RESUMO
The clinical-immunological spectrum of human Leishmania (L.) infantum chagasi-infections in the Brazilian Amazon has been defined using DTH/IFAT-IgG immune assays and the clinical statuses of infected individuals, revealing five profiles: three asymptomatic [Asymptomatic Infection (AI), Subclinical Resistant Infection (SRI), and Indeterminate Initial Infection (III)], and two symptomatic profiles [Subclinical Oligosymptomatic Infection (SOI) and Symptomatic Infection (SI = American visceral leishmaniasis/AVL)]. We evaluated the diagnostic potential of urine qPCR over the entire spectrum of infection. Resine Instagene Matrix® was used for DNA extraction from urinary sediment, with amplification carried out using SYBR® Green Taq with the RV1 and RV2 primers. We examined urine samples from 151 individuals from an endemic area of AVL in Pará State in the Brazilian Amazon, including: 91 (60.3%) with diagnoses of previous infections [13 (14.3%) sharing the AI profile, 13 (14.3%) with the SRI profile, 43 (47.2%) with III, 12 (13.2%) with SI (treated AVL), and 10 (11%) with SI (untreated AVL)]; sixty (39.7%) were DTH(-)/IFAT-IgG(-) (the uninfected group). The urine qPCR was positive in 61.5% of both the AI and SRI profiles, 65% of the III profile, 50% of treated AVL, 100% of untreated AVL, and 6.7% of the uninfected group. Those results confirmed the urine qPCR diagnosis in 100% of untreated AVL cases as well as in more than 60% of the cases with asymptomatic AI, SRI, and III profiles - indicating it as a promising tool for monitoring the evolution of human L. (L.) infantum chagasi-infections in endemic areas.
Assuntos
Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Infecções Assintomáticas , Brasil , Feminino , Humanos , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/urina , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Adulto JovemRESUMO
Visceral leishmaniasis is a serious and debilitating infection with high fatality rate in tropical and subtropical countries. As clinical symptoms of visceral leishmaniasis are not so specific, confirmatory diagnostic methods with high sensitivity and specificity are needed. Noninvasive methods have been developed using urine as a clinical sample for visceral leishmaniasis diagnosis. In fact, there is a clear correlation between kidney impairment and Leishmania DNA in urine. However, it has been proved that Leishmania nucleic acid may also be isolated from patients without any sign of renal involvement. Even though urine has become a promissing biological sample, it is still not widely used due to several issues, such as (i) incomprehension of the whole renal pathophysiology process in visceral leishmaniasis, (ii) presence of many amplification inhibitors in urine, and (iii) lack of an efficient urinary DNA extraction method. In this article, we performed a literature review to bring a new perspective for Leishmania DNA isolation in urine.
Assuntos
DNA de Protozoário/urina , Leishmania/genética , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/urina , DNA de Protozoário/isolamento & purificação , Humanos , Leishmania/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
ABSTRACT Visceral leishmaniasis is a serious and debilitating infection with high fatality rate in tropical and subtropical countries. As clinical symptoms of visceral leishmaniasis are not so specific, confirmatory diagnostic methods with high sensitivity and specificity are needed. Noninvasive methods have been developed using urine as a clinical sample for visceral leishmaniasis diagnosis. In fact, there is a clear correlation between kidney impairment and Leishmania DNA in urine. However, it has been proved that Leishmania nucleic acid may also be isolated from patients without any sign of renal involvement. Even though urine has become a promissing biological sample, it is still not widely used due to several issues, such as (i) incomprehension of the whole renal pathophysiology process in visceral leishmaniasis, (ii) presence of many amplification inhibitors in urine, and (iii) lack of an efficient urinary DNA extraction method. In this article, we performed a literature review to bring a new perspective for Leishmania DNA isolation in urine.
Assuntos
Humanos , DNA de Protozoário/urina , Leishmania/genética , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/urina , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos Testes , DNA de Protozoário/isolamento & purificação , Sensibilidade e Especificidade , Leishmania/isolamento & purificaçãoRESUMO
Visceral leishmaniasis (VL) is a serious and fatal disease caused by the parasites Leishmania infantum and Leishmania donovani The gold standard diagnostic test for VL is the demonstration of parasites or their DNA in spleen, lymph node, or bone marrow aspirates. Serological tests exist but cannot distinguish active VL from either prior exposure to the parasites or previously treated VL disease. Using mass spectroscopy, we have previously identified three L. infantum protein biomarkers (Li-isd1, Li-txn1, and Li-ntf2) in the urine of VL patients and developed a sensitive and specific urine-based antigen detection assay for the diagnosis of VL that occurs in Brazil (where VL is caused by L. infantum). However, unpublished observations from our laboratory at DetectoGen showed that these biomarkers were detected in only 55% to 60% of VL patients from India and Kenya, where the disease is caused by L. donovani Here, we report the discovery and characterization of two new biomarkers of L. donovani (Ld-mao1 and Ld-ppi1) present in the urine of VL patients from these two countries. Capture enzyme-linked immunosorbent assays using specific rabbit IgG and chicken IgY were developed, and the assays had sensitivities of 44.4% and 28.8% for the detection of Ld-mao1 and Ld-ppi1, respectively. In contrast, a multiplexed assay designed to simultaneously detect all five leishmanial biomarkers markedly increased the assay sensitivity to 82.2%. These results validate the utility of leishmanial protein biomarkers found in the urine of VL patients as powerful tools for the development of an accurate diagnostic test for this disease.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/urina , Proteínas de Protozoários/urina , Adolescente , Adulto , Idoso , Anticorpos Antiprotozoários , Biomarcadores/urina , Brasil , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Índia , Quênia , Leishmania donovani/isolamento & purificação , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/parasitologia , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Adulto JovemRESUMO
A leishmaniose visceral canina (LVC) é considerada uma zoonose importante, que tem se propagado com grande agilidade, e em regiões metropolitanas, como o município deCaucaia, no Ceará, a doença já é considerada endêmica. Conceituada como uma enfermidade de caráter crônico, a mesma é capaz de progredir para um quadro fatal, e oscães são os principais reservatórios domésticos, o que implica o contato íntimo do parasita e homem. Apesar de a patologia ser sistemicamente severa, não há até o momento teste específico para seu diagnóstico. Assim, associação de várias técnicas de diagnóstico pode ser benéfica e permitir o diagnóstico mais precoce devido a sinalizações de necessidade de pesquisa de cães assintomáticos. Neste contexto, há poucos estudos que relatam sobre as alterações ultrassonográficas presentes em animais positivos para leishmaniose. Este trabalho tem como objetivo fazer uma revisão acerca dos principais aspectos de diagnósticos da leishmaniose desde os principais sintomas clínicos e dos achados laboratoriais, tais como hemograma, bioquímicas sérias, sumário de urina, achados ultrassonográficos, histopatológicos de animais naturalmente infectados pela Leishmaniose Visceral Canina.
Visceral canine leishmaniasis (LCV) is considered an important zoonosis, which hasspread with great agility, and in metropolitan regions, such as the municipality of CaucaiaCeará, the disease is already considered endemic. Conceptualized as a chronic disease, it is able to progress to a fatal condition, and dogs are the main domestic reservoirs, which implies the intimate contact of the parasite and man. Although the pathology is systemically severe, there is no up to now specific test for its diagnosis. Thus, association of various diagnostic techniques may be beneficial and allow earlier diagnosis due to signs of need for asymptomatic dog research. In this context, few studies report on sonographicchanges present in animals positive for Leishmaniasis. This work aims to review the main diagnostic aspects of Leishmaniasis from the main clinical symptoms and laboratory findings such as hemogram, serious biochemistry, urine summary, ultrasonographic and histopathological findings of animals naturally infected with Visceral Canine Leishmaniasis.
Assuntos
Animais , Cães , Leishmaniose Visceral/diagnóstico por imagem , Leishmaniose Visceral/sangue , Leishmaniose Visceral/urina , Leishmaniose Visceral/veterinária , Ultrassonografia/veterináriaRESUMO
A leishmaniose visceral canina (LVC) é considerada uma zoonose importante, que tem se propagado com grande agilidade, e em regiões metropolitanas, como o município deCaucaia, no Ceará, a doença já é considerada endêmica. Conceituada como uma enfermidade de caráter crônico, a mesma é capaz de progredir para um quadro fatal, e oscães são os principais reservatórios domésticos, o que implica o contato íntimo do parasita e homem. Apesar de a patologia ser sistemicamente severa, não há até o momento teste específico para seu diagnóstico. Assim, associação de várias técnicas de diagnóstico pode ser benéfica e permitir o diagnóstico mais precoce devido a sinalizações de necessidade de pesquisa de cães assintomáticos. Neste contexto, há poucos estudos que relatam sobre as alterações ultrassonográficas presentes em animais positivos para leishmaniose. Este trabalho tem como objetivo fazer uma revisão acerca dos principais aspectos de diagnósticos da leishmaniose desde os principais sintomas clínicos e dos achados laboratoriais, tais como hemograma, bioquímicas sérias, sumário de urina, achados ultrassonográficos, histopatológicos de animais naturalmente infectados pela Leishmaniose Visceral Canina.(AU)
Visceral canine leishmaniasis (LCV) is considered an important zoonosis, which hasspread with great agility, and in metropolitan regions, such as the municipality of CaucaiaCeará, the disease is already considered endemic. Conceptualized as a chronic disease, it is able to progress to a fatal condition, and dogs are the main domestic reservoirs, which implies the intimate contact of the parasite and man. Although the pathology is systemically severe, there is no up to now specific test for its diagnosis. Thus, association of various diagnostic techniques may be beneficial and allow earlier diagnosis due to signs of need for asymptomatic dog research. In this context, few studies report on sonographicchanges present in animals positive for Leishmaniasis. This work aims to review the main diagnostic aspects of Leishmaniasis from the main clinical symptoms and laboratory findings such as hemogram, serious biochemistry, urine summary, ultrasonographic and histopathological findings of animals naturally infected with Visceral Canine Leishmaniasis.(AU)
Assuntos
Animais , Cães , Leishmaniose Visceral/sangue , Leishmaniose Visceral/diagnóstico por imagem , Leishmaniose Visceral/veterinária , Leishmaniose Visceral/urina , Ultrassonografia/veterináriaRESUMO
OBJECTIVE: To evaluate the usefulness of early acute kidney injury (AKI) biomarkers in clinical management of visceral leishmaniasis. METHODS: Prospective study with 50 hospitalised VL patients. AKI biomarkers, that is, serum and urinary neutrophil gelatinase-associated lipocalin (sNGAL, uNGAL, respectively), urinary kidney injury molecule-1 (uKIM-1) and urinary monocyte chemotactic protein-1 (uMCP-1), were quantified by immunoassay (ELISA). Also, interferon-gamma (INF-y) and C-reactive protein (CRP) were evaluated as inflammatory biomarkers possibly related to VL severity. RESULTS: VL patients had hyponatremia, hypoalbuminemia, hypergammaglobulinemia, haematologic and hepatic disorders. AKI was found in 46%, and one death (2%) occurred. The AKI group had significant longer hospital stay, lower levels of IFN-y and higher levels of CRP, more clinical renal abnormalities and higher levels of sNGAL, uNGAL, uKIM-1 and uMCP-1. Overall, sNGAL, uKIM-1 and uMCP-1 showed correlations with important clinical renal abnormalities, such as proteinuria, albuminuria, serum creatinine and glomerular filtration rate using adjusted correlations with CRP and IFN-y. Only sNGAL showed an early association with AKI development (OR = 2.78, 95% CI = 1.429-5.428, per each increase of 50 ng/ml), even after adjusting for clinical signals of VL severity and for immune biomarkers. Moreover, sNGAL showed a better performance in predicting AKI development (AUC-ROC = 0.81, 95% CI = 0.69-0.93; cut-off = 154 ng/ml, sensitivity = 82.6%, specificity = 74.1%, P < 0.001). CONCLUSIONS: Visceral leishmaniasis-associated nephropathy showed important proximal tubular injury and glomerular inflammation. Serum NGAL showed an early association with VL-associated nephropathy and may be used to improve clinical management strategies and decrease morbimortality in VL patients.
Assuntos
Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/parasitologia , Leishmaniose Visceral/sangue , Leishmaniose Visceral/urina , Proteínas de Fase Aguda/metabolismo , Adulto , Biomarcadores/sangue , Biomarcadores/urina , Brasil , Proteína C-Reativa/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Interferon gama/metabolismo , Lipocalina-2/metabolismo , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
Leishmania infantum is a protozoan that causes visceral leishmaniasis, a potentially deadly neglected tropical disease. The gold standard for diagnosis has traditionally been detection of amastigotes in bone marrow or spleen aspirates, but this is an invasive procedure that carries the risk of serious complications. Newer PCR techniques are opening new avenues and tissues for testing. Therefore, we tested if amastigotes and DNA from L. infantum could be detected in patient urine. We detected L. infantum DNA in six out of 30 urine samples from patients with visceral leishmaniasis and the promastigotes were isolated in culture from the urine of one patient. These results suggest the feasibility of using urine samples to diagnose visceral leishmaniasis, especially in acute cases or renal infection, providing a valuable tool for doctors and clinicians to use for screening and diagnosis of leishmaniasis in patients.
Assuntos
Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/urina , Animais , Brasil , Humanos , Leishmaniose Visceral/parasitologia , Reação em Cadeia da Polimerase/métodosRESUMO
The availability of some sorts of biological samples which require noninvasive collection methods has led to an even greater interest in applying molecular biology on visceral leishmaniasis (VL) diagnosis, since these samples increase the safety and comfort of both patients and health professionals. In this context, this work aimed to evaluate the suitability of the urine as a specimen for Leishmania infantum kinetoplast DNA detection by real-time quantitative PCR (qPCR). Subsequent to the reproducibility analysis, the detection limit of the qPCR assay was set at 5fg (~0.025 parasites) per µL of urine. From the comparative analysis performed with a set of diagnostic criteria (serological and molecular reference tests), concordance value of 96.08% was obtained (VL-suspected and HIV/AIDS patients, n=51) (P>0.05). Kappa coefficient (95% CI) indicated a good agreement between the test and the set of diagnostic criteria (k=0.778±0.151). The detection of Leishmania DNA in urine by qPCR was possible in untreated individuals, and in those with or without suggestive renal impairment. Fast depletion of the parasite's DNA in urine after treatment (from one dose of meglumine antimoniate) was suggested by negative qPCR results, thus indicating it as a potential alternative specimen to follow up the efficacy of therapeutic approaches. Even when evaluated in a clinically heterogeneous set of patients, the urine showed good prospect as sample for VL diagnosis by qPCR, also indicating a good negative predictive value for untreated suspected patients.
Assuntos
DNA de Cinetoplasto/isolamento & purificação , DNA de Cinetoplasto/urina , Leishmania infantum/genética , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/urina , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Urina/parasitologia , Síndrome da Imunodeficiência Adquirida/complicações , Adolescente , Adulto , Idoso , Brasil , Criança , Creatinina/sangue , Creatinina/urina , DNA de Cinetoplasto/sangue , DNA de Cinetoplasto/genética , DNA de Protozoário/sangue , DNA de Protozoário/isolamento & purificação , DNA de Protozoário/urina , Feminino , HIV/patogenicidade , Humanos , Leishmania infantum/patogenicidade , Leishmaniose Visceral/sangue , Leishmaniose Visceral/parasitologia , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Ureia/sangue , Ureia/urina , Adulto JovemRESUMO
BACKGROUND: Visceral leishmaniasis (VL) can be fatal without timely diagnosis and treatment. Treatment efficacies vary due to drug resistance, drug toxicity and co-morbidities. It is important to monitor treatment responsiveness to confirm cure and curtail relapse. Currently, microscopy of spleen, bone marrow or lymph node biopsies is the only definitive method to evaluate cure. A less invasive test for treatment success is a high priority for VL management. METHODS: In this study, we describe the development of a capture ELISA based on detecting Leishmania donovani antigens in urine samples and comparison with the Leishmania Antigen ELISA, also developed for the same purpose. Both were developed as prototype kits and tested on patient urine samples from Sudan, Ethiopia, Bangladesh and Brazil, along with appropriate control samples from endemic and non-endemic regions. Sensitivity and specificity were assessed based on accurate detection of patients compared to control samples. One-Way ANOVA was used to assess the discrimination capacity of the tests and Cohen's kappa was used to assess their correlation. RESULTS: The Leishmania Antigen Detect ELISA demonstrated >90% sensitivity on VL patient samples from Sudan, Bangladesh and Ethiopia and 88% on samples from Brazil. The Leishmania Antigen ELISA was comparable in performance except for lower sensitivity on Sudanese samples. Both were highly specific. To confirm utility in monitoring treatment, urine samples were collected from VL patients at days 0, 30 and 180 post-treatment. For the Leishmania Antigen Detect ELISA, positivity was high at day 0 at 95%, falling to 21% at day 30. At day 180, all samples were negative, corresponding well with clinical cure. A similar trend was also seen for the Leishmania Antigen ELISA albeit; with lower positivity of 91% at Day 0 and more patients, remaining positive at Days 30 and 180. DISCUSSION: The Leishmania Antigen Detect and the Leishmania Antigen ELISAs are standardized, user- friendly, quantitative and direct tests to detect Leishmania during acute VL as well as to monitor parasite clearance during treatment. They are a clear improvement over existing options. CONCLUSION: The ELISAs provide a non-invasive method to detect parasite antigens during acute infection and monitor its clearance upon cure, filling an unmet need in VL management. Further refinement of the tests with more samples from endemic regions will define their utility in monitoring treatment.