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A Flow Cytometry-Based Serological Assay to Detect Visceral Leishmaniasis in HIV-Infected Patients.

ABSTRACT: Visceral Leishmaniasis (VL) is a severe parasitic disease that has emerged as an important opportunistic condition in HIV-infected patients and whose control is impaired by inaccurate identification. This is mainly due to the serological tests used for VL having a reduced performance in cases of VL-HIV coinfection due to a low humoral response. In this situation, however, a positive test has even greater diagnostic value when combined with the clinical status. This study aimed to evaluate the application and performance of flow cytometry to detect anti-Leishmania infantum antibodies in HIV-infected patients. Sera from VL/HIV coinfected patients, characterized using "gold standard" techniques, were compared with sera from healthy controls plus sera from HIV-infected individuals. The flow cytometry results were expressed as levels of IgG reactivity, based on the percentage of positive fluorescent parasites (PPFP). A ROC curve analysis of a serum titration indicated a PPFP of 1.26% as being the cutoff point to segregate positive and negative results. At the 1:2,048 dilution, with 89% sensitivity and 83% specificity, flow cytometry showed greater sensitivity in relation to the serological tests evaluated. Futhermore, flow cytometry was the only assay that positively identified all VL-HIV patients with quantified HIV load. Together, these findings suggest that flow cytometry may be used as an alternative serological approach for VL identification and as a tool to characterize the humoral response against Leishmania infantum in HIV-infected patients.

SUBMITTER: da Silva ED

PROVIDER: S-EPMC8119745 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background:Human visceral leishmaniasis (VL) is a life-threatening protozoan disease caused by parasites belonging to the Leishmania donovani complex. Ethiopia has the highest VL-HIV co-infection rate in the world, with several of these patients presenting with repeated episodes of VL disease (ie, relapse). However, we lack data on how HIV patients with multiple VL relapse present clinically, and whether they continue to respond to currently available medicines. Methods:The medical records of VL-HIV co-infected patients with multiple VL relapses at the Leishmaniasis Treatment and Research Center in Gondar, Ethiopia, between June 2012 and June 2016 were retrieved. Variables on their clinical and laboratory profiles were collected. Descriptive analysis was done to show the characteristics of the VL episodes. Result:A total of 48 VL episodes in 12 patients were identified, the median number of episodes per patient was 5 (interquartile range, 4-8 episodes). The median time to relapse was 5 months (interquartile range, 3-5.5 months). Splenomegaly was present in 47 of the episodes (98%), fever or other accompanying symptoms were present in only 66% (32 out of 48). The median tissue parasite grade at VL diagnosis was 6+ (interquartile range, 5+- 6+). All patients were on antiretroviral therapy. The median duration of treatment per episode was 2 months (interquartile range, 2-2 months). All patients achieved parasitological cure at discharge at each episode. Conclusions:Multiple recurrences of VL diseases were observed in HIV co-infected patients. With recurrent episodes, splenomegaly was found to be the main manifestation, whereas fever was less common. These patients came with recurrence of diseases in <6 months and required prolonged treatment to achieve cure.Further research on prediction, prevention, and better management options for recurrent VL is needed. ORCID ID: https://orcid.org/0000-0002-1410-0454. (Curr Ther Res Clin Exp. 2020; 81:XXX-XXX).

Project description:The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients.We conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and immunohistochemistry (IHC) assays. Finally, a sample handling method was developed to maintain the integrity of phospho signal during sample shipping and storage to ensure clinical application.The phospho flow assay provided single-cell PD monitoring of S6 phosphorylation in tumor and surrogate cells using fixed BMA and PB, assessing pathway modulation in response to GDC-0941 with sensitivity similar to that of MSD assay. The one-shot sample fixation and handling protocol herein demonstrated exceptional preservation of protein phosphorylation. In contrast, the IHC assay was less sensitive in terms of signal quantification while the biochemical approach (MSD) was less suitable to assess PD activities due to the undesirable impact associated with cell isolation on the protein phosphorylation in tumor cells.We developed a robust PD biomarker assay for the clinical evaluation of PI3K inhibitors in MM, allowing one to decipher the PD response in a relevant cell population. To our knowledge, this is the first report of an easily implemented clinical PD assay that incorporates an unbiased one-shot sample handling protocol, all (staining)-in-one (tube) phospho flow staining protocol, and an integrated modified data analysis for PD monitoring of kinase inhibitors in relevant cell populations in BMA and PB. The methods described here ensure a real-time, reliable and reproducible PD readout, which can provide information for dose selection as well as help to identify optimal combinations of targeted agents in early clinical trials.

Project description:Visceral leishmaniasis (VL) still represents a serious public health problem in Brazil due to the inefficiency of the control measures currently employed, that included early diagnosis and treatment of human cases, vector control, euthanasia of infected dogs and, recently approved in Brazil, treatment with Milteforam drug. Effective clinical management depend largely on early and unequivocal diagnosis, however, cross-reactivity have also been described in serological tests, especially when it refers to individuals from areas where Chagas' disease is also present. Thus, to discover new antigens to improve the current serological tests for VL diagnosis is urgently needed. Here, we performed an immunogenomic screen strategy to identify conserved linear B-cell epitopes in the predicted L. infantum proteome using the following criteria: i) proteins expressed in the stages found in the vertebrate host, amastigote stage, and secreted/excreted, to guarantee greater exposure to the immune system; ii) divergent from proteins present in other infectious disease pathogens with incidence in endemic areas for VL, as T. cruzi; iii) highly antigenic to humans with different genetic backgrounds, independently of the clinical stage of the disease; iv) stable and adaptable to quality-control tests to guarantee reproducibility; v) using statistical analysis to determine a suitable sample size to evaluate accuracy of diagnostic tests established by receiver operating characteristic strategy. We selected six predicted linear B-cell epitopes from three proteins of L. infantum parasite. The results demonstrated that a mixture of peptides (Mix IV: peptides 3+6) were able to identify VL cases and simultaneously able to discriminate infections caused by T. cruzi parasite with high accuracy (100.00%) and perfect agreement (Kappa index = 1.000) with direct methods performed by laboratories in Brazil. The results also demonstrated that peptide-6, Mix III (peptides 2+6) and I (peptides 2+3+6) are potential antigens able to used in VL diagnosis, represented by high accuracy (Ac = 99.52%, 99.52% and 98.56%, respectively). This study represents an interesting strategy for discovery new antigens applied to serologic diagnosis which will contribute to the improvement of the diagnosis of VL and, consequently, may help in the prevention, control and treatment of the disease in endemic areas of Brazil.

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A Flow Cytometry-Based Serological Assay to Detect Visceral Leishmaniasis in HIV-Infected Patients.

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