ABSTRACT
BACKGROUND: Coronavirus disease 2019 originated in China and swiftly spread worldwide, posing a significant threat to public health. Caused by SARS-CoV-2, it manifests as a flu-like illness that can escalate to Acute Respiratory Distress Syndrome, potentially resulting in fatalities. In countries where HIV/Leishmania infantum is endemic, the occurrence of concurrent SARS-CoV-2/HIV/Leishmania infantum infections is a reality, prompting inquiries into appropriate clinical management. CASE PRESENTATION: We present the case of a 48-year-old woman who was hospitalized for 36 days across three different hospitals in the state of Pernambuco, Brazil. She was diagnosed with SARS-CoV-2/HIV/L. infantum coinfection. The patient exhibited severe COVID-19 symptoms, including fever, productive cough, and dyspnea. Throughout her hospitalization, she experienced oxygen saturation levels of ≤ 93%, along with fluctuations in blood pressure, respiratory rate, and heart rate. Her blood tests revealed lymphopenia, leukopenia, and neutropenia, while laboratory results indicated abnormal levels of d-dimer, AST, ALT, lactate dehydrogenase, ferritin, and C-reactive protein. A computed tomography scan revealed 75% involvement of the lung parenchyma with patchy ground-glass opacities. CONCLUSION: Against all odds, the patient was discharged. The leukopenia associated with HIV/L. infantum may have played a decisive role. Further studies are necessary to better understand diagnostic strategies and clinical management measures for HIV/L. infantum coinfected patients who are susceptible to SARS-CoV-2 infection.
Subject(s)
COVID-19 , Coinfection , HIV Infections , Leishmania infantum , SARS-CoV-2 , Humans , Female , COVID-19/complications , Middle Aged , Coinfection/virology , Coinfection/parasitology , HIV Infections/complications , Leishmania infantum/isolation & purification , Leishmaniasis, Visceral/drug therapy , Leishmaniasis, Visceral/complications , Leishmaniasis, Visceral/diagnosis , BrazilABSTRACT
According to the World Health Organization (WHO), leishmaniasis is a zoonotic/anthroponotic parasitic disease endemic in 99 countries. It is estimated that approximately 12 million people are infected with Leishmania spp. and 350 million people live at risk. Every year, two million new cases are added to these figures. One and a half million cases of zoonotic/anthroponotic cutaneous leishmaniasis and 500 000 cases of visceral leishmaniasis are reported annually. One person is estimated to to be infected with cutaneous leishmaniasis in every 20 seconds and visceral leishmaniasis causes 60 000 deaths. In this report, two pediatric cases diagnosed with visceral leishmaniasis were presented. In the study, bone marrow aspirations were performed to determine the etiology of the disease in an eight-month-old male patient with fever and hepatosplenomegaly who had been followed up in Manisa Celal Bayar University, Department of Pediatrics, Division of Pediatric Hematology with the diagnosis of severe glucose-6-phosphate dehydrogenase (G-6PD) deficiency since the neonatal period and in a nine-month-old female patient who had had a high fever and bicytopenia for two weeks. Bone marrow aspirations were cultured in NNN medium and their smears were stained and examined with Giemsa. rk-39 and Leishmania IFAT tests were performed by using patients' sera. Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) analysis was also performed for Leishmania spp. Leishmania spp. amastigotes were observed in Giemsa-stained smear preparations, Leishmania spp. promastigotes were grown in NNN medium, rk39 rapid diagnostic kit was weakly positive, Leishmania IFAT was positive at a titer of 1/1024 and Leishmania tropica was identified as the causative agent by RT-qPCR analysis for both cases. These two cases suggested that fatal cases of visceral leishmaniasis may increase with the spread of visceralized isolates of L.tropica, the most common causative agent of cutaneous leishmaniasis in Türkiye, and this issue may create a significant public health problem.
Subject(s)
Leishmania tropica , Leishmaniasis, Visceral , Humans , Male , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/parasitology , Leishmania tropica/isolation & purification , Leishmania tropica/genetics , Female , Infant , Bone Marrow/parasitology , Glucosephosphate Dehydrogenase Deficiency/complications , AnimalsABSTRACT
Canine leishmaniosis (CanL) is caused by the protozoal parasite Leishmania infantum, which is transmitted by sand flies in warm climates across the world. Because dogs are considered a primary domestic reservoir for the parasite that causes leishmaniosis in humans, it is important from a One Health perspective that CanL be properly managed. In endemic regions, CanL is a common differential diagnosis in sick dogs because the clinical signs and clinicopathological disorders of the disease are non-specific, variable, and may overlap those of other common conditions. Diagnosis is based on the presence of compatible clinical signs, laboratory abnormalities, and confirmation by serological and parasitological evidence of infection. Here, we describe the performance of a point-of-care (POC) immunoassay that uses recombinant antigens to detect canine anti- L. infantum antibodies in a convenience sample set from a diagnostic laboratory, a group of canine patients with clinical staging, and in apparently healthy dogs from endemic areas. An immunofluorescence antibody test (IFAT) was used as the semiquantitative reference method. In the convenience sample set with high IFAT titers (≥ 1:800), the POC immunoassay demonstrated perfect agreement with IFAT (100%; 90/90). Using samples from dogs staged as either LeishVet Stage 2 or 3 or LeishVet Stage 1, positive agreement of the POC immunoassay with the IFAT was 98.8% (82/83) and 83.8% (31/37), respectively. The negative agreement with IFAT was 98.9% (272/275) in apparently healthy dogs from endemic areas of Greece and Italy. Since the performance of the POC immunoassay was associated with IFAT titer and clinical stage of CanL, the test may help veterinarians when determining if CanL is likely responsible for a patient's clinical picture or when evaluating an apparently healthy patient prior to vaccination.
Subject(s)
Antibodies, Protozoan , Dog Diseases , Leishmania infantum , Leishmaniasis, Visceral , Dogs , Animals , Dog Diseases/diagnosis , Dog Diseases/parasitology , Dog Diseases/epidemiology , Leishmania infantum/immunology , Leishmania infantum/isolation & purification , Leishmaniasis, Visceral/veterinary , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/epidemiology , Leishmaniasis, Visceral/parasitology , Antibodies, Protozoan/blood , Point-of-Care Systems , Fluorescent Antibody Technique/veterinary , Sensitivity and Specificity , Male , Female , Endemic Diseases/veterinaryABSTRACT
Accurate detection of viable Leishmania parasites is critical for evaluating visceral leishmaniasis (VL) treatment response at an early timepoint. We compared the decay of kinetoplast DNA (kDNA) and spliced-leader RNA (SL-RNA) in vitro, in vivo, and in a VL patient cohort. An optimized combination of blood preservation and nucleic acid extraction improved efficiency for both targets. SL-RNA degraded more rapidly during treatment than kDNA, and correlated better with microscopic examination. SL-RNA quantitative polymerase chain reaction emerges as a superior method for dynamic monitoring of viable Leishmania parasites. It enables individualized treatment monitoring for improved prognoses and has potential as an early surrogate endpoint in clinical trials.
Subject(s)
DNA, Kinetoplast , Leishmaniasis, Visceral , RNA, Spliced Leader , Humans , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/drug therapy , Leishmaniasis, Visceral/parasitology , DNA, Kinetoplast/genetics , RNA, Spliced Leader/genetics , RNA, Spliced Leader/metabolism , RNA, Protozoan/genetics , RNA, Protozoan/analysis , Animals , Leishmania/genetics , Antiprotozoal Agents/therapeutic use , BiomarkersABSTRACT
Surveillance and sustained control of visceral leishmaniasis (VL) require reliable serodiagnostic tools. rK39, the gold standard antigen for VL diagnosis, is limited by its documented poor sensitivity in certain endemic regions, such as East Africa, and by the longevity of its antibodies, making it difficult to distinguish active from cured infections. In a recent publication in mBio, Roberts et al. (A. J. Roberts, H.B. Ong, S. Clare, C. Brandt, et al., mBio 15:e00859-24, 2024, https://doi.org/10.1128/mbio.00859-24) identified new immunogenic Leishmania candidates in dogs and humans. In dogs, combined antigens LdBPK_290790.1 + LdBPK_362700.1 (D4 +D46) distinguished symptomatic from asymptomatic infections. For humans, LdBPK_323600.1 (D36) antigen produced short-lived antibodies and performed well in patient cohorts from Bangladesh and Ethiopia, but not Kenya. This study adds promising new candidates to our serodiagnostic toolbox but highlights the need for more antigen discovery studies that may have to be focused on regional performance.
Subject(s)
Antibodies, Protozoan , Antigens, Protozoan , Dog Diseases , Leishmaniasis, Visceral , Serologic Tests , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/veterinary , Leishmaniasis, Visceral/immunology , Dogs , Animals , Antigens, Protozoan/immunology , Antigens, Protozoan/genetics , Serologic Tests/methods , Humans , Dog Diseases/diagnosis , Dog Diseases/parasitology , Dog Diseases/immunology , Antibodies, Protozoan/blood , Sensitivity and Specificity , EthiopiaABSTRACT
Human visceral leishmaniasis (VL) is a severe disease whose diagnosis comprises immunological tests, microscopic biopsy examination, and biomolecular assays. In veterinary medicine, conjunctival swabs are widely used for detection of parasite DNA. Here, we describe the case of human VL in which conjunctival swabs were successfully used for Leishmania detection.
Subject(s)
Conjunctiva , Leishmaniasis, Visceral , Humans , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/parasitology , Conjunctiva/parasitology , Specimen Handling/methods , Male , DNA, Protozoan/geneticsABSTRACT
Visceral Leishmaniasis, caused by Leishmania infantum, is a tropical neglected disease and the most dangerous form of Leishmaniasis. It occurs zoonotically, with domestic transmission posing risks to humans as dogs have high susceptibility and are natural reservoirs of the parasite. Given their epidemiological role, improvements are needed in diagnosing Canine Visceral Leishmaniasis (CVL). Thus, we mapped linear epitopes from the rLiNTPDase2 antigen through peptide microarray and identified six positive epitopes. Validation through peptide ELISA revealed three promising peptides with accuracies of 78.6%, 85.92%, and 79.59%. Their combination yielded 97.58% accuracy. Negative epitopes were also found, which interacted with CVL-negative and Chagas Disease positive samples. Their removal from the rLiNTPDase2 sequence resulted in the rNT2.neg, which obtained enhanced specificity over rLiNTPDase2. The rNT2.neg validation achieved 87.50% sensitivity, 90.55% specificity, and 93.5% accuracy within 127 CVL-positive and 96 CVL-negative samples. Therefore, three peptides and rNT2.neg show significant promise for CVL diagnosis.
Subject(s)
Antigens, Protozoan , Dog Diseases , Epitope Mapping , Leishmania infantum , Leishmaniasis, Visceral , Animals , Antigens, Protozoan/immunology , Dogs , Leishmania infantum/immunology , Dog Diseases/diagnosis , Dog Diseases/parasitology , Leishmaniasis, Visceral/diagnosis , Sensitivity and Specificity , Epitopes/immunology , Peptides/immunology , Peptides/chemistry , Enzyme-Linked Immunosorbent Assay/methods , Protozoan Proteins/immunologyABSTRACT
A systematic review (SR) of published efficacy studies in visceral leishmaniasis (VL) was undertaken to describe methodological aspects of design, conduct, analysis, and reporting. Studies published during 2000-2021 and indexed in the Infectious Diseases Data Observatory VL library of clinical studies were eligible for inclusion (N = 89 studies). Of the 89 studies, 40 (44.9%) were randomized, 33 (37.1%) were single-armed, 14 (15.7%) were nonrandomized multiarmed studies, and randomization status was unclear in two (2.2%). After initial screening, disease confirmation was done by microscopy in 26 (29.2%) and by a combination of serology and microscopy in 63 (70.8%). Post-treatment follow-up duration was <6 months in three (3.3%) studies, 6 months in 75 (84.3%), and >6 months in 11 (12.4%) studies. Confirmation of relapse was solely based on clinical suspicion in four (4.5%) studies, parasitological demonstration in 64 (71.9%), using molecular/serological/parasitological method in 6 (6.7%), and there was no information in 15 (16.9%). Of the 40 randomized studies, sample size calculation was reported in only 22 (55.0%) studies. This review highlights substantial variations in definitions adopted for disease diagnosis and therapeutic outcomes suggesting a need for a harmonized trials protocol.
Subject(s)
Antiprotozoal Agents , Leishmaniasis, Visceral , Leishmaniasis, Visceral/drug therapy , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/epidemiology , Humans , Antiprotozoal Agents/therapeutic use , Research Design , Treatment Outcome , Randomized Controlled Trials as TopicABSTRACT
In endemic areas for canine visceral leishmaniasis (CVL), the occurrence of coinfection with other pathogens, such as Ehrlichia spp., has been associated with worsening of the clinical condition. The study aimed to evaluate the occurrence of histological changes in the myocardia of dogs naturally infected with Leishmania chagasi with or without coinfection with Ehrlichia spp.. We evaluated paraffin-embedded myocardial sections from 31 dogs, affected by either L. chagasi alone or coinfected with L. chagasi and Ehrlichia spp., to compare the extent and degree of cardiac damage. The blocks were divided into two groups. G1 (dogs infected only by L. chagasi) and G2 (dogs coinfected with L. chagasi and Ehrlichia spp.). The right atrium free wall, right ventricle free wall, left ventricle, and interventricular septum of all groups were evaluated. Cardiac alterations were observed in 41.93% (52/124) of the fragments evaluated and inflammatory infiltrate was the most common pattern found. The G2 group showed a higher incidence of myocarditis, with 61.53% (32/52), compared to the G1 group, in which 20 out of 72 cases (27.7%) exhibited histopathological changes (p <0.05). These findings confirmed that coinfection can potentiate cardiac damage in dogs.
Subject(s)
Dog Diseases , Ehrlichiosis , Leishmaniasis, Visceral , Animals , Dogs , Leishmaniasis, Visceral/veterinary , Leishmaniasis, Visceral/complications , Leishmaniasis, Visceral/diagnosis , Dog Diseases/parasitology , Dog Diseases/microbiology , Male , Ehrlichiosis/veterinary , Ehrlichiosis/complications , Ehrlichiosis/diagnosis , Coinfection/veterinary , Female , Myocarditis/veterinary , Myocarditis/microbiology , Myocarditis/parasitology , Ehrlichia/isolation & purification , Myocardium/pathologyABSTRACT
Leishmaniasis, a parasitic disease found in many parts of southern Europe, is transmitted in both humans and canines through the bite of phlebotomine sandflies, and can present in a variety of ways, such as cutaneous, mucocutaneous, diffuse, and visceral. In Bulgaria there are endemic areas of canine leishmaniasis, with sporadic cases in humans. However, no detailed studies of the animal population and vectors have been performed. Here we describe a few clinical cases of canine visceral leishmaniasis in two districts in western Bulgaria: one endemic and one without previously detected cases in humans or dogs. Diagnosis was confirmed serologically and molecularly using both real time and conventional PCR. Specific anti-leishmanial antibodies were confirmed in three of the cases via ELISA, with 50% of them returning extremely high values. In the majority of the cases DNA fragments were detected in the skin or lymph node aspirate but not in the blood. This paper highlights the need for further studies updating the current knowledge on the epidemiology, diagnosis, and control of visceral leishmaniasis in the reservoir host population.
Subject(s)
Dog Diseases , Leishmaniasis, Visceral , Dogs , Animals , Bulgaria/epidemiology , Dog Diseases/diagnosis , Dog Diseases/epidemiology , Dog Diseases/parasitology , Leishmaniasis, Visceral/veterinary , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/epidemiology , Male , Female , Leishmaniasis/veterinary , Leishmaniasis/diagnosis , Leishmaniasis/epidemiologyABSTRACT
BACKGROUND: Post kala-azar dermal leishmaniasis (PKDL) is a consequential dermal manifestation of visceral leishmaniasis (VL), serving as a parasite reservoir. The traditional diagnostic approach, which requires an invasive skin biopsy is associated with inherent risks and necessitates skilled healthcare practitioners in sterile settings. There is a critical need for a rapid, less invasive method for Leishmania detection. The main objective of this study was to evaluate and compare the diagnostic efficacy of PCR and qPCR in detecting PKDL, utilizing both skin and blood samples and to assess the utility of blood samples for molecular diagnosis. METHODS AND RESULTS: 73 individuals exhibiting clinical symptoms of PKDL and who had tested positive for rK39 rapid diagnostic test (RDT) were enrolled in this study. For the diagnosis of PKDL, both PCR and real-time quantitative PCR (qPCR), employing SYBR Green and TaqMan assays, were performed on blood and skin matched samples. qPCR results using both TaqMan and SYBR Green assay, indicated higher parasite loads in the skin compared to blood, as evident by the Ct values. Importantly, when blood samples were used for PKDL diagnosis by qPCR, an encouraging sensitivity of 69.35% (TaqMan assay) and 79.36% (SYBR Green) were obtained, compared to 8.2% with conventional PCR. CONCLUSION: The findings of the study suggest the potential utility of blood for molecular diagnosis by qPCR, offering a less invasive alternative to skin biopsies in field setting for the early detection of parasitaemia in PKDL patients and effective management and control of the disease.
Subject(s)
Leishmaniasis, Cutaneous , Leishmaniasis, Visceral , Real-Time Polymerase Chain Reaction , Humans , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/blood , Leishmaniasis, Visceral/parasitology , Leishmaniasis, Cutaneous/diagnosis , Leishmaniasis, Cutaneous/parasitology , Leishmaniasis, Cutaneous/blood , Leishmaniasis, Cutaneous/genetics , Real-Time Polymerase Chain Reaction/methods , Male , Female , Adult , Adolescent , Skin/parasitology , Skin/pathology , Sensitivity and Specificity , Middle Aged , Parasite Load/methods , Molecular Diagnostic Techniques/methods , Young Adult , Child , DNA, Protozoan/genetics , DNA, Protozoan/bloodABSTRACT
BACKGROUND: Leishmania infantum is endemic in the Mediterranean region, presenting mostly as visceral leishmaniasis (VL). In Portugal, reporting of VL cases to public health authorities is mandatory, but significant underreporting is likely. This study aimed to describe the epidemiological and clinical aspects of the VL cases diagnosed in hospitals of the Portuguese National Health Service (NHS), between 2010 and 2020. METHODS: Collaboration was requested to every hospital of the Portuguese NHS in Mainland Portugal. Cases were screened through a search of diagnostic discharge codes or, if not available, by a search of positive laboratory results for Leishmania infection. Sociodemographic and clinical data was retrieved from medical records. Simultaneously, the National Health authority was contacted to request access to data of notified cases of VL between 2010 and 2020. Descriptive, hypothesis testing and multiple binary logistic regression models were performed. RESULTS: A total of 221 VL cases were identified. A significant increase in estimated national incidence was seen in the years after 2016 (P = 0.030). VL was predominantly diagnosed in people living with HIV (PLWH) and in children (representing around 60% of the new cases), but the outcome was generally poorer in non-HIV patients with associated immunosuppression, with significantly lower rates of clinical improvement at 7 (P = 0.003) and 30 days (P = 0.008) after treatment. Atypical presentations, with gastrointestinal and/or respiratory involvement, were seen in 8.5% of VL cases. Hemophagocytic lymphohistiocytosis was diagnosed in 40.0% of children under 5 years of age. Only 49.7% of incident VL cases were reported. Simultaneous involvement of the skin was confirmed in 5.9% of patients. CONCLUSIONS: VL presents a continuing threat in Portugal, especially to PLWH and children, and an increasing threat to other immunosuppressed groups. Recent increases in incidence should be closely monitored to allow prompt interventions. Programs to control the disease should focus on providing tools for earlier diagnosis and on reducing underreporting and promoting an integrated surveillance of human and animal disease. These data should be combined with asymptomatic infection and vector information, following a One Health approach.
Subject(s)
Hospitals, Public , Leishmaniasis, Visceral , Leishmaniasis, Visceral/epidemiology , Leishmaniasis, Visceral/diagnosis , Humans , Portugal/epidemiology , Male , Female , Child , Retrospective Studies , Child, Preschool , Adolescent , Infant , Adult , Middle Aged , Hospitals, Public/statistics & numerical data , Incidence , Young Adult , Leishmania infantum/isolation & purification , Aged , HIV Infections/epidemiology , Infant, NewbornABSTRACT
This case report summarizes the experience from diagnosis and treatment of a patient with repeated high fever, hepatosplenomegaly and pancytopenia. Following exclusion of bacterial, viral, fungal infections and hematological diseases, metagenomic next-generation sequencing of the patient's peripheral blood revealed Leishmania infantum infection, and rK39 rapid diagnostic test showed positive for anti-Leishmania antibody, while microscopic examination of bone marrow smears identified Leishmania amastigotes. Therefore, the case was definitively diagnosed as visceral leishmaniasis, and given anti-infective treatment with sodium antimony gluconate and hormone, hepatoprotection, elevation of white blood cell counts and personalized nursing. Then, the case was cured and discharged from hospital. Metagenomic next-generation sequencing is of great value in etiological detection of fever patients with unknown causes, which deserves widespread clinical applications.
Subject(s)
High-Throughput Nucleotide Sequencing , Leishmaniasis, Visceral , Humans , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/drug therapy , Leishmaniasis, Visceral/parasitology , Male , Metagenomics/methods , Adult , Middle AgedABSTRACT
BACKGROUND: Diagnosis of visceral leishmaniasis (VL) in resource-limited endemic regions is currently based on serological testing with rK39 immunochromatographic tests (ICTs). However, rK39 ICT frequently has suboptimal diagnostic accuracy. Furthermore, treatment monitoring and detection of VL relapses is reliant on insensitive and highly invasive tissue aspirate microscopy. Miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) is an innovative and user-friendly molecular tool which does not require DNA extraction and uses a lateral flow strip for result read-out. This assay could be an interesting candidate for more reliable VL diagnosis and safer test of cure at the point of care. METHODOLOGY/PRINCIPLE FINDINGS: The performance of mini-dbPCR-NALFIA for diagnosis of VL in blood was assessed in a laboratory evaluation and compared with the accuracy of rK39 ICTs Kalazar Detect in Spain and IT LEISH in East Africa. Limit of detection of mini-dbPCR-NALFIA was 650 and 500 parasites per mL of blood for Leishmania donovani and Leishmania infantum, respectively. In 146 blood samples from VL-suspected patients from Spain, mini-dbPCR-NALFIA had a sensitivity of 95.8% and specificity 97.2%, while Kalazar Detect had a sensitivity of 71.2% and specificity of 94.5%, compared to a nested PCR reference. For a sample set from 58 VL patients, 10 malaria patients and 68 healthy controls from Ethiopia and Kenya, mini-dbPCR-NALFIA had a pooled sensitivity of 87.9% and pooled specificity of 100% using quantitative PCR as reference standard. IT LEISH sensitivity and specificity in the East African samples were 87.9% and 97.4%, respectively. CONCLUSIONS/SIGNIFICANCE: Mini-dbPCR-NALFIA is a promising tool for simplified molecular diagnosis of VL and follow-up of treated patients in blood samples. Future studies should evaluate its use in endemic, resource-limited settings, where mini-dbPCR-NALFIA may provide an accurate and versatile alternative to rK39 ICTs and aspirate microscopy.
Subject(s)
Leishmania donovani , Leishmaniasis, Visceral , Sensitivity and Specificity , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/parasitology , Humans , Leishmania donovani/genetics , Leishmania donovani/isolation & purification , Immunoassay/methods , Leishmania infantum/genetics , Leishmania infantum/isolation & purification , Polymerase Chain Reaction/methods , Spain , Molecular Diagnostic Techniques/methods , Female , Male , Adult , Adolescent , Child , Young Adult , Middle Aged , Africa, Eastern , DNA, Protozoan/genetics , DNA, Protozoan/blood , Child, PreschoolABSTRACT
Leishmaniasis is a vector-borne disease caused by many Leishmania spp. which infect humans and other mammalian hosts. Leishmania infantum is the main agent of canine leishmaniasis (CanL) whose diagnosis is usually confirmed by serological and molecular tests. This study aimed to evaluate the clinical and analytical sensitivities of a lab-on-chip (LOC) real-time PCR applied on the portable Q3-Plus V2 platform (Q3 qPCR) in the detection of L. infantum. The Q3 qPCR performance was assessed by comparing the quantification cycle (Cq) values with those obtained using the qPCR run on a CFX96 Real-Time System (CFX96 qPCR). A total of 173 DNA samples (extracted from bone marrow, lymph node, blood, buffy coat, conjunctival swab, and skin) as well as 93 non-extracted samples (NES) (bone marrow, lymph node, blood, and buffy coat) collected from dogs were tested with both systems. Serial dilutions of each representative DNA and NES sample were used to assess the analytical sensitivity of the Q3 qPCR system. Overlapping Cq values were obtained with the Q3 qPCR and CFX96 qPCR, both using DNA extracted from L. infantum promastigotes (limit of detection, <1 promastigote per milliliter) and from biological samples as well as with NES. However, the Q3 qPCR system showed a higher sensitivity in detecting L. infantum in NES as compared with the CFX96 qPCR. Our data indicate that the Q3 qPCR system could be a reliable tool for detecting L. infantum DNA in biological samples, bypassing the DNA extraction step, which represents an advance in the point-of-care diagnostic of CanL.
Subject(s)
Dog Diseases , Leishmania infantum , Leishmaniasis, Visceral , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity , Dogs , Animals , Leishmania infantum/genetics , Leishmania infantum/isolation & purification , Real-Time Polymerase Chain Reaction/methods , Dog Diseases/diagnosis , Dog Diseases/parasitology , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/veterinary , Leishmaniasis, Visceral/parasitology , Lab-On-A-Chip Devices , Molecular Diagnostic Techniques/methods , DNA, Protozoan/geneticsABSTRACT
Visceral leishmaniasis is a deadly infectious disease and is one of the world's major neglected health problems. Because the symptoms of infection are similar to other endemic diseases, accurate diagnosis is crucial for appropriate treatment. Definitive diagnosis using splenic or bone marrow aspirates is highly invasive, and so, serological assays are preferred, including the direct agglutination test (DAT) or rK39 strip test. These tests, however, are either difficult to perform in the field (DAT) or lack specificity in some endemic regions (rK39), making the development of new tests a research priority. The availability of Leishmania spp. genomes presents an opportunity to identify new diagnostic targets. Here, we use genome data and a mammalian protein expression system to create a panel of 93 proteins consisting of the extracellular ectodomains of the Leishmania donovani cell surface and secreted proteins. We use these panel and sera from murine experimental infection models and natural human and canine infections to identify new candidates for serological diagnosis. We observed a concordance between the most immunoreactive antigens in different host species and transmission settings. The antigen encoded by the LdBPK_323600.1 gene can diagnose Leishmania infections with high sensitivity and specificity in patient cohorts from different endemic regions including Bangladesh and Ethiopia. In longitudinal sampling of treated patients, we observed reductions in immunoreactivity to LdBPK_323600.1 suggesting it could be used to diagnose treatment success. In summary, we have identified new antigens that could contribute to improved serological diagnostic tests to help control the impact of this deadly tropical infectious disease. IMPORTANCE: Visceral leishmaniasis is fatal if left untreated with patients often displaying mild and non-specific symptoms during the early stages of infection making accurate diagnosis important. Current methods for diagnosis require highly trained medical staff to perform highly invasive biopsies of the liver or bone marrow which pose risks to the patient. Less invasive molecular tests are available but can suffer from regional variations in their ability to accurately diagnose an infection. To identify new diagnostic markers of visceral leishmaniasis, we produced and tested a panel of 93 proteins identified from the genome of the parasite responsible for this disease. We found that the pattern of host antibody reactivity to these proteins was broadly consistent across naturally acquired infections in both human patients and dogs, as well as experimental rodent infections. We identified a new protein called LdBPK_323600.1 that could accurately diagnose visceral leishmaniasis infections in humans.
Subject(s)
Antibodies, Protozoan , Antigens, Protozoan , Leishmania donovani , Leishmaniasis, Visceral , Protozoan Proteins , Serologic Tests , Leishmania donovani/genetics , Leishmania donovani/immunology , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/immunology , Leishmaniasis, Visceral/parasitology , Animals , Humans , Mice , Dogs , Antigens, Protozoan/genetics , Antigens, Protozoan/immunology , Antibodies, Protozoan/blood , Antibodies, Protozoan/immunology , Protozoan Proteins/genetics , Protozoan Proteins/immunology , Serologic Tests/methods , Biomarkers/blood , Female , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Mice, Inbred BALB C , Membrane Proteins/genetics , Membrane Proteins/immunology , Sensitivity and Specificity , Dog Diseases/diagnosis , Dog Diseases/parasitologyABSTRACT
Infection and clinical cases of leishmaniasis caused by Leishmania infantum in cats have been increasingly reported in several countries, including Brazil. In this study, we used an enzyme-linked immunosorbent assay (ELISA) and an immunochromatographic test (ICT) based on a recombinant antigen (rKDDR-plus) to detect anti-Leishmania antibodies in cats from an animal shelter in northeastern Brazil. We compared the results with an ELISA using L. infantum crude antigen (ELISA-CA). We also investigated the presence of Leishmania DNA in blood or ocular conjunctival samples as well as the association between Leishmania PCR positivity and serological positivity to feline immunodeficiency virus (FIV), feline leukemia virus (FeLV) and Toxoplasma gondii. Concerning serological assays, a higher positivity was detected using the ICT-rKDDR-plus (7.5%; 7/93) as compared to ELISA-rKDDR-plus (5.4%; 5/93) and ELISA-CA (4.3%; 4/93). Upon PCR testing, 52.7% (49/93) of the ocular conjunctival swabs and 48.3% (44/91) of the blood samples were positive. Together, PCR and serological testing revealed overall positivities of 73.1% (68/93) and 12.9% (12/93), respectively. Among PCR-positive samples, 45.5% (31/68) showed co-infection with FIV, 17.6% (12/68) with FeLV, and 82.3% (56/68) with T. gondii. More than half of the PCR-positive cats showed at least one clinical sign suggestive of leishmaniasis (58.8%; 40/68) and dermatological signs were the most frequent ones (45.5%; 31/68). Both tests employing the recombinant antigen rKDDR-plus (i.e., ICT-rKDDR-plus and ELISA-rKDDR-plus) detected more positive cats than the ELISA-CA but presented low overall accuracy. PCR testing using either blood or ocular conjunctival samples detected much more positive cats than serological tests.
Subject(s)
Cat Diseases , Coinfection , Enzyme-Linked Immunosorbent Assay , Immunodeficiency Virus, Feline , Leishmania infantum , Leukemia Virus, Feline , Recombinant Proteins , Cats , Animals , Cat Diseases/diagnosis , Cat Diseases/parasitology , Cat Diseases/virology , Cat Diseases/blood , Cat Diseases/epidemiology , Brazil/epidemiology , Enzyme-Linked Immunosorbent Assay/veterinary , Immunodeficiency Virus, Feline/isolation & purification , Coinfection/veterinary , Coinfection/parasitology , Coinfection/epidemiology , Coinfection/virology , Leishmania infantum/isolation & purification , Leukemia Virus, Feline/genetics , Leukemia Virus, Feline/immunology , Male , Female , Toxoplasma , Antibodies, Protozoan/blood , Leishmaniasis, Visceral/veterinary , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/epidemiology , Leishmaniasis, Visceral/blood , Polymerase Chain Reaction/veterinary , Toxoplasmosis, Animal/diagnosis , Toxoplasmosis, Animal/epidemiology , Toxoplasmosis, Animal/bloodABSTRACT
Maintenance chemotherapy is a standard treatment in patients with non-progressive advance staged IV non-squamous non-small cell lung cancer after induction therapy. Here, we report the case of a 53-year-old man undergoing a maintenance monotherapy with pemetrexed who presented prolonged pancytopenia despite filgrastim injections. A bone marrow aspiration revealed a macrophage activation syndrome with Leishmania amastigotes. A Polymerase Chest Reaction testing confirmed the diagnosis of visceral leishmaniasis. Treatment with liposomal amphotericin B was started. Oncologists should bear in mind that visceral leishmaniasis in endemic areas can potentially induce severe and prolonged pancytopenia in immunosuppressed patients, during chemotherapy in particular.
Subject(s)
Leishmaniasis, Visceral , Lung Neoplasms , Pancytopenia , Humans , Pancytopenia/chemically induced , Leishmaniasis, Visceral/drug therapy , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/complications , Male , Middle Aged , Lung Neoplasms/drug therapy , Carcinoma, Non-Small-Cell Lung/drug therapy , Diagnosis, Differential , Pemetrexed/therapeutic use , Pemetrexed/adverse effects , Antineoplastic Agents/adverse effects , Antineoplastic Agents/therapeutic use , Antiprotozoal Agents/therapeutic use , Amphotericin B/therapeutic useABSTRACT
Visceral leishmaniasis-associated hemophagocytic lymphohistiocytosis (VL-HLH) is indistinguishable from those of HLH of other etiologies due to the overlap symptoms, posing a serious threat to life. In this study, we aimed to provide insights for early diagnosis and improve outcomes in pediatric patients with VL-HLH. We retrospectively analyzed the clinical and laboratory data of 10 pediatric patients with VL-HLH and 58 pediatric patients with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH). The median time from symptom onset to cytopenia in patients with VL-HLH and EBV-HLH was 11 days (interquartile range, 7-15 days) and five days (interquartile range, 3.75-9.25 days) (P = 0.005). Both groups showed liver injury and increased lactate dehydrogenase levels; however the levels of aspartate aminotransferase, alanine aminotransferase, direct bilirubin, and lactate dehydrogenase in patients with VL-HLH were significantly lower than those in patients with EBV-HLH (P < 0.05). The fibrinogen and triglyceride levels were almost normal in VL-HLH patients but were significantly altered in EBV-HLH cases ( P < 0.05). The positive rate of first bone marrow microscopy examination, anti-rK39 IgG detection, and blood metagenomic next-generation sequencing was 50%, 100%, and 100%, respectively. After VL diagnosis, eight patients were treated with sodium stibogluconate and two were treated with liposomal amphotericin B. All the patients with VL-HLH recovered. Our study demonstrates that regular triglyceride and fibrinogen levels in pediatric patients with VL-HLH may help in differential diagnosis from EBV-HLH. VL-HLH is milder than EBV-HLH, with less severe liver injury and inflammatory responses, and timely treatment with antileishmanial agents is essential to improve the outcomes of pediatric patients with VL-HLH.
Subject(s)
Epstein-Barr Virus Infections , Leishmaniasis, Visceral , Lymphohistiocytosis, Hemophagocytic , Child , Humans , Epstein-Barr Virus Infections/complications , Epstein-Barr Virus Infections/drug therapy , Epstein-Barr Virus Infections/diagnosis , Lymphohistiocytosis, Hemophagocytic/diagnosis , Lymphohistiocytosis, Hemophagocytic/drug therapy , Lymphohistiocytosis, Hemophagocytic/etiology , Herpesvirus 4, Human , Leishmaniasis, Visceral/complications , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/drug therapy , Retrospective Studies , Fibrinogen , Triglycerides/therapeutic use , Lactate DehydrogenasesABSTRACT
Leishmaniases are a group of diseases under the category of neglected tropical diseases targeted for global elimination. However, they continue to pose major clinical and public health problems, especially among those living in poor socioeconomic conditions. Here, we summarize leishmaniasis elimination efforts in Bhutan. Between 1994 and 2022, Bhutan recorded 54 cases of leishmaniasis across 14 of its 20 districts. There are seven species of Phlebotomus and three species of Sergentomyia sand flies documented in the country. At a subnational level, all endemic districts recorded a visceral leishmaniasis annual incidence <1 per 10,000 population, meeting the regional elimination targets. Serological testing with ELISA and molecular testing with polymerase chain reaction were established at the Royal Center for Disease Control in 2022. A leishmaniasis prevention and management guideline was adopted in 2023 to aid clinicians in diagnosis and management. Active and passive case surveillance was integrated with the national infectious disease early warning and response system. Risk-based entomological surveillance and control have also been prioritized. Climate change may play a major role in rendering districts in the temperate zone favorable for vector proliferation. The country's medical university introduced a diploma course in medical entomology in 2023 to augment the human resources needed for vector surveillance efforts. However, leishmaniasis elimination lacks dedicated programmatic management amid competing priorities for resources against other infectious diseases. Leishmaniasis elimination requires a targeted and programmatic approach in Bhutan, including cross-border collaborative efforts with neighboring Indian states. Bhutan remains highly committed to achieving leishmaniasis elimination targets.