Recurrence of visceral and muco-cutaneous leishmaniasis in a patient under immunosuppressive therapy

Ensuring accuracy in the diagnosis of leishmaniasis is crucial due to the myriad of potential differential diagnoses. Given the inherent limitations of serological techniques, real-time polymerase chain reaction (qPCR) emerges as a superior alternative. Furthermore, parasitological methods, conventionally regarded as the gold standard owing to their high specificity, encounter challenges concerning sensitivity and invasiveness for patients. In this context, the present study aims to assess, via meta-analysis, the performance of qPCR in diagnosing visceral and cutaneous leishmaniasis. This meta-analysis encompassed studies published between January 2011 and December 2022, sourced from six databases (PubMed, LILACS, Scopus, Scielo, EMBASE, and Web of Science), utilizing the keywords "qPCR," "molecular diagnosis," and "leishmaniasis." Epidemiological studies focusing on the efficacy of qPCR for leishmaniasis diagnosis were included. Data such as study demographics, geographic locations, sampling techniques, and the number of positive qPCR results were aggregated and analyzed to derive overall positivity rates, sensitivity, and specificity values associated with qPCR. Heterogeneity analysis was conducted on the data to select appropriate models, and the collective efficacy data of qPCR were illustrated in forest plots. Fifty-four studies met all inclusion criteria. The positivity rates for human visceral and cutaneous leishmaniasis were 27.07% (95% CI: 17.81-36.33%) and 60.40% (95% CI: 30.23-90.57%), respectively. In cases of visceral leishmaniasis in dogs, cats, and wild animals, the positivity rates were 26.55% (95% CI: 21.40-31.70%), 0.92% (95% CI: 0.09-1.75%), and 28.98% (95% CI: 21.86-35.10%), respectively. Analysis of the selected studies revealed high overall sensitivity and specificity values achieved with qPCR, at 91.08% (95% CI: 81.77-100.39%) and 98.08% (95% CI: 97.13-99.03%), respectively. This study indicates that qPCR is a highly sensitive and specific tool, adequately suitable for the diagnosis of human visceral and cutaneous leishmaniasis, as well as visceral leishmaniasis in animals.

Parasitic Infections in Solid Organ Transplant Recipients

First Report of Visceral Leishmaniasis In An Orthotopic Heart Transplant Recipient

To overcome some of the limitations of conventional microbiologic techniques, polymerase chain reaction (PCR)-based assays are proposed as useful tools for the diagnosis of visceral leishmaniasis. A comparative study using conventional microbiologic techniques (i.e., serologic testing, microscopic examination, and culture) and a Leishmania species-specific PCR assay, using peripheral blood and bone marrow aspirate samples as templates, was conducted during an 8-year period. The study cohort consisted of 594 Italian immunocompetent (adult and pediatric) and immunocompromised (adult) patients experiencing febrile syndromes associated with hematologic alterations and/or hepatosplenomegaly. Identification of the infecting protozoa at the species level was directly obtained by PCR of peripheral blood samples, followed by restriction fragment-length polymorphism analysis of the amplified products, and the results were compared with those of isoenzyme typing of Leishmania species strains from patients, which were isolated in vitro. Sixty-eight patients (11.4%) had a confirmed diagnosis of visceral leishmaniasis. Eleven cases were observed in human immunodeficiency virus (HIV)-uninfected adults, 20 cases were observed in HIV-infected adults, and the remaining 37 cases were diagnosed in HIV-uninfected children. In the diagnosis of primary visceral leishmaniasis, the sensitivities of the Leishmania species-specific PCR were 95.7% for bone marrow aspirate samples and 98.5% for peripheral blood samples versus sensitivities of 76.2%, 85.5%, and 90.2% for bone marrow aspirate isolation, serologic testing, and microscopic examination of bone marrow biopsy specimens, respectively. None of 229 healthy blood donors or 25 patients with imported malaria who were used as negative control subjects had PCR results positive for Leishmania species in peripheral blood samples (i.e., specificity of Leishmania species-specific PCR, 100%). PCR and restriction fragment-length polymorphism analysis for Leishmania species identification revealed 100% concordance with isoenzyme typing in the 19 patients for whom the latter data were available. PCR assay is a highly sensitive and specific tool for the diagnosis of visceral leishmaniasis in both immunocompetent and immunocompromised patients and can be reliably used for rapid parasite identification at the species level.

In general, the conventional techniques available for the diagnosis of leishmaniasis have relatively low sensitivity. This means that parasite-rich samples (which can usually only be collected by very invasive methods, such as bone-marrow aspiration) must be employed. This problem has not yet been solved even by use of the PCR-based techniques currently available. However, a new PCR-ELISA has been developed for the diagnosis of cutaneous (CL) and visceral leishmaniasis (VL) caused by Leishmania infantum. This assay appears to have sufficient sensitivity to be effective in the diagnosis of VL not only when bone-marrow aspirates are investigated but also when the samples are of peripheral blood. Overall, the ability of the PCR-ELISA to detect Leishmania, in 76 samples (22 of peripheral blood, 36 bone-marrow aspirates and 18 skin samples) from 72 patients living in a endemic region, was better than that of culture or the examination of Giemsa-stained smears. For example, L. infantum kDNA was detected by PCR-ELISA in 15 (83%) of the 18 skin samples from suspected cases of CL, whereas the combined use of several classical techniques only confirmed the presence of amastigotes in five (28%) of these samples. Similarly, only 21 individuals were diagnosed as having VL by conventional techniques whereas 30 were found Leishmania-positive in the PCR-ELISA. The new PCR-ELISA also appears to be a suitable technique for detecting leishmanial kDNA in samples of peripheral blood from cases of L. infantum-HIV co-infection. The assay is more sensitive than the combined use of several conventional techniques in the diagnosis of subclinical VL, probably because those with subclinical infection have relatively low parasitic loads that are generally undetectable using the other techniques.

Introduction: Leishmaniasis is a disease caused by a protozoan parasite of the genus Leishmania, which is transmitted through bites of infected sandflies. It has been reported that the polymerase chain reaction (PCR) is a more sensitive and specific test for the diagnosis of visceral leishmaniasis than bone marrow examination. This recent study is a renewed effort to validate the role of PCR in the diagnosis of visceral leishmaniasis. Objective: The objective of this study was to determine the sensitivity and specificity of PCR in the diagnosis of visceral leishmaniasis. Study design: Cross sectional (validation) study carried out in the Haematology department, Armed Forces Institute of Pathology, Rawalpindi. from 25 th March 2011 to 24 th March 2012. Subjects and Methods: A total number of 59 patients with visceral leishmaniasis diagnosed on microscopic bone marrow examination with equal number of negative controls were studied. The subjects were tested for the presence of visceral leishmaniasis by the polymerase chain reaction. Results: All the 59 patients were found to be positive for visceral leishmaniasis by PCR. None of the negative controls were positive by PCR. Conclusion: The study validates that PCR is equal to microscopic bone marrow examination in the diagnosis of visceral leishmaniasis. DOI: http://dx.doi.org/10.4038/sljid.v4i1.6036

Diagnosis of visceral leishmaniasis (VL) through the detection of its causative agents namely Leishmania donovani and L. infantum is traditionally based on immunochromatographic tests, microscopy of bone marrow, spleen aspirates, liver or lymph node and differential diagnosis. While the first process has low specificity, the later one carries the risk of fatal hemorrhage. Over the last decade, multiple Polymerase Chain Reaction (PCR) based diagnosis has been developed using blood and urine sample with a varying degree of sensitivity and specificity, an issue worth improving for precision diagnosis. Earlier, we reported a PCR-based diagnosis of L. donovani in peripheral blood using a novel set of PCR primers with absolute specificity. Using the same set of primers and PCR conditions, here we describe diagnosis of L. donovani from urine, for a non-invasive, rapid and safe diagnosis. Diagnosis of VL was carried out using urine samples collected from clinically diagnosed VL patients (n = 23) of Bangladesh in Real Time PCR. Test results were validated by comparing blood samples from the same set of patients. Sensitivity and specificity of this diagnosis was analyzed using retrospective bone marrow samples, collected earlier from confirmed VL patients (n = 19). The method showed 100% sensitivity in detecting L. donovani in urine and corresponding blood and retrospective bone marrow samples, as well as 100% specificity in control groups. A Real Time PCR-based molecular detection system using urine sample is hereafter presented what could be a, non-invasive approach for VL detection with precision and perfection.

A comparative analysis of different molecular targets using PCR for diagnosis of old world leishmaniasis

In this phase III trial for diagnostics for visceral leishmaniasis (VL) in India, we compared parasitological diagnosis with several serological tests: direct agglutination test (freeze dried; DAT-FD), rK-39 strip test, rK-26 strip test and a latex agglutination test for antigen detection in urine (KAtex) in 452 subjects from the endemic regions of Bihar, India. The subjects were segregated into four categories: 230 confirmed patients, 52 probable cases, 70 non-cases and 100 healthy endemic controls. The first two groups were used for estimating sensitivity, the latter two for specificity. Sensitivity of DAT-FD was 98.9%, rK-39: 98.9%, KAtex: 67.0% and rK-26: 21.3%. Sensitivity of DAT-FD on blood taken on filter paper (DAT-FDF) was 99.3%, which was comparable with that using serum. Specificity of serological tests was comparable and high (DAT-FD and DAT-FDF: 94%, rK-39 strip test: 97%, KAtex: 99% and rK-26 strip test: 100%). The classical 'gold standard' parasitological demonstration in splenic smear performed poorly as it missed 18.4% of cases that benefited from VL treatment. Reproducibility of the serological tests between field and central laboratories was excellent (kappa = 1.0, 0.99, 0.96 and 0.94 respectively for microscopy, DAT-FD, rK-39 strip test and rK-26 strip test). A high degree of agreement was observed between DAT-FD and rK-39 strip test (kappa = 0.986). Although DAT-FD and rK-39 strip test were highly sensitive with excellent specificity, the ease of use of the latter makes it most suitable for the diagnosis of VL in the field conditions.

Visceral leishmaniasis treated with liposomal amphotericin B.

Background Leishmaniasis (LI) is a vector-borne illness caused by a protozoan of the genus Leishmania. Data on the features of LI in patients with inflammatory bowel disease (IBD) are scarce. Aim To describe the characteristics of patients with IBD who present LI, the infection outcomes and the risk factors associated with developing visceral leishmaniasis (VL). Methods Observational, retrospective study performed in 26 hospitals in Spain, including all adult patients with IBD who developed LI from 2012 to 2022. Results A total of 73 patients were included. The clinical and demographic characteristics areshown in Table 1. Sixty patients (82%) presented localized cutaneous LI (CL) 2 (2.7%) diffuse CL, 3 (4%) mucocutaneous L (MCL) and 8 (11%) VL.The time-lapse between the starting of symptoms and the diagnosis of LI was 4.7±4.5 months (range 0-24). All patients were under biologics [69(94%)] or immunosuppressants (IMM) [4(6%)] at LI diagnosis. AntiTNF was the biologic drug used in 97%, while 2 patients (3%) were receiving ustekinumab. Twenty-seven out of 69 patients (39%) received combotherapy with IMM and 6 (8%) with corticosteroids. LI resolution was achieved by 35 (48%) and 70 (96%) patients at 1 and 12 months, respectively. Biologic withdrawal after LI diagnosis was not statistically related with increased rates of infection resolution among patients with localized CL. Biologic therapy was permanently discontinued in 21 patients (30%) among the whole cohort. Age was the only risk factor associated with VL (OR 1.2, 95%CI 1.04-1.39; p=0.012), while biological therapy was associated with an increased risk of CL or MCL (OR 39.7, 95%CI 1.2-1220.2; p=0.035). Conclusion LI in patients with IBD presents a favourable clinical outcome, even in those who continue on biological therapy, being age the only risk factor for developing VL. This infection should be considered for immunosuppressed patients with IBD dwelling in endemic areas who exhibit compatible symptoms.

A Novel Semiquantitative Fluorescence-Based Multiplex Polymerase Chain Reaction Assay for Rapid Simultaneous Detection of Bacterial and Parasitic Pathogens from Blood

Leishmaniasis is a set of complex and multifaceted syndromes, with different clinical manifestations, caused by different species of the genus Leishmania spp. that can be characterized by at least four syndromes: visceral leishmaniasis (VL, also known as kala-azar), post-kala-azar dermal leishmaniasis (PKDL), cutaneous leishmaniasis (CL), and mucocutaneous leishmaniasis (MCL). Among the most serious clinical forms, VL stands out, which causes the death of around 59,000 people annually. Fast and accurate diagnosis in VL is essential to reduce the disease's morbidity and mortality. There are a large number of diagnostic tests for leishmaniasis, however they do cross-react with other protozoa and their sensitivity changes according to the clinical form of the disease. Thus, it is essential and necessary to provide a diagnosis that is sufficiently sensitive to detect asymptomatic infected individuals and specific to discriminate individuals with other infectious and parasitic diseases, thus enabling more accurate diagnostic tools than those currently used. In this context, the aim of this review is to summarize the conventional diagnostic tools and point out the new advances and strategies on visceral and cutaneous leishmaniasis diagnosis.

Leishmaniasis is a major health problem and its diagnosis still represents a challenge. Since consistent evidence on the comparison of serological methods is lacking, our work aims to compare five serological tests for the diagnosis of visceral and asymptomatic leishmaniasis in southern France, a region where leishmaniasis is endemic. Serum samples from 75 patients living in Nice, France were retrospectively analyzed. They included patients affected by visceral leishmaniasis (VL; n = 25), asymptomatic carriers (AC; n = 25) and negative controls (n = 25). Each sample was tested using two immunochromatographic tests (ICT; IT LEISH® and TruQuick IgG/IgM®), an indirect fluorescent antibody test (IFAT) and two Western Blotting (WB; LDBio BIORAD® and an in-house method). Diagnosis of VL with IFAT and TruQuick® showed the highest diagnostic performance parameters. IFAT had 100% sensitivity and specificity, while TruQuick had 96% sensitivity and 100% specificity. Finally, the two tests showed high accuracy (100% for IFAT and 98% for TruQuick) for the AC group. WB LDBio® was the only method able to detect Leishmania latent infection, with a sensitivity of 92%, and a specificity of 100%, with a Negative Predictive Value (NPV) of 93%. This performance is reflected in the high accuracy of the test. The data obtained with TruQuick® supports its application in the rapid diagnosis of leishmaniasis in endemic areas, a feature not shown by IFAT despite its high diagnostic performance. Regarding the diagnosis of asymptomatic leishmaniasis, the best results were obtained with WB LDBio®, confirming previous studies.

ConclusionsRapid diagnosis of visceral, cutaneous, and mucocutaneous leishmaniasis may be made by slide agglutination. “H” and “O”'. agglutinogens are present in all species tested. The homologous organism is agglutinated to a high titer, but co-agglutinins are present in low titer to the heterologous organisms. L. donovani and T. cruzi, responsible for systemic infections, produce higher titers than L. tropica and L. brasiliensis, which have only cutaneous or mucocutaneous manifestations.