Leishmaniasis recidivans by Leishmania tropica in Central Rift Valley Region in Kenya

International Journal of Infectious Diseases 74 (2018) 109–116

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Leishmaniasis recidivans by Leishmania tropica in Central Rift Valley Region in Kenya Joseph Wambugu Gitaria , Samson Muuo Nzoub,c,* , Fred Wamunyokolia , Esther Kinyerud, Yoshito Fujiie , Satoshi Kanekoc,e , Matilu Mwaub a

Department of Molecular Biology and Biotechnology, Pan African University Institute of Basic Sciences, Technology and Innovation, Nairobi, Kenya Center for Infectious Parasitic Diseases Research, Kenya Medical Research Institute, Nairobi, Kenya Nagasaki University, Institute of Tropical Medicine, Kenya Medical Research Institute Project (NUITM-KEMRI Project), Nairobi, Kenya d Ministry of Health, Nakuru County Government, Kenya e Institute of Tropical Medicine, Eco-epidemiology Department (NEKKEN), Nagasaki University, Japan b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 23 March 2018 Received in revised form 5 July 2018 Accepted 5 July 2018 Corresponding Editor: Eskild Petersen, Aarhus, Denmark

Objectives: This study sought to determine the endemic Leishmania species, the clinical features of cutaneous leishmaniasis (CL) in the Central Rift Valley in Kenya and to give an account on unresponsiveness to treatment in the region. Methods: Participants were clinically identified and grouped into untreated, classical and recidivate based on clinical manifestation and clinical data. Leishmaniasis recidivans lesions were scaly hyperemic papules that appeared before the classic lesion had healed or after healing. The demographics and socioeconomic data were recorded and lesion scraping samples screened through microscopy and Internal Transcribed Spacer 1-PCR. Leishmania species were identified using Restriction Fragment Length Polymorphism. Results: A total of 52 participants were sampled, of which, 44.2% of the cases were recidivate and L. tropica the only species identified. All patients had been treated using sodium stibogluconate (SSG) which is the recommended first-line drug in Kenya. 60% of the patients experienced prolonged exposure to the drug (>30 days). Conclusion: L. tropica is the endemic Leishmania species for CL leading to classical and leishmaniasis recidivans. Treatment of CL in the area is not effective hence, alternative measures/therapy should be considered to cope with the unresponsiveness. © 2018 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).

Keywords: Cutaneous leishmaniasis ITS1-PCR RFLP Leishmaniasis recidivans Sodium stibogluconate Central Rift Valley Kenya

Introduction Leishmaniasis is a neglected disease caused by Leishmania species, transmitted by female sandflies of the genera Phlebotomus and Lutzomyia (World Health Organization, 2010). The parasites are single-celled protozoa of Kinetoplastid origin. An estimated 350 million people in 98 countries are at risk of Leishmania infections. The global prevalence is 12 million cases and an average of 2 to 2.5 million new infections occur annually (Alvar et al., 2012; World Health Organization/Department of Control of Neglected Tropical Diseases, 2017). There are more than 20 Leishmania species that cause leishmaniasis (Desjeux, 1991), where the

* Corresponding author at: Nagasaki University Institute of Tropical MedicineKenya Medical Research Institute Project (NUITM-KEMRI-Project), Nairobi, Kenya. E-mail address: [email protected] (S.M. Nzou).

resulting infection depends on the infecting species. Leishmania infections are classified as visceral or kala-azar, cutaneous and mucocutaneous (Mcgwire and Satoskar, 2014). Cutaneous leishmaniasis (CL) is the most common and affects the epidermal layer of the skin resulting in disfiguring lesions (Bailey and Lockwood, 2007). Some of the species known to cause CL include; L. tropica, L. major, L. aethiopica, L. infantum, L. mexicana, L. amazonensis, L. braziliensis (Ashford, 2000) and L. donovani (Karunaweera et al., 2003). In the East Africa region, CL is caused by L. tropica, L. major and L. aethiopica (Bensoussan et al., 2006). According to World Health Organization, incidences of CL could be higher than the reported figures if proper diagnosis and consistent reporting guidelines are followed (Karimkhani et al., 2016). Control of CL is geared towards chemotherapy where the mainstay drugs are pentavalent antimonials (Rai et al., 2013; de Vries et al., 2015). Sodium stibogluconate (SSG) has been used

https://doi.org/10.1016/j.ijid.2018.07.008 1201-9712/© 2018 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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worldwide for the treatment of CL for many decades (Frézard et al., 2014), however, parasite resistance against the drug and other antimonials has recently been reported in different parts of the world (Croft et al., 2006; Sundar et al., 2000; Perez-Franco et al., 2016; Lira et al., 1999). In CL endemic regions, more than one species may coexist, warranting the use of molecular methods for precise identification of etiological species. Internal Transcribed Spacer 1-Polymerase Chain Reaction (ITS1 PCR) in combination with Restriction Fragment Length Polymorphism (RFLP) method is a desirable tool due to its specificity and rapid characterization of Leishmania species (Schönian et al., 2003) cost-effectively (Monroy-Ostria et al., 2014). Reports on the incidences of CL in Kenya were documented over three decades ago in different parts of the country such as Naivasha, Laikipia plateau, Baringo and Mount Elgon (Desjeux, 1991; Malaria Consortium, 2010). However, the disease burden is not well documented (Hotez et al., 2006; Odiwuor et al., 2012). The etiological species in some regions are not well known and most of the data is largely extrapolated. Unpublished reports show an increased number of infections that have spread to areas that were previously non-endemic. In a diagnostic screening conducted in 2009 after a CL outbreak in the Kenyan Rift Valley, L. tropica and L. donovani were reported in CL patients (Odiwuor et al., 2012). Leishmaniasis recidivans is an unusual clinical variant of cutaneous leishmaniasis disease. It typically recurs at the site of an original ulcer within 2 years, often at the edge of the scar and the lesions maybe difficult to treat (Bittencourt Achiléa et al., 1993). A treatment program involving the use of SSG was introduced in the region. However, unresponsiveness to treatment and relapses are currently experienced by some patients despite prolonged treatment. In addition, challenges related to the availability of drugs and compliance are experienced in the region. Leishmania species circulating in some parts of the Kenyan Rift Valley have been reported (Odiwuor et al., 2012) but the clinical aspects have never been documented. Cases of resistance have been reported in different parts of the world, but, in Kenya, it is yet to be documented (Sundar et al., 2000; Perez-Franco et al., 2016; Lira et al., 1999). This study, therefore, sought to determine the clinical aspects of L. tropica, to provide an account on leishmaniasis recidivans and cases of unresponsiveness to treatment in the Central Rift Valley in Kenya. Methodology Study area This study was done in Gilgil Sub-county, a rural area in the Central Rift Valley in Kenya situated at an altitude of 2616 meters above sea level, a latitude of 0
290 32.1900 S and longitude of 36
190 2.2800 E. the area is located approximately 133 kilometers from the Kenyan capital, Nairobi. Community health volunteers mobilized the people with signs and symptoms resembling CL to outreach clinics where they were clinically identified by a clinician based on clinical history and cutaneous lesions presenting in form of papules, nodules or ulcers on the face, neck, arms and legs. All the participants were consecutively sampled after identification and were residents of the rural villages; Gitare, Jika, Kambi Turkana, Sogonoi and Njeru. Identification of patients, inclusion and exclusion Clinicians identified the patients through clinical examination of the cutaneous lesions. All participants who were clinically identified with CL symptoms were included in the study if they agreed to participate by signing informed consent forms while

those with healed lesion or those who had lesions that did not conform to the diagnosis of CL or failed to sign informed consent forms were excluded. Collection of data Demographic and clinical data were collected using a structured interview guide where information on age, sex, residence, occupation, awareness on vectors, type of housing, the onset of disease, stage of infection, site and number of lesions, duration of treatment and compliance to treatment was recorded. Collection of lesion samples Skin scraping samples were collected from the edges of active lesions by a clinician. The lesions were first cleaned using alcohol swabs then gently opened using sterile scalpels and the samples collected by gentle scraping on the open lesions. Part of the samples was smeared on microscope slides, air dried and stored in a microscope slide box. Duplicate samples were collected in 1.5 ml Eppendorf tubes, stored in a cooler box containing ice packs and transported to the laboratory. On arrival, the samples were stored at 30
C awaiting analysis. Sample screening to confirm CL infection The samples were analyzed through microscopy and ITS1 PCR for visualization of amastigotes and detection of Leishmania DNA respectively. Microscopy The prepared slide smears were first fixed with absolute methanol, air dried, stained using 10% Giemsa stain for 20 minutes, washed with running tap water and air dried again for 15 minutes. They were examined under x100 oil immersion objective and the amastigote counts graded according to the Chulay and Bryceson method of quantitating Leishmania parasites (Chulay and Bryceson, 1983). Molecular analyses DNA extraction Genomic DNA was extracted using Quick-DNATM Miniprep Plus Kit (Zymo Research, USA) following the manufacturer’s instructions. The DNA samples were quantitated using NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, USA) at 260/280 nm before PCR analysis. ITS1-PCR assay The DNA samples were subjected to ITS1 PCR to confirm the infection of Leishmania. The region between the small subunit (SSU) ribosomal RNA and the 5.8S ribosomal RNA genes in the parasite genomic DNA was amplified using LITSR 50 CTGGATCATTTTCCGATG30 and L5.8S 50 TGATACCACTTATCG CACTT30 primers as previously described (Bensoussan et al., 2006; Schönian et al., 2003; Monroy-Ostria et al., 2014). 5 ml of the PCR products were loaded on 2.5% agarose gel for electrophoresis analysis. The gel was stained with 2x Gel Red for 30 minutes while shaking and images visualized using UltraSlim UV Transilluminator (Maestrogen, Taiwan). Identification of Leishmania species

III

The ITS1-PCR products were subjected to RFLP assay using Hae restriction endonuclease enzyme (Promega, USA) for

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identification of Leishmania species as previously described (Schönian et al., 2003; Monroy-Ostria et al., 2014; Mouttaki et al., 2014; Kazemi-Rad et al., 2008). Briefly, volumes of 20 ml consisting of 14.8 ml of nuclease-free water, 2.2 ml of restriction buffer, 1 ml of the restriction enzyme and 2 ml of PCR products were incubated at 37
C for 2 hours. 10 ml of the products were loaded on 3% agarose for electrophoresis analysis. The gel was stained with 2x Gel Red for 30 minutes and the images observed using UltraSlim UV Transilluminator (Maestrogen, Taiwan).

lesions while others had a combination of nodules and papules and ulcerations (Table 1). 92% of the participants had facial lesions while 8% had lesions on ears and extremities. 10% of the participants acquired the disease before the year 2011, 19% were infected between 2011 and 2014, while the majority (71%) acquired the infection between 2015 and 2017. Treatment durations were prolonged ranging between 1 to 36 months among different participants (Table 1). Despite prolonged treatment, more than 30% of the patents were not cured.

Results

Classification of participants

Demographic factors of the study participants

Based on clinical features of the lesions, the participants were grouped into three categories as follows; classical leishmaniasis, leishmaniasis recidivans and untreated leishmaniasis (Figure 1), which accounted for 23.0%, 44.2% and 36.2% respectively. Participants were grouped under classical cutaneous leishmaniasis (CL) if they had well defined ulcers or nodules without satellite papules which responds to treatment characterized by re-epithelialization of the skin during the course of treatment. Those who experienced recrudescence of lesions on initially healed scars after completing the treatment course or had plaques with satellite papules were classified under leishmaniasis recidivans (Figure 2). Patients who had been exposed to SSG for six months or longer accounted for 78.4% of total recidivate cases (Table 2). Those who had not received any formal treatment at the time of data collection were classified as untreated. The untreated CL cases had not been exposed to SSG and the clinical characteristics resembled those of the classical CL and recidivans (Table 3). A total of 90% of participants from Njeru had not been treated at the time of data collection.

A total of fifty-two participants constituting 40% (21) males and 60% (31) females from Gitare, Jika, Kambi Turkana, Njeru, and Sogonoi participated in this study (Table 1). Their age was between 5 to 52 years and the average age was 16 years. Children <18 years of age constituted 75% of the participants while 25% were adults 18 years of age. 67% (14) of the males were positively diagnosed compared to 81% (25) of the females. Individuals between 5 to 20 years accounted for 78.8% of the total infections. The participants engaged in multiple occupations including subsistence farming, provision of casual labor in plantations, herding livestock and charcoal burning (Table 1). All the study sites were in rural settings and the participants were permanent residents in the region. The study sites had thick vegetation cover near living houses, numerous caves and rocky crevices. All the sites except Gitare were forested and houses were poorly constructed with earthen floors, gaps and cracks. The participants from Njeru had established settlements within or adjacent to the Mau forest and carried out subsistence farming within the forested escarpment. 89% of participants engaging in charcoal burning dwelled in caves (Figure S1). More than 40% of children participated in herding livestock (Table 1) while others engaged in shooing monkeys from the farms. Herding of livestock was mainly conducted in forests, bush lands and near homesteads. Majority of the participants (79%) did not have knowledge and awareness of sandflies and their role in CL transmission. Clinical manifestations and clinical data Cutaneous lesions were manifested in the form of papules, nodules or ulcerations. Most of the participants (54%) had nodular

Treatment of patients In all the sites where treatment programs were on course, patients were treated on weekly basis through intralesional infiltration of SSG 1–5 ml (100 mg/ml) with a dosage of 20 mg/ kg/day for 6-12 weeks until healing. For patients with single lesions, I-2 ml of the drug is usually administered while those with multiple lesions, 3-5 mls are administered. Usually, 6 injections are considered adequate unless in cases of multiple lesions and patients with leishmaniasis recidivans, where treatment is done up to 12 weeks. In leishmaniasis recidivans, symptoms of recrudescence of lesions on initially healed scars reappeared 30-60 days

Table 1 Demographic and clinical characteristics of the participants. Characteristics Gender Age Residence Awareness on vectors Type of housing Occupation

Males 40% (21) <18 years 75% (39) Gitare 26.9% (14) Yes 21% (11)

Females 60% (31) 18 years 25%(13) Jika 21.1% (11) No 79% (41)

Timber 75% (39) Subsistence farming 92%(48)

Mud 25% (13) Casual labor 67%(35)

Stage of infection

Papules 8%(4)

Site of lesion Number of lesions Onset of disease Duration of treatment Clinical manifestation Microscopy results ITS1 PCR results

Average age 16 years Kambi Turkana 3.9% (2)

Mode 13 Njeru 19.2% (10)

Median 13 Sogonoi 28.9%(15)

Herding livestock 42% (22) Nodules + papules 25% (13)

Pupils & students 79% (41)

Nodules 54%(28)

Burning charcoal 33% (17) Ulcerations 13%(7)

Facial 92%(42) 1-2, 60% (31) Before 2011, 10% (5) Untreated 32% (17)

Ears 4%(2) 3-4, 23% (12) 2011-2014, 19% (10) 1-5 months 21% (11)

Extremities 4%(2) 5, 17%(9) 2015-2017, 71% (37) 6-12 months 10% (5)

13-24 months 31% (16)

24 months 6% (3)

Leishmaniasis recidivans 44.2% (23) Positive 13% (7) Positive 75% (39)

Classical leishmaniasis 23.1% (12) Negative 82%(32) Negative 25%(13)

Untreated cases 32.7% (17)

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Figure 1. The distribution of recidivate, classical and untreated cases of CL.

Figure 2. Manifestation of leishmaniasis recidivans. Sogonoi (A) Gitare (B and D), and Njeru (C). Leishmaniasis recidivans was characterized by recrudescence of papules or nodules on previously healed scars. Table 2 The percentage cases of leishmaniasis recidivans in comparison to durations of SSG exposure. Treatment duration (months)

% recidivate (n = 23)

Untreated 1 2-5 6-12 13-24 24

8.6% 13.0% 0.0% 47.8% 17.6% 13.0%

after completing the 12-week treatment course. Treatment was however occasionally interrupted for 1-3 weeks in some patients due to lack/late drug supplies or non-compliance. The incidences of irregular treatment observed in the region were 40% (14) of all the treated cases (n = 35). Among the 14

patients who had irregular treatment, 86% (12) were characterized by development of signs and symptoms of leishmaniasis recidivans. This indicates a relationship of irregular treatment with the emergence of treatment failure/leishmaniasis recidivans. Unresponsiveness to treatment Unresponsiveness to treatment was denoted by treatment failure among patients who were treated with SSG for 12 weeks or more. The number of positively identified patients was high despite treatment spanning more than 12 weeks. Many of the patients treated for 5 months or more were not cured (Table 3). Patients who were unresponsive to the treatment, symptoms such as increased size and number of nodules, plaque or ulceration and lack of complete re-epithelialization were observed.

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Table 3 Proportions of patients that were unresponsive to treatment after treatment with SSG for various durations. Duration of treatment (Months)

Number of CL cases treated

Positively diagnosed cases

% positive

5 6-10 11-15 16-20 21-25 26-30 31-35 >36

11 1 3 6 7 0 0 7

10 1 2 4 3 0 0 2

91% 100% 67% 67% 43% 0% 0% 29%

Microscopy and grading of counts Amastigotes were visualized in 13.4% of Giemsa stained slides examined under x100 oil immersion objective. Based on the method described by Chulay and Bryceson (1983), 4% of the Giemsa stained slides were graded 2+, indicating an average of 110 amastigotes in 100 fields. 10% of slides were graded 1+ representing an average of 1-10 amastigotes in 1000 fields while 86% of slides were graded zero since no parasites were observed on the entire slides (Table S1). Screening for leishmaniasis using ITS1-PCR 75% of the samples were positively identified through ITS1 PCR. Single bands of approximately 350 base pairs were observed after electrophoresis analysis on 2.5% agarose gel (Figure 3A). Identification of Leishmania species through ITS1-PCR RFLP assay When the ITS1 PCR products were subjected to RFLP assay using Hae III restriction endonuclease, two fragments of approximately

200 base pairs and 60 base pairs were visualized on 3% agarose gel for all the Leishmania positive samples (Figure 3B). DNA extracted from in vitro cultures of L. major and L. tropica was used as positive control. The ITS1 PCR products from L. major were digested into two fragments of approximately 220 base pairs and 130 base pairs while L. tropica yielded two fragments of approximately 200 base pairs and 60 base pairs. Discussion This study reports leishmaniasis recidivans and classical leishmaniasis as the clinical types of CL associated with L. tropica in the Central Rift Valley in Kenya. L. tropica was identified as the endemic Leishmania species in the region. This finding partly agrees with the report of a previous study where L. tropica was identified as the predominant Leishmania species while L. donovani was isolated in a CL patient (Odiwuor et al., 2012). There was also evidence of overexposure and underexposure of SSG among CL patients in the region that resulted from prolonged treatment and drug unavailability or non-compliance respectively. Underexposure to SSG means that some of the patients did not complete the

Figure 3. (A) Internal Transcribed Spacer-1 PCR results for diagnosis of leishmaniasis. Bands showing the ITS1 region of Leishmania parasites on 2.5% agarose gel. MW represent 100 bp molecular weight ladder, lanes 1 to 8 are the positive samples (approximately 350 bp), L.m and L.t represent L. major and L. tropica positive controls respectively and NC is the negative control. (B) Restriction Fragment Length Polymorphism of the ITS1 PCR products. DNA banding on 3% agarose gel after restriction of the ITS1-PCR products using Hae III enzyme. MW represent 50 bp molecular weight ladder, lanes 1 to 8 represent the restricted L. tropica ITS1-PCR products, L.m and L.t represent L. major and L. tropica ITS1-PCR products used as positive controls respectively and NC is the negative control.

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recommended treatment course (20 mg/kg/day for 6-12weeks). Overexposure on the other hand means that there was prolonged exposure to SSG with some patients being exposed for more than 6 months. Classical leishmaniasis was characterized by healing lesions due to treatment while leishmaniasis recidivans was associated with recrudescence of papules or nodules from previously healed lesions (Rubeiz, 1999). The latter also had minimal response to SSG and persisted for years leading to serious disfiguring effects on the skin. This is similar to the other reported cases of this type of infection in other parts of the world (Marovich et al., 2001; Dassoni et al., 2017). The treatment carried out in the region was not continuous as recommended, either due to unavailability of drugs or failure to comply with treatment. This could be a contributing factor to the emergence of drug resistance and leishmaniasis recidivans. Although infections caused by L. tropica tend to be difficult to treat (Dassoni et al., 2017; Kumar et al., 2007; Sharifi et al., 2010), the problem could be compounded with irregular exposure to drugs occasioned by failure in dose compliance and drug unavailability. This could be the reason why 29% of the patients showed unresponsiveness to treatment for more than 36 months of irregular exposure to SSG. Treatment was only conducted when drug supplies were available and interrupted when the stocks run out. 91.4% of leishmaniasis recidivans cases were experienced by patients who had previously been exposed to SSG. On the other hand, in other areas such as Njeru, where treatment programs had not been initiated at the time of data collection, and only one case of leishmaniasis recidivans was recorded. This prompts further investigations to explain whether exposure to SSG could lead to the emergence of leishmaniasis recidivans. 36.2% of positively diagnosed patients had not undergone treatment because of different reasons such as the absence of treatment programs in their local areas, others cited painful intra-lesion injections as the reason why they avoided treatment while others opted for complementary herbal medication. Emergence of drug-resistant Leishmania parasites has previously been reported in different leishmaniasis endemic areas where pentavalent antimony drugs have been used (Abdo et al., 2003; Monte-Neto et al., 2015). Several studies have explained how the resistance may arise as follows; overexposure of SSG for longer durations than the recommended ones (Croft et al., 2006), the frequent contact of the drug and parasites in a human host and drug exposure in sub-optimal levels especially where drug supply is a challenge such as among poor communities (Rijal et al., 2003). The high percentage of leishmaniasis recidivans cases in the study region could as well be associated with non-compliance to treatment either due to inconsistent or prolonged exposure to SSG which could be a sign of emerging parasite resistance against the drug. Similar experiences have been reported in other studies (Croft et al., 2006; Rijal et al., 2003). For the situation in Kenya, the recommendation is to change the current medication in order to cope with the suspected resistant Leishmania parasites. Additionally, development of a new drug is recommended to tackle resistance in Leishmania. Factors associated with CL infection were identical to the ones reported in other parts of the world (Ghatee et al., 2017; Manomat et al., 2017; De Araujo et al., 2016). Lack of knowledge and awareness of the disease and the socio-economic activities among the patients were the major factors contributing to the infection. Engagement of the patients in multiple occupations (Table 1) could increase the number of infections due to the enhanced frequency of contact with vectors. A total of 78.8% of the infection were recorded among participants between 5 and 20 years, including school going children, perhaps because of the frequent contact with vectors

while grazing animals in forested areas, shooing monkeys from the farms and the frequent walking trips to and from school along forested paths. Sand flies are nocturnal or crepuscular, meaning that they can bite at night or early in the morning when the sun is rising or late in the evening just before sunset. They also bite during the day when distributed in their habitats (Anjili et al., 2011; Abdallah et al., 2017). Therefore, it is possible that children were bitten during these times. Generally, the factors associated with CL infections reported in this study were almost similar to the ones reported in leishmaniasis endemic areas in other parts of the world (Ghatee et al., 2017; Manomat et al., 2017; De Araujo et al., 2016). Microscopy is a specific and direct method for diagnosis but lacks sensitivity (Sagi et al., 2017). The low sensitivity (13.4%) recorded in this study may be attributed to low parasite density, the difficulty in observing amastigotes from samples originating from leishmaniasis recidivans cases (Sharifi et al., 2010; OliveiraNeto et al., 1998) or the low parasite density due to the healing process. Since more than one Leishmania species had previously been reported more than eight years ago among CL patients in the region (Odiwuor et al., 2012), it was, therefore, necessary to identify the current circulating species for the purpose of determining the best clinical interventions. Through clinical examination of lesions and patient history data, all the participants were clinically identified as positive. This was based on physical signs on the lesions that included crustaceous ulcerative lesions on the face and extremities, the presence of satellite lesions and/or lymphangitis (de Vries et al., 2015). This was backed up with patients living in leishmania endemic region (Votýpka et al., 2012; Saberi et al., 2012). However, 75% of the samples were positively confirmed through ITS1 PCR. This could imply low sensitivity while using ITS1 PCR method (Bensoussan et al., 2006) or clinical mis-diagnosis (Swain et al., 2016; Seyhan et al., 2009). The advantage of ITS1 PCR method is the specificity and rapid identification of Leishmania species upon subjecting the ITS1 PCR products to RFLP analysis using Hae III restriction endonuclease (Bensoussan et al., 2006; Schönian et al., 2003; Yehia et al., 2012). All CL positive samples underwent Hae III enzyme restriction, resulting in two fragments of approximately 200 bp and 60 bp, therefore, identified as L. tropica. These results were in consistent with the findings of other studies (Schönian et al., 2003; Mouttaki et al., 2014; Kazemi-Rad et al., 2008). L. donovani was not detected from any participant as previously reported (Odiwuor et al., 2012). Conclusion Leishmaniasis recidivans was found in the areas where standard treatment was initiated except for one area. Treatment should be carried out with strict adherence to the recommended doses of treatment and clinical follow-up. Studies to assess parasite resistance against SSG would be necessary. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements The authors appreciate the infrastructural support provided by the Nagasaki University Institute of Tropical Medicine - Kenya Medical Research Institute project (NUITM-KEMRI project). We also appreciate the efforts of staff members at Gilgil Sub-county hospital for their invaluable input in this study. This manuscript has been submitted for publication with the permission of the Director of KEMRI.

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