Multilocus sequence and microsatellite identification of intra-specific hybrids and ancestor-like donors among natural Ethiopian isolates of Leishmania donovani.

Protozoan parasites of the genus Leishmania (Kinetoplastida: Trypanosomatidae) cause widespread and devastating human diseases. Visceral leishmaniasis is endemic in Ethiopia where it has also been responsible for fatal epidemics. It is postulated that genetic exchange in Leishmania has implications for heterosis (hybrid vigour), spread of virulent strains, resistance to chemotherapeutics, and exploitation of different hosts and vectors. Here we analyse 11 natural Ethiopian Leishmania donovani isolates consisting of four putative hybrids, seven parent-like isolates and over 90 derived biological clones. We apply a novel combination of high resolution multilocus microsatellite typing (five loci) and multilocus sequence typing (four loci) that together distinguish parent-like and hybrid L. donovani strains. Results indicate that the four isolates (and their associated biological clones) are genetic hybrids, not the results of mixed infections, each possessing heterozygous markers consistent with inheritance of divergent alleles from genetically distinct Ethiopian L. donovani lineages. The allelic profiles of the putative hybrids may have arisen from a single hybridisation event followed by inbreeding or gene conversion, or alternatively from two or more hybridisation events. Mitochondrial sequencing showed uniparental maxicircle inheritance for all of the hybrids, each possessing a single mitochondrial genotype. Fluorescence activated cell sorting analysis of DNA content demonstrated that all hybrids and their associated clones were diploid. Together the data imply that intra-specific genetic exchange is a recurrent feature of natural L. donovani populations, with substantial implications for the phyloepidemiology of Leishmania

Genomic analysis of natural intra-specific hybrids among Ethiopian isolates of Leishmania donovani.

Parasites of the genus Leishmania (Kinetoplastida: Trypanosomatidae) cause widespread and devastating human diseases. Visceral leishmaniasis due to Leishmania donovani is endemic in Ethiopia where it has also been responsible for major epidemics. The presence of hybrid genotypes has been widely reported in surveys of natural populations, genetic variation reported in a number of Leishmania species, and the extant capacity for genetic exchange demonstrated in laboratory experiments. However, patterns of recombination and the evolutionary history of admixture that produced these hybrid populations remain unclear. Here, we use whole-genome sequence data to investigate Ethiopian L. donovani isolates previously characterized as hybrids by microsatellite and multi-locus sequencing. To date there is only one previous study on a natural population of Leishmania hybrids based on whole-genome sequences. We propose that these hybrids originate from recombination between two different lineages of Ethiopian L. donovani occurring in the same region. Patterns of inheritance are more complex than previously reported with multiple, apparently independent, origins from similar parents that include backcrossing with parental types. Analysis indicates that hybrids are representative of at least three different histories. Furthermore, isolates were highly polysomic at the level of chromosomes with differences between parasites recovered from a recrudescent infection from a previously treated individual. The results demonstrate that recombination is a significant feature of natural populations and contributes to the growing body of data that shows how recombination, and gene flow, shape natural populations of Leishmania

High seroprevalence of anti-SARS-CoV-2 antibodies among Ethiopian healthcare workers

BACKGROUND: COVID-19 pandemic has a devastating impact on the economies and health care system of sub-Saharan Africa. Healthcare workers (HWs), the main actors of the health system, are at higher risk because of their occupation. Serology-based estimates of SARS-CoV-2 infection among HWs represent a measure of HWs' exposure to the virus and could be used as a guide to the prevalence of SARS-CoV-2 in the community and valuable in combating COVID-19. This information is currently lacking in Ethiopia and other African countries. This study aimed to develop an in-house antibody testing assay, assess the prevalence of SARS-CoV-2 antibodies among Ethiopian high-risk frontline HWs. METHODS: We developed and validated an in-house Enzyme-Linked Immunosorbent Assay (ELISA) for specific detection of anti-SARS-CoV-2 receptor binding domain immunoglobin G (IgG) antibodies. We then used this assay to assess the seroprevalence among HWs in five public hospitals located in different geographic regions of Ethiopia. From consenting HWs, blood samples were collected between December 2020 and February 2021, the period between the two peaks of COVID-19 in Ethiopia. Socio-demographic and clinical data were collected using questionnaire-based interviews. Descriptive statistics and bivariate and multivariate logistic regression were used to determine the overall and post-stratified seroprevalence and the association between seropositivity and potential risk factors. RESULTS: Our successfully developed in-house assay sensitivity was 100% in serum samples collected 2- weeks after the first onset of symptoms whereas its specificity in pre-COVID-19 pandemic sera was 97.7%. Using this assay, we analyzed a total of 1997 sera collected from HWs. Of 1997 HWs who provided a blood sample, and demographic and clinical data, 51.7% were females, 74.0% had no symptoms compatible with COVID-19, and 29.0% had a history of contact with suspected or confirmed patients with SARS-CoV-2 infection. The overall seroprevalence was 39.6%. The lowest (24.5%) and the highest (48.0%) seroprevalence rates were found in Hiwot Fana Specialized Hospital in Harar and ALERT Hospital in Addis Ababa, respectively. Of the 821 seropositive HWs, 224(27.3%) of them had a history of symptoms consistent with COVID-19 while 436 (> 53%) of them had no contact with COVID-19 cases as well as no history of COVID-19 like symptoms. A history of close contact with suspected/confirmed COVID-19 cases is associated with seropositivity (Adjusted Odds Ratio (AOR) = 1.4, 95% CI 1.1-1.8; p = 0.015). CONCLUSION: High SARS-CoV-2 seroprevalence levels were observed in the five Ethiopian hospitals. These findings highlight the significant burden of asymptomatic infection in Ethiopia and may reflect the scale of transmission in the general population

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Molekulare Epidemiologie und Populationsgenetik von Leishmania donovani in verschiedenen äthiopischen Gebieten endemisch für viszerale Leishmaniose

Visceral leishmaniasis (VL) is a systemic disease caused by parasites of the Leishmania donovani complex and fatal if left untreated. In East Africa, Ethiopia is the second most affected country next to Sudan. Despite knowledge of the molecular epidemiology and population genetics of the etiologic agents of the VL is crucial for better understanding the dynamics of the disease as well as for implementing effective control measures little is known about the population structure of Ethiopian L. donovani. Whether the occurrence of VL epidemic outbreaks in highland areas, which were formerly considered non- endemic for VL, was related to introduction of parasites, epidemiological changes or a combination of both is also not yet clear. Through a very thorough analysis of L. donovani strains from two main geographical areas in Ethiopia: (i) northwest foci (Humera and Metema) and (ii) south foci (Konso and Negele Borena) using 14 unlinked microsatellite markers, we found a hierarchical genetic population structuring with considerable genetic diversity. When compared with L. donovani strains from the neighbouring countries Sudan and Kenya, all strains from northwest Ethiopia grouped with strains from Sudan in one population named NE/SD whereas all strains from south Ethiopia and Kenya were assigned to another population named SE/KE. These two main East African L. donovani populations seem to correlate with the geographic distribution of the two principal sand fly vector species involved in the transmission of the parasite in East Africa. Both populations were further subdivided into two subpopulations which in turn consisted of 2 to 3 genetic clusters. Interestingly, one of the subpopulations in the NE/SD population contained all but three of the strains newly isolated from HIV-VL co-infected patients. In contrast to the NE/SD population, high levels of inbreeding were observed in the SE/KE population. Also, we detected putative hybrids resulting from natural hybridization/recombination events among the sympatric subpopulations in the NE/SD population. Most interestingly, almost all of these putative hybrid strains were isolated from HIV-VL co-infected patients. High inbreeding, implying recombination between similar and/or closely related strains, as well as the presence of natural hybrids at intra- species level represent strong arguments against the so far suggested strict clonality of L. donovani parasites. In order to track the possible origins of the parasites responsible for an outbreak of VL in the highland of Ethiopia called Libo Kemkem, we analysed a panel of strains collected during and after the outbreak. Molecular characterization of strains from this newly emerging focus using highly polymorphic microsatellite markers revealed a striking genetic heterogeneity among these strains which could be assigned to at least three genetically distinct clusters. All but three strains from this focus grouped together with strains from other foci of VL in northwest Ethiopia bordering Sudan. These findings support the hypothesis that the epidemic was related to multiple introductions of the parasite and not to the spread of a particular virulent parasite. An alternative explanation for the remarkable genetic variability could be that these parasites have been circulated locally for decades without being noticed. In this case it remains, however, an enigma why a sudden epidemic outbreak had occurred in 2004. Disseminated cutaneous leishmaniasis (CL) caused by L. donovani in HIV/VL co-infected patients can be associated with immuno-suppression. Such cases can easily be misdiagnosed as diffuse CL, PKDL, PKDL/VL or lepromatous leprosy. Molecular characterization of strains isolated from both the viscera and skin lesions using ITS1-PCR-RFLP and MLMT, together with clinical, immunological and virological data, demonstrated that the skin lesions in these HIV-positive VL patients were due to dissemination of the viscerotropic L. donovani parasites as a consequence of the severe immuno-suppression. On the other hand, a case of PKDL/VL was diagnosed six months after ART commencement in a VL/HIV co-infected patient with stage IV disease as a consequence of immune restoration. Further studies using a combination of clinical, virological and molecular methods are required for a better management of HIV co-infected patients showing different clinical symptoms.Die viszerale Leishmaniose (VL) ist eine systemische Erkrankung, die durch Parasiten des Leishmania donovani Komplex hervorgerufen wird und unbehandelt tödlich verlaufen kann. Äthiopien ist in Ostafrika am zweithäufigsten, nach dem Sudan, betroffen. Obwohl Kenntnisse über die molekulare Epidemiologie und die Populationsgenetik des Erregers der VL essentiell für ein besseres Verständnis der Dynamik der Erkrankung und für die Implementierung effektiver Kontrollmaßnahmen sind, ist bisher wenig über die Populationsstrukturen von L. donovani in Äthiopien bekannt. Auch ob ein epidemischer Ausbruch in einem der Hochlandgebiete, die bisher als nicht endemisch für VL angesehen wurden, auf den Import von Parasiten, epidemiologische Veränderungen oder eine Kombination von beidem zurückzuführen ist, ist noch nicht klar. Mit Hilfe einer detaillierten Analyse von 14 molekularen Markern bei L. donovani-Isolaten aus zwei geographischen Regionen in Äthiopien, i) aus den nordwestlichen Foci (Humera und Metema) und ii) den südlichen Foci (Konso und Negele Borena), haben wir hierarchische Populationsstrukturen und eine bemerkenswerte genetische Diversität von L. donovani in Äthiopien festgestellt. Bei einem Vergleich mit L. donovani-Stämmen aus den Nachbarländern Sudan und Kenia gruppierten alle Stämme aus Nordwest-Äthiopien mit den Stämmen aus dem Sudan in einer Population (NE/SD), während alle Stämme aus dem Süden Äthiopiens und aus Kenia einer anderen Population (SE/KE) zugeordnet wurden. Diese beiden hauptsächlichen L. donovani-Populationen in Ostafrika scheinen mit der Verbreitung der beiden prinzipiellen Vektorenarten (Sandmücken) zu korrelieren, die an der Übertragung der Parasiten in Ostafrika beteiligt sind. Beide Populationen lassen sich weiter in je zwei Subpopulationen aufteilen, die wiederum aus 2 bis 3 genetischen Gruppen bestehen. Es ist interessant, dass bis auf drei Stämme alle anderen Stämme, die von HIV-VL ko-infizierten Patienten isoliert wurden, zu der derselben Subpopulation in der NE/SD Population gehören. In der Population SE/KE wurde, im Gegensatz zu der Population NE/SD, eine hohe Inzuchtrate gefunden. Wir konnten auch die Existenz natürlicher Hybride nachweisen, die wahrscheinlich durch natürliche Hybridsierung/Rekombination zwischen den sympatrischen Subpopulationen in der NE/SD Population entstanden sind. Von großem Interesse ist, dass fast alle dieser Hybridstämme von HIV-VL ko-infizierten Patienten stammen. Eine hohe Inzuchtrate, die auf Rekombinationen zwischen ähnlichen und/oder eng verwandten Stämmen zurückzuführen ist, sowie die Existenz natürlicher intra- spezifischer Hybride sind deutliche Argumente gegen die bisherige Annahme strikter Klonalität bei L. donovani-Parasiten. Um die mögliche Herkunft von Parasiten, die für einen VL Ausbruch im Hochland von Äthiopien verantwortlich waren, zu klären, wurde eine Reihe von Stämmen analysiert, die während oder nach dem Ausbruch isoliert worden waren. Die molekulare Charakterisierung der Stämme von diesem neuen Fokus mit Hilfe von hochpolymorphen Mikrosatellitenmarkern zeigte eine überraschende genetische Heterogenität dieser Stämme, die mindestens drei genetisch distinkten Gruppen zugeordnet werden konnten. Mit Ausnahme von drei Stämmen gruppierten alle Stämme von diesem Fokus mit Stämmen aus anderen Endemiegebieten für VL in Nordwest- Äthiopien an der Grenze zum Sudan. Dieses Ergebnis unterstützt die Hypothese, dass die Epidemie durch mehrfache Importe von Parasiten verursacht wurde und nicht durch die Ausbreitung eines spezifischen, besonders virulenten Stamms. Eine alternative Erklärung für die bemerkenswerte genetische Variabilität könnte jedoch auch sein, dass diese Stämme schon mehrere Dekaden unbemerkt in diesem Gebiet zirkulieren. In diesem Fall wäre es aber ein Rätsel, warum es im Jahr 2004 zu einem plötzlichen epidemischen Ausbruch kam. Disseminierte kutane Leishmaniosen (CL) können im Zusammenhang mit Immunsuppression stehen, wie bei HIV/VL koinfizierten Patienten beobachtet wurde. Solche Fälle können leicht als diffuse CL, PKDL, PKDL/VL oder Lepra falsch diagnostiziert werden. Die molekulare Charakterisierung mit Hilfe der ITS1-PCR-RFLP und des MLMT von Stämmen, die aus viszeralen Organen und Hautläsionen des gleichen Patienten isoliert wurden, zusammen mit klinischen, immunologischen und virologischen Daten hat ergeben, dass die Hautläsionen bei den HIV-positiven VL-Patienten durch die Ausbreitung der viszerotropen L. donovani-Parasiten als Folge der schweren Immunsuppression bedingt waren. Andererseits wurde sechs Monate nach Beginn von ART bei einem VL/HIV-Patienten mit Phase IV AIDS eine PKDL/VL infolge der Immunrestoration diagnostiziert. Weitere kombinierte klinische, virologische und molekulare Untersuchungen sind für ein besseres Management von HIV-koinfizierten Patienten mit unterschiedlicher klinischer Symptomatik notwendig

Development and characterization of serotype-specific monoclonal antibodies against the dengue virus-4 (DENV-4) non-structural protein (NS1)

Abstract Background Dengue, caused by one of the four serologically distinct dengue viruses (DENV-1 to − 4), is a mosquito-borne disease of serious global health significance. Reliable and cost-effective diagnostic tests, along with effective vaccines and vector-control strategies, are highly required to reduce dengue morbidity and mortality. Evaluation studies revealed that many commercially available NS1 antigen (Ag) tests have limited sensitivity to DENV-4 serotype compared to the other three serotypes. These studies indicated the need for development of new NS1 Ag detection test with improved sensitivity to DENV-4. An NS1 capture enzyme linked immunoassay (ELISA) specific to DENV-4 may improve the detection of DENV-4 cases worldwide. In addition, a serotype-specific NS1 Ag test identifies both DENV and the infecting serotype. Methods In this study, we used a small-ubiquitin-like modifier (SUMO*) cloning vector to express a SUMO*-DENV-4 rNS1 fusion protein to develop NS1 DENV-4 specific monoclonal antibodies (MAbs). These newly developed MAbs were then optimized for use in an anti-NS1 DENV-4 capture ELISA. The serotype specificity and sensitivity of this ELISA was evaluated using (i) supernatants from DENV (1–4)-infected Vero cell cultures, (ii) rNS1s from all the four DENV (1–4) and, (iii) rNS1s of related flaviviruses (yellow fever virus; YFV and West Nile virus; WNV). Results From the evaluation studies of the newly developed MAbs, we identified three DENV-4 specific anti-NS1 MAbs: 3H7A9, 8A6F2 and 6D4B10. Two of these MAbs were optimal for use in a DENV-4 serotype-specific NS1 capture ELISA: MAb 8A6F2 as the capture antibody and 6D4B10 as a detection antibody. Conclusion This ELISA was sensitive and specific to DENV-4 with no cross-reactivity to other three DENV (1–3) serotypes and other heterologous flaviviruses. Taken together these data indicated that our MAbs are useful reagents for the development of DENV-4 immunodiagnostic tests

High Seroprevalence of anti-SARS-CoV-2 antibodies among Ethiopian healthcare workers [preprint]

Background COVID-19 pandemic has a devastating impact on the economies and health care system of sub-Saharan Africa. Healthcare workers (HWs), the main actors of the health system, are at higher-risk because of their occupation. Serology-based estimates of SARS-CoV-2 infection among HWs represent a measure of HWs’ exposure to the virus and a guide to the prevalence of SARS-CoV-2 in the community. This information is currently lacking in Ethiopia and other African countries. This study aimed to develop an in-house antibody testing assay, assess the prevalence of SARS-CoV-2 antibodies among Ethiopian high-risk frontline HWs. Methods and findings A cross-sectional seroprevalence study was conducted among HWs in five public hospitals located in different geographic regions of Ethiopia. Socio-demographic and clinical data were collected using questionnaire-based interviews. From consenting HWs, blood samples were collected between December 2020 and February 2021, the period between the two peaks of COVID-19 in Ethiopia. The collected sera were tested using an in-house immunoglobin G (IgG) enzyme-linked immunosorbent assay (ELISA) for SARS-CoV-2 specific antibodies on sera collected from HWs. Of 1,997 HWs who provided a blood sample, demographic and clinical data, 50.5% were female, 74.0% had no symptoms compatible with COVID-19, and 29.0% had history of contact with suspected or confirmed patient with SARS-CoV-2 infection. The overall seroprevalence was 39.6%. The lowest (24.5%) and the highest (48.0%) seroprevalence rates were found in Hiwot Fana Specialized Hospital in Harar and ALERT Hospital in Addis Ababa, respectively. Of the 821 seropositive HWs, 224(27.3%) had history of symptoms consistent with COVID-19. A history of close contact with suspected/confirmed COVID-19 cases was strongly associated with seropositivity (Adjusted odds Ratio (AOR) =1.4, 95% CI 1.1-1.8; p=0.015). Conclusion High SARS-CoV-2 seroprevalence levels were observed in the five Ethiopian hospitals. These findings highlight the significant burden of asymptomatic infection in Ethiopia, and may reflect the scale of transmission in the general population

Prevalence of oncogenic human papillomavirus (HPV 16/18) infection, cervical lesions and its associated factors among women aged 21-49 years in Amhara region, Northern Ethiopia.

BackgroundHuman papillomavirus (HPV) infection is considered as the major risk factor for the development of cervical cancer, second most frequent cancer in Ethiopia. However, the magnitude of the problem and the associated factors remain unrevealed in the Amhara region. This study aimed to determine the prevalence of HPV infection and factors contributing to the progression of HPV infection to cervical cancer.MethodsFacility-based cross-sectional study design was employed among women aged 21 to 49 years of age who came for routine cervical cancer screening to 4 randomly selected hospitals (2 general and 2 referral) of Amhara region from May to October, 2019. The sample size was calculated by using the single population proportion formula, proportionated to hospitals, and women were recruited consecutively. Socio demographic and clinical data were collected using a pretested questionnaire and detection of HPV infection was done using HPV test (OncoE6TM Cervical Test) specific to HPV16/18 in cervical swabs. Visual inspection with acetic acid (VIA) was used to determine cervical lesions (precancerous and cancerous). Descriptive statistics and bivariate and multivariate logistic regression were used to describe HR-HPV and cervical lesions burden and association between HR-HPV, and cervical lesions and potential risk factors.ResultsAmong 337 women 21 to 49 years (median age of 35 years ±SD = 7.1 years) of age enrolled in the study, The overall prevalence of oncogenic HPVs (HPV16/18) and the VIA-positivity rate, possible an indicative of cervical lesions, were 7.1% and 13.1%, respectively. Logistic regression analysis showed a significant association between early age of first sexual intercourse (COR = 2.26; 95% CI: 1.0-5.05) and level of education (COR = 0.31; 95% CI: 0.12-0.78) with cervical lesions. Higher odds of HPV positivity (COR = 1.56; 95% CI: 0.59-4.11, p = 0.36) and VIA positivity (COR = 1.39; 95% CI: 0.64-3.00, p = 0.39) were observed among participants who had a history of sexually transmitted illnesses (STIs).ConclusionsThere was a relatively low prevalence of oncogenic HPV 16/18 and VIA-positivity in women attending four hospitals in the Amhara Region. Early age sexual contact, high parity, and being uneducated/low level of education were independently associated factors with HR-HPV infection and development of cervical lesions, highlighting the importance of prioritizing the limited HPV testing to those risk groups

Inference of population structure of Leishmania donovani strains isolated from different Ethiopian visceral leishmaniasis endemic areas.

BACKGROUND: Parasites' evolution in response to parasite-targeted control strategies, such as vaccines and drugs, is known to be influenced by their population genetic structure. The aim of this study was to describe the population structure of Ethiopian strains of Leishmania donovani derived from different areas endemic for visceral leishmaniasis (VL) as a prerequisite for the design of effective control strategies against the disease. METHODOLOGY/PRINCIPAL FINDINGS: Sixty-three strains of L. donovani newly isolated from VL cases in the two main Ethiopian foci, in the north Ethiopia (NE) and south Ethiopia (SE) of the country were investigated by using 14 highly polymorphic microsatellite markers. The microsatellite profiles of 60 previously analysed L. donovani strains from Sudan, Kenya and India were included for comparison. Multilocus microsatellite typing placed strains from SE and Kenya (n = 30) in one population and strains from NE and Sudan (n = 65) in another. These two East African populations corresponded to the areas of distribution of two different sand fly vectors. In NE and Sudan Phlebotomus orientalis has been implicated to transmit the parasites and in SE and Kenya P. martini. The genetic differences between parasites from NE and SE are also congruent with some phenotypic differences. Each of these populations was further divided into two subpopulations. Interestingly, in one of the subpopulations of the population NE we observed predominance of strains isolated from HIV-VL co-infected patients and of strains with putative hybrid genotypes. Furthermore, high inbreeding irreconcilable from strict clonal reproduction was found for strains from SE and Kenya indicating a mixed-mating system. CONCLUSIONS/SIGNIFICANCE: This study identified a hierarchical population structure of L. donovani in East Africa. The existence of two main, genetically and geographically separated, populations could reflect different parasite-vector associations, different ecologies and varying host backgrounds and should be further investigated