Distinct pattern of immunophenotypic features of innate and adaptive immunity as a putative signature of clinical and laboratorial status of patients with localized cutaneous leishmaniasis.

In this study, we have analysed the phenotypic features of innate/adaptive immunity of patients with localized cutaneous leishmaniasis (LCL), categorized according to their clinical/laboratorial status, including number of lesion (L1; L2­4), days of illness duration (≤60;>60) and positivity in the Montenegro skin test (MT−;MT+). Our findings highlighted a range of phenotypic features observed in patients with LCL (↑%HLA-DR+ neutrophils; ↑CD8+ HLA-DR+/CD4+ HLA-DR+ T cell ratio; ↑HLA-DR in B lymphocytes, ↑%CD23+ neutrophils, monocytes and B cells; ↑α-Leishmania IgG and ↑serum NO2⁻ + NO3⁻). Selective changes were observed in L1 (↑%HLA-DR+ neutrophils, ↑CD8+ HLA-DR+/CD4+ HLA-DR+ T cell ratio and ↑serum NO2⁻ + NO3⁻) as compared to L2­4 (↑%CD5− B cells; ↑CD23+ B cells and ↑α-Leishmania IgG). Whilst ≤60 presented a mixed profile of innate/adaptive immunity (↓%CD28+ neutrophils and ↑%CD4+ T cells), >60 showed a well-known leishmanicidal events (↑CD8+ T cells; ↑serum NO2⁻ + NO3⁻ and ↑α-Leishmania IgG). MT+ patients showed increased putative leishmanicidal capacity (↑%HLA-DR+ neutrophils; ↑%CD23+ monocytes; ↑CD8+ HLA-DR+/CD4+ HLA-DR+ T cell ratio and ↑ serum NO2⁻ + NO3⁻). Overall, a range of immunological biomarkers illustrates the complex immunological network associated with distinct clinical/laboratorial features of LCL with applicability in clinical studies.

Citometria de fluxo no diagnóstico da leishmaniose visceral canina / Flow cytometry used in canine visceral leishmaniasis diagnosis

Descreve-se a padronização de nova metodologia para detecção de anticorpos antiformas promastigotas fixadas de L. (L.) chagasi, por citometria de fluxo (AAPF-IgG), sua aplicabilidade e desempenho na identificação de casos de leishmaniose visceral canina (LVC). Foram avaliados dois grupos de cães classificados pela reação de imunofluorescência indireta (RIFI), como: não reatores (NR, n=10) e reatores (R, n=50) dos quais foram coletadas amostras de sangue (soro) para realização dos testes laboratoriais. Os resultados relacionados ao estabelecimento, aplicabilidade e desempenho da metodologia AAPF-IgG demonstraram que essa metodologia possibilita a identificação de uma região de reatividade diferencial entre cães NR e R, no soro diluído a 1:2048 e o valor de 20 por cento de parasitos fluorescentes positivos (PPFP) como ponto de corte entre resultados positivos e negativos, mostrando que a AAPF-IgG aplica-se na identificação de casos de LVC, possibilitando distinguir 96 por cento de cães R como positivos e 100 por cento de cães NR como negativos. Esses resultados em conjunto sugerem que a utilização da AAPF-IgG pode ser um novo instrumento para ensaios clínicos de diagnóstico sorológico da LVC.

The current study evaluated the standardization of a new methodology for detection of anti-fixed L. (L.) chagasi promastigote antibodies by flow cytometry (AAPF-IgG), as well its applicability and performance in the identification of cases of Canine Visceral Leishmaniasis (CVL). Two groups of dogs were classified by RIFI (gold standard) as no reactors (NR, n=10) and reactors (R, n=50). Blood samples were collected and used for the laboratorial tests (RIFI and AAPF-IgG). The results showed that the new AAPF-IgG assay makes possible the identification of an area of differential reactivity between dogs NR and R at the dilution of 1:2048 and 20 percent of percentage of positive fluorescent parasite as the cut point among positive and negative results. The AAPF-IgG assay was able to distinguish 96 percent of R dogs as positive and 100 percent of NR dogs as negative. Hence, those data support the applicability of flow cytometry AAPF-IgG method as a new instrument for serological diagnosis of CVL.

Clinical value of anti-live Leishmania (Viannia) braziliensis immunoglobulin G subclasses, detected by flow cytometry, for diagnosing active localized cutaneous leishmaniasis.

OBJECTIVE: To evaluate the clinical value of flow cytometry anti-live promastigate antibody (FC-ALPA), for diagnosing active cutaneous leishmaniasis. METHOD: Serum samples from 145 individuals living in endemic areas for localized cutaneous leishmaniasis (population 1) were classified as having the disease or not and then tested for their IgG reactivity by indirect immunofluorescence assay and FC-ALPA-IgG. The results of FC-ALPA-IgG were expressed as percentage of positive fluorescent parasite. Both tests were also evaluated in serum samples of people with visceral leishmaniasis and Chagas disease (population 1A). RESULTS: In population 1, FC-ALPA-IgG performed better than the immunofluorescence assay regarding sensitivity, specificity and predictive values. Analysis of the results according to the likelihood ratios indicated that a percentage of positive fluorescent parasite 60% it reinforces diagnosis of the disease (likelihood ratio = 7.0). Immunofluorescent assay is of little value (likelihood ratio=2.04). In population 1A, both tests performed worse, but FC-ALPA-IgG achieved better statistical indexes than immunofluorescent assay. CONCLUSION: The FC-ALPA-IgG is a valuable method for serological diagnosis of localized cutaneous leishmaniasis. FC-ALPA-IgG1/ALPA-IgG2 combined analysis is an additional serological tool for discriminating localized visceral leishmaniasis, Chagas disease and visceral leishmaniasis in areas where these infections co-exist.

Establishing phenotypic features associated with morbidity in human T-cell lymphotropic virus type 1 infection.

The human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HT). Although it is widely believed that virus infection and host immune response are involved in the pathogenic mechanisms, the role of the immune system in the development and/or maintenance of HT remains unknown. We performed an analysis of the peripheral blood leukocyte phenotype for two different subcohorts of HTLV-1-infected individuals to verify the existence of similar immunological alterations, possible laboratory markers for HT. The leukocyte population balance, the activation status of the T lymphocytes, and the cellular migratory potential of T lymphocytes, monocytes, and neutrophils were evaluated in the peripheral blood of HTLV-1-infected individuals classified as asymptomatic individuals, oligosymptomatic individuals, and individuals with HT. Data analysis demonstrated that a decreased percentage of B cells, resulting in an increased T cell/B cell ratio and an increase in the CD8+ HLA-DR+ T lymphocytes, exclusively in the HT group could be identified in both subcohorts, suggesting its possible use as a potential immunological marker for HT for use in the laboratory. Moreover, analysis of likelihood ratios showed that if an HTLV-1-infected individual demonstrated B-cell percentages lower than 7.0%, a T cell/B cell ratio higher than 11, or a percentage of CD8+ HLA-DR+ T lymphocytes higher than 70.0%, this individual would have, respectively, a 12-, 13-, or 22-times-greater chance of belonging to the HT group. Based on these data, we propose that the T cell/B cell ratios and percentages of circulating B cells and activated CD8+ T lymphocytes in HTLV-1-infected patients are important immunological indicators which could help clinicians monitor HTLV-1 infection and differentiate the HT group from the asymptomatic and oligosymptomatic groups.