Ana Rabello

Predictors of Visceral Leishmaniasis Relapse in HIV-Infected Patients: A Systematic Review

Background and Objectives Visceral leishmaniasis (VL) is a common complication in AIDS patients living in Leishmania-endemic areas. Although antiretroviral therapy has changed the clinical course of HIV infection and its associated illnesses, the prevention of VL relapses remains a challenge for the care of HIV and Leishmania co-infected patients. This work is a systematic review of previous studies that have described predictors of VL relapse in HIV-infected patients. Review Methods We searched the electronic databases of MEDLINE, LILACS, and the Cochrane Central Register of Controlled Trials. Studies were selected if they included HIV-infected individuals with a VL diagnosis and patient follow-up after the leishmaniasis treatment with an analysis of the clearly defined outcome of prediction of relapse. Results Eighteen out 178 studies satisfied the specified inclusion criteria. Most patients were males between 30 and 40 years of age, and HIV transmission was primarily via intravenous drug use. Previous VL episodes were identified as risk factors for relapse in 3 studies. Two studies found that baseline CD4+ T cell count above 100 cells/mL was associated with a decreased relapse rate. The observation of an increase in CD4+ T cells at patient follow-up was associated with protection from relapse in 5 of 7 studies. Meta-analysis of all studies assessing secondary prophylaxis showed significant reduction of VL relapse rate following prophylaxis. None of the five observational studies evaluating the impact of highly active antiretroviral therapy use found a reduction in the risk of VL relapse upon patient follow-up. Conclusion Some predictors of VL relapse could be identified: a) the absence of an increase in CD4+ cells at follow-up; b) lack of secondary prophylaxis; and c) previous history of VL relapse. CD4+ counts below 100 cells/mL at the time of primary VL diagnosis may also be a predictive factor for VL relapse.

Mixed inflammatory/regulatory cytokine profile marked by simultaneous raise of interferon-gamma and interleukin-10 and low frequency of tumour necrosis factor-alpha(+) monocytes are hallmarks of active human visceral Leishmaniasis due to Leishmania chagasi infection.

Considering the complexity of the immunological events triggered during active visceral Leishmaniasis (VL), the relevance of the segregation of the immune response during human VL into type 1 and type 2 still remains unclear. For this purpose, in individuals living in risk areas for VL, we have evaluated especially asymptomatic individuals and patients with active VL, the plasmatic levels of cytokines and reactive nitrogen species under ex vivo conditions. In addition, we have also performed an analysis of intracellular cytokine patterns of circulating leucocytes after short‐term culture, particularly in the absence of antigenic‐specific stimulation, in order to reflect dynamic events of immune response in vivo during Leishmania chagasi infection. Although asymptomatic individuals and non‐infected subjects presented a similar immunological profile, an outstanding inflammatory/regulatory profile, based on higher plasmatic levels of cytokines such as interleukin (IL)‐8, interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α, IL‐6 and IL‐10, was associated with clinical status observed in active VL. In this context, we hypothesize that IL‐10, through its ability to inhibit anti‐leishmanial macrophage activation, associated with the lower frequency of TNF‐α+ monocytes and ordinary levels of nitrite and nitrate are the major mechanisms associated with disease onset.

Efficacy of anti-leishmania therapy in visceral leishmaniasis among HIV infected patients: a systematic review with indirect comparison.

Objective We conducted a systematic literature review with indirect comparison of studies evaluating therapeutic efficacy and toxicity associated to visceral leishmaniasis (VL) therapy among HIV infected individuals. Main outcome measurements The outcomes of interest were clinical and parasitological cure, mortality, and adverse events. Methods PRISMA guidelines for systematic reviews and Cochrane manual were followed. Sources were MEDLINE, LILACS, EMBASE, Web of Knowledge databases and manual search of references from evaluated studies. We included all studies reporting outcomes after VL treatment, regardless of their design. Study quality was evaluated systematically by using the Newcastle-Ottawa Scale (NOS) for assessing the quality of nonrandomized studies in meta-analyses. Comprehensive Meta-Analysis software v.2.2.048 was used to perform one-group meta-analysis of study arms with the same drug to estimate global rates of success and adverse events with each drug. These estimates were used, when possible, to indirectly compare treatment options, adjusted for CD4 count. Direct comparison was pooled when available. Results Seventeen studies reporting five treatment regimens and outcome of 920 VL episodes occurring in HIV infected individuals were included. The main outstanding difference in outcome among the treatment regimens was observed in mortality rate: it was around 3 times higher with high-dose antimony use (18.4%, CI 95% 13.3–25%), indirectly compared to lipid formulations of amphotericin B treatment (6.1%, CI 95% 3.9–9.4%). It was observed, also by indirect comparison, higher rates of clinical improvement in study arms using amphotericin B than in study arms using pentavalent antimonial therapy (Sbv). The parasitological cure, an outcome that presented some degree of risk of selection and verification bias, had rates that varied widely within the same treatment arm, with high heterogeneity, hampering any formal comparison among drugs. One direct comparison of amphotericin and antimoniate was possible combining results of two studies and confirming the superiority of amphotericin. Conclusions Available evidence suggests that amphotericin is superior to antimony treatment. Death rate using antimoniate high dose is unacceptably high. Randomized controlled trials are necessary to compare different formulations and doses of amphotericin, alternative therapies and drug combinations.

Diagnosis of American visceral leishmaniasis in humans and dogs using the recombinant Leishmania donovani A2 antigen.

A2 proteins are expressed in the amastigote stage of Leishmania donovani and are composed predominantely by a conserved repetitive element. Here, we have investigated the presence of anti-A2 antibodies in a panel of American Visceral Leishmaniasis (VL) sera. Anti-A2 antibodies were detected by ELISA, using a recombinant A2 protein containing a tag of six histidine residues (A2-HIS), in 77% of patients sera with symptomatic VL and in 87% of sera from dogs that tested positive in Leishmania immunofluorescent-antibody test (IFAT) or in the parasitological evaluation. Anti-A2 antibodies were also detected in 14 out of 15 symptomatic and in 10 out of 13 asymptomatic dogs. In addition, among the asymptomatic/anti-A2 positive animals, 9 were also positive for the presence of parasites. No significant cross reactivity was observed with sera of animals with other common canine diseases. Our findings suggest that A2 protein is a potential tool for the diagnosis of VL in the New World, and will be particularly useful for diagnosis of dogs.

A Global Comparative Evaluation of Commercial Immunochromatographic Rapid Diagnostic Tests for Visceral Leishmaniasis

Accuracy of rapid diagnostic tests was high in the Indian subcontinent; however, in Brazilian and East African samples, reduced sensitivity suggests that several cannot be used alone to exclude visceral leishmaniasis. Data on ease of use and performance using whole blood and in human immunodeficiency virus coinfections is needed.

Development and Evaluation of a Sensitive PCR-ELISA System for Detection of Schistosoma Infection in Feces

Background A PCR-enzyme-linked immunosorbent assay (PCR-ELISA) was developed to overcome the need for sensitive techniques for the efficient diagnosis of Schistosoma infection in endemic settings with low parasitic burden. Methodology/Principal Findings This system amplifies a 121-base pair tandem repeat DNA sequence, immobilizes the resultant 5′ biotinylated product on streptavidin-coated strip-well microplates and uses anti-fluorescein antibodies conjugated to horseradish peroxidase to detect the hybridized fluorescein-labeled oligonucleotide probe. The detection limit of the Schistosoma PCR-ELISA system was determined to be 1.3 fg of S. mansoni genomic DNA (less than the amount found in a single cell) and estimated to be 0.15 S. mansoni eggs per gram of feces (fractions of an egg). The system showed good precision and genus specificity since the DNA target was found in seven Schistosoma DNA samples: S. mansoni, S. haematobium, S. bovis, S. intercalatum, S. japonicum, S. magrebowiei and S. rhodaini. By evaluating 206 patients living in an endemic area in Brazil, the prevalence of S. mansoni infection was determined to be 18% by examining 12 Kato-Katz slides (41.7 mg/smear, 500 mg total) of a single fecal sample from each person, while the Schistosoma PCR-ELISA identified a 30% rate of infection using 500-mg of the same fecal sample. When considering the Kato-Katz method as the reference test, artificial sensitivity and specificity rates of the PCR-ELISA system were 97.4% and 85.1%, respectively. The potential for estimating parasitic load by DNA detection in feces was assessed by comparing absorbance values and eggs per gram of feces, with a Spearman correlation coefficient of 0.700 (P<0.0001). Conclusions/Significance This study reports the development and field evaluation of a sensitive Schistosoma PCR-ELISA, a system that may serve as an alternative for diagnosing Schistosoma infection.

The Diagnostic Accuracy of Serologic and Molecular Methods for Detecting Visceral Leishmaniasis in HIV Infected Patients: Meta-Analysis

Background Human visceral leishmaniasis (VL), a potentially fatal disease, has emerged as an important opportunistic condition in HIV infected patients. In immunocompromised patients, serological investigation is considered not an accurate diagnostic method for VL diagnosis and molecular techniques seem especially promising. Objective This work is a comprehensive systematic review and meta-analysis to evaluate the accuracy of serologic and molecular tests for VL diagnosis specifically in HIV-infected patients. Methods Two independent reviewers searched PubMed and LILACS databases. The quality of studies was assessed by QUADAS score. Sensitivity and specificity were pooled separately and compared with overall accuracy measures: diagnostic odds ratio (DOR) and symmetric summary receiver operating characteristic (sROC). Results Thirty three studies recruiting 1,489 patients were included. The following tests were evaluated: Immunofluorescence Antibody Test (IFAT), Enzyme linked immunosorbent assay (ELISA), immunoblotting (Blot), direct agglutination test (DAT) and polimerase chain reaction (PCR) in whole blood and bone marrow. Most studies were carried out in Europe. Serological tests varied widely in performance, but with overall limited sensitivity. IFAT had poor sensitivity ranging from 11% to 82%. DOR (95% confidence interval) was higher for DAT 36.01 (9.95–130.29) and Blot 27.51 (9.27–81.66) than for IFAT 7.43 (3.08–1791) and ELISA 3.06 (0.71–13.10). PCR in whole blood had the highest DOR: 400.35 (58.47–2741.42). The accuracy of PCR based on Q-point was 0.95; 95%CI 0.92–0.97, which means good overall performance. Conclusion Based mainly on evidence gained by infection with Leishmania infantum chagasi, serological tests should not be used to rule out a diagnosis of VL among the HIV-infected, but a positive test at even low titers has diagnostic value when combined with the clinical case definition. Considering the available evidence, tests based on DNA detection are highly sensitive and may contribute to a diagnostic workup.

Analysis of anti-keyhole limpet haemocyanin antibody in Brazilians supports its use for the diagnosis of acute schistosomiasis mansoni

Antibody (immunoglobulin (Ig) G) to the haemocyanin of the keyhole limpet (KLH) (Megathura crenulata), which shares a well defined carbohydrate epitope with the surface of schistosomula of Schistosoma mansoni, was determined by enzyme-linked immunosorbent assay (ELISA) in the sera of Brazilians with acute schistosomiasis. Of 53 such individuals tested, 51 had a level of KLH reactivity in excess of the mean +2 standard deviations of that exhibited by chronically infected individuals. This difference in reactivity allowed the acute cases to be readily identified by visual inspection of ELISA plates. The levels of IgG in patients with hepatointestinal and hepatosplenic schistosomiasis, as well as in non-infected, seropositive residents of endemic areas and infected children from endemic areas, were not statistically different from those of intestinal patients. Significant levels of anti-KLH IgG were not detected in patients with leishmaniasis, Chagas disease, ancylostomiasis or ascariasis. The results support the use of KLH as a means of rapidly and easily identifying individuals with acute schistosomiasis.

A urbanização das leishmanioses e a baixa resolutividade diagnóstica em municípios da Região Metropolitana de Belo Horizonte

During the period from 1994 to 1999 cutaneous leishmaniasis was reported in 32 (89%) out of 36 municipalities in the Metropolitan Region of Belo Horizonte, Brazil, of which one (2,8%) municipality was classified as a very high risk area, 16 (44,5%) as high risk, seven (19,4%) as moderate risk areas and 12 (33,3%) as low risk. From 1994 to 1995, visceral leishmaniasis was reported in six (16%) municipalities whereas in 1998 --1999 this number increased to 15 (42%). Annual numbers of cases during 1994 to 1999 were 30, 53, 64, 53 and 84, respectively. In 19 (61.3%) municipalities no reference center for the diagnosis of the infection was available, so that most of the patients (80%) were referred to Belo Horizonte. Twelve (39%) municipalities have a center for leishmaniasis evaluation, however in only eight (67%) of these basic specific diagnostic tests were available. Rapid and extensive increase of leishmaniasis associated with low diagnosis capacity has been observed in the metropolitan area of Belo Horizonte.

Immune Response in Human Visceral Leishmaniasis: Analysis of the Correlation Between Innate Immunity Cytokine Profile and Disease Outcome

We investigated the cytokine profile of cells of the innate immune response and its association with active (ACT), asymptomatic (AS) and cured (CUR) human visceral leishmaniasis (VL), as well as noninfected (NI) subjects. The frequency of cytokine‐producing cells was determined after short‐term in vitro incubation of whole peripheral blood samples with soluble Leishmania antigen (SLA). Our data demonstrated a predominant type 2 cytokine profile in NI and ACT. In NI, we observed an increase of IL‐4+ neutrophils, IL‐10+ eosinophils besides a decrease of tumour necrosis factor (TNF)‐α+ eosinophils/monocytes. Yet in ACT, we observed an increase of IL‐4+ neutrophils and natural killer (NK) cells and IL‐10+ monocytes, a reduced frequency of IL‐12+ and IFN‐γ+ eosinophils and lower levels of TNF‐α+ and IL‐12+ monocytes. AS presented a mixed profile, characterized by an increase of IFN‐γ+ neutrophils/eosinophils and NK cells, of IL‐12+ eosinophils/monocytes, as well as increase of IL‐4+ neutrophils and NK cells and IL‐10+ eosinophils/monocytes. In contrast, CUR was characterized by a type 1 response with an increase of IFN‐γ+ neutrophils/eosinophils and NK cells, associated with an increase in IL‐12+ monocytes. In conclusion, we show a correlation between innate immune cytokine patterns and clinical status of VL, suggesting that these cells, in addition to other factors, may contribute to the cytokine microenvironment in which Leishmania‐specific T cells are primed and to disease outcome.