Introduction

Leishmaniasis is a neglected tropical vector-borne disease transmitted by infected female Phlebotomus sandflies and is caused by protozoan parasites of more than 20 species1,2. Approximately 70 species of sandflies can transmit Leishmania. They belong to the genus Phlebotomus in the Old World (Europe, Asia, and Africa), while in the New World (Americas) they are classified under the genus Lutzomyia3.

Leishmania has different forms, including visceral leishmaniasis (VL), the most severe form, and cutaneous leishmaniasis (CL), the most common4. CL is endemic in 100 countries across six continents, with an estimated annual prevalence of 12 million people, including 2 million new cases globally1,5,6. In Ethiopia, the first CL cases were recorded in 1913; since then, it has been reported to be prevalent in highland areas, particularly at altitudes ranging from 1,400 to 2,700 m (approximately 1.68 mi) above sea level (ASL)7. Different findings have reported CL incidences ranging from 5.6 to 65.8% in different areas of Ethiopia1,8,9,10,11,12,13,14,15, showing its significant importance for public health, including in Northeast Ethiopia16.

In Ethiopia, CL manifests in three clinical forms: localized cutaneous leishmaniasis (LCL), mucosal leishmaniasis (ML), and diffuse cutaneous leishmaniasis (DCL). Although few studies have reported Leishmania tropica, Leishmania aethiopica is the primary causative agent of CL in Ethiopia17.

This disease, commonly known as “poor man’s disease,” affects millions of people worldwide. The clinical manifestations range from papules to nodules, resulting in atrophic scars that lead to disfigurement and stigma that persist even after treatment18. Clinical features are associated with several factors, such as age, proximity to farmland, availability of reservoir hosts (hyrax), and housing conditions11,13,14,15,19.

Despite the availability of different methods for the detection of leishmaniasis, molecular techniques have become increasingly relevant because of their improved sensitivity, specificity, and applicability to a variety of clinical samples in different areas20,21,22,23,24. Real-time PCR with LC kDNA PCR showed a high sensitivity for detecting L. aethiopica. However, their application is limited22 and has not yet been validated. The internal transcribed spacer region-1 (ITS1) gene is a highly variable region of the Leishmania genome used for identifying and differentiating Leishmania species.

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is used to detect and identify specific genetic sequences25. The ITS1 polymerase chain reaction-restriction analysis technique has been commonly used in different studies for Leishmania parasite profiling (ITS1-PCR)26,27,28,29.

Because of the growing burden of CL, different stakeholders and disease control programs in endemic regions have begun implementing strategic action plans to combat the disease by implementing regular national surveillance, improving diagnostic facilities, ensuring access to effective treatment, and public health awareness programs for the prevention and early detection of CL. In the road map for neglected tropical diseases (NTDs), the World Health Organization (WHO) set targets for 85% of all CL cases to be detected and reported and 95% of reported cases to be treated by 203030. The Ethiopian Ministry of Health (MoH) collaborates with governmental and nongovernmental organizations to align their mutual initiatives with global health targets. These collaborative and integrated actions reflect the commitment to combating the burden of CL and improving the health outcomes of affected individuals. However, CL is still on the WHO list of emerging and uncontrolled NTDs, with an estimated 350 million people at risk5,31.

Cutaneous leishmaniasis is one of the most common NTDs in Ethiopia. Although it is endemic to various parts of the country, the full burden and distribution of the disease are not well established. Compared with other NTDs, 170 cases are suspected to be endemic, and only 80 cases are officially confirmed32 as endemic to CL. While nearly 29 million people are estimated to be at risk and the annual incidence ranges from 20,000 to 50,000 cases32, less than 10% of these cases are reported to have received treatment in the last five years1 owing to limited care seeking through formal health services33. Despite being known in Ethiopia since 1913, CL has been highly neglected1, with only 14 treatment centers currently known, which underscores the importance of targeted interventions and control measures to mitigate its burden.

The Ethiopian Ministry of Health collaborates with governmental and nongovernmental organizations to align their mutual initiatives with global health targets. These collaborative and integrated actions reflect the commitment to combating the burden of CL and improving the health outcomes of affected individuals. However, CL is still on the WHO list of emerging and uncontrolled NTDs, with an estimated 350 million people at risk6,34. Among all neglected tropical diseases, CL is the most overlooked disease in Ethiopia. Thus, the epidemiology of CL in Ethiopia may underscore the importance of targeted interventions and control measures to mitigate this burden.

For evidence-based public health programs targeting CL interventions, earlier studies reported estimates of CL incidence or incidence via retrospective record reviews, institution- or facility-based prospective approaches, smaller samples, and community-based active surveillance for CL lesions, which were rare at the community level and overlooked in hard-to-reach rural areas. These findings were based on clinical examinations and conventional detection methods34, except for a few studies that used molecular techniques for confirmatory tests. Furthermore, earlier evidence was significantly affected by the low health-seeking behavior of patients due to the perceived self-healing nature of CL, poor access to health facilities (as most affected people are rural and remote dwellers), and poor access to diagnosis and treatment. Thus, a community-based study addressing hard-to-reach rural areas with a large sample size and molecular techniques is crucial for evidence-based intervention. Therefore, this study aimed to estimate CL underreported hotspot areas. Unlike earlier studies, this study allowed us to understand the distribution of CL at the kebele level, which is crucial for targeted, evidence-based public health interventions. This study overcomes the methodological limitations of earlier studies in which ITS1 locus markers were used throughout the study. Moreover, the findings of this study could serve as a basis for the entomological study of sandfly vectors in this area. Therefore, this study aimed to investigate the molecular epidemiology, distribution, and determinants of CL at the kebele level via the ITS1 marker in clinical samples from Northeast Ethiopia.

Results

Demographic and clinical characteristics of the study participants

Among the 384 suspected cases, 356 were included in the study, resulting in a response rate of 92.7%. Among the 356 suspected cases, 253 (71.1%) were positive for Leishmania parasites. The median age of the participants was 15 years (IQR ± 12). 57% of the patients were male, and 70.4% of the males had LCL. Families with fewer than five household members accounted for 60.4% of the sample. With respect to the patterns of different clinical features of CL, 75.0%, 19.1%, 5.3%, and 0.56% of suspected cases presented with LCL, ML, MCL, and leishmaniasis recidivans (LRs), respectively. Examples of the clinical presentations of CL in the study area are shown in Fig. 1. Most patients had a single lesion (263, 73.9%), and the face was the most common active lesion site (269, 75.6%). More than one-quarter of the 107 (29.7%) patients with suspected CL had lesions for more than 1 year, which may indicate delayed diagnosis or treatment (P < 0.001) (Table 1).

Fig. 1
figure 1

Clinical manifestations of cutaneous leishmaniasis from PCR-confirmed CL cases in the study are: (a) and (b) localized cutaneous leishmaniasis; (c–f) mucocutaneous leishmaniasis.

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Table 1 Sociodemographic and clinical characteristics of the study participants in Northeast Ethiopia (2022–2023).
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Molecular epidemiology of CL, distribution, and detection of Leishmania species

Molecular epidemiology of CL

Among the 356 suspected cases, 253 (71.1%; 95% CI: 66.1–75.6) were confirmed to have Leishmania parasites. The polymerase chain reaction results revealed that the highest number of confirmed cases occurred in the 9 to 19 years age group (80.3%; 139/173), whereas 57.3% (82/143) of those older than 19 years of age were confirmed. The prevalence of CL infection was similar in men (71.4%) and women (70.6%). With respect to living conditions, the CL infection rates of the study participants living with and without animals in the house were 76.2% (163/214) and 63.4% (90/142), respectively (Table 2). The prevalence rates of CL infection among the study participants living in houses with cracked walls and floors were 68.8% (212/308) and 68.7% (204/297), respectively (Fig. 2).

Fig. 2
figure 2

Housing conditions of the CL cases.

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Distribution of cutaneous leishmaniasis across different Kebeles

The distribution of confirmed CL infections across selected kebeles ranged from 0.3 to 4.5%. A greater proportion of confirmed CL cases were reported from Satero (4.5%), TidGebeya (4.49%), Berara (3.37%), Berara Jerjero (2.8%), Kundi Nazarjor (2.8%), and Amdework (2.8%). In contrast, Woyis Kisat, Tembeko, Ayda Segor, Amumo, Sulula, Chacha, and Ketema Zuria had a lower number of CL cases (Fig. 3 and Supplementary Table S1).

The number of CL cases in each kebele was marked to provide a visual representation of their distribution across the kebeles (Fig. 3). The choropleth map revealed that CL infection was more common among Kebeles in Dissie-Zuria and Kutaber Werda. The distribution of cases across the mentioned kebeles revealed that the burden of CL differed across study kebeles in the respective Woreda administrations in Northeast Ethiopia (Fig. 3 and Supplementary Table S1).

Fig. 3
figure 3

Map of surveyed kebeles showing the number and distribution of CL cases in northeast Ethiopia.

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The color changes represent the burden or case number in each kebele, and the light blue dots represent the size, which represents the size (case count) of each kebele. The number shows the name of the kebele included in the study as follows: 1-Ketema zuria; 2- Amdework; 3- Weyiso kisat; 4- Kisat kola; 5- Beseka; 6- Tembeko; 7- Wegeltena; 8- Gosh Meda; 9- Keda Misitinkir; 10- Wteyo Aberkut; 11- Ayda Segora; 12- Chacha; 13- Amba Mariyam; 14- Kundi Najarjor; 15- Asecha Abo; 16- Abaselama; 17- Manos; 18- Dashengena Elsa; 19- Haroye; 20- Beshelo; 21- Liwicho; 22- Goro Mender; 23- Satiro; 24- KedejJerjero; 25- Ayata; 26- Asgedo; 27- Kolamote; 28- KeyGedel; 29- Degamote; 30- Arsis Amba; 31- Berara; 32- Berara Jerjero; 33- Tid Gebeya; 34- Hara Webelo; 35- Atari Mesk; 36- Nbara Ager; 37-Bosena; 38- Keteteya; 39- Adame; 40- Sulula Sulula Town; and 41- Amumo.

Detection and identification of Leishmania parasites

PCR was used to target a Leishmania-specific ITS1 gene, which was amplified via the primers LITS and L5.8 S to generate PCR products between 300 and 350 bp in length. Among all the samples, amastigotes were observed in 89 (25%) Giemsa-stained positive control and clinical samples (Fig. 4).

Fig. 4
figure 4

Agarose gel electrophoresis for the detection of L. aethiopica using PCR. Lanes 16 and 28: 100 bp DNA ladders. Lanes 2 and 15: negative controls; Lane 27, L. aethiopica(positive control); Lanes 3, 4, 11, 12, 13, 22, 23, 24, and 26: no amplified results; Lanes 5, 5, 6, 8, 10, 14, 17, 20, 21, 25, 14, and 15: positive results. Lanes 18 and 19 show positive results with weak amplification (confirmed after repeated experiments).

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When HaeIII digested the ITS-1PCR product, L. aethiopica produced three bands with sizes of 200–220 bp, 65 bp, and 23 bp. When the ITS-1PCR-digested product was subjected to 2% agarose gel electrophoresis, two well-resolved bands of approximately 215 bp and 65 bp and a poorly resolved 23 bp band in a sample similar to the positive control of L. aethiopica were detected(Fig. 5).

Fig. 5
figure 5

L. aethiopica species detected by ITS1 PCR-RFLP after HaeIII digestion. Lanes 1 & 16, 100 bp DNA ladder; lanes 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, and 14, L. aethiopica from samples; lane 15, L. aethiopica (positive control).

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Factors associated with cutaneous leishmaniasis infection

Factors associated with CL were determined via a binary logistic regression model. The model fitness test was performed via the Hosmer–Lemeshow test (P = 0.479), which revealed that the assumptions were met. In the binary logistic regression analysis, nine explanatory variables, including the presence of animals in the house, age less than 9 years, family size, age group between 9 and 19 years, presence of a cave near human settlement and gorge within 300 m, hyrax near 300 m, animal burrow near home, animal dung near home, immediate neighbor with ulcerating disease, and sharing residence with a household member with typical skin lesions (P < 0.2), were included.

The variables independently associated with CL, after controlling for confounding effects in the multivariable logistic regression analysis, were the presence of animals in the house (AOR: 2.06; 95% CI: 1.23–3.43), age group < 9 years (AOR: 2.94; 95% CI: 1.21–7.14), age group 9–19 years (AOR: 3.23; 95% CI: 1.89–5.51), and household size of five or more members (AOR: 2.78; 95% CI: 1.60–4.84), which were significantly associated with CL infection (Table 2). Among the 314 study participants living in houses with cracked floors, 216 (68.8%) had CL infection, whereas among the 277 study participants living in houses with grass roofs, 181 (65.3%) had CL infection (Fig. 2).

Table 2 Bivariable and multivariable logistic regression analyses for factors associated with CL infection in Northeast Ethiopia (March 2022-May 2023).
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Discussion

This study was conducted to assess the molecular epidemiology, distribution, and determinants of CL at the kebele level in Northeast Ethiopia. The overall prevalence of CL among the suspected cases via PCR was 71%. The presence of animals in the house, among the age group < 9 years, an age group between 9 and 19 years, and a household size of five years and above, was significantly associated with CL infection. Our study is the first molecular epidemiological study with a sufficient sample size at the kebele level, the lowest administrative unit in Ethiopia.

Localized cutaneous leishmaniasis (LCL) is the most prevalent type of cutaneous leishmaniasis, accounting for 75% of cases. This result is in line with reports from health facilities in Nefas Mewucha (79.1% and 70.2%)35,36, Bahir Dar (48.9%)37, and various regions of Ethiopia10,19,38,39,40,41,42. Most of the patients (75.8%) had a single lesion, which is supported by an earlier report from northeastern Ethiopia19. In this study, the predominant site of the lesion was the face (34.0%), with common presentations including swelling, erythema, and crusting/scaling (31.7%). This finding is supported by earlier evidence reported in Bahir Dar health facilities37. Localized cutaneous leishmaniasis caused by L. aethiopica often manifests on exposed skin, particularly on the face, where it is exposed to sandfly bites38.

With respect to the distribution of cutaneous leishmaniasis at the kebele level, Satero 21 (4.5%), Tid Gebeya 20 (4.49%), Berara 15 (3.37%), Berara Jerjero 13 (2.8%), Kundi Nazarjor (2.8%), and Amdework (2.8%) had more CL cases. This could be due to their ecological and environmental factors, which favor vectors that facilitate the transmission dynamics of CL. Additionally, these areas are topographically characterized by gorges and caves that harbor hyrax, and most of the households are within a 300-meter radius of the reservoir host. These findings highlight that the transmission of CL is not uniform across kebeles, providing valuable information on risk factor differences and sources of infection.

Although L. tropica and L. donovani have been reported in the Awash Valley of the Ethiopian Rift Valley43 and known endemic areas of VL in northwest Ethiopia44, all CL infections confirmed with PCR-RFLP were identified as L. aethiopica, which is consistent with the findings of another similar study conducted at Boru Meda Hospital, northeast Ethiopia19.

The overall prevalence of CL among the suspected cases through PCR-RFLP was 71.1%, indicating that, among the ten-suspected cases, at least seven individuals had CL infection.

The prevalence is higher than that previously reported in endemic areas of Ethiopia, such as 3.6–4.0% in Ochollo39, 4.8% in Siliti Woreda40, 22.4% in Boru Meda Hospital19, and 31.7% in Tefera Hailu Memorial Hospital in Sekota45. This is due to differences in the geographical areas39,40 and diagnostic tools used, where earlier studies used less sensitive conventional microscopy and/or culture results19 and/or health facility-based12 retrospective data45,46. This might be due to environmental factors such as higher temperature and humidity, which could increase the sand fly population density and, in turn, increase CL transmission in the area.

Polymerase chain reaction amplicon based techniques, such as sequencing and RFLP, have advantages that they can be applied directly to biological samples and avoiding parasite culture. The methods are more reliable and accurate for the identification of Leishmania parasite in epidemiological studies47. Next generation sequencing have emerged for identification of vector‒parasite‒reservoir association co-infections in the endemic areas48.

Our study involved extensive molecular analysis with a large sample size to estimate the burden of CL in the area. We employed commercial tissue extraction kits that increased the DNA yield for our PCR experiments.To our knowledge, there has been no molecular epidemiological study with a sufficient sample size at the kebele level, which is the lowest administrative unit in Ethiopia. Our study effectively determined the magnitude and spatial distribution of CL at this level, in which CL cases are clustered in areas where reservoir hosts and favorable environmental conditions exist.Additionally, the difference in study area and period contributes to the existing differences; the current study was conducted in a war-torn area where people lived for up to a month in a cave during the internal conflict, which further worsened the risk of exposure to biting by sandflies. In addition, Leishmania control and prevention strategies have not been properly implemented owing to internal conflicts.Furthermore, methodological approaches and study population type have been other influential factors for the existing differences, where earlier studies in different areas relied on a facility-based approach and retrospective findings. Most CL cases are characterized by a delay in diagnosis, and most patients seek traditional healers rather than healthcare services. Thus, the rate of detection in earlier studies can be significantly underestimated, whereas the present study provides a real estimate of CL. A community-based study in which suspected cases of healthcare-seeking and low healthcare-seeking behavior were addressed revealed that these cases significantly affected the detection ability of CL. This variability highlights the importance of area-specific investigations to assess the true burden of CL accurately and to guide targeted interventions.

The higher infection rate among children (< 9 years) and adolescents (9–19 years) is consistent with several studies conducted elsewhere in the country10,39, possibly because domestic animals, where their associated animal dung and organic materials are available, increase the density of the vector population. On the other hand, keeping animals in rural areas of Ethiopia might increase exposure to sandfly bites around caves and gorges, where reservoir hosts with relatively high vector populations are available. Another risk could be from schoolyards, which may have vegetation that harbors sandflies and increases the number of sandfly bites.

In this study, a household size of five or more had statistically significant associations with CL infection. A large household size and high population density are considered proxy indicators of poverty, which has been associated with CL49. Higher household sizes could predispose households to frequent sandfly–human contact with active disease.

Home sharing with domestic animals is associated with a more than twofold increase in the risk of CL infection. The presence of animals in the same house could increase the degree of human infection caused by increasing sandfly density, thereby increasing the degree of human-vector contact. This finding aligns with earlier studies conducted in Ethiopia50,51. This might be because livestock and their dung create ideal breeding conditions for sandflies in areas around homes. Sandflies often breed and gather in organic waste, such as dung, rodent burrows, and cracks in walls. This finding underscores the need for health education about living with domestic animals and sanitation practices around the home to reduce CL infections in the study area. Another explanation could be living with animals in the house, which is related to the presence of a high vector density, the presence of a reservoir host, and the environmental conditions/habitats that are ideal for the reproduction of sandflies. Additionally, the socioeconomic characteristics of residents, poor waste disposal systems, house sharing with domestic animals, poor housing conditions, and human activities, such as outdoor activities, increase sandfly contact52.

The positivity rate was higher in those living in poor housing conditions (cracked walls, floors, and roofing materials other than concrete or solid materials), revealing the role of housing conditions in CL transmission dynamics. Poverty is associated with environmental factors that increase risk, such as poorly constructed houses with mud walls, cracked floors, nearby animal dung, and thatched roofs that provide suitable habitats for sandflies and increase the risk of CL transmission49,53, which is supported by our observation that living close to or with pets is common. This provides sources of blood meal and decaying organic material for breeding sandflies and increases leishmaniasis infection. This might be because livestock and their dung create ideal conditions for sandflies in areas around homes. Sandflies often breed and gather in organic waste, such as dung, rodent burrows, and cracks in walls. This finding underscores the need for health education about living with domestic animals and sanitation practices around the home to reduce CL infections in the study area. Another explanation could be living with animals in the house, which relate to the presence of a high vector density, the presence of a reservoir host, and the environmental conditions/habitats that are ideal for the reproduction of sandflies. Additionally, the socioeconomic characteristics of residents, poor waste disposal systems, house sharing with domestic animals, poor housing conditions, and human activities, such as outdoor activities, increase sandfly contact49. Related results suggest that living with infected people increases the risk of transmission, revealing the role of social and community factors in CL transmission19,39.

Furthermore, living within 300 m of a farm and the presence of animal burrows, dung, and rock hyrax could increase the risk of Leishmania infection, although these factors were not significantly associated with CL. This high proportion of infections was supported by earlier studies that reported the role of these factors in CL transmission risk10,14,28,38,39,54,55,56. This could be due to the potential role of repositories of Leishmania parasites by rock hyrax and the ideal environment of burrows in rocky areas for sandflies with food sources and moisture. Moreover, animal burrows and burrows in rocky areas are rich in humidity and organic matter because decaying materials provide breeding grounds for sandflies. Additionally, these habitats provide shelter and resting places for sandflies during the day, support their Leishmania parasites, attract sandflies, contribute to the persistence of the parasite in the environment, and increase the risk of CL transmission to humans in Ethiopia38,56.

The present study was strengthened by the use of advanced molecular techniques for the detection of parasites and multiple study settings with a community-based prospective study at the kebele level, which captures different ecological and environmental contexts in the three zones of northeastern Ethiopia. However, our study was not without limitations, as we faced a scarcity of resources, and sequencing for the identification of potential strains was not performed. Some of the previously identified cases were not included in the data collection because some were unavailable at nearby health facilities. Moreover, we lacked observational assessments at the household level for risk factors such as waste disposal, proximity to agricultural land, the presence of nearby animal burrows, general housing conditions, and elevation differences that could influence our results.

Conclusions

The overall prevalence of CL is relatively high, indicating that this disease is a public health problem in this area. Leishmania aethiopica was detected as the causative agent of CL in northeastern Ethiopia. A greater proportion of cases were found in Satero, Tid Gebeya, Berara, Berara Jerjero, Kundi Nazarjor, and Amdework. Cutaneous leishmaniasis infection was significantly more common among participants from larger families, those living in households with animals, those with children less than 9 years old, and those aged 9–19 years. These findings highlight the need for tailored, evidence-based public health interventions, which will contribute to the national cutaneous leishmaniasis control strategy through the consideration of local ecological and environmental conditions. Furthermore, these findings underscore the need to improve health outcomes and quality of life in affected communities. This study also revealed that CL is a public health problem in northeastern Ethiopia, highlighting the need for routine vector surveillance at the kebele level to better understand the transmission dynamics of CL and targeted control strategies to mitigate its burden in the region.

Materials and methods

Study area and period

A cross-sectional study was conducted from March 2022 to May 2023 in three CL endemic zones (Wag Hamra, North Wollo, and South Wollo) in northeastern Ethiopia. According to the Central Statistical Agency of Ethiopia (CSA) 2021/22 census, the total populations of Wag Hamra, North Wollo, and South Wollo were 548,884, 1,989,563, and 4,518,862, respectively. The study was conducted at the kebele level (the smallest administrative unit in Ethiopia) in eight woredas (two in Dehana, eight in Delanta district, 11 in Kutaber district, 13 in Dessie, two in Tehuledere, one in Dawunt, one in Tenta, and three in Kalu Woredas) from March 2022 to May 2023 (Fig. 3).

Dessie Zuria is in the South Wollo Zone of the Amhara Region, at a latitude and longitude of 11°8′N 39°38′E, with an altitude between 2,470 and 2,550 m above sea level (masl). Kutaber is a populated place in the Amhara regional state of Ethiopia. It is 3,058 masl. It is located at an altitude of 3,058 masl. Its coordinates are 11°16’0” N and 39°31’60” E. Kalu is at an elevation of 3,327 m above sea level. Its coordinates are 11°28’0” N and 39°31’60” E. Tehuledere is found at an elevation of 1,948 masl. Its coordinates are 11°18’0” N and 39°40’60” E. Delanta is found at 2,984 masl. Its coordinates are 11°34’60” N and 39°10’0” E. Amdewerq (Amdework) (Amharic “a pillar of gold”) is a town in northern Ethiopia and is found in the Wag Hamra Zone of the Amhara region. The town has a latitude and longitude of 12°20 N, 38°45 E, and an elevation of 2421 masl. Amdewerq is the administrative center of Dehana District. Skin lesions were common in these areas. From the selected records, all suspected cutaneous leishmaniasis cases fulfilling the inclusion criteria were included in this study. In contrast, patients with suspected CL who started treatment were excluded.

Sample size determination and sampling strategy

The sample size was determined via a single population proportion formula by taking the 50% prevalence at the molecular level and by considering a 95% confidence interval (CI) and 5% margin of error57.

$$n=\frac{{\left(z\propto/2\right)}^{2}P\:(1-P)}{d^{2}}\:n=\frac{{\left(1.96\right)}^{2}0.5\:(1-0.5)}{\left(0.05\right)^{2}}=384$$

where n is the sample population, p is the molecular epidemiology of CL (0.5), d is the margin of error (0.05), and Z (∝/2) is the reliability coefficient of 95%, which is 1.96.

First, the number of cases at the Kebele level from February to September 2022 was determined in collaboration with the Kebele health office staff and administrators via data from CL treatment centers, local health department reports, local health facility records, and reports from health extension workers. All patients with corresponding numbers of suspected CL cases with at least one skin ulcer are listed. Kebeles were listed and selected on the basis of the number of suspected cases, resources, accessibility, distance, and environmental characteristics.

Variables

Leishmaniasis infection, defined by the ITS1-PCR positive rate (coded as 1 if ITS1-PCR and/or microscopy positive and 0 if ITS1-PCR negative), was the dependent variable, whereas sociodemographic characteristics (age, sex, place of residence, family size, marital status, educational status, occupation), clinical characteristics (duration of the oldest lesion, number, and location of lesions, presentation index of the lesion), environmental and living conditions (e.g., cave within 300 m, rock hyrax near the house within 300 m, animal den near the house, animal manure near the house, farm near the house within 300 m, an immediate neighbor with an ulcerative disease, presence of a nearby forest or thicket, shared living with a household member with typical skin lesions, living in a house with cracked walls and floors including clay or soil with visible cracks, and living in a house with a roof made of grass, leaves, earth, or rocky caves) were the independent variables.

Data collection and sample analysis

Preliminary survey and data collection

All necessary information was collected from participants who met the inclusion criteria via a semistructured questionnaire aimed at collecting sociodemographic characteristics, clinical characteristics, laboratory data, and other potential risk factor data after written informed consent was obtained. Before actual data collection began, a house-to-house visit was conducted to find suspected clusters of CL cases. The community often called the skin lesion by the colloquial name “kunchir,” which is often a clinical manifestation of CL. All suspected CL cases from selected Kebeles who did not receive treatment were sent to nearby local health facilities by experienced dermatologists to record their clinical manifestations. Unique, nonpigmented, mottled, and depressed scars of healed lesions were used as operative criteria to diagnose past CL. Papular, nodular, and ulcerative lesions were noted and used for the clinical diagnosis of localized cutaneous leishmaniasis (LCL). Patients with initial congestion or bleeding and progressive destruction of the mucous membranes of the nose, mouth, and throat were found to have mucosal leishmaniasis (ML); however, mucosal and skin lesions were simultaneously identified as mucocutaneous leishmaniasis (MCL). Patients with multiple nonulcerative nodular lesions, often larger than those with LCL, are often diagnosed with diffuse cutaneous leishmaniasis (DCL)27. All suspected CL cases from selected kebeles without current treatment were sent to nearby local health facilities by experienced dermatologists to record their clinical characteristics. Unique, nonpigmented, mottled, and depressed scars of healed lesions were used as operative criteria to diagnose past CL. Papular, nodular, and ulcerative lesions were noted and used for the clinical diagnosis of localized cutaneous leishmaniasis (LCL). Patients with first congestion or bleeding and progressive ulcerative destruction of the mucous membranes of the nose, mouth, and throat were found to have mucosal leishmaniasis; however, mucosal and skin lesions were simultaneously identified as mucocutaneous leishmaniasis. Patients with multiple nonulcerative nodular lesions, often larger than those with LCL, were diagnosed with diffuse cutaneous leishmaniasis (DCL).

Skin scraping samples were collected from all 356 suspected CL cases during the door-to-door visit at a nearby health facility. A structured questionnaire (prelabeled during house-to-house visits) that included demographic and clinical characteristics and risk factors was completed.

Skin scrap sample collection and processing

Following the detailed sampling instructions, two skin-slit samples were collected from the skin lesions of each suspected CL patient. After the lesions/nodules were disinfected with absolute ethanol, a small incision was made at the edge of the lesion via a sterile disposable scalpel blade, and the sample was mounted on two glass slides. The skin scraping smears (SSSs) were air-dried and fixed in methanol, the slides were stained with 10% Giemsa, and one slide was examined for the presence of amastigotes via light microscopy (Fig. 6).

Fig. 6
figure 6

Leishmania amastigotes (a,b) from Giemsa-stained skin scraping smears under 100x magnification.

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DNA extraction

To detect the Leishmania parasite, Leishmania-specific ribosomal internal transcribed spacer 1 (ITS1) gene amplification and digestion of the ITS1 PCR product with the restriction enzyme HaeIII from swabs were performed. DNA was extracted via the Animal Tissue DNA Isolation Kit (SIMEGEN, China, Cat. No. 3101060, Lot No. 20230221) according to the manufacturer’s instructions for the animal tissue protocol. Briefly, the smear was incubated in absolute ethanol for 10 min, dried at room temperature, covered with 1 ml of sterile distilled water, and incubated at room temperature for 10 min. The swabs were completely removed with sterile blades, transferred to a 1.5 ml reaction tube, centrifuged at 8000 × g for 1 min, and washed three times in sterile physiological saline. Finally, the supernatant was discarded, and the pellets were used for DNA extraction58,59. The resulting final volume of 50 µL was stored at − 20 °C until use.

Leishmania parasite detection

The ITS1 gene was amplified with the primer pair L5.8 S and LITSR, which produces a 300– to 350–bp PCR product as described by33 to detect Leishmania infection. Amplification was performed in a total volume of 50 µl with 25 µl of master mix (10 × Perfecta, USA), 6 µl of DNA, 2 µl of each primer, and sterile DNase/RNase-free water. PCR amplification was carried out in a Bio-Rad MyCycler with PCR conditions such as denaturation at 95 °C for 5 min, denaturation at 94 °C for 1 min, annealing at 48 °C for 30 s, extension at 72 °C for 1 min, a final extension at 72 °C for 5 min, and a final hold at 4 °C for 35 cycles. The PCR products were electrophoretically analyzed on a 1.5% agarose gel (GSTM agarose, Geneshun Biotech, lot number: 1905824) in 0.5× Tris-acetate buffer and visualized under UV light. A reaction was considered positive if a band between 300 and 350 bp in size was observed (Figs. 5 and 6).

A PCR restriction fragment length polymorphism (RFLP) approach was used to identify the Leishmania parasites. The ITS1 PCR products positive for Leishmania species and the control Leishmania strains (L. aethiopica, ID NO_1368347) were digested with HaeIII enzyme (Biolab, UK) at 37 °C for four hours according to the manufacturer’s instructions. The restriction fragments were analyzed via 2% agarose electrophoresis and visualized under UV light. The results of the digestion of the ITS1 products from the DNA isolates and reference strains revealed two visible bands and one low-resolution band for L. aethiopica(220 bp, 65 bp, and 23 bp). Fragment sizes were compared to bands of 100-bp DNA size ladders (Fig. 6).

Quality assurance

To ensure quality and data reliability, all quality control checks were performed before, during, and after data collection. The semistructured questionnaire was prepared and translated into the local language (Amharic). The content and face validity of the questionnaire were prepared by experts. Preliminary testing was conducted on the CL cases in Kobo City. During data collection, questionnaires were properly categorized and coded, and the data were checked daily for completeness and accuracy. The quality of the laboratory tests was ensured through standard procedures and internal quality control of the reagents. DNA yield and quality were checked via a NanoDrop 2000c spectrophotometer at 260/280 nm after setting the background reading to zero with an elution solution. A 260/280 ratio of ~ 1.8 corresponds to pure DNA. Our NanoDrop spectrophotometer results were confirmed by observing intact DNA fragments near the sample well after electrophoresis.

For PCR-RFLP, negative and positive controls with distilled water and L. aethiopica DNA, respectively, were included during the DNA extraction and PCR runs to ensure reliability and validity and to check for contamination or inhibition. L. aethiopica was obtained from the Amaure Hansen Research Institute (AHRI), Ethiopia, as a positive control (PC). In each extraction batch, one negative control for extraction (NC: distilled water) and a PC were included. All PCs, extraction NCs, no template controls (nuclease-free water), and clinical samples were subjected to PCR under the same conditions. The analysis, reporting, and interpretation of this study followed the STROBE guidelines for cross-sectional studies60.

Data processing and analysis

The data were entered into a computer via SPSS statistical package software version 25.0 (Chicago, IL, USA). Descriptive statistics were calculated and are presented in tables and figures. The relationships between CL infection and the independent variables were assessed via bivariate logistic regression, and variables with p values < 0.2 were subjected to multivariable binary logistic regression after the model fitness test with the Hosmer–Lemeshow test was performed. Finally, variables with an adjusted odds ratio (AOR) and p-value < 0.05 were included.