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Visceral leishmaniasis (VL) requires diagnostic assays to complement clinical suspicion. However, there is no standardization of a diagnostic flow using available assays. This study aimed to evaluate the performance of parasitological, molecular, and serological assays for diagnosing VL and propose a diagnostic flow based on performance, practicality, and invasiveness. We conducted a study of 10-year (2010-2020) routine diagnoses of VL at the Brazilian National Reference Laboratory. We propose a diagnostic flow where individuals suspected of VL are initially screened for antibodies using an immunochromatographic test (ICT) with rK39 antigen on the nitrocellulose membrane. This is followed by a blood polymerase chain reaction (PCR) for Leishmania sp. kDNA and direct parasitological exam and/or PCR in bone marrow aspirate. A positive result in any of these assays can define a VL case. If clinical suspicion persists in negative individuals, the diagnostic flow should be repeated. The proposed flow has the potential to standardize and improve the diagnosis of VL. It reduces the need for invasive tests without compromising diagnostic accuracy.
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Leishmaniose Visceral , Humanos , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/epidemiologia , Brasil/epidemiologia , Algoritmos , Anticorpos , BioensaioRESUMO
Tests for visceral leishmaniasis (VL) are not uniformly effective for all endemic regions. In a serological assay, a novel antigen, otubain cysteine peptidase, compared with rK39, showed comparable sensitivity with Indian VL serum samples and prominently increased sensitivity with Brazilian samples, as well as improved monitoring of the treatment response.
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Leishmania donovani , Leishmaniose Visceral , Anticorpos Antiprotozoários , Antígenos de Protozoários , Cisteína , Ensaio de Imunoadsorção Enzimática , Humanos , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/tratamento farmacológico , Peptídeo Hidrolases , Sensibilidade e Especificidade , Testes SorológicosRESUMO
BACKGROUND: Visceral Leishmaniasis (VL) is a severely neglected disease affecting millions of people with high mortality if left untreated. In Ethiopia, the primary laboratory diagnosis of VL is by using an antigen from a 39-amino acid sequence repeat of a kinesin-related (rK39) of leishmania donovani complex (L. donovani), rapid diagnostic tests (RDT). Different rk39 RDT brands are available with very variable performance and studies from Ethiopia showed a very wide range of sensitivity and specificity. Therefore, a systematic review and meta-analysis were conducted to determine the pooled sensitivity and specificity of rk39 RDT in Ethiopia. METHOD: PUBMED, EMBASE, and other sources were searched using predefined search terms to retrieve all relevant articles from 2007 to 2020. Heterogeneity was assessed by visually inspecting summary receiver operating curves (SROC), Spearman correlation coefficient (rs), Cochran Q test statistics, inconsistency square (I2) and subgroup analysis. The presence and statistical significance of publication bias were assessed by Egger's test at p < 0.05, and all the measurements showed the presence of considerable heterogeneity. Quality assessment of diagnostic accuracy studies (QUADAS-2) checklists was used to check the qualities of the study. RESULTS: A total of 664 articles were retrieved, and of this 12 articles were included in the meta-analysis. Overall pooled sensitivity and specificity of the rk39 RDT to diagnose VL in Ethiopia were 88.0% (95% CI 86.0% to 89.0%) and 84.0% (95% CI 82.0% to 86.0%), respectively. The sensitivity and specificity of the rk39 RDT commercial test kits were DiaMed: 86.9% (95% CI 84.3% to 89.1%) and 82.2% (95% CI 79.3% to 85.0%), and InBios: 80.0% (95% CI 77.0% to 82.8%) and 97.4% (95% CI 95.0% to 98.8%), respectively. CONCLUSION: Referring to our result, rk39 RDT considered an essential rapid diagnostic test for VL diagnosis. Besides to the diagnostic accuracy, the features such as easy to perform, quick (10-20 min), cheap, equipment-free, electric and cold chain free, and result reproducibility, rk39 RDT is advisable to remains in practice as a diagnostic test at least in the remote VL endemic localities till a better test will come.
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Leishmania donovani , Leishmaniose Visceral , Antígenos de Protozoários , Etiópia , Humanos , Leishmaniose Visceral/diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Leishmania infantum is the most common cause of visceral leishmaniasis (VL) in Iran, where mainly the patients are children under the age of 5 years. Timely, less invasive, and accurate diagnosis and proper treatment of the disease are necessary. This retrospective study aimed to search for a less invasive but robust algorithm on VL diagnostic tests in children. Four hundred and fifteen patients with clinical suspicion of VL, 50 healthy children from VL endemic areas, 46 healthy individuals from non-endemic VL areas, and 47 non-VL diseases were tested using three diagnostic tests: indirect immunofluorescent antibody test (IFAT), rK39-rapid diagnostic test (rK39-RDT), and quantitative PCR (qPCR). One hundred and two suspected VL cases were positive in at least one test and were cured after receiving appropriate treatment. Of these 102 VL patients, 94 were positive in qPCR, 84 in IFAT, and 79 in rK39-RDT. None of the tests detected all the patients, but overall, qPCR is capable of detecting more VL patients than serological tests, i.e., 92.2%, compared to IFAT, 82.4%, and rK39, 77.5%. There was only a significant difference between the sensitivity of qPCR and rK39-RDT (p = 0.024). The specificity was 100% for qPCR and IFAT (≥128) and 98.6% for rK39-RDT. qPCR alone is capable of detecting most of the VL-suspected children. Serological tests like IFAT and rk39-RDT are recommended to increase the overall sensitivity of detection in patients with a negative molecular test. Combining qPCR with a serological test (IFAT or rK39-RDT) can help diagnose 98% of VL. In laboratories without molecular facilities, we recommend testing with the combination of rK39-RDT and IFAT yielding a combined sensitivity of 93.1% equivalent to that of qPCR in our study.
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Algoritmos , Testes Diagnósticos de Rotina/métodos , Leishmaniose Visceral/diagnóstico , Adolescente , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Criança , Pré-Escolar , DNA de Cinetoplasto/isolamento & purificação , Testes Diagnósticos de Rotina/normas , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Lactente , Irã (Geográfico)/epidemiologia , Leishmania infantum/genética , Leishmania infantum/imunologia , Leishmaniose Visceral/epidemiologia , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
Because of visceral leishmaniasis (VL) urbanization and spreading of the human immunodeficiency virus (HIV) infection to rural areas, coinfection has become more common. Here, we compared the accuracy of Kalazar Detect® (KD), an rK39-based immunochromatographic (IC) test, and OrangeLife® (OL), an rK39 + rK28 IC test, for diagnosing VL in patients coinfected with HIV in an endemic area in Brazil. Seventy-six VL patients and 40 patients with other diseases, of which 31 and 21 patients, respectively, were infected with HIV, were examined. The sensitivity of OL and KD tests was 88.89 and 95.45%, respectively, in patients without HIV. The sensitivity dropped to 67.74 and 61.29%, respectively, in coinfected patients. The decrease in sensitivity was not related to a decrease in the production of Leishmania-specific IgG. Because of the low sensitivity of rk39 test in HIV-infected patients, we suggest that patients with negative rK39 results should undergo further investigation with additional serological tests that are not based only on the rK39 antigen and examination of bone marrow aspirates.
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Cromatografia de Afinidade/métodos , Infecções por HIV/complicações , Leishmaniose Visceral/diagnóstico , Adulto , Antígenos de Protozoários/imunologia , Brasil , Coinfecção , Feminino , Humanos , Leishmaniose Visceral/complicações , Masculino , Proteínas de Protozoários/imunologia , Sensibilidade e EspecificidadeRESUMO
Canine leishmaniasis (CanL) caused by Leishmania infantum (L. infantum) is considered as a zoonotic disease and within the last few decades, studies have identified the parasite as a major causative agent of human visceral leishmaniasis. However, in dogs, few recent studies have determined L. major as a cause of cutaneous manifestations and L. tropica as an etiological agent for cutaneous lesions involving mucosa. Interestingly, current study has found canine cutaneous lesions with mucosal involvement in a dog diagnosed with L. major, for the first time, in a focused area of human cutaneous leishmaniasis (CL) in the borderline between northern and central Iraq. Both molecular and phylogenetic studies showed that the dog L. major strain is closely related to that previously isolated from human CL in the same area. Moreover, serological study using rK39 identified IgG response against Leishmania, and the histological finding revealed the infiltration of inflammatory cells around the infection sites. These data will broaden our knowledge about CanL concerning the appearance of cutaneous clinical manifestations with mucocutaneous lesions caused by L. major. Further study on other animal reservoirs and vectors will shed the light on the epidemiology of this disease.
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Doenças do Cão/parasitologia , Leishmania infantum/isolamento & purificação , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/veterinária , Animais , Doenças do Cão/diagnóstico , Cães , Humanos , Iraque , Leishmania infantum/classificação , Leishmania infantum/genética , Leishmania infantum/fisiologia , Leishmaniose Cutânea/diagnóstico , Leishmaniose Visceral/parasitologia , Filogenia , Zoonoses/diagnóstico , Zoonoses/parasitologia , Zoonoses/transmissãoRESUMO
Canine leishmaniosis (CanL) is a major veterinary concern and a public health issue. Serological data are essential for disease management. Several antigens used in serological assays have specificity related problems preventing relevant seropositivity values establishment. Herein we report significant seropositivity level disparity in a study cohort with 384 dogs from eight countries, for antigens traditionally used in CanL - soluble promastigote Leishmania antigens (SPLA) and K39 recombinant protein (rK39): 43·8 and 2·9% for SPLA and rK39, respectively. To better understand the reasons for this disparity, CanL-associated serological response was characterized using, for complement serological evaluation, a ubiquitous antigen - soluble Escherichia coli antigens (SECAs). Using cohorts of CanL dogs and dogs without clinical evidences of CanL from non-endemic regions of Portugal, the serological response of CanL animals followed specific trend of seropositivity rK39 > SPLA > SECA absent in non-diseased animals. Using receiver operating characteristic curve analysis, these characteristic trends were converted in ratios, SPLA/SECA, rK39/SECA and rK39/SPLA, that presented high predictive for discriminating the CanL cohort that was potentiated when applied in a scoring system involving positivity to four out of five predictors (rK39, SPLA, SPLA/SECA, rK39/SECA and rK39/SPLA). In fact, this approach discriminated CanL with similar sensitivity/specificity as reference antigens, diminishing seropositivity in European cohort to 1·8%. Ultimately, non-related antigens like SECA and seropositivity ratios between antigens enable different perspectives into serological data focusing on the search of characteristic serological signatures and not simple absolute serology values contributing to comprehensive serological status characterization.
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Adenosina Trifosfatases/sangue , Antígenos de Bactérias/sangue , Antígenos de Protozoários/sangue , Proteínas de Bactérias/sangue , Doenças do Cão/diagnóstico , Escherichia coli/imunologia , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/veterinária , Canais de Translocação SEC/sangue , Animais , Doenças do Cão/imunologia , Cães , Ensaio de Imunoadsorção Enzimática/veterinária , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/imunologia , Portugal , Proteínas de Protozoários/sangue , Proteínas Recombinantes/sangue , Proteínas SecA , Sensibilidade e EspecificidadeRESUMO
Introduction: Visceral leishmaniasis (VL), a neglected tropical disease that causes substantial morbidity and mortality, is a serious health problem in Ethiopia. Infections are caused by Leishmania (L.) donovani parasites. Most individuals remain asymptomatic, but some develop VL, which is generally fatal if not treated. We identified the area of Metema-Humera in Northwest Ethiopia as a setting in which we could follow migrant workers when they arrived in an endemic area. The demographic characteristics of this population and factors associated with their risk of asymptomatic infection are poorly characterised. Methods: We divided our cohort into individuals who visited this area for the first time (first comers, FC) and those who had already been in this area (repeat comers, RC). We followed them from the beginning (Time 1, T1) to the end of the agricultural season (Time 2, T2), performing tests for sand fly bite exposure (anti-sand fly saliva antibody ELISA) and serology for Leishmania infection (rK39 rapid diagnostic test and the direct agglutination test) at each time point and collecting information on risk factors for infection. Results: Our results show that most migrant workers come from non-endemic areas, are male, young (median age of 20 years) and are farmers or students. At T1, >80% of them had been already exposed to sand fly bites, as shown by the presence of anti-saliva antibodies. However, due to seasonality of sand flies there was no difference in exposure between FC and RC, or between T1 and T2. The serology data showed that at T1, but not at T2, a significantly higher proportion of RC were asymptomatic. Furthermore, 28.6% of FC became asymptomatic between T1 and T2. Over the duration of this study, one FC and one RC developed VL. In multivariable logistic regression of asymptomatic infection at T1, only age and the number of visits to Metema/Humera were significantly associated with asymptomatic infection. Conclusion: A better understanding of the dynamics of parasite transmission and the risk factors associated with the development of asymptomatic infections and potentially VL will be essential for the development of new strategies to prevent leishmaniasis.
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The lack of accurate and feasible diagnostic tests poses a significant challenge to visceral leishmaniasis (VL) healthcare services in endemic areas. To date, various VL diagnostic tests have been or are being developed, and their diagnostic performances need to be assessed. In the present study, the diagnostic performances of rk39 RDT, the direct agglutination test (DAT), microscopy, loop-mediated isothermal amplification (LAMP), and miniature direct-on-blood polymerase chain reaction-nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) were assessed using quantitative polymerase chain reaction (qPCR) as the reference test in an endemic region of Ethiopia. In this study, 235 suspected VL cases and 104 non-endemic healthy controls (NEHCs) were recruited. Among the suspected VL cases, 144 (61.28%) tested positive with qPCR. The sensitivities for rk39 RDT, DAT, microscopy, LAMP assay, and mini-dbPCR-NALFIA were 88.11%, 96.50%, 76.58%, 94.33%, and 95.80%, respectively. The specificities were 83.33%, 97.96%, 100%, 97.38%, and 98.92% for rk39 RDT, DAT, microscopy, LAMP assay, and mini-dbPCR-NALFIA, respectively. In conclusion, rk39 RDT and microscopy exhibited lower sensitivities, while DAT demonstrated excellent performance. LAMP and mini-dbPCR-NALFIA showed excellent performances with feasibility for implementation in remote endemic areas, although the latter requires further evaluation in such regions.
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Visceral leishmaniasis (VL) was widely prevalent in Henan Province in the 1950s. Through active efforts by the government, there were no local cases reported from 1984 to 2015. In 2016, local VL cases reoccurred, and there was an increasing trend of VL cases in Henan Province. To provide a scientific control of VL, an investigation was conducted in Henan Province from 2016 to 2021. The data from VL cases were obtained from the Disease Surveillance Reporting System of the Chinese Center for Disease Control and Prevention. The rK39 immunochromatographic test (ICT) and PCR assay were performed among high-risk residents and all dogs in the patients' village. ITS1 was amplified, sequenced, and subjected to phylogenetic analyses. A total of 47 VL cases were reported in Henan Province during 2016-2021. Of the cases, 35 were local, and they were distributed in Zhengzhou, Luoyang, and Anyang. The annual average incidence was 0.008/100,000, showing an upward trend year by year (χ2 = 3.987, p = 0.046). Their ages ranged from 7 months to 71 years, with 44.68% (21/47) in the age group of 0-3 years and 46.81% (22/47) in the age group ≥15 years. The cases occurred throughout the year. The high-risk populations were infants and young children (age ≤3), accounting for 51.06% (24/47), followed by farmers at 36.17% (17/47). The ratio of males to females was 2.13:1. The positive rates of rK39 ICT and PCR were 0.35% (4/1130) and 0.21% (1/468) in the residents. The positive rates of rK39 ICT and PCR were 18.79% (440/2342) and 14.92% (139/929) in the dogs. The ITS1 amplification products in the patients and positive dogs were sequenced. The homology between the target sequence and Leishmania infantum was more than 98%. The phylogenetic analysis indicated that the patients and the positive dogs were infected by the same type of Leishmania, which was consistent with the strains in the hilly endemic areas in China. This paper showed that patients and domestic dogs were infected by the same type of L. infantum and that the positive rate in dogs was relatively high in Henan Province. Because the measures of patient treatment and culling of infected dogs were not effective in reducing VL incidence in Henan Province, it is urgent to develop new approaches for the control of VL, such as wearing insecticide-impregnated collars on dogs, treating the positive dogs, spraying insecticide for sandflies control, and improving residents' self-protection awareness to prevent the further spread of VL in Henan Province.
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Sri Lanka reports a focus of L. donovani induced cutaneous leishmaniasis (CL). Our more recent parasite and clinical studies and historical evidence point towards long term existence of Leishmania in the country, indicating a possible evolution leading to antigenic heterogenicity as well. In-house enzyme-linked immunosorbent assay (ELISA) that was developed during phase 1 study indicated >80% sero-positivity in local CL, while visceral leishmaniasis (VL) remained very rare with majority being negative when tested with rK39 assay. A novel serological tool was developed and sero-positivity of VL was assessed for the first time. The assay showed 100.0% sensitivity and 98.3% specificity for detection of VL. Samples were showed less positivity with established direct agglutination test (DAT) and rK39 strip test. The assay was less expensive than that of established rapid diagnostic tests (RDTs), culture and PCR assays. This assay may be useful in diagnosing clinical VL infections, detection of light microscopy (LM) negative patients, tracking post treatment stages, field screening of asymptomatic cases and in further serological studies.
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Leishmania donovani , Leishmaniose Cutânea , Leishmaniose Visceral , Humanos , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia , Antígenos de Protozoários , Testes Sorológicos , Ensaio de Imunoadsorção Enzimática , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/epidemiologia , Anticorpos Antiprotozoários , Sensibilidade e EspecificidadeRESUMO
Introduction: During several years of work in Sudan, we occasionally had been confronted with patients who presented clinical features highly suggestive of visceral leishmaniasis (VL) however direct agglutination test (DAT) readings that were either at the high negative or low positive titre range. Inquiries on the fate of those particular patients revealed mortality, undetermined diagnosis or that in some of them leukaemia was finally diagnosed. Gap statement: Investigate as to what extent haematological malignancies (HMs) interfere with VL diagnosis. Aim: Evaluate specificity of DAT version newly developed in this study wherein sodium dodecyle sulphate (SDS) was incorporated as a test sample denaturant in comparison with a standard reference wherein ß-mercaptoethanol (ß-ME) was used in test execution. Methodology: Seventy plasma samples from patients with HMs were collected and tested in a primary DAT version (P-DAT). The results obtained were compared with those of the rK39 strip test as VL reference diagnostic. HM samples revealing titres higher than the start dilution (1â:â100) in P-DAT were further tested in a ß-ME- and urea-modified DAT versions. The specificity of the newly developed SDS-DAT was assessed against that of ß-ME-DAT and rK39 strip tests as current reference diagnostics for VL. Results: Seven out of 70 patients with HMs scored positive outcomes (titre ≥1â:â3200) in P-DAT and four in the reference rK39 strip test. Of the seven that tested positive in P-DAT or four in the reference rK39, none reacted at titre >1â:â100 in the SDS-DAT. Significant reduction in non-specific agglutination reactions was achieved as a result in respect to the HM plasma samples (P value <0.05). Conclusion: To establish desired specificity for VL diagnosis in respect to HMs and subsequently minimize or avoid serious side effects due to unjustified anti-leishmanials prescription the combined application of the SDS-DAT here described and an improved version of the rK39 for confirmation is recommended.
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Visceral leishmaniasis (VL) is a disease caused by species of the Leishmania donovani complex that is mainly transmitted through the urban cycle involving dogs as the primary reservoir. In Colombia, the incidence of VL is increasing, along with the spread of potential vectors. This study aims to investigate the eco-epidemiological factors associated with Leishmania spp. infection in dogs from the Metropolitan Area of Bucaramanga (MAB), Santander, eastern Colombia, which is a region at risk for VL. We conducted molecular and serological surveillance of Leishmania spp. in 207 dogs from MAB to determine the epidemiological factors associated with infection. Subsequently, we carried out a molecular and serological analysis of phlebotomine and humans, respectively, in areas with a higher prevalence of infection, aiming to describe the main features associated with the transmission cycle. Out of the 207 dogs tested, 37 (17.8%, 95% CI = 12.6-23.1%) were positive for the presence of Leishmania antibodies by the IFAT test, and only 9 (4.3%, 95% CI = 1.55-7.15%) were positive for L. infantum by PCR. Multivariate analyses indicated that canine shelters and dogs with clinical signs commonly associated with canine VL had a higher prevalence of infection (P < 0.05). In the entomological survey, 69 blood-fed female phlebotomine of the genus Lutzomyia were captured in canine shelters, among them, 55% were identified as Lutzomyia camposi, 29% as Lu. ovallesi, 7% as Lu. dubitans, 6% as Lu. torvida, and 3% as Lu. cayennensis. The identified meal sources of the phlebotomine included human, pig, avian, cattle, and porcupine (Coendou quichua) blood. However, no phlebotomine positive for Leishmania spp. were detected by molecular analyses. Finally, 14 humans who had frequent contact with L. infantum-positive dogs were analyzed through rK39 test, but none tested was positive for IgG/IgM antibodies. The molecular and serological analyses indicate the circulation of L. infantum in dogs from MAB, with canine shelters having the highest prevalence of infection. The entomological survey of canine shelters showed a significant diversity of phlebotomine without potential vectors of L. infantum, suggesting the presence of infection in dogs from these areas could take place in other locations or through other transmission routes. The circulation of L. infantum in multiple dogs from MAB suggests a latent risk of zoonotic transmission of VL in these cities.
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BACKGROUND: Ethiopia is one of the top 10 countries in the world where 90% visceral leishmaniasis cases are reported. Metema-Humera lowlands are the most important foci in Ethiopia. Blood transfusion in visceral leishmaniasis endemic foci in Ethiopia does not consider screening of visceral leishmaniasis during blood donation. The aim of this study is therefore, was to assess asymptomatic Leishmania donovani infection and associated factors among blood donors attending at Metema district Blood Bank, Northwest Ethiopia. METHODS: A Health facility based cross-sectional study was conducted at Metema Blood Bank from February to March 2020. A total of 205 blood donors were eligible and participated in this study. Structured questionnaire were used to collect data on socio-demographic characteristics and perceived risk factors associated with asymptomatic visceral leishmaniasis among blood donors. Blood donors were screened using both rK39 and direct agglutination tests based on the manufactures' instructions. Data were analyzed using SPSS version 20.0. Chi-square test was used to assess associations of Leishmania donovani infection with predisposing factors. Associations were considered statstically significant on P-value < 0.05 at 95% confidence level. RESULTS: Of the total 205 participants, 32(15.6%) were positive for asymptomatic Leishmania donovani infection at least by one of the diagnostic tests used. Eight (3.9%) and 30(14.6%) of the participants` were positive by the rK39 and direct agglutination tests, respectively. Six (2.9%) donors were tested positive by both diagnostic tests. Family history of visceral leishmaniasis (X²=11.334, P = 0.003) and having neighbors with history of visceral leishmaniasis (X²=5.923, P = 0.015) were significantly associated with asymptomatic Leishmania donovani infection among blood donors. CONCLUSIONS: The prevalence of asymptomatic Leishmania donovani infection was 15.6%. Asymptomatic visceral leishmaniasis was significantly associated with donors' family and neighbors' history of infection. Therefore, laboratory screening of blood donors for visceral leishmaniasis in endemic areas will be mandatory. Moreover, this study will give base line information for future study in the country.
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Leishmania infantum, the sand fly-transmitted protozoan parasite responsible for leishmaniasis in humans, dogs, and cats, is endemic in the Iberian Peninsula. However, the impact of L. infantum infection on the conservation of the endangered Iberian lynx (Lynx pardinus) is unknown. Herein, we describe for the first time the occurrence of L. infantum infection among a population of reintroduced and wild-born L. pardinus living in the Portuguese Guadiana Valley Park. The presence of infection was addressed by molecular detection of Leishmania kinetoplast DNA (kDNA) in 35 lynxes, with further confirmation of L. infantum species performed by an internally transcribed spacer (ITS)-1 sequencing. Eight blood samples were positive for kDNA, and ITS-1 sequencing confirmed the presence of L. infantum in two of those samples. Exposure to Leishmania was screened in a group of 36 lynxes using an immunofluorescence antibody test (IFAT) and a multi-antigen enzyme-linked immunosorbent assay (ELISA), using SPLA, rK39, and CPX as Leishmania-specific antigens. Four animals presented a positive IFAT at a dilution of 1:40. Eight samples were considered seropositive to all ELISA Leishmania-specific antigens. Agreement between PCR, IFAT, and all ELISA antigens was found for 1 in 27 samples. These results highlight the susceptibility of autochthonous L. pardinus to L. infantum infection. Further investigation is required to assess the impact of L. infantum infection on this wild species conservation.
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Canine leishmaniosis (CanL) is a vector-borne disease caused by Leishmania infantum. Infection in dogs can result in a disease with non-specific clinical signs or in a subclinical condition. Infection diagnosis is crucial to guide public health measures considering the zoonotic potential of L. infantum. Serological approaches to detect infection with a reduced antigen panel potentially limit the quality of the information obtained. To evaluate the impact of using distinct antigens in a serological survey, a cohort with 390 dogs from endemic regions in Portugal was subjected to a serological evaluation using ELISA and DAT. Using ELISA, six Leishmania-specific antigens in conjunction with a non-related antigen, Escherichia coli soluble antigens, were evaluated. The global seroprevalence was 10.5% for DAT and 15.4 to 23.1% for ELISA, depending on the antigen for the latter. Still, only 8.2% of the animals were seropositive to all Leishmania-specific antigens. Importantly, a further 31.0% presented antigen-dependent seropositivity. Considering this observation, a serological score system was proposed and validated to address the complex serology results. With this system, the overall dog seropositivity was 26.9%. This work highlights the limitations of single-antigen serological surveys and presents an approach that might contribute to the establishment of CanL-specific serological profiles.
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Visceral leishmaniasis (VL) is on the verge of elimination on the Indian subcontinent. Nonetheless, the currently low VL-incidence setting brings along new challenges, one of which is the validity of the diagnostic algorithm, based on a combination of suggestive clinical symptoms in combination with a positive rK39 Rapid Diagnostic Test (RDT). With this study, we aimed to assess the positive predictive value of the diagnostic algorithm in the current low-endemic setting in India by re-assessing newly diagnosed VL patients with a qPCR analysis on venous blood as the reference test. In addition, we evaluated the specificity of the rK39 RDT by testing non-VL cases with the rK39 RDT. Participants were recruited in Bihar and Uttar Pradesh, India. VL patients diagnosed based on the diagnostic algorithm were recruited through six primary health care centers (PHCs); non-VL cases were identified through a door-to-door survey in currently endemic, previously endemic, and non-endemic clusters, and tested with rK39 RDT, as well as-if positive-with qPCR on peripheral blood. We found that 95% (70/74; 95% CI 87-99%) of incident VL cases diagnosed at the PHC level using the current diagnostic algorithm were confirmed by qPCR. Among 15,422 non-VL cases, 39 were rK39 RDT positive, reflecting a specificity of the test of 99.7% (95% CI 99.7-99.8%). The current diagnostic algorithm combining suggestive clinical features with a positive rK39 RDT still seems valid in the current low-endemic setting in India.
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Clinical diagnosis has become a challenge amidst a surge of cutaneous leishmaniasis in Southern Sri Lanka. The routine diagnostic method, slit-skin smear (SSS), has variable sensitivity, leading to undiagnosed cases. Improved diagnostics are urgently needed. We assessed a new in-house ELISA method for its diagnostic capabilities against ITS-1 nested PCR (gold standardGs). A cohort of 190 clinical CL cases was examined by SSS microscopy, anti-rKRP42 IgG ELISA (serum- and urine-based), and rK39-Immunochromatographic strip test. Validation was done using non-endemic sera, and cutoffs were developed using the receiver operating curve. The sensitivity of SSS for case detection was 77.9% (authors) and 76.3% (technicians). ELISA vs. Gs demonstrated sensitivity (Sn) = 94.4%; specificity (Sp) = 50.0%; positive predictive value (PPV) = 97.1%; negative predictive value (NPV) = 33.3%; Kappa agreement (Kp) = 0.39/p < 0.01. Comparison of the combination method (SSS by technicians and ELISA) vs. Gs showed: Sn = 98.9%; Sp = 30.0; PPV = 96.2; NPV 60.0%; Kp = 0.378/p < 0.01. All methods performed better compared to SSS (29.4%) where the clinical diagnosis was doubtful (PCR = 94.15%; serum ELISA = 88.2%; combination = 94.1%; p < 0.01 for all). High serum anti-rKRP42 titers were seen in those with multiple lesions. Anti-rKRP42 urine ELISA was suboptimal as a diagnostic test. A 9% rate of positivity was seen for rk39-ICT, and positives recorded high anti-rKRP42 titers. The diagnostic accuracy can be increased above the level of the Gs by combining SSS and ELISA. Advanced studies are required to understand the association between rk39-ICT positivity and high anti-rKRP42 titers.
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BACKGROUND: Recombinant antigens rK39 (based on kinesin sequence) and rK28 (comprising kinesin and HASPB sequences) are a mainstay of serological diagnosis for visceral leishmaniasis (VL). However, their key epitopes and the significance of their structural conformation are not clearly defined, particularly in relation to reported cross-reactivity with sera from patients with malaria, schistosomiasis, and tuberculosis. METHODS: To assess the effect of conformation on antigenicity with Sudanese VL sera, antigens rK39 and rK28 were heat-denatured at 95 °C for 10 min and then assayed by enzyme-linked immunosorbent assay (ELISA). Amino acid sequences of rK39 and rK28 were submitted to NCBI BLASTp to assess homology with Plasmodium, Schistosoma, and Mycobacterium. RESULTS: Heat denaturation significantly diminished the antigenicity of rK39 compared to non-denatured antigen (P = 0.001), but not for rK28 (P = 0.275). In BLASTp searches, HASPB sequences from rK28 had similarities with sequences from Plasmodium, encompassing software-predicted B-cell epitopes. CONCLUSIONS: The antigenicity of rK39 appears to be dependent on structural conformation, whereas that of rK28 depends on linear sequence. HASPB sequence homology with Plasmodium may be responsible for the reported cross-reactivity of rK28 with malaria sera. Further work is warranted to refine the specificity of these antigens.
Assuntos
Leishmaniose Visceral , Humanos , Leishmaniose Visceral/diagnóstico , Antígenos de Protozoários/genética , Cinesinas , Epitopos de Linfócito B/genética , Proteínas de Protozoários , Sensibilidade e EspecificidadeRESUMO
Introduction: A range of assays have been developed to detect specific antileishmanial antibody, such as rK 39 immunochromatographic test (ICT), KE 16 ICT, ELISA test, and indirect immunofluorescent antibody test (IFAT), which play a crucial role in serological diagnosis of visceral leishmaniasis (VL). However, limited published reports are available on the utility of serological test (IFAT test/rk 39), smear examination, and culture in the diagnosis of VL and post-kala-azar dermal leishmaniasis (PKDL) in our country. Materials and Methods: We present utility of serological test (IFAT test/rK 39), smear examination for Leishmania donovani (LD) bodies, and culture in 2589 samples from 2294 VL/PKDL suspected patients (January 2009-December 2019) tested in Centre for Arboviral and Zoonotic diseases, National Centre for Disease Control, New Delhi, India, for laboratory diagnosis of VL/PKDL. Results: A total of 80/553 (14.4%) cases were confirmed of VL (74/522 cases by demonstration of LD bodies in bone marrow smear examination, 5/12 in splenic smear examination 1/19 by culture) and 4/21 (19.0%) cases were confirmed of PKDL (demonstration of LD bodies in slit skin smear examination. In our study 197/1368 (14.4%) cases were diagnosed positive by IFAT, 34/646 (5.2%) cases by rk 39 ICT for VL/PKDL by demonstration of specific antileishmanial antibodies. Conclusion: As the goal of elimination of VL as a public health problem is approaching, apart from serological tests such as rk 39 and IFAT, direct methods of detection such as (parasitic demonstration in BM smear, culture, and molecular tests) for Leishmania may play a crucial role for achieving a correct diagnosis and treatment. We also concluded that IFAT though not field-friendly, its optimal use as an adjunct test with BM smear in all stages of infections may be required. Further rk39 is a simple, reliable, noninvasive, and field-friendly test for diagnosis VL, especially in endemic areas.