro de pacientes portadores de LCL(L+) daquela observada para indivíduos sem manifestação clínica de LCL(L-), empregando-se em cada teste: a) 500.000 promastigotas vivas de 10 dias de cultivo; b) soro teste na diluição 1:1.024; c) anticorpo secundário anti-IgG humano-FITC na diluição 1:400; d) ambas incubações a 37ºC por 30 minutos e e) o valor do percentual de parasito fluorescentes positivos-PPFP de 60 por cento como ponto de corte entre resultados positivos e negativos. Em uma segunda etapa, foi feita a avaliação comparativa do desempenho do método proposto e de um método tradicional, a Reação de Imunofluorescência Indireta-RIFI. A análise dos índices de desempenho expressos em percentual (sensibilidade, especificidade, valores preditivos, probabilidade de doença pós-teste negativo, taxa de falso-positivo, acurácia e Índice J de Youden) e a Razão de Verossimilhança-RV demonstrou a melhor performance da AAPV-IgG em relação à RIFI, na identificação de casos de LCL em atividade clínica. A análise das RVs demonstrou que os títulos da RIFI foram desprovidos de valor diagnóstico enquanto que na AAPV-IgG, valores de PPFP>60 por cento contribuem para a confirmação do diagnóstico da LCL e valores de PPFP£60 por cento praticamente excluem o diagnóstico de LCL. Posteriormente, foram feitas simulações com situações clínicas que podem concorrer para falso-positividade ou falso-negatividade do teste em estudo. O tempo de duração da lesão na LCL, casos de leishmaniose visceral-LV e doença de Chagas-DC foram identificados como situações clínicas capazes de interferir no desempenho da pesquisa AAPV-IgG aplicada ao diagnóstico da LCL, uma vez que podem apresentar resultados falso-negativos e falso-positivos, respectivamente. Considerando a queda no desempenho da AAPV-IgG na identificação de LCL numa amostragem populacional que inclui LV e DC, foi proposta a avaliação do desempenho da quantificação das subclasses de IgG no esclarecimento de situações positivas no teste original. A análise combinada da AAPV-IgG1/IgG2 mostrou-se útil no esclarecimento de situações positivas no teste original, quando aplicado numa amostragem populacional que inclui LV e DC. O estabelecimento do ponto de corte de 60 por cento de PPFP para AAPV-IgG1 e de 50 por cento de PPFP para AAPV-IgG2 permitiu o esclarecimento de 93 por cento das situações AAPV-IgG positivas que não apresentavam LCL e confirmar 100 por cento dos casos de LCL em atividade clínica. A análise das RVs demonstrou que um resultado positivo na análise combinada praticamente confirma a presença de LCL e que um resultado negativo praticamente confirma a ausência de LCL. Finalmente, foi demonstrada a utilidade da AAPV-IgG no monitoramento precoce e tardio da cura pós-terapêutica específica da LCL. Observou-se que, que 71 por cento dos pacientes sem recidivas após 2 anos do tratamento foram identificados precocemente (30d-PT), com queda na reatividade de PPFP, superior a 10 por cento, na diluição do soro 1:4.096.

Anticorpos antipromastigotas vivas de Leishmania (Viannia) braziliensis, detectados pela citometria de fluxo, para identificaçäo da infecção ativa na leishmaniose tegumentar americana / Anti-live Leishmania (Viannia) braziliensis promastigote antibodies, detected by flow cytometry, to identify active infection in american cutaneous leishmaniasis

Neste estudo, descrevemos etapas iniciais de padronizaçäo de uma nova metodologia para detecçäo de anticorpos antipromastigotas vivas de Leishmania (Viannia) braziliensis, pela citometria de fluxo e a análise de sua aplicabilidade para estudos clínicos. Foram avaliados 39 indivíduos com sorologia convencional (RIFI) positiva para leishmaniose, classificados quanto à ausência/presença de lesäo (L- e L+). Os resultados foram expressos sob a forma de percentual de parasitas fluorescentes positivos (PPFP). A análise dos dados, na diluiçäo 1:1.024, permitiu distinguir 95 por cento dos pacientes L+ como um grupo de alta reatividade (PPFP>50 por cento) e 72 por cento dos indivíduos L- como um grupo de baixa reatividade (PPFP<=50 por cento). A análise dos títulos da reaçäo de imunofluorescência indireta näo demonstrou nenhuma relaçäo com a ausência/presença de lesäo. Em conjunto, nossos dados sugerem a aplicabilidade da citometria de fluxo na identificaçäo dos casos de infecçäo ativa, o que näo tem sido possível através das reaçöes sorológicas convencionais

[Anti-live Leishmania (Viannia) braziliensis promastigote antibodies, detected by flow cytometry, to identify active infection in american cutaneous leishmaniasis]. / Anticorpos antipromastigotas vivas de Leishmania (Viannia) braziliensis, detectados pela citometria de fluxo, para identificação da infecção ativa na leishmaniose tegumentar americana.

In the current study we described initial standardization steps of a new methodology to detect anti-live Leishmania (Viannia) braziliensis promastigote antibodies by flow cytometry, followed by analysis of its applicability to clinical studies. We have studied 39 individuals with positive conventional serology to leishmaniasis, classified according to the absence/presence of cutaneous lesions (L- and L+). The results were expressed as percentage of positive fluorescent parasites (PPFP). Data analysis at dilution of 1:1,024, allowed the distinction of 95% of L+ patients as a group of high reactivity (PPFP>50%) and 72% of L- individuals as a group of low reactivity (PPFP<50%). The analysis of immunofluorescence assay titers did not show any relationship with the absence/presence of lesion. Together, our data support the applicability of flow cytometry to identify cases of active infection, which has not been possible through conventional serological reactions.