Urgent considerations for booster vaccination strategies against Ebola virus disease.

With two endorsed and prophylactic vaccines against Zaire ebolavirus (referred to hereafter as EBOV), the number of individuals vaccinated against EBOV worldwide is estimated to range between 500 000 and 1 000 000 individuals, increasing with every renewed EBOV threat and vaccination campaign. Therefore, re-exposure of previously vaccinated health-care workers, and possibly community members, could become more frequent. In the absence of long-term data on vaccine efficacy and duration of protection, we urgently need to understand revaccination strategies that could maximise the level of protection. In this Personal View, we highlight the scarcity of available evidence to guide revaccination recommendations for the accumulating groups of previously vaccinated communities or front-line health-care workers that could be redeployed or re-exposed in the next EBOV outbreak(s). This evidence base is crucial to identify optimal target populations and the frequency of booster doses, and guide vaccine interchangeability (especially in settings with limited or unpredictable vaccine supplies), while preventing vaccine mistrust, equity concerns, and exclusion of vulnerable populations. We discuss five priority gaps (to whom, when, and how frequently, to provide booster doses; long-term correlates and thresholds of protection; the effect of vector-directed immunity and viral variant protection; comparative research in mix-and-match schedules; and implementation concerns) that should be urgently tackled to adapt the initial EBOV prophylactic vaccination strategies considering potential booster dose vaccinations.

Persistent T cell unresponsiveness associated with chronic visceral leishmaniasis in HIV-coinfected patients.

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.

Lack of functional TCR-epitope interaction is associated with herpes zoster through reduced downstream T cell activation.

The role of T cell receptor (TCR) diversity in infectious disease susceptibility is not well understood. We use a systems immunology approach on three cohorts of herpes zoster (HZ) patients and controls to investigate whether TCR diversity against varicella-zoster virus (VZV) influences the risk of HZ. We show that CD4+ T cell TCR diversity against VZV glycoprotein E (gE) and immediate early 63 protein (IE63) after 1-week culture is more restricted in HZ patients. Single-cell RNA and TCR sequencing of VZV-specific T cells shows that T cell activation pathways are significantly decreased after stimulation with VZV peptides in convalescent HZ patients. TCR clustering indicates that TCRs from HZ patients co-cluster more often together than TCRs from controls. Collectively, our results suggest that not only lower VZV-specific TCR diversity but also reduced functional TCR affinity for VZV-specific proteins in HZ patients leads to lower T cell activation and consequently affects the susceptibility for viral reactivation.

Chronic high level parasitemia in HIV-infected individuals with or without visceral leishmaniasis in an endemic area in North-West Ethiopia: potential superspreaders?

BACKGROUND: HIV patients 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 PreLeish prospective cohort study, following a total of 490 HIV infected individuals free of VL at enrollment for upto 24-37 months in North-West Ethiopia. Blood Leishmania PCR was done systematically. This case series reports on ten HIV-coinfected individuals with chronic VL (≥3 VL episodes during follow-up) for upto 37 months, and three individuals with asymptomatic Leishmania infection for upto 24 months. RESULTS: All ten chronic VL cases were male, on antiretroviral treatment, with 0-11 relapses before enrollment. Median baseline CD4 counts were 82 cells/µL. They displayed three to six VL treatment episodes over a period upto 37 months. Leishmania blood PCR levels were strongly positive for almost the entire follow-up time (median Ct value 26 (IQR 23-30), including during periods between VL treatment. Additionally, we describe three HIV-infected individuals with asymptomatic Leishmania infection and without VL history, with equally strong Leishmania parasitemia over a period of upto 24 months without developing VL. All were on antiretroviral treatment at enrollment, with baseline CD4 counts ranging from 78 to 350 cells/µL. CONCLUSION: These are the first data on chronic parasitemia in HIV-infected individuals from L donovani endemic areas. HIV patients with asymptomatic and symptomatic Leishmania infection could potentially be highly infectious and constitute Leishmania superspreaders. Xenodiagnosis studies are required to confirm infectiousness.

Diagnosing Viral Infections Through T-Cell Receptor Sequencing of Activated CD8+ T Cells.

T-cell-based diagnostic tools identify pathogen exposure but lack differentiation between recent and historical exposures in acute infectious diseases. Here, T-cell receptor (TCR) RNA sequencing was performed on HLA-DR+/CD38+CD8+ T-cell subsets of hospitalized coronavirus disease 2019 (COVID-19) patients (n = 30) and healthy controls (n = 30; 10 of whom had previously been exposed to severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]). CDR3α and CDR3ß TCR regions were clustered separately before epitope specificity annotation using a database of SARS-CoV-2-associated CDR3α and CDR3ß sequences corresponding to >1000 SARS-CoV-2 epitopes. The depth of the SARS-CoV-2-associated CDR3α/ß sequences differentiated COVID-19 patients from the healthy controls with a receiver operating characteristic area under the curve of 0.84 ± 0.10. Hence, annotating TCR sequences of activated CD8+ T cells can be used to diagnose an acute viral infection and discriminate it from historical exposure. In essence, this work presents a new paradigm for applying the T-cell repertoire to accomplish TCR-based diagnostics.

Leveraging T-cell receptor - epitope recognition models to disentangle unique and cross-reactive T-cell response to SARS-CoV-2 during COVID-19 progression/resolution.

Despite the general agreement on the significance of T cells during SARS-CoV-2 infection, the clinical impact of specific and cross-reactive T-cell responses remains uncertain. Understanding this aspect could provide insights for adjusting vaccines and maintaining robust long-term protection against continuously emerging variants. To characterize CD8+ T-cell response to SARS-CoV-2 epitopes unique to the virus (SC2-unique) or shared with other coronaviruses (CoV-common), we trained a large number of T-cell receptor (TCR) - epitope recognition models for MHC-I-presented SARS-CoV-2 epitopes from publicly available data. These models were then applied to longitudinal CD8+ TCR repertoires from critical and non-critical COVID-19 patients. In spite of comparable initial CoV-common TCR repertoire depth and CD8+ T-cell depletion, the temporal dynamics of SC2-unique TCRs differed depending on the disease severity. Specifically, while non-critical patients demonstrated a large and diverse SC2-unique TCR repertoire by the second week of the disease, critical patients did not. Furthermore, only non-critical patients exhibited redundancy in the CD8+ T-cell response to both groups of epitopes, SC2-unique and CoV-common. These findings indicate a valuable contribution of the SC2-unique CD8+ TCR repertoires. Therefore, a combination of specific and cross-reactive CD8+ T-cell responses may offer a stronger clinical advantage. Besides tracking the specific and cross-reactive SARS-CoV-2 CD8+ T cells in any TCR repertoire, our analytical framework can be expanded to more epitopes and assist in the assessment and monitoring of CD8+ T-cell response to other infections.

Surgical output within the Fast Track Initiative against trachoma in Amhara region, Ethiopia.

INTRODUCTION: Trachomatous trichiasis (TT) is the advanced stage of trachoma where lashes touch the globe of the eye causing permanent damage. Without eyelid surgery, TT can lead to irreversible blindness. In 2015 the Ethiopian Ministry of Health launched the Fast Track Initiative with the aim of enhancing the provision of surgical services for TT. The aims of this study were to determine the productivity of individual surgeons during the 2017 Initiative, to compare this productivity with the Ministry's annual target indicator of ≥ 200 surgeries, and to assess the factors associated with surgical output. METHODOLOGY: This retrospective cross-sectional study utilized programmatic data on surgical output from 140 surgeons active from January 2017 through December 2017 in the eastern half of Amhara region, Ethiopia. Data were collected from a surgery monitoring dataset, analyzed, and compared to the performance targets set by the Ministry. RESULTS: The mean annual number of surgeries carried out per surgeon was 169 (standard deviation: 111) for a total of 23,616 surgeries. Among the 140 surgeons, 38% achieved the target set by the Ministry. Location of surgical training site and estimated surgical backlog were signficantly associated with a higher surgery output. CONCLUSIONS: An increase in surgical output was observed compared to productivity prior to the Initiative, although the average annual output during the 2017 Fast Track Initiative was lower than the Ministry's target. Using data driven approaches to setting annual productivity goals should be considered, particularly in light of fewer remaining TT cases as a result of the successful Initiative.

Comparison of serum cytokine levels in symptomatic and asymptomatic HIV-Leishmania coinfected individuals from a Brazilian visceral leishmaniasis endemic area.

BACKGROUND: Visceral leishmaniasis (VL) remains an important infectious disease worldwide. VL-HIV coinfected individuals can present with atypical clinical forms of VL and have a high risk of VL relapse. Some cytokines have been described as potential markers to diagnose active VL and to predict the severity of the cases. However, few studies have included VL-HIV coinfected patients. We aimed to characterize the levels of several cytokines among VL-HIV coinfected individuals living in a VL-endemic area in Northeast Brazil. METHODS: This was a retrospective, cross-sectional study, aiming to estimate the levels of various cytokines in symptomatic and asymptomatic VL-HIV coinfected individuals. There were 134 study participants (35 symptomatic VL-HIV, 75 asymptomatic VL-HIV, and 24 healthy controls), all ≥ 18 years-old. Serum cytokine levels (interferon-γ, tumor necrosis factor, and interleukins 2, 4, 6, 10, and 17A) were quantified using the Becton Dickinson-BD's Cytometric Bead Array (CBA) system. RESULTS: The population mainly consisted of men (64.9%), with a median age of 35 (27-41) years. Asymptomatic individuals were younger (p = 0.013), with more years of education (p < 0.001), and were more often on antiretroviral therapy (p < 0.001) than those in the symptomatic group. Hemoglobin levels (p < 0.001), lymphocytes (p < 0.001) and CD4 count (p < 0.001) were lower in symptomatic individuals, while HIV viral loads were higher (p < 0.001). In the symptomatic VL-HIV coinfected group, we observed increased serum levels of IL-17A, IL-6, and IL-10 compared to asymptomatic patients and the healthy controls. There were no differences in the levels of all cytokines between asymptomatic VL-HIV coinfected individuals and the healthy controls. CONCLUSIONS: Higher serum levels of IL-17A, IL-6, and IL-10 cytokines were observed in symptomatic coinfected individuals but not in asymptomatically infected individuals. More studies among HIV-positive persons are needed to better understand the role of serum cytokines for prognosis, to define cure and predict VL relapses in VL-HIV coinfected individuals.

Aetiologies of acute undifferentiated febrile illness at the emergency ward of the University of Gondar Hospital, Ethiopia.

OBJECTIVE: Causes of acute febrile illness (AFI) often remain undetermined in developing countries, due to overlap of symptoms and limited available diagnostics. We aimed to assess the aetiology of AFI in adults in a referral hospital in northwest Ethiopia. METHODS: While all participants were tested for malaria by rapid diagnostic test (RDT), microscopy was only done on physician's request. Dengue virus (DENV) infections were detected using an RDT and ELISAs and dengue, yellow fever and chikungunya cases were identified by PCR. Bacterial aetiologies were investigated using blood culture and PCR. RESULTS: The aetiology of acute infection was identified for 20.5% of 200 patients enrolled. Eleven percent tested positive for Plasmodium, while microscopy was only requested for half of the identified malaria cases. For 4.0% of the Plasmodium-infected patients, an acute or past DENV (co-)infection was detected. We found 7.5% acute and 13.0% past DENV - all serotype 3 - infections. Bacterial infections were observed in 4.5% of the patients. CONCLUSION: Malaria is still a considerable aetiology of AFI and dengue is underrecognised. There are areas where both diseases occur concomitantly, and the DENV-3 serotype presumably spreads from Sudan to northern Ethiopia. As only 20.5% of the aetiologies were identified, a broader testing platform is required.

HLA-DRB1 Alleles Associated with Lower Leishmaniasis Susceptibility Share Common Amino Acid Polymorphisms and Epitope Binding Repertoires.

Susceptibility for leishmaniasis is largely dependent on host genetic and immune factors. Despite the previously described association of human leukocyte antigen (HLA) gene cluster variants as genetic susceptibility factors for leishmaniasis, little is known regarding the mechanisms that underpin these associations. To better understand this underlying functionality, we first collected all known leishmaniasis-associated HLA variants in a thorough literature review. Next, we aligned and compared the protection- and risk-associated HLA-DRB1 allele sequences. This identified several amino acid polymorphisms that distinguish protection- from risk-associated HLA-DRB1 alleles. Subsequently, T cell epitope binding predictions were carried out across these alleles to map the impact of these polymorphisms on the epitope binding repertoires. For these predictions, we used epitopes derived from entire proteomes of multiple Leishmania species. Epitopes binding to protection-associated HLA-DRB1 alleles shared common binding core motifs, mapping to the identified HLA-DRB1 amino acid polymorphisms. These results strongly suggest that HLA polymorphism, resulting in differential antigen presentation, affects the association between HLA and leishmaniasis disease development. Finally, we established a valuable open-access resource of putative epitopes. A set of 14 HLA-unrestricted strong-binding epitopes, conserved across species, was prioritized for further epitope discovery in the search for novel subunit-based vaccines.

Cellular Immune Phenotypes and Worsening Scores of Frailty-Associated Parameters Over an 18-Month Period in the Very Old.

Frailty has been related to inflammaging and certain immune parameters. In previous analyses of participants older than 80 years of age in the longitudinal BELFRAIL cohort study, the main focus was on T-cell phenotypes and the association with cytomegalovirus (CMV) serostatus and survival, finding that a CD4:CD8 ratio greater than 5 was associated with frailty, impaired activities of daily living (ADLs), and mortality (but only in women). Here, we phenotyped peripheral blood immune cells via multicolor flow cytometry and correlated these with the dynamics of changes in ADL, geriatric depression score, Mini-Mental State Examination, and Short Physical Performance Battery from baseline values over 18 months follow-up. We found that higher frequencies of B cells and late-differentiated CD8+ T cells at 18 months from baseline were associated with ADL impairment that had worsened over the preceding 18 months. There were no significant associations with monocyte, dendritic cell, or natural killer (NK) cell phenotypes. No associations with the Geriatric Depression Scale, the Mini-Mental State Examination, or the Short Physical Performance Battery were found. Thus, while these results do not establish causality, they suggest that certain adaptive immune, but not innate immune, parameters are associated with a worsened ADL in the very old.

Diagnostic accuracy of direct agglutination test, rK39 ELISA and six rapid diagnostic tests among visceral leishmaniasis patients with and without HIV coinfection in Ethiopia.

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.

Evaluation of C-reactive protein and myxovirus resistance protein A to guide the rational use of antibiotics among acute febrile adult patients in Northwest Ethiopia.

OBJECTIVES: In low-resource settings, treatment is often given empirically without knowledge of the aetiology due to a lack of diagnostics. In the search for reliable rapid tests to guide treatment work-up, this study was performed to determine whether two biomarkers could differentiate bacterial from non-bacterial infections in acute febrile patients. METHODS: Adults with acute fever were recruited at a referral hospital in Ethiopia. The QuikRead Go test was used to quantify C-reactive protein (qCRP) and the FebriDx test was used for combined qualitative detection of the bacterial CRP marker with myxovirus resistance protein A (MxA), a viral biomarker. RESULTS: Of the 200 patients included in this study, most presented with 2-3 days of fever, headache, and joint pain. Antibiotics were prescribed for 83.5% and antimalarials for 36.5%, while a bacterial infection was only confirmed in 5% and malaria in 11%. The median qCRP level for confirmed bacterial infections was 128 mg/l. The FebriDx and QuikRead Go test had an overall agreement of 72.0%. CONCLUSIONS: An over-prescription of antibiotics for febrile patients was observed, even for those with low CRP levels and without a confirmed bacterial infection. The added value of the FebriDx was limited, while the combined use of rapid tests for qCRP and malaria should be considered for the management of acute febrile illness and antibiotic stewardship.

Sexual Transmission of Visceral Leishmaniasis: A Neglected Story.

For visceral leishmaniasis (VL), a major vector-borne parasitic disease, an alternative sexual transmission route is well documented in dogs but evidence is lacking in humans. Here, we discuss the current knowledge and key questions to be answered as it may be an additional obstacle in ongoing VL elimination programs.

Minimally Invasive Microbiopsies as an Improved Sampling Method for the Diagnosis of Cutaneous Leishmaniasis.

Current sampling methods to diagnose cutaneous leishmaniasis are invasive and painful. An alternative and minimally invasive microbiopsy device was evaluated in a diverse range of cutaneous leishmaniasis lesions in Ethiopia. Using polymerase chain reaction-based diagnosis, the microbiopsy outperformed the routine skin slit sample by detecting more patients while pain scores were significantly lower.

Towards the trachoma elimination target in the Southern region of Ethiopia: How well is the SAFE strategy being implemented?

INTRODUCTION: Trachoma is one of the 20 neglected tropical diseases and a serious public health problem in Ethiopia. To reach the WHO elimination target by 2020, SAFE (Surgery, Antibiotics, Facial cleanliness, Environmental improvement) strategy has been implemented in the Southern Nations, Nationalities, and Peoples' Region (SNNPRs), Ethiopia. Scarce evidence exists regarding recent progress in achieving elimination of active trachoma (< 5%) and how well the SAFE strategy implemented. METHODOLOGY: A retrospective analysis of programmatic data in the period 2013-2018 was used. All trachoma endemic districts in SNNPR were included. Data collected from the Federal Ministry of Health on trachoma prevalence and SAFE strategy were analyzed. RESULTS: Out of 134 endemic districts, only 35 had their planned impact survey, of which only 11 districts achieved the elimination target. Six districts reverted backwards from eliminated status to low (1) or moderate (5) level. The median prevalence of active trachoma in these 35 districts was 10% in 2017/18. In 2017, the mean antibiotic treatment coverage was 90%, but only 56% and 68% of districts implemented and reported on "F" and "E" components, respectively. In the high prevalence districts, only 10% delivered their planned five rounds of Zithromax® mass distribution. CONCLUSIONS: These data showed a lack in planned impact surveys with only a limited number of districts reached the WHO elimination threshold by 2018. Lack of attention on high prevalent districts, and recent reversal of trachoma eliminated districts to moderate or low prevalence levels argue for urgent and prioritized implementation of the SAFE strategy.

Whole Blood Stimulation Assay as a Treatment Outcome Monitoring Tool for VL Patients in Ethiopia: A Pilot Evaluation.

Visceral leishmaniasis (VL) is a lethal disease if left untreated. Current treatments produce variable rates of treatment failure and toxicity without sterile cure, rendering treatment efficacy monitoring essential. To avoid repeated invasive tissue aspirates as well as empirical treatment, there is a need for new tools that allow a less-invasive and early assessment of treatment efficacy in the field. Cross-sectional studies have suggested levels of cytokines/chemokines after whole blood stimulation as good markers of cure, but longitudinal studies are lacking. In this study, we followed 13 active VL cases in an endemic area in Ethiopia by measuring the production of IFN-γ, TNF-α, IP-10, IL-2, IL-10, MCP-1, and MIG before, during, and at the end of treatment. After 24 hours of stimulation of whole blood with soluble Leishmania antigen, we observed an early, robust, and incremental increase of IFN-γ, TNF-α, and IP-10 levels in all patients during treatment. Moreover, based on the IFN-γ levels that showed an average 13-fold increase from the time of diagnosis until the end of treatment, we could almost perfectly discriminate active from cured status. Similar concentrations and patterns were found in stimulation assays with the two main Leishmania species. The levels of IFN-γ, IP-10, or TNF-α also seemed to be inversely associated with the parasite load at baseline. Despite a 1/10 drop in concentrations, similar patterns were observed in IFN-γ and IP-10 levels when dried plasma spots were stored at 4°C for an average of 225 days. All the above evidence suggests a detectable restoration of cell-mediated immunity in VL and its association with parasite clearance. With a potential application in rural settings by means of dried plasma spots, we recommend to further explore the early diagnostic value of such assays for treatment efficacy monitoring in large cohort studies including treatment failure cases.

Host transcriptomic signature as alternative test-of-cure in visceral leishmaniasis patients co-infected with HIV.

BACKGROUND: Visceral leishmaniasis (VL) treatment in HIV patients very often fails and is followed by high relapse and case-fatality rates. Hence, treatment efficacy assessment is imperative but based on invasive organ aspiration for parasite detection. In the search of a less-invasive alternative and because the host immune response is pivotal for treatment outcome in immunocompromised VL patients, we studied changes in the whole blood transcriptional profile of VL-HIV patients during treatment. METHODS: Embedded in a clinical trial in Northwest Ethiopia, RNA-Seq was performed on whole blood samples of 28 VL-HIV patients before and after completion of a 29-day treatment regimen of AmBisome or AmBisome/miltefosine. Pathway analyses were combined with a machine learning approach to establish a clinically-useful 4-gene set. FINDINGS: Distinct signatures of differentially expressed genes between D0 and D29 were identified for patients who failed treatment and were successfully treated. Pathway analyses in the latter highlighted a downregulation of genes associated with host cellular activity and immunity, and upregulation of antimicrobial peptide activity in phagolysosomes. No signs of disease remission nor pathway enrichment were observed in treatment failure patients. Next, we identified a 4-gene pre-post signature (PRSS33, IL10, SLFN14, HRH4) that could accurately discriminate treatment outcome at end of treatment (D29), displaying an average area-under-the-ROC-curve of 0.95 (CI: 0.75-1.00). INTERPRETATION: A simple blood-based signature thus holds significant promise to facilitate treatment efficacy monitoring and provide an alternative test-of-cure to guide patient management in VL-HIV patients. FUNDING: Project funding was provided by the AfricoLeish project, supported by the European Union Seventh Framework Programme (EU FP7).

Longitudinal evaluation of asymptomatic Leishmania infection in HIV-infected individuals in North-West Ethiopia: A pilot study.

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.