Evaluation of Less Invasive Sampling Tools for the Diagnosis of Cutaneous Leishmaniasis.

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.

Ecology and Infection Status of Sand Flies in Rural and Urban Cutaneous Leishmaniasis Endemic Areas in Northwest Ethiopia.

Cutaneous leishmaniasis (CL) caused by Leishmania aethiopica is transmitted by Phlebotomus longipes in northern Ethiopia. No studies have been conducted to investigate the transmission dynamics of CL, despite its high endemicity in both rural and urban settings. Evidence on the ecology and behavior of the vector from this area are required to develop integrated disease control strategies. Sand flies were collected in the dry and wet seasons in 2021 in CL-endemic rural Gindmeteaye and urban Addis-Alem in northwest Ethiopia. Trapping was performed with sticky and Centers for Disease Control and Prevention (CDC) light traps in three habitats, including inside patients' houses, peridomestic areasand in caves/rocky areas. Sand flies were morphologically identified to species level. Female Phlebotomus species were categorized according to blood feeding status and tested by spliced-leader (SL-) ribonucleic acid (RNA) polymerase chain reaction (PCR) to screen for Leishmania infection. Of 1161 sand flies, the majority (77%) were P. longipes, six (0.5%) were P. orientalis and the remaining were Sergentomyia. The abundance of the 430 female P. longipes was significantly linked to seasonality (p < 0.001), with the majority in the dry season occurring in the outdoor rocky (37%) and peridomestic (34%) sites, while, in the wet season, most (62%) were captured indoors. This seasonality was more pronounced in rural Gindmeteaye, where housing construction is poor. The number of blood-fed and gravid P. longipes was significantly higher in the wet (31%; 22%), compared to the dry season (13%; 8%), and their proportion was highest indoors. Eighteen (4%) female P. longipes were Leishmania positive, with highest infection prevalence in caves (7% compared to 3% indoors, p = 0.022), and in the dry season (6%, p < 0.001). Phlebotomus orientalis specimens were all captured in May in rural Gindmeteaye, five indoors and one in a peridomestic site. Further research should be conducted to investigate the absolute contribution of humans and indoor transmission to the transmission cycle of CL. Inhabitants of endemic villages should be made aware that evening outdoor activities near caves may increase their exposure to infectious sand flies. Whether P. orientalis can breed and become infected at high altitudes should be further studied.

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.

Anopheles arabiensis continues to be the primary vector of Plasmodium falciparum after decades of malaria control in southwestern Ethiopia.

BACKGROUND: Investigating the species distribution and their role in malaria transmission is important as it varies from place to place and is highly needed to design interventions appropriate to the site. The current study aimed to investigate the Anopheles mosquito species distribution and their infection rate in southwestern Ethiopia. METHODS: The study was conducted in 14 malaria-endemic kebeles (the smallest administrative unit), which were situated in eight different malaria-endemic districts and four zones in southwestern Ethiopia. Ten per cent of households in each village were visited to collect adult mosquitoes using Centers for Disease Control and Prevention (CDC) light traps. The larval and pupal collection was done from breeding sites within the villages, and reared to adults. Female mosquitoes were morphologically identified. The head and thorax of adult Anopheles mosquitoes were tested for circumsporozoite proteins (CSPs) using ELISA. At the same time, legs, wings, and abdomen were used to identify sibling species using PCR targeting the rDNA intergenic spacers region for species typing of the Anopheles funestus group and the internal transcribed spacer 2 region genes for Anopheles gambiae complex. RESULTS: A total of 1445 Anopheles mosquitoes comprising eight species were collected. Of 813 An. gambiae complex tested by PCR, 785 (97%) were Anopheles arabiensis, and the remaining 28 (3%) were not amplified. There were 133 An. funestus group captured and tested to identify the species, of which 117 (88%) were positive for Anopheles parensis, and 15 (11%) were not amplified. A single specimen (1%) showed a band with a different base pair length from the known An. funestus group species. Sequencing revealed this was Anopheles sergentii. Among 1399 Anopheles tested for CSPs by ELISA, 5 (0.4%) An. arabiensis were positive for Plasmodium falciparum and a single (0.07%) was positive for Plasmodium vivax. CONCLUSIONS: Anopheles arabiensis continues to play the principal role in malaria transmission despite implementing indoor-based interventions for decades. Sequencing results suggest that An. sergentii was amplified by the An. funestus group primer, producing PCR amplicon size of different length. Therefore, relying solely on amplifying a specific gene of interest in grouping species could be misleading, as different species may share the same gene.

Reemergence of Human African Trypanosomiasis Caused by Trypanosoma brucei rhodesiense, Ethiopia.

We report 4 cases of human African trypanosomiasis that occurred in Ethiopia in 2022, thirty years after the last previously reported case in the country. Two of 4 patients died before medicine became available. We identified the infecting parasite as Trypanosoma brucei rhodesiense. Those cases imply human African trypanosomiasis has reemerged.

First report of cutaneous leishmaniasis caused by Leishmania donovani in Ethiopia.

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.

Community-based treatment of cutaneous leishmaniasis using cryotherapy and miltefosine in Southwest Ethiopia: the way forward?

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.

Detection of asymptomatic Leishmania infection in blood donors at two blood banks in Ethiopia.

Visceral leishmaniasis (VL) is a disease caused by Leishmania parasites. While predominantly transmitted by sandflies, cases of VL transmitted through blood transfusion have been reported, particularly in immunocompromised recipients. Although Leishmania parasites have been found in blood donors in some VL endemic areas, this has never been studied in East-Africa, where HIV prevalence is relatively high. We established the prevalence of asymptomatic Leishmania infection and associated socio-demographic factors among blood donors presenting at two blood bank sites (Metema and Gondar) in northwest Ethiopia between June and December 2020. Metema is located in a VL-endemic area; Gondar has historically been considered VL non-endemic but as an outbreak of VL has occurred around Gondar, it was defined as previously VL non-endemic. Blood samples were tested by the rK39 rapid diagnostic test (RDT), rK39 ELISA, direct agglutination test (DAT) and qPCR targeting kinetoplast DNA (kDNA). Asymptomatic infection was defined as positive by any of these tests in a healthy person. A total of 426 voluntary blood donors were included. The median age was 22 years (IQR, 19-28 years); 59% were male and 81% resided in urban areas. Only one participant had a history of VL and three had a family history of VL. Asymptomatic infection was detected in 15.0% (n = 32/213) in Metema and 4.2% (n = 9/213) in Gondar. The rK39 ELISA was positive in 5.4% (n = 23/426), the rK39 RDT in 2.6% (11/426), PCR in 2.6% (11/420) and DAT in 0.5% (2/426). There were six individuals with two positive tests: one positive on rK39 RDT and PCR and five positive on rK39 RDT and ELISA. The prevalence of asymptomatic infection was higher in Metema (VL-endemic) and males but was not associated with age, a history of VL amongst family members or living in a rural area. Antibodies against Leishmania and parasite DNA was detected in a substantial number of blood donors. Future research should be directed at better defining the risk to recipients, including parasite viability studies and longitudinal studies amongst recipients.

PCR for detection of Leishmania donovani from microscopically negative tissue smears of suspected patients in Gondar, Ethiopia.

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.

COVID-19 vaccine acceptability among healthcare workers in Ethiopia: Do we practice what we preach?

OBJECTIVE: We assessed healthcare workers (HCWs) COVID-19 vaccine acceptability in Ethiopia. METHODS: We carried out a cross-sectional survey from February to April 2021 in HCWs from five teaching hospitals. HCWs were selected using convenient sampling, and data were collected through a survey link. Descriptive analysis and mixed-effect logistic regression were performed. A total of 1,314 HCWs participated in the study. RESULTS: We found that 25.5% (n = 332) of the HCWs would not accept a COVID-19 vaccine and 20.2% (n = 264) were not willing to recommend COVID-19 vaccination to others. Factors associated with vaccine non-acceptance were female sex (AOR = 1.8; 95% CI: 1.3-2.5), the perception that vaccines are unsafe (AOR = 15.0; 95% CI: 8.7-25.9), not considering COVID-19 as health risk (AOR = 4.4; 95% CI: 2.0-9.5) and being unconcerned about contracting COVID-19 at work (AOR = 3.5; 95% CI: 1.5-8.4). Physicians were more willing to accept vaccination than other HCWs. Higher vaccine acceptability was also noted with increasing age. Participants most often indicated safety concerns as the determining factor on their decision to get vaccinated or not. CONCLUSION: Overall, a quarter of HCWs would not accept a COVID-19 vaccine. Communications and training should address vaccine safety concerns. Additionally, emphasis should be given to showing current and future impact of COVID-19 on the personal, public and country level unless control efforts are improved. Interventions aimed to increase vaccine uptake should focus their efforts on younger and non-physician HCWs.

Evaluation of the CL Detect Rapid Test in Ethiopian patients suspected for Cutaneous Leishmaniasis.

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.

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.

Miltefosine for the treatment of cutaneous leishmaniasis-A pilot study from Ethiopia.

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.

Detection of Cutaneous Leishmaniasis Foci in South Ethiopia.

Cutaneous leishmaniasis (CL) is a major public health problem in Ethiopia. The disease is endemic in Ochollo, a village in southern Ethiopia, but there are no reports of CL in the wider area, although it is ecologically very similar. We conducted a rapid assessment survey in the South Ethiopian Rift Valley and found 100 parasitologically confirmed CL cases in 38 villages not reported endemic for CL. Approximately half of the cases were children (57%), and most lesions occurred on the face (78%) and were older than 6 months (77%). Only 2% of the people was aware of the mode of transmission, and 9% sought modern treatment at a hospital. These preliminary data indicate that CL is much more widespread than previously reported and that the disease might have a large psychosocial impact. Hence, this study calls for larger surveys across the Ethiopian highlands. Additionally, health education and treatment capacity need to be implemented.

Evaluation of conventional and four real-time PCR methods for the detection of Leishmania on field-collected samples in Ethiopia.

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.

An integrative approach to identify sand fly vectors of leishmaniasis in Ethiopia by morphological and molecular techniques.

BACKGROUND: Ethiopia is affected by human leishmaniasis caused by several Leishmania species and transmitted by a variety of sand fly vectors of the genus Phlebotomus. The sand fly fauna in Ethiopia is highly diverse and some species are closely related and similar in morphology, resulting in difficulties with species identification that requires deployment of molecular techniques. DNA barcoding entails high costs, requires time and lacks reference sequences for many Ethiopian species. Yet, proper species identification is pivotal for epidemiological surveillance as species differ in their actual involvement in transmission cycles. Recently, protein profiling using MALDI-TOF mass spectrometry has been introduced as a promising technique for sand fly identification. METHODS: In our study, we used an integrative taxonomic approach to identify most of the important sand fly vectors of leishmaniasis in Ethiopia, applying three complementary methods: morphological assessment, sequencing analysis of two genetic markers, and MALDI-TOF MS protein profiling. RESULTS: Although morphological assessment resulted in some inconclusive identifications, both DNA- and protein-based techniques performed well, providing a similar hierarchical clustering pattern for the analyzed species. Both methods generated species-specific sequences or protein patterns for all species except for Phlebotomus pedifer and P. longipes, the two presumed vectors of Leishmania aethiopica, suggesting that they may represent a single species, P. longipes Parrot & Martin. All three approaches also revealed that the collected specimens of Adlerius sp. differ from P. (Adlerius) arabicus, the only species of Adlerius currently reported in Ethiopia, and molecular comparisons indicate that it may represent a yet undescribed new species. CONCLUSIONS: Our study uses three complementary taxonomical methods for species identification of taxonomically challenging and yet medically import Ethiopian sand flies. The generated MALDI-TOF MS protein profiles resulted in unambiguous identifications, hence showing suitability of this technique for sand fly species identification. Furthermore, our results contribute to the still inadequate knowledge of the sand fly fauna of Ethiopia, a country severely burdened with human leishmaniasis.

High-resolution habitat suitability model for Phlebotomus pedifer, the vector of cutaneous leishmaniasis in southwestern Ethiopia.

BACKGROUND: Phlebotomus pedifer is the vector for Leishmania aethiopica causing cutaneous leishmaniasis (CL) in southwestern Ethiopia. Previous research on the transmission dynamics of CL resulted in recommendations for vector control. In order to target these interventions towards affected areas, a comprehensive understanding of the spatial distribution of P. pedifer at high spatial resolution is required. Therefore, this study determined the environmental predictors that facilitate the distribution of P. pedifer and created a map indicating the areas where conditions are suitable for survival of the vector in southwestern Ethiopia with high spatial resolution. METHODS: Phlebotomus pedifer presence points were collected during two entomological surveys. Climate, vegetation and topographic variables were assembled. Climate variables were interpolated with variables derived from high-resolution digital elevation models to generate topoclimatic layers representing the climate conditions in the highlands. A Maximum Entropy model was run with the presence points, predicting variables and background points, which were selected based on a bias file. RESULTS: Phlebotomus pedifer was the only captured Phlebotomus species in the study area and was collected at altitudes ranging between 1685 and 2892 m. Model projections indicated areas with suitable conditions in a 'belt' surrounding the high mountain peaks. Model performance was high, with train and test AUC values being 0.93 and 0.90, respectively. A multivariate environmental similarity surface (MESS) analysis showed that the model projection was only slightly extrapolated for some of the variables. The mean annual temperature was the environmental variable, which contributed most to the model predictions (60.0%) followed by the seasonality in rainfall (13.2%). Variables representing steep slopes showed very low importance to model predictions. CONCLUSIONS: Our findings indicate that the suitable habitats for P. pedifer correspond well with the altitudes at which CL was reported previously, but the predictions are more widely distributed, in contrast with the description of CL to occur in particular foci. Moreover, we confirm that vector distribution is driven by climate factors, suggesting inclusion of topoclimate in sand fly distribution models. Overall, our model provides a map with a high spatial resolution that can be used to target sand fly control measures in southwestern Ethiopia.

Evaluation of a pan-Leishmania SL RNA qPCR assay for parasite detection in laboratory-reared and field-collected sand flies and reservoir hosts.

BACKGROUND: In eco-epidemiological studies, Leishmania detection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-Leishmania SYBR green quantitative PCR (qPCR) assay which detects the conserved spliced-leader RNA (SL RNA) sequence was developed recently. This study assessed the SL RNA assay performance combined with a crude extraction method for the detection of Leishmania in field-collected and laboratory-reared sand flies and in tissue samples from hyraxes as reservoir hosts. METHODS: Field-collected and laboratory-infected sand fly and hyrax extracts were subjected to three different qPCR approaches to assess the suitability of the SL RNA target for Leishmania detection. Nucleic acids of experimentally infected sand flies were isolated with a crude extraction buffer with ethanol precipitation and a commercial kit and tested for downstream DNA and RNA detection. Promastigotes were isolated from culture and sand fly midguts to assess whether there was difference in SL RNA and kDNA copy numbers. Naive sand flies were spiked with a serial dilution of promastigotes to make a standard curve. RESULTS: The qPCR targeting SL RNA performed well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL RNA and kDNA detection. Copy numbers of kDNA were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL RNA levels were slightly lower in sand fly promastigotes (ΔCq 1.7). The theoretical limit of detection and quantification of the SL RNA qPCR respectively reached down to 10-3 and 10 parasite equivalents. SL RNA detection in stored hyrax samples was less efficient with some false-negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents. CONCLUSIONS: This study shows that a crude extraction method in combination with the SL RNA qPCR assay is suitable for the detection and quantification of Leishmania in sand flies. The assay is inexpensive, sensitive and pan-Leishmania specific, and accordingly an excellent assay for high-throughput screening in entomological research.

Feeding behavior and activity of Phlebotomus pedifer and potential reservoir hosts of Leishmania aethiopica in southwestern Ethiopia.

BACKGROUND: Cutaneous leishmaniasis (CL) is a major public health concern in Ethiopia. However, knowledge about the complex zoonotic transmission cycle is limited, hampering implementation of control strategies. We explored the feeding behavior and activity of the vector (Phlebotomus pedifer) and studied the role of livestock in CL transmission in southwestern Ethiopia. METHODS: Blood meal origins of engorged sand flies were determined by sequencing host DNA. A host choice experiment was performed to assess the feeding preference of P. pedifer when humans and hyraxes are equally accessible. Ear and nose biopsies from livestock were screened for the presence of Leishmania parasites. Sand flies were captured indoor and outdoor with human landing catches and CDC light traps to determine at which time and where P. pedifer is mostly active. PRINCIPAL FINDINGS: A total of 180 P. pedifer sand flies were found to bite hosts of 12 genera. Humans were the predominant blood meal source indoors (65.9%, p < 0.001), while no significant differences were determined outdoors and in caves. In caves, hyraxes were represented in blood meals equally as humans (45.5% and 42.4%, respectively), but the host choice experiment revealed that sand flies have a significant preference for feeding on hyraxes (p = 0.009). Only a single goat nose biopsy from 412 animal samples was found with Leishmania RNA. We found that P. pedifer is predominantly endophagic (p = 0.003), but occurs both indoors and outdoors. A substantial number of sand flies was active in the early evening, which increased over time reaching its maximum around midnight. CONCLUSION: In contrast to earlier suggestions of exclusive zoonotic Leishmania transmission, we propose that there is also human-to-human transmission of CL in southwestern Ethiopia. Livestock does not play a role in CL transmission and combined indoor and outdoor vector control measures at night are required for efficient vector control.

Ecology and seasonality of sandflies and potential reservoirs of cutaneous leishmaniasis in Ochollo, a hotspot in southern Ethiopia.

BACKGROUND: Ochollo is a village in southern Ethiopia burdened with cutaneous leishmaniasis (CL), where Phlebotomus pedifer is the only vector for Leishmania aethiopica and hyraxes are confirmed reservoir hosts. A detailed description of the different players of transmission, and the ecology and seasonality of the vector needs to be established in order to accomplish efficient control programs. METHODS AND FINDINGS: Between March 2017 and February 2018, a monthly sandfly collection was carried out in different habitats and records of temperature and humidity were taken. Rodents and hyraxes were trapped in the dry and wet season. All samples were screened for Leishmania kinetoplast DNA (kDNA). Positive samples were further processed for determination of the Leishmania species and the species of the sandfly/small mammal that was found infected. Additionally, the species of 400 sandfly specimens from different habitats and seasons was identified. 17,190 Sergentomyia and Phlebotomus sandflies were caught and showed an overall kDNA prevalence of 2.6%, all were L. aethiopica infections only found in P. pedifer. The overall sandfly and P. pedifer abundance peaked in the dry season and was negatively correlated with the %RH. The kDNA prevalence varied over the months and was negatively correlated with the temperature. Total sandfly abundance did not differ between the sampled habitats, but P. pedifer was the distinct predominant species only in caves. Moreover, significantly more infected sandflies were found in caves. Only 1/192 rodents were kDNA positive, while 20.0% (5/25) of Heterohyrax brucei were found infected. CONCLUSIONS: This study suggests that caves may be a source of multiplication of the infection. If an outdoor control program would be considered, it would be useful to focus on caves in the wet season, when the sandfly abundance is lowest. The captured rodent species appear not important for transmission and the contribution of hyraxes in transmission should be further investigated.