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BACKGROUND: People with human immunodeficiency virus (PWH) with recurrent visceral leishmaniasis (VL) could potentially drive Leishmania transmission in areas with anthroponotic transmission such as East Africa, but studies are lacking. Leishmania parasitemia has been used as proxy for infectiousness. METHODS: This study is nested within the Predicting Visceral Leishmaniasis in HIV-InfectedPatients (PreLeisH) prospective cohort study, following 490 PWH free of VL at enrollment for up to 24-37 months in northwest Ethiopia. Blood Leishmania polymerase chain reaction (PCR) was done systematically. This case series reports on 10 PWH with chronic VL (≥3 VL episodes during follow-up) for up to 37 months, and 3 individuals with asymptomatic Leishmania infection for up to 24 months. RESULTS: All 10 chronic VL cases were male, on antiretroviral treatment, with 0-11 relapses before enrollment. Median baseline CD4 count was 82â cells/µL. They displayed 3-6 VL treatment episodes over a period up to 37 months. Leishmania blood PCR levels were strongly positive for almost the entire follow-up (median cycle threshold value, 26 [interquartile range, 23-30]), including during periods between VL treatment. Additionally, we describe 3 PWH with asymptomatic Leishmania infection and without VL history, with equally strong Leishmania parasitemia over a period of up to 24 months without developing VL. All were on antiretroviral treatment at enrollment, with baseline CD4 counts ranging from 78 to 350â cells/µL. CONCLUSIONS: These are the first data on chronic parasitemia in PWH from Leishmania donovani-endemic areas. PWH with asymptomatic and symptomatic Leishmania infection could potentially be highly infectious and constitute Leishmania superspreaders. Xenodiagnosis studies are required to confirm infectiousness.
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Infecções por HIV , Leishmaniose Visceral , Parasitemia , Humanos , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/transmissão , Etiópia/epidemiologia , Masculino , Infecções por HIV/complicações , Infecções por HIV/epidemiologia , Adulto , Parasitemia/epidemiologia , Parasitemia/parasitologia , Estudos Prospectivos , Pessoa de Meia-Idade , Doenças Endêmicas , Contagem de Linfócito CD4 , Reação em Cadeia da PolimeraseRESUMO
Background: Diagnosis of cutaneous leishmaniasis (CL) usually relies on invasive samples, but it is unclear whether more patient-friendly tools are good alternatives for diverse lesions when used with polymerase chain reaction (PCR). Methods: Patients with suspected CL were enrolled consecutively in a prospective diagnostic accuracy study. We compared dental broach, tape disc, and microbiopsy samples with PCR as index tests, using PCR with skin slit samples as reference test. Subsequently, we constructed a composite reference test including microscopy, the 3 index tests and skin slit PCR, and we compared these same tests with the composite reference test. We assessed diagnostic accuracy parameters with 95% confidence intervals for all comparisons. Results: Among 344 included patients, 282 (82.0%) had CL diagnosed, and 62 (18.0%) CL absence, by skin slit PCR. The sensitivity and specificity by PCR were 89.0% (95% confidence interval, 84.8%-92.1%) and 58.1% (45.7%-69.5%), respectively, for dental broach, 96.1% (93.2%-97.8%) and 27.4% (17.9%-39.6%) for tape disc, and 74.8% (66.3%-81.7%) and 72.7% (51.8%-86.8%) for microbiopsy. Several reference test-negative patients were consistently positive with the index tests. Using the composite reference test, dental broach, and skin slit had similar diagnostic performance. Discussion: Dental broach seems a less invasive but similarly accurate alternative to skin slit for diagnosing CL when using PCR. Tape discs lack specificity and seem unsuitable for CL diagnosis without cutoff. Reference tests for CL are problematic, since using a single reference test is likely to miss true cases, while composite reference tests are often biased and impractical as they require multiple tests.
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A large proportion of HIV-coinfected visceral leishmaniasis (VL-HIV) patients exhibit chronic disease with frequent VL recurrence. However, knowledge on immunological determinants underlying the disease course is scarce. We longitudinally profiled the circulatory cellular immunity of an Ethiopian HIV cohort that included VL developers. We show that chronic VL-HIV patients exhibit high and persistent levels of TIGIT and PD-1 on CD8+/CD8- T cells, in addition to a lower frequency of IFN-γ+ TIGIT- CD8+/CD8- T cells, suggestive of impaired T cell functionality. At single T cell transcriptome and clonal resolution, the patients show CD4+ T cell anergy, characterised by a lack of T cell activation and lymphoproliferative response. These findings suggest that PD-1 and TIGIT play a pivotal role in VL-HIV chronicity, and may be further explored for patient risk stratification. Our findings provide a strong rationale for adjunctive immunotherapy for the treatment of chronic VL-HIV patients to break the recurrent disease cycle.
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Coinfecção , Infecções por HIV , Leishmaniose Visceral , Humanos , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/complicações , Leishmaniose Visceral/parasitologia , Infecções por HIV/imunologia , Infecções por HIV/complicações , Coinfecção/imunologia , Masculino , Adulto , Feminino , Linfócitos T CD8-Positivos/imunologia , Pessoa de Meia-Idade , Doença Crônica , Linfócitos T CD4-Positivos/imunologia , EtiópiaRESUMO
BACKGROUND: As untreated visceral leishmaniasis (VL) is fatal, reliable diagnostics are pivotal for accurate treatment allocation. The current diagnostic algorithm for VL in Ethiopia, which is based on the rK39 rapid diagnostic test and microscopy of tissue smears, lacks sensitivity. This probably leads to missed cases and patients not receiving treatment. METHODOLOGY: We conducted a retrospective study on stored microscopically negative spleen and bone marrow smears from suspected VL patients collected at the Leishmaniasis Research and Treatment Center (LRTC) in Gondar, northern Ethiopia between June 2019 and November 2020. Sociodemographic, clinical and treatment data were collected and samples were tested by real-time PCR targeting kinetoplast DNA. PRINCIPLE FINDINGS: Among the 191 eligible samples (135 spleen and 56 bone marrow) with a microscopically negative and valid PCR result, 119 (62.3%) were positive by PCR, although Ct values for some were high (median 33.0). Approximately three quarters of these undiagnosed primary VL (77.3%) and relapse (69.6%) patients did not receive antileishmanial treatment. Of the 56 microscopically negative bone marrow samples, 46 (82.1%) were PCR positive, which is considerably higher compared to the microscopically negative spleen samples, for which 73 out of 135 (54.1%) were PCR positive. The odds of being PCR positive were significantly higher for bone marrow aspirates and higher when white blood cell values were lower and splenomegaly (in cm) was more pronounced. CONCLUSIONS: This study demonstrates that a lot of suspected VL patients remain undiagnosed and untreated. This indicates the urgent need for better diagnostics for VL in the East-African region. The outcomes of PCR positive should be closely monitored and treatment should be provided if the patient deteriorates. In resource limited settings, implementation of PCR on bone marrow aspirate smears of patients with low WBC values and splenomegaly could lead to considerable improvements in patient management.
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Leishmania donovani , Leishmaniose Visceral , Humanos , Leishmania donovani/genética , Estudos Retrospectivos , Esplenomegalia , Etiópia , Sensibilidade e Especificidade , Leishmaniose Visceral/diagnóstico , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Background: Cutaneous leishmaniasis (CL) is a common, yet massively underreported skin morbidity in Ethiopia. Most patients never seek treatment, as this is offered only in specialized treatment centers. Early diagnosis and treatment through decentralization is crucial to decrease transmission and to reach the NTD roadmap goals. However, little information is available on outcomes and challenges of community-based treatment initiatives. Methods: A community-based prospective cohort study was conducted in Ochollo. Patients with clinically or microscopy confirmed CL were included. Cryotherapy was (to be) given weekly with at least four sessions for uncomplicated lesions, and miltefosine was given for 4 weeks for complicated lesions. Miltefosine adherence was assessed by counting pill strips. Clinical and patient-reported outcomes (dermatological life quality index and patient-global assessment) were assessed at month 6 (M6). Results: A total of 107 patients were included, with a median age of 6 years. Two patients refused, and 15 could not be treated as they were too young (<4 years) for miltefosine. Giving cryotherapy to patients weekly was not feasible due to long wound healing times and required use of topical antibiotics. Only 52.4% of miltefosine patients finished >90% of their tablets by M1. Among 46 patients treated with cryotherapy, 24 (52.2%) were cured at M6, and 9 (19.6%) had substantial improvement. The cure rate was 16/39 (41.0%) for miltefosine with 28.2% (11/39) substantial improvement. Before treatment, more than half (57.8%) of patients reported that CL did not negatively impact their life, which significantly increased to 95.2% at M6. At this time, 61.7% of patients said their lesion was clear, which was 1% before treatment. Conclusion: Our study is the first to identify the challenges and opportunities of miltefosine and cryotherapy for community treatment of CL. Although overall cure rates were lower than expected, patient-reported outcomes were generally positive and quite some patients had good improvement.
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BACKGROUND: Leishmaniasis is a common neglected tropical disease in Ethiopia. Visceral leishmaniasis (VL) caused by Leishmania donovani presents in the lowlands, while cutaneous leishmaniasis (CL) affects people living in the highlands. Although CL is described as being caused by Leishmania aethiopica, there is also evidence of L. tropica and L. major isolated from a patient, sand flies and potential reservoirs. Information on species causing CL in Ethiopia is patchy, and no nation-wide study has ever been done. Understanding which species are causing CL in Ethiopia can have important implications for patient management and disease prevention. METHODS: We analyzed stored routine samples and biobanked DNA isolates from previously conducted studies of CL patients from different centers in the north, center and south of Ethiopia. Species typing was performed using ITS-1 PCR with high-resolution melt (HRM) analysis, followed by HSP70 amplicon sequencing on a selection of the samples. Additionally, sociodemographic, clinical and laboratory data of patients were analyzed. RESULTS: Of the 226 CL samples collected, the Leishmania species could be determined for 105 (45.5%). Leishmania aethiopica was identified in 101 (96.2%) samples from across the country. In four samples originating from Amhara region, northwestern Ethiopia, L. donovani was identified by ITS-1 HRM PCR, of which two were confirmed with HSP70 sequences. While none of these four patients had symptoms of VL, two originated from known VL endemic areas. CONCLUSIONS: The majority of CL was caused by L. aethiopica, but CL due to L. tropica and L. major cannot be ruled out. Our study is the first to our knowledge to demonstrate CL patients caused by L. donovani in Ethiopia. This should spark future research to investigate where, how and to which extent such transmission takes place, how it differs genetically from L. donovani causing VL and whether such patients can be diagnosed and treated successfully with the currently available tools and drugs.
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Leishmania donovani , Leishmaniose Cutânea , Leishmaniose Visceral , Humanos , Leishmania donovani/genética , Etiópia/epidemiologia , Leishmaniose Cutânea/epidemiologia , Leishmaniose Visceral/epidemiologia , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: Cutaneous leishmaniasis (CL) is common in Ethiopia, mainly affecting impoverished populations in rural areas with poor access to health care. CL is routinely diagnosed using skin slit smear microscopy, which requires skilled staff and appropriately equipped laboratories. We evaluated the CL Detect Rapid Test (InBios, Washington, USA), which is supplied with a dental broach sampling device, as a diagnostic alternative which could be used in field settings. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the diagnostic accuracy of the CL Detect Rapid Test on skin slit and dental broach samples from suspected CL patients at the Leishmaniasis Research and Treatment Center in Gondar, Ethiopia. A combined reference test of microscopy and PCR on the skin slit sample was used, which was considered positive if one of the two tests was positive. We recruited 165 patients consecutively, of which 128 (77.6%) were confirmed as CL. All microscopy-positive results (n = 71) were also PCR-positive, and 57 patients were only positive for PCR. Sensitivity of the CL Detect Rapid Test on the skin slit was 31.3% (95% confidence interval (CI) 23.9-39.7), which was significantly higher (p = 0.010) than for the dental broach (22.7%, 95% CI 16.3-30.6). Sensitivity for both methods was significantly lower than for the routinely used microscopy, which had a sensitivity of 55.5% (IQR 46.8-63.8) compared to PCR as a reference. CONCLUSIONS/SIGNIFICANCE: The diagnostic accuracy of the CL Detect Rapid Test was low for skin slit and dental broach samples. Therefore, we do not recommend its use neither in hospital nor field settings. TRIAL REGISTRATION: This study is registered at ClinicalTrials.gov as NCT03837431.
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Antígenos de Protozoários/análise , Imunoensaio/métodos , Leishmania/imunologia , Leishmaniose Cutânea/diagnóstico , Pele/parasitologia , Adolescente , Adulto , Estudos Transversais , DNA de Protozoário/genética , Etiópia , Feminino , Humanos , Leishmania/classificação , Leishmania/genética , Masculino , Peroxirredoxinas/imunologia , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Pele/patologia , Adulto JovemRESUMO
BACKGROUND: Cutaneous leishmaniasis (CL) in Ethiopia, caused by Leishmania aethiopica, is often severe and hard to treat compared to CL caused by other species elsewhere. Miltefosine is the only oral anti-leishmanial drug, with a favorable side-effect profile compared to routinely available sodium stibogluconate (SSG), but evidence about its use for L. aethiopica is lacking. METHODOLOGY AND PRINCIPAL FINDINGS: In an observational cohort study, treatment outcomes, safety and adherence among CL patients who required systemic treatment and received miltefosine for 28 days in Boru Meda Hospital and University of Gondar Hospital were studied. Patient cure was defined as 100% flattening for non-ulcerated lesions and 100% flattening and 100% re-epithelization for ulcerated lesions. Outcomes were documented for day 28, 90 and 180, both per site, and pooled, adjusting for site as a fixed effect with effect coding. Among 94 included patients (32 in Gondar, 62 in Boru Meda), median lesion duration was 12 months, median size six cm, and mucosal involvement (46.8%) and diffuse (30.9%) lesions were common. Adherence to miltefosine was good, and side-effects were tolerable. Initial outcomes at day 28 were promising, with 68.8% and 94.0% of patients having good improvement or cure in Gondar and Boru Meda respectively. In Boru Meda, outcomes were good with 72.7% and 72.9% cure at day 90 and day 180 respectively. In Gondar, results were less promising, with only 12.5% and 26.7% cure at day 90 and day 180, although confidence intervals were wide. In pooled estimates, 48.7% of patients reached cure at day 180, and 32.3% relapsed. Outcomes were better in Boru Meda Hospital, for smaller lesions and for mucosal lesions. CONCLUSIONS/SIGNIFICANCE: Based on miltefosine's good initial response, tolerable side-effects, tablet-form, we propose to include miltefosine for future clinical trials using extended treatment schedules, combination therapy, or targeting specific subgroups. TRIAL REGISTRATION: ClinicalTrials.gov NCT04004754.
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Antiprotozoários/uso terapêutico , Leishmaniose Cutânea/tratamento farmacológico , Fosforilcolina/análogos & derivados , Administração Oral , Adolescente , Adulto , Antiprotozoários/administração & dosagem , Antiprotozoários/efeitos adversos , Estudos de Coortes , Etiópia , Feminino , Humanos , Leishmania/efeitos dos fármacos , Masculino , Fosforilcolina/administração & dosagem , Fosforilcolina/efeitos adversos , Fosforilcolina/uso terapêutico , Projetos Piloto , Cooperação e Adesão ao Tratamento/estatística & dados numéricos , Resultado do TratamentoRESUMO
In most low-resource settings, microscopy still is the standard method for diagnosis of cutaneous leishmaniasis, despite its limited sensitivity. In Ethiopia, the more sensitive molecular methods are not yet routinely used. This study compared five PCR methods with microscopy on two sample types collected from patients with a suspected lesion to advise on optimal diagnosis of Leishmania aethiopica. Between May and July 2018, skin scrapings (SS) and blood exudate from the lesion spotted on filter paper (dry blood spot, DBS) were collected for PCR from 111 patients of four zones in Southern Ethiopia. DNA and RNA were simultaneously extracted from both sample types. DNA was evaluated by a conventional PCR targeting ITS-1 and three probe-based real-time PCRs: one targeting the SSU 18S rRNA and two targeting the kDNA minicircle sequence (the 'Mary kDNA PCR' and a newly designed 'LC kDNA PCR' for improved L. aethiopica detection). RNAs were tested with a SYBR Green-based RT-PCR targeting spliced leader (SL) RNA. Giemsa-stained SS smears were examined by microscopy. Of the 111 SS, 100 were positive with at least two methods. Sensitivity of microscopy, ITS PCR, SSU PCR, Mary kDNA PCR, LC kDNA PCR and SL RNA PCR were respectively 52%, 22%, 64%, 99%, 100% and 94%. Microscopy-based parasite load correlated well with real-time PCR Ct-values. Despite suboptimal sample storage for RNA detection, the SL RNA PCR resulted in congruent results with low Ct-values. DBS collected from the same lesion showed lower PCR positivity rates compared to SS. The kDNA PCRs showed excellent performance for diagnosis of L. aethiopica on SS. Lower-cost SL RNA detection can be a complementary high-throughput tool. DBS can be used for PCR in case microscopy is negative, the SS sample can be sent to the referral health facility where kDNA PCR method is available.
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Leishmania/genética , Leishmania/isolamento & purificação , Leishmaniose/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Algoritmos , DNA de Cinetoplasto/genética , DNA de Protozoário/genética , Etiópia , Leishmaniose/parasitologia , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/parasitologia , Carga Parasitária , Sensibilidade e Especificidade , Pele/parasitologia , Manejo de Espécimes , Inquéritos e QuestionáriosRESUMO
Diagnosis of a first-time visceral leishmaniasis (VL) infection in Ethiopia is established by use of a rapid diagnostic test (RDT) detecting antibodies against rK39, direct agglutination test (DAT) and microscopy according to the national algorithm. The performance of individual tests and algorithm is variable and depends on several factors, one being HIV status. Limited data are available on the performance of tests in VL-HIV coinfected patients. Assessment of the performance of DAT (ITM-A), rK39 ELISA (Serion) and six RDT (Onsite Leishmania Ab CTK, Antigen ICT Xinjier, IT Leish Biorad, Kalazar Detect Inbios, rK39 IgG1 Coris, rk28 IgG1 Coris) for the diagnosis of VL was done on a panel of 91 stored serum and plasma samples of 'first-episode' suspected VL patients, with HIV coinfection (n = 51) and without (n = 40). A combined reference standard was used: either positive microscopy on tissue aspirates, or in case of negative microscopy, positive PCR results on the aspirate slide. Additionally, endemic healthy controls (n = 20), non-endemic controls (n = 10) and patients with confirmed malaria infection (n = 10) were tested for specificity evaluation. Sensitivities ranged from 69.2% for DAT (applied cut-off ≥ 1/3200) to 92.2% for the Onsite RDT, whereas specificities ranged from 20.0% for Kalazar Antigen ICT to 100% for IT Leish and rK39 IgG1. Sensitivities from all assays decreased upon stratification according to HIV status but was only significantly different for rK39 Serion ELISA (p-value 0.0084) and the Onsite RDT (p-value 0.0159). In conclusion, performance of commercially available assays for VL on samples from Northern-Ethiopian patients varied widely with a substantial decrease in sensitivity in the VL-HIV coinfected group. Clear guidelines on minimal performance criteria of individual tests and algorithms are needed, as well as which reference standard should be used to determine the performance.
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Antígenos de Protozoários/imunologia , Infecções por HIV/complicações , Leishmania/imunologia , Leishmaniose Visceral/diagnóstico , Testes de Aglutinação , Testes Diagnósticos de Rotina , Ensaio de Imunoadsorção Enzimática , Etiópia/epidemiologia , Humanos , Leishmaniose Visceral/parasitologia , Padrões de ReferênciaRESUMO
BACKGROUND: In endemic regions, asymptomatic Leishmania infection is common. In HIV patients, detection of asymptomatic Leishmania infection could potentially identify those at risk of visceral leishmaniasis (VL). However, data on the prevalence, incidence, and determinants of asymptomatic infection, and the risk of VL are lacking. METHODS: We conducted a cross-sectional survey at a single ART centre, followed by a prospective cohort study amongst HIV-infected adults in HIV care in a district hospital in a VL-endemic area in North-West Ethiopia (9/2015-8/2016). Asymptomatic Leishmania infection was detected using the direct agglutination test (DAT), rK39-rapid diagnostic test (RDT)), PCR on peripheral blood and the KAtex urine antigen test, and defined as positivity on any Leishmania marker. All individuals were followed longitudinally (irrespective of the Leishmania test results). Risk factors for asymptomatic Leishmania infection were determined using logistic regression. RESULTS: A total of 534 HIV-infected individuals enrolled in HIV care were included in the study. After excluding 13 patients with a history of VL and an 10 patients with incomplete baseline Leishmania tests, 511 were included in analysis. The median age was 38 years (interquartile range (IQR) 30-45), 62.6% were male. The median follow-up time was 12 months (IQR 9-12). No deaths were reported during the study period. Most (95.5%) were on antiretroviral treatment at enrolment, for a median of 52 months (IQR 27-79). The median CD4 count at enrolment was 377 cells/mm3 (IQR 250-518). The baseline prevalence of Leishmania infection was 12.8% in males and 4.2% in females. Overall, 7.4% tested positive for rK39, 4.3% for DAT, 0.2% for PCR and 0.2% for KAtex. Independent risk factors for a prevalent infection were male sex (odds ratio (OR) 3.2; 95% confidence intervals (CI) 14-7.0) and concurrent malaria infection (OR 6.1; 95% CI 1.9-18.9). Amongst the 49 prevalent (baseline) infections with further follow-up, the cumulative incidence of losing the Leishmania markers by one year was 40.1%. There were 36 incident infections during the course of the study, with a cumulative one-year risk of 9.5%. Only one case of VL was detected during follow-up. CONCLUSIONS: We found a high prevalence of asymptomatic Leishmania infection, persisting in most cases. The incidence was more modest and overt VL was rare. Larger and longer studies with more complete follow-up may help to decide whether a test and treat strategy would be justified in this context. TRIAL REGISTRATION: ClinicalTrials.gov NCT02839603.