Burden and risk factors of cutaneous leishmaniasis in a peri-urban settlement in Kenya, 2016.

BACKGROUND: Cutaneous leishmaniasis is a neglected disease known to cause significant morbidity among the poor. We investigated a suspected outbreak to determine the magnitude of cases, characterize the cases and identify risk factors of cutaneous leishmaniasis in Gilgil, a peri-urban settlement in Central Kenya.
METHODS: Hospital records for the period 2010-2016 were reviewed and additional cases were identified through active case search. Clinical diagnosis of cutaneous leishmaniasis was made based on presence of ulcerative, nodular or papular skin lesion. The study enrolled 58 cases matched by age and neighbourhood to 116 controls in a case control study. Data was collected using structured questionnaires and simple proportions, means and medians were computed, and logistic regression models were constructed for analysis of individual, indoor and outdoor risk factors.
RESULTS: Of the 255 suspected cases of cutaneous leishmaniasis identified, females constituted 56% (142/255) and the median age was 7 years (IQR 7-21). Cases occurred in clusters and up to 43% of cases originated from Gitare (73/255) and Kambi-Turkana (36/255) villages. A continuous transmission pattern was depicted throughout the period under review. Individual risk factors included staying outside the residence in the evening after sunset (OR 4.1, CI 1.2-16.2) and visiting forests (OR 4.56, CI 2.04-10.22). Sharing residence with a case (OR 14.4, CI 3.8-79.3), residing in a thatched house (OR 7.9, CI 1.9-45.7) and cracked walls (OR 2.3, CI 1.0-4.9) were identified among indoor factors while sighting rock hyraxes near residence (OR 5.3, CI 2.2-12.7), residing near a forest (OR 7.8, CI 2.8-26.4) and having a close neighbour with cutaneous leishmaniasis (OR 6.8, CI 2.8-16.0) were identified among outdoor factors.
CONCLUSIONS: We identify a large burden of cutaneous leishmaniasis in Gilgil with evidence of individual, indoor and outdoor factors of disease spread. The role of environmental factors and rodents in disease transmission should be investigated further.

Introduction

Leishmaniasis is a disease caused by a protozoan, Leishmania and is transmitted to humans and other mammals by the bite of a female phlebotomite sand-fly (Phlebotomus species). Three forms of the disease affect humans; cutaneous, muco-cutaneous and visceral forms (Kala-azar). The disease is considered a neglected tropical disease mainly affecting the rural poor. Cutaneous leishmaniasis (CL) commonly occurs in clusters among destabilized or migrant populations in low socio-economic settings with the current trend in distribution of new infections indicating a progressive spread of the disease to previously non-endemic areas [1-5].

Worldwide, over 350 million people are estimated to be at risk of CL and up to 1.5 million new infections are reported annually [6]. Owing to challenges in surveillance and reporting, the burden of CL is grossly underestimated [4]. Though disfiguring and debilitating in the affected people, the disease is rarely fatal, hence little attention has been given to prevention and control measures by health authorities [4,5]. Despite that, proven control strategies including vector eradication and early treatment of insect bites in endemic areas have been shown to be successful. Insect vector control activities such as indoor insecticide residual sprays, insecticide impregnated barriers (bed nets, curtains, clothes, carpets), environmental spraying, and control of reservoir hosts (rodents) are effective but expensive when rolled out on a large scale [4]. Therefore, targeted control programs guided by an understanding of local drivers of the disease including lifestyle and environmental factors would provide significant cost savings and value for money in addition to achieving disease control [5,7].

Recurrent outbreaks of CL in Kenya, Ethiopia and South Sudan have been reported in the past and are often associated with high morbidity. Kenya is classified by WHO as endemic for CL. However, there is relative scarcity of published data on the extent, burden and risk factors of CL [8]. In other parts of the world, urbanization and expansion of farming and other human activities into forests is often associated with disease outbreaks [5]. In Kenya, areas around the Rift Valley escarpments and major mountains are known natural habitats for sand-flies [9-11]. More than 48 species of sand-flies, including the Phlebotomus species that are vectors for CL (P. duboscqi, P. guggisbergi, P. pedifer and P. acleatus), have been identified in various habitats [12]. Recently, the areas around the Rift Valley in Kenya have been experiencing rapid population growth and increased environmental pressure resulting from in-migration and increased human activities in forests [13,14].

In early 2016, the health ministry of Kenya received notification about increase in cases of a skin disease suspected to be CL in Nakuru county, in south-eastern Rift Valley. We report the findings of a records review and a follow-up case control study conducted to determine the magnitude of the disease, characterize the cases and identify factors associated with the spread of the disease.

Methods

Study site

The study was conducted in Gilgil sub-county, a rapidly growing peri-urban settlement located in south-eastern part of the Great Rift Valley in Kenya, between 20th January and 3rd February 2016 ( The terrain of Gilgil sub-county is generally mountainous to the north with plenty of rocky escarpments. The southern end of the sub-county is mainly composed of undulating plains, flat grazing lands and solidified lava that form large crevices and rocky caves infested with wild mammals and rodents [9,11]. The area is sparsely populated (population density of <200 persons per square Km) and typical scattered housing characterizes the settlement pattern this area [15]. Of late, the area has experienced an influx of new settlements since it is regarded as a high potential area yet a cheaper alternative to the urban life in neighbouring Nairobi city or Nakuru town [14]. The sub-county is traversed by a busy highway and is a preferred destination for potential peri-urban home-owners due to its ease of access from the surrounding urban centres. Previous studies in this area have identified the insect vector (Sand-fly) and the agent (Leishmania donovani, Leishmania infantum and Leishmania chagasi) to be prevalent in this area[9-11].

Map of the investigation area, Gilgil, Kenya 2016.

This map was drawn on QGIS Version 2.18.15 using mapping resources from International Livestock Research Institute (ILRI) [16,17].

Study design

Hospital records were reviewed by trained staff and additional cases were identified through door-to-door case search. Cases were then enrolled in a population-based case control study.

Review of records

A standard data abstraction tool was used to review records covering nine health facilities in Murindati and Mbaruk/Eburru wards to develop an outbreak line-list. The facilities included Afya medical clinic, Camp Brethren medical clinic, Eburru dispensary, Mbaruk dispensary, Ol-Jorai health centre, Rhine Valley health center, Karunga dispensary, Langa-Langa dispensary and Anti-Stock Theft Unit dispensary. The total catchment population for these facilities is approximately 54,000 persons [18]. Entries made in outpatient, inpatient, laboratory, and specialist clinic registers between January 2010 and January 2016 were included in the records review.

A suspected case of CL was defined based on clinical diagnosis recorded in hospital records as ‘skin ulcer’, ‘skin wound’, ‘plaque’, ‘dermatitis’, ‘skin infection’ or ‘cutaneous leishmaniasis’. The outbreak line-list was updated through addition of probable cases. A probable case was defined as a resident with a typical skin lesion (a skin ulcer with typical raised edges and depressed centre or a skin plaque-a circumscribed, nodular or palpable skin lesion) on physical examination by a medical officer in the study team during the study period. Due to delays in receiving sample collection and laboratory testing supplies, no laboratory confirmation for CL was done on the suspected or the probable cases. All entries that matched ‘suspected’, ‘probable’ or ‘confirmed’ case definitions were included in the outbreak line list. The line list also included other information such as name, sex, age, date seen at the facility, residence, signs and symptoms, diagnosis and treatment given. Patients whose clinical diagnosis and contact information (physical address or phone contact) were missing were excluded from the line list.

Enrolment of cases and controls into a case control study

To determine the risk factors of CL infection in the study population, we conducted a follow-up case-control study. Cases and controls consisted of eligible residents found in the study area during the study period (20th January-3rd February 2016).

Case and control recruitment

We approached and enrolled 59 cases for the case control study: 41 cases who could be reached during the study period were selected from the outbreak line-list and were first ascertained to be probable CL cases by experienced medical officers in the study team on the basis of typical skin lesions (a skin ulcer with raised edges and depressed centre or a skin plaque, described as a circumscribed, nodular or papular skin lesion). A further 18 probable cases were identified from the community during active house-to-house survey upon examination by the medical officers ( The investigation team comprising a field epidemiologist, 2 medical doctors, a laboratory scientist and 2 public health specialists, worked with community-based locators (community health volunteers and local chiefs) and the recruited cases to locate additional cases for inclusion in the case control study in a respondent-driven sampling process. In each village, the number of cases that were recruited in the case control study was allocated by probability proportional to size sampling approach based on the proportion of residents from that village with suspected CL from the outbreak line-list.

Each of the enrolled cases were matched to two community-based controls by age using the following criteria: Cases less than two years of age were matched to controls within two years, cases 2−4 years old were matched to controls within 3 years, cases 5−19 years to controls within 5 years, cases 20−59 year’s old to controls within10 years, and cases more than 60 years old to controls within 20 years. One case was dropped in the final analysis owing to lack of suitable controls, bringing the total number of cases and controls included in the study to 174 (58 cases and 116 controls) (

Controls were selected from among residents of the same age group and living in the same or neighbouring village(s) as the case patients, and had no typical skin lesions (ulcer, plaque, wound or scar) upon inquiry and examination by medical doctors in the study team. To locate a possible control, a member of the investigation team would spin a bottle while standing afront the case’s residence to determine the direction of movement. A random number between 2 and 5 was drawn to indicate the number of houses in the chosen direction to be passed before the team would attempt to recruit a control. This process was repeated until two eligible controls were recruited for each case. Five potential controls were dropped from the study on account of having old healed scars upon examination ().

Sample size

The sample size was determined by the number of eligible cases present in the study area during the study period. A sample size of 58 patients and 116 controls (2 controls per case) was adopted to give the study at least 80% power at the 5% significance level and able to detect an odds ratio (OR) of 0.3 for an exposure present in 31% of controls [19,20]. The exposure chosen was use of mosquito nets.

Data collection and analysis

A structured questionnaire (study questionnaire in S1 Questionnaire) was developed and pretested in three randomly selected non-study households on the first day of field work. The questionnaire was administered to cases and controls through face to face interviews. Information on demographic profile, clinical presentation, risk factor profile and environmental exposures among cases and controls regarding CL were collected. Exposure data in both cases and controls was collected in relation to the year of onset of symptoms in the case patients. Environmental observations around the home were made by the study personnel and recorded in the questionnaires.

All data from the questionnaires were entered into a database and cleaned using Epi info version 7 (CDC, Atlanta GA, USA) and Microsoft Excel (2010). Simple proportions, means and medians were calculated for categorical data and continuous data respectively. To identify factors associated with the outbreak, chi-square tests were conducted for categorical data, and odds ratios and 95% confidence intervals (95% CI) computed. Risk factors were categorised into three groups in the analysis: factors relating to the individual, factors relating to the indoor environment and factors relating to the outdoor environment. Factors with a P≤0.05 in the bivariate analysis were considered significant and were included in the ‘group model’ for each category of factors. To develop the final model, regression analysis was conducted using backward elimination method, starting with all factors that had a P≤0.2 in the group model to determine independent risk factors.

Human subject’s protection

This study was approved by Ministry of Health (MOH) in Kenya and was conducted as part of public health response to an acute event and as such was not reviewed by an ethical review body. Oral consent was obtained from the case-control study subjects and was documented in the study questionnaires. Study information was provided in form of written leaflets given to all study participants and displayed at strategic locations in health facilities in the study area (example in S1 Leaflet). An outbreak response protocol was developed and authorisation to conduct the study was given by the MOH through the Field Epidemiology and Laboratory Training Program (Approval letter Ref No. MOH/DPH/DSRU/REG/07/Vol. 1) (letter in S1 Letter). Permission to conduct the study was also sought from the department of health in Nakuru county and respective health facilities where records were abstracted. Measures taken to assure confidentiality of the information provided during these interviews included conducting interviews in a private place convenient for respondents, storage of paper questionnaires in lockable cabinets accessible only with authorization of the principal investigator and password protection of de-identified data in electronic databases. Review of surveillance data and active case finding in the community were conducted as part of routine surveillance by the MOH, and all the data collected in the line-list was anonymized by dropping all personal identifiers (patient names, in-patient/out-patient numbers and phone contact) before analysis. Individuals who had active lesions at the time of the study were referred for free treatment at Nakuru county referral hospital

Results

From the review of health facility records and house-to-house survey, a total of 255 cases of suspected CL were identified between 2010–2016. There was one death documented from a 67-year old woman on treatment for CL over the period of review. Of the identified cases, females constituted 48.6% (124/255) and the median age was 7 years (IQR 3–17). Cases occurred in clusters and up to 43% of cases originated from Gitare (73/255) and Kambi-Turkana (36/255) villages (

Cases of suspected CL were recorded continuously throughout the period between 2010 and 2016 with occasional peaks in June of 2010 and December of 2014. Most (23.5%) cases of suspected CL were recorded in 2014 while the least (11.8%) cases were recorded in 2011. (

Case control study

summarizes the demographic characteristics of cases and controls enrolled in the case control study. Males constituted 55.2% (32/58) of the cases and 49.1% (57/116) of the controls. Cases were predominantly young persons aged below 15 years (56.9%). The youngest case was 2 years while the oldest was 86 years old. There was significant difference in the distribution of cases and controls by type of occupation. However, education level was not significantly different between the two groups.

Among the cases, the median duration of illness was 2 years (range 1–4 years). Cases had multiple lesions with the majority (84.5% or 49/58) of cases presenting with both ulcerative and nodular lesions. The majority of lesions were located on the head and neck regions (81.6%, or 40/49) while 6.1% (3/49) were located in the hands and 2.0% (1/49) were in the foot. In thirteen cases (22.4%), both active ulcerative lesions and scars were found. Other symptoms observed among cases included pruiritus (15.5%), rash (5.2%), bruising (5.2%), skin infections (5.2%) and nasal stuffiness (3.4%). Various wound treatment remedies were cited by the cases including herbal medication (72.1%), skin ointments (41.9%) and antibiotics (25.6%).

In terms of household ownership and use of mosquito-nets, 10.3% (6/58) of the cases and 18.1% (21/116) of the controls owned and slept under a mosquito-net every night. Household indoor residual spraying was reported by less than 5% of both cases and controls. Sighting of wild mammals around homes was common among both cases and controls: Some 87.9% of cases and 57.8% of controls reported sighting rock hyraxes and 75.9% of the cases and 69.0% of the controls reported sighting mongooses near their homes. In terms of housing condition, 82.8% of cases and 97.4% of the controls lived in houses with roofing made of corrugated iron sheet, 56.9% of cases and 59.5% of the controls lived in houses with earthen floor, 27.6% of cases and 21.6% of the controls lived in houses with cracked floors. The majority of cases (91.4%) lived in houses located near a forest compared to 57.8% controls.

Risk factor analysis

Potential risk factors were categorised into three groups for purposes of analysis: factors related to the individual, factors related to indoor dwelling environment and factors related to outdoor environment. Table 3 shows the distribution of cases and controls with corresponding crude and adjusted odds ratios for the variables analysed in the 3 ‘group models.

Table 3

Analysis of the risk factors associated with cutaneous leishmaniasis in Gilgil-Kenya, 2016.

Variables	Cases N = 58		Controls N = 116		Crude OR (95% CI)	Adjusted OR (95% CI)	P value
	n	%	n	%
A. Factors related to the individual
Participant is male	32	55.2	57	49.1	1.3 (0.7–2.4)	1.0 (0.5–1.9)	0.892
Education level is primary level and below	55	94.8	103	88.8	2.3 (0.6–13.1)	2.5 (0.6–9.6)	0.190
Spending time outside home after sunset	9	15.5	5	4.3	4.1 (1.2–16.2)	2.1 (0.6–7.7)	0.284
Individual using bed net	6	10.3	21	18.1	0.5 (0.2–1.5)	0.6 (0.2–1.7)	0.366
Occupation involves forest visit d	20	34.5	12	10.3	4.6 (2.0–10.2)	3.8 (1.6–9.0)	0.003
History of travel	9	18.4	15	12.9	1.2 (0.5–3.0)	1.1 (0.4–2.9)	0.916
B. Factors related to indoor dwelling environment
Roofing made of other materials e	10	8.6	3	2.7	7.9 (1.9–45.7)	13.6 (2.5–74.7)	0.003
Living in a house with cracked walls f	48	82.8	79	68.1	2.3 (1.0–4.9)	3.9 (1.1–13.6)	0.032
Living in a house with cracked floors g	49	84.5	94	81.0	1.3 (0.5–3.0)	1.4 (0.3–7.1)	0.707
5 or fewer regular household members	37	63.8	60	51.7	1.6 (0.9–3.1)	1.8 (0.8–3.8)	0.131
Sharing residence with household member with ulcerating disease	16	27.6	3	2.3	14.4 (3.8–79.3)	16.0 (4.1–62.8)	<0.001
C. Factors related to outdoor environment
Sighting rock hyrax near residence	51	87.9	67	57.8	5.3 (2.2–12.7)	3.0 (0.9–9.3)	0.065
Sighting wild jackal near residence	11	19.0	19	16.4	1.2 (0.5–2.7)	1.2 (0.4–3.6)	0.683
Sighting porcupine near residence	43	74.1	79	68.1	1.3 (0.7–2.8)	0.5 (0.2–1.3)	0.159
Sighting mongoose near residence	44	75.9	80	69.0	1.4 (0.7–2.9)	1.1 (0.4–2.7)	0.846
Domestic dogs in the residence	36	62.1	83	71.6	0.7 (0.3–1.3)	1.3 (0.5–3.2)	0.553
Presence of a nearby forest or thicket	53	91.4	67	57.8	7.8 (2.8–26.4)	7.0 (2.0–24.7)	0.003
Presence of a nearby open water source h	5	8.6	24	20.7	0.4 (0.1–1.1)	0.4 (0.1–1.1)	0.081
Immediate neighbour has typical skin lesions i	21	36.2	9	7.8	6.8 (2.8–16.0)	3.1 (1.1–8.8)	0.031
Distant neighbour has typical skin lesions i	17	29.3	41	35.3	0.8 (0.4–1.5)	1.0 (0.4–2.4)	0.937
Presence of garbage mound near residence					0.6 (0.3–1.1)	0.6 (0.2–1.5)	0.278
D. Protective factor
Presence of cultivated crop farm near residence	46	79.3	113	97.4	0.1 (0.0–0.4)	0.1 (0.0–0.5)	0.006

Occupation that involves forest visit included those who were engaged in charcoal burning, herding, hunting, stone masonry and mining.

Other roofing materials included grass, leaves, earthen, wood or rocky caves.

Cracked wall type included earthen walls, rocky caves or walls have visible cracks and crevices.

Cracked floors included earthen, rocky caves, or floors with visible crevices.

Open water sources included river, dam, well, bore hole, pond or spring.

Typical skin lesions included a skin ulcer with typical raised edges and depressed centre or a skin plaque (a circumscribed, nodular or palpable skin lesion) on physical examination by a medical officer

In the first category, individuals who preferred staying outside their residence in the evening after sunset (OR 4.1, CI 1.2–16.2) and those whose primary occupation involved visiting forests (OR 4.6, CI 2.0–10.2) had significant associations with disease in the bivariate analysis. After adjusting for other factors in the multivariate model, only occupations involving forest visit remained significant with a reduced odds ratio of 3.8. Activities such as charcoal burning, hunting, herding, stone masonry and mining were included among the occupations involving forest visits. When assessed separately, these occupations had significantly large odds ratios, but this analysis is not reported here due to possible close link between each of these occupations with forest visits. Other individual attributes such as sex, level of education, history of travel or use of mosquito nets did not have any significant association with disease.

Three of the five factors that were fitted in the second category of factors related to indoor transmission remained statistically significant in the multivariate analysis. This included sharing residence with a household member with typical skin lesions (OR 14.4, CI 3.8–79.3), residing in a house with alternative roofing materials (OR 7.9, CI 1.9–45.7) and residing in a house with cracked walls (OR 2.3, CI 1.0–4.9). These factors also showed significant associations with increased odds ratios after allowing for other factors in the multivariate model.

In the third category of factors related to outdoor environment, four of the eleven factors included in the analysis were significant: sighting rock hyraxes near residence (OR 5.3, CI 2.2–12.7), residing near a forest (OR 7.8, CI 2.8–26.4) and living close to a neighbour with typical skin lesion (OR 6.8, CI 2.8–16.0) had increased likelihood of CL. Three of these remained significant in the multivariate model but with reduced odds ratios. Having a cultivated crop farm surrounding the residence (OR 0.1, CI 0.0–0.4) appeared protective. This association remained significant in the multivariate model.

In the final model, seven factors remained significant after controlling for all factors. Occupations that involve visit to the forest (aOR 3.4, CI 1.1–10.7), living in a house with cracked walls (aOR 5.5, CI 1.6–19.3), five or fewer household occupants (aOR 2.8, CI 1.1–7.1), sharing residence with a household member with typical ulcerating disease (aOR 26.7, CI 5.2–135.8), having of a forest in the neighbourhood of residence (aOR 5.8, CI 1.7–19.6) and having a neighbour with typical skin lesions (aOR 5.3, CI 1.8–15.7) were significant in the final model. Having a cultivated crop farm near the residence (aOR 0.1, CI 0.0–0.5) remained protective. The odds ratios for these associations are as shown in .

Discussion

Review of hospital records

This study has highlighted the burden of CL in Gilgil through records review and house-to-house survey. Young children of school-going age were disproportionately affected, and cases mainly originated from two geographical locations. The study area is relatively sparsely populated and a finding of 255 cases in this area is significant. However, barring the quality of hospital records, the number of CL cases in this area could be higher. CL has been known to be present in this geographical area since the early 1990’s [9,21]. It is therefore not surprising that we found evidence of continuous transmission of CL between 2010–2016. The official health reporting portal of Kenya MOH through the Kenya Health Information System (KHIS) platform does not incorporate CL among the monthly aggregate reports sent from health facilities across the country [22]. In this portal, reports of CL are ordinarily lumped and reported as “diseases of the skin” making it impossible to compute cases of CL at the national level. Coupled with inadequate diagnosis at health facility level, there is potential underreporting of CL over the period.

CL cases were clustered around two geographical locations in the study area: cases originated either from Gitare, the northern part of Gilgil along the rocky cliffs on the wall of the Great Rift Valley or from the southern end of Gilgil in Útut forest which is located in an area of solidified volcanic lava on the floor of the Great Rift Valley. Based on observations by the study team, both regions are remote, largely inaccessible, lack basic infrastructure and are inhabited by small scale farmers (Gitare Village) or forest dwellers (Útut Village). In Gitare village, crop farmers were observed to be encroaching the thickets around rocky escarpments near their homes for farmland and new homes were seen to be constructed in areas that were previously under forest cover. In Útut village, charcoal burning, stone mining, beekeeping and herding are majorly carried out in the forests by many residents.

The finding of clustering of cases in the two foci could point at accelerated CL transmission in these areas. Indeed, Sang et al described Útut forest, among other regions, as a focus of CL on the floor of the Great Rift Valley with the sand-flies also identified in this region [11]. This focalized transmission pattern could be attributed to three possible explanations. First, the sand-fly vector implicated in transmission of CL has a restricted flight radius, mostly flying within a range of 50 meters around their habitat and would therefore only bite and transmit CL in a localized geographical area [23]. Secondly, there is abundance of mammal reservoirs in these localities as evidenced by the increased likelihood (three-fold) of sighting of rock hyraxes by the majority of CL patients in the study area. Wild rodents including rock hyraxes have been described in literature as animal reservoirs of CL and could facilitate transmission of CL in this locality [11]. Lastly and importantly, these two localities epitomize high rate of human encroachment to previously non-inhabited land. In Gitare village, crop farming is expanding while in Útut forest, more people are visiting the forest to burn charcoal, harvest honey, mine stones and herd livestock.

Risk factors of cutaneous leishmaniasis

Through analysis of demographic, behavioural and environmental attributes, we have been able to identify independent risk factors that are associated with CL in the study area. Significant associations seen in analysis of risk factors in this study suggest an overlap of factors that promote the likelihood of occurrence of CL at individual, indoor or outdoor levels. Individual factors included behavioural (spending time outside residence in the evening after sunset) and occupational factors (involving visit to the forest). Indoor risk factors included houses with cracked walls, households with fewer inhabitants and households with at least one member infected with CL. Outdoor transmission was associated with individuals residing close to a forest, close to a neighbour with CL or individuals who sighted wild rodents such as the rock hyrax near their residence.

The sand-fly vector for CL is described in literature as naturally anthropophilic and crepuscular, preferring to bite both indoors and outdoors in the evenings and early mornings [4,12,24]. In previous studies, the sand-fly has been identified in parts on Kenya, including Gilgil [12,23,25,26]. Among the indoor factors, we observed almost four-fold increased likelihood of CL among residents of houses with cracked walls. It is possible that large cracks and crevices on walls of residences provided daytime hiding environment for the sand-fly after a blood meal. Perhaps a more direct evidence of indoor transmission is supported by our finding of 16-fold increased risk of CL in households where at least one member had typical ulcers. This observation could be explained in part by familial clustering tendencies of CL that have been reported in previous studies [27-29] or by a possible presence of high sand-fly vector density within these residential units [4].

Our finding of increased risk of CL in households with 5 or less inhabitants marks a departure from what has been observed in most studies since a large house-hold size (number of regular residents of a household) and high population density are considered as proxy indicators of poverty which has in turn been associated with CL [2]. One possible explanation for this reverse association could be that small household sizes could potentially predispose the few household occupants to frequent sand-fly-human contact through bites [4]. Such an inverse relationship has also been observed in the case of common arthropod-vector borne diseases such as malaria [30,31].

Presence of forest near residences and residents engaged in occupations that involved visit to the forests including charcoal burning, herding, hunting, wild honey harvesting and stone mining, all had increased likelihood of CL. Forest and thickets are likely to provide suitable habitats to immature and mature forms of the sand-fly vector. Additionally, the rocky caves and thickets around Gilgil have also been known to be infested with hyraxes and other mammals that are natural reservoirs of CL, and were likely to be sighted around homes among those with CL [21,32]. As a result of increased population pressure, changes in land use (from forestry to agriculture) and deforestation, reservoir mammals have been known to migrate closer to human residences [2,4,24]. Therefore, one plausible explanation for increased risk observed among residents who frequent forests for livelihood could be that such exposures would increase the frequency and duration of sand-fly-human contact. It appears that frequent vector-human contact either due to increased density of sand-fly in the human dwelling environment or as a result of humans venturing in sand-fly infested habitats in the forests could explain the increased risk of transmission in both indoor and outdoor settings, with the constant being increased exposure to the vector.

Studies have consistently shown more cases of CL among poor, neglected populations who are likely to be less educated and mostly unemployed [2,33]. In our study, most of the sampled population (94.8%) had primary level of education or less, 20% were unemployed and relied on menial jobs to occupy their time and generate income and 72.1% used herbal medication for treatment of skin lesions. All these findings reinforce this pattern of association. Other publications have also shown similar findings [34,35]. However, analysis of level of education and employment status did not show significant associations in the bivariate analysis in our study.

As expected, cases were younger, a finding that is consistent with available literature that has shown that prevalence of CL increases with age in from early childhood then levels off by 15 years of age [4,34]. Even though this observation has been explained by a gradual acquisition of immunity among susceptible persons with increasing age, the explanation that appears more plausible is that as children grow up and become more mobile, they are likely to be exposed to bites when they visit forests around their homes. Perhaps, an interplay of these two factors would be very likely. Some cases had both active lesions (typical ulcer) and old scars at various stages of healing. This could imply repeated infections in the same individual, a possible indication that immunity developed following primary infection could not likely be life-long as seen elsewhere in literature [36].

Skin ulcers were frequently located in head and neck regions of the body and less frequently in hands and feet. Similar findings have been recorded in other studies [11,37]. The wounds mainly affected exposed areas of the body which are commonly bitten by the sand-fly. Mouth and nostril ulcers were also observed among some of the cases, possibly because such areas of the body are also exposed to insect bites. Ordinarily, mucosal lesions are typical of muco-cutaneous leishmaniasis (MCL), however, MCL would be highly unlikely to occur in this region given that the agent (L. tropica) that is known to be present in this area is mainly associated with CL [11].

Despite its strengths, this study had some limitations. Hospital records available for the review were either incomplete or inaccurate. Lack of a proper hospital records could lead to underestimating/overestimating the burden of CL. Secondly, laboratory confirmation was not done for the identified cases. Therefore, some of the chosen controls could actually be incubating cases or false positives. Thirdly, it was also not possible to compute the incidence of disease as some of the cases enlisted reported multiple infections over time. Moreover, CL has a long latency period hence some cases identified in the hospital records could be prevalent cases as opposed to incident cases. Owing to the long duration of disease, there is potential for recall bias could affect the quality of responses that we got from respondents. Despite our finding of strong association between individual, indoor and peri-domestic factors in the transmission of CL, our study did not estimate the relative contribution of each of these factors in transmission of CL.

Conclusion and recommendations

This study has highlighted the burden of CL in Gilgil. However, due to sparse data in the visited facilities, the true burden of disease could be higher. Cases were reported throughout the years, consistent with a locally endemic disease transmitted continuously throughout the period. This study highlighted indoor and outdoor risk factors that promoted clustering of cases among household members and focalized transmission pattern in specific neighbourhoods in the study area. Occupations and activities that involve visiting forests, residing near forests or sharing a house or neighbourhood with a person who has CL were identified as significant exposures of the disease. CL lesions mostly affected younger or older residents with lesions mainly located in exposed parts of the body.

There is need to strengthen diagnosis and reporting for CL in Gilgil in order to provide a better estimate of the disease burden. Emphasis should be put on quality of data collected in the affected health facilities. Tailored control interventions including indoor residual spraying, barrier methods (such as insecticide treated nets, insect repellents and wearing protective clothing) and destruction of vector breeding grounds would be effective when deployed in this area given the focalized pattern of transmission of CL driven by human interaction with known sand-fly hotspots such as forests. However, owing to the anticipated difficulties in deployment of these control measures, we recommend that studies be conducted to establish the feasibility and effectiveness of these interventions among residents of Gilgil. Modification of human behaviour in areas of known transmission risks would appear to be potentially effective as a disease control strategy. Discouraging practices such as hunting and charcoal burning would in theory be effective but would not be practical without alternative sources of income. However, the role of environmental factors and wild mammals in disease transmission should be investigated further.

STROBE checklist.

(DOC)

Click here for additional data file.

Cutaneous leishmaniasis questionnaire.

(PDF)

Click here for additional data file.

Cutaneous leishmaniasis information leaflet.

(PDF)

Click here for additional data file.

Ministry of Health (MOH) approval letter.

(PDF)

Click here for additional data file.

Cutaneous leishmaniasis dataset.

(XLSX)

Click here for additional data file.

5 Sep 2019

PONE-D-19-19202

Cases of Cutaneous Leishmaniasis in a peri-urban settlement in Kenya, 2016

PLOS ONE

Dear Dr Ngere,

Thank you very much for submitting your manuscript "Cases of Cutaneous Leishmaniasis in a peri-urban settlement in Kenya, 2016" (#PONE-D-19-19202) for review by PLOS ONE. As with all papers submitted to the journal, your manuscript was fully evaluated by academic editor (myself) and by independent peer reviewers. The reviewers appreciated the attention to an important health topic, but they raised substantial concerns about the paper that must be addressed before this manuscript can be accurately assessed for meeting the PLOS ONE criteria. Therefore, if you feel these issues can be adequately addressed, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. We can’t, of course, promise publication at that time.

We would appreciate receiving your revised manuscript by Oct 20 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Abdallah M. Samy, PhD

Academic Editor

PLOS ONE

Additional Editor Comments:

I invited and received four reviews for your manuscript. All reviews raised some substantial concerns about your manuscript as it currently stands. I read through their comments and found that they coincided on several points, and that their reviews were uniformly solid and detailed. I read the manuscript myself, and I must say that I coincide with the reviewers' points entirely. As such, I would recommend “major revision”. I would kindly ask you to go through all comments raised by each reviewer and address them properly before sending a revised version of this manuscript. Please check all PLOS ONE style requirements available via https://journals.plos.org/plosone/s/submission-guidelines before submitting the revised version. Finally, please consider a careful language revision for your manuscript.

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information. Moreover, please include more details on how the questionnaire was pre-tested, and whether it was validated.

3. Please correct your reference to "p=0.000" to "p<0.001" or as similarly appropriate, as p values cannot equal zero.

4. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially identifying or sensitive patient information) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. Please see http://www.bmj.com/content/340/bmj.c181.long for guidelines on how to de-identify and prepare clinical data for publication. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Partly

Reviewer #3: Yes

Reviewer #4: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

Reviewer #3: Yes

Reviewer #4: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The efforts of the authors are appreciated. However, there is a grave concern about the cases recruited in the study. Suspected cases, with the definition authors provided, must not be included in the study.

Reviewer #2: Cases of Cutaneous Leishmaniasis in a peri-urban settlement in Kenya, 2016

Ngere, et al.

1. The time period the clinical reports that were reviewed covered 2010-2016. This is clearly indicated in the paper text but should be indicated in the initial paper Summary Section as well.

2. Ethical review of the study was not required as this was a MOH response to an acute situation. It seems however, that the appropriate measures were taken to assure patient protections (confidentiality and consent). Was MOH number (approval) given to the Study—might this be cited?

3. The authors state that cases are higher in June-December period. However, this is really only the case for 2014. This may be more a case of continuous transmission. It is not clear why there is a peak for June 2010 and then little disease reported subsequently. In 2011, 2012 and even 2013- disease appears continuous.

4. In reporting factors relevant to disease—some are positively related (forest time, neighbors with disease) while others are negatively associated (protective- crop growing). These are not really distinguished in the tables—although the text is clear. It would be useful to make this clearer in the Tables presented.

5. It is unclear to this reviewer that some of the hypotheses concerning the epidemiological features of the leishmaniasis outbreak are reasonable. 1) The authors suggest (Discussion Section) that as many lesions are on the nose and mouth that mucocutaneous leishmaniasis may be occurring. This would be unusual for the species likely transmitting in the area- which is likely (based on the reservoir hosts implicated), L. tropica. Actually a report in 1994 (Trans R Soc Trop Med Hyg. 1994 . 88:35-7; note this reference (9) has been miscited in the Reference Section) from this area indicated that this species is in circulation in this area of Kenya. No mention is made of this point though other findings of this paper were mentioned in the text. MCL is not straightforward (as the authors appear to realize) and these really sound more like these lesions are where the insect bite occurred and not really a dissemination/metastasis from a cutaneous site to mucosal tissue. L. tropica (to date) is most commonly associated with CL rather than MCL. 2) The authors (Discussion) mention that there is a higher susceptibility at the ends of the age spectrum – older and younger. They suggest that the susceptibilities of these populations might reflect impaired immune responses associated with these groups. However, this does not match their data. The lower immune responses of children are generally associated with infants and the group here with higher level of infection was the >5-15 year olds and not the <5 year old group. In fact the highest incidence of cases (48% of total) is in this group. Few cases appear in the >60 years group. This group (5-15 years) would likely be more mobile and in and out of the nearby forests. Certainly, the data available could be used to examine this point. However, immune impairment does not seem reasonable. 3) In Table 4, it is indicated that one of the “interior” factors for disease is “Living in a house with cracked walls”. This sounds like the barrier to the outside is broken. Would this not reflect the exterior conditions then? In these cases, were the houses closer to the forests (another disease factor) as well? It would be useful to assess this in terms of identifying intervenable control measures.

Minor Points:

1. On the Title Page- it is not clear what the symbol & represents.

2. Line 114- Leishmania donovani, the d should not be capitalized; line 115, the same for the c in L. chagasi. The Word program is annoying in that it does this- capitalizes letters after periods (.).

3. Map in Figure 1 is useful but could be improved with the addition of specific geographical landmarks (City, river).

Reviewer #3: First

The study is within the scope of the journal considered about one of the neglected diseases in the world.

The title should be matched the objective, methodology and conclusion, it better to be as

“Risk factors of cutaneous leishmaniasis in a pre-urban settlement in Kenia, 2016”

Abstract should not include any abbreviations and not exceed more than 300 wards. So the abbreviation “CL” should be dropped from the conclusion section of the abstract and the abstract wards (389) should be decreased to match the journal criteria.

Really, the study has a clear design of a case control study type however, all the cases of the study group either suspected or probably cases of cutaneous leishmaniasis. All cases diagnosed clinically so in conclusion section, it is better to use “there is evidence” rather than “there is strong evidence”

second

The pronoun “we” was used several time through the paper, it is better to avoid using the subjective pronouns and it should be dropped from the text.

-In the line number 93, the abbreviation “CL” was not previously defined and should be written as cutaneous leishmaniasis rather than the short form “CL”.

-In the line number 81, “intermediate hosted” it is better to be “reservoir” rather than intermediate.

-In the line number 182, drop the wards “at the” from the sentence

-In the line number 228, the ward “while” it is better to be dropped.

-In the line number 262, it is better to add word “Kenia” at the end of the title of table 3.

-In the line number 290, it is better to add the time of the study “2016” at the end of the title of the table 4.

-In the line, number 337, it is better to uses “reservoir mammals” rather than “infected wild rodents”

-In the line number 474, the references 15 should be corrected by 17

-In the line number 481, the reference 18 should be corrected number 20

Finlay

The map should be clearer illustrating all valleys, villages and cities which were mentioned through the paper.

Reviewer #4: It is a well written manuscript that provides further information on CL in Kenya, which is an understudied topic. However, there are some important issues in the methodology (e.g. definition of cases, how matching was done) and I also have my concerns about the ethical section. More details are provided below.

General

Abbreviations should be used consistently. CL and cutaneous leishmaniasis are used throughout the manuscript (e.g. in full in line 293). After the first time (line 70?) using the full name the abbreviation should be used.

Introduction

Line 93, there are several vectors for CL, which one is meant here?

Comments methods

General

To make what was done easier to understand the paper should consequently discuss the hospital review first (and separately) and the case control study after.

Study site:

The total catchment area of the health facilities should be indicated. Is 255 cases a lot? Understanding the context will help judge that.

The health facilities in Morendat and Mbaruk should be described in more detail.

Why was the gilgil sub-county chosen?

Line 113-114: which sandfly is meant? Did they really identify Leishmania infantum in Kenya? The agents described here seem wrong, especially for CL in Kenya.

Study population:

As a whole, the selection of the study population should be more clearly described. Specifically, the relationship between the line list made based on the hospital records to the case control study. From the 255 only 41 were included as cases. How were the 41 selected? What happened to the rest? Could there have been a bias in selecting these 41? Also for the controls, did all the controls agree to participate in the study?

How was the house to house survey used to detect the additional 18 cases carried out? What was the strategy? What were the exact criteria to define cases in the house to house survey.

There is mention of suspected, probable or confirmed cases, and that all are included as cases. However, is this correct? How many were actually suspected, probably and confirmed?

Were there any patients with missing data? Did this lead to any exclusions?

Definition of cases and control:

I have a big issue with the definition of cases, as I fear the definition of a CL case is too broad, and therefore the cases contain false positives. Patients with ‘dermatitis’, ‘skin infection’, or ‘skin wound’ now all seem to be recorded as cases, which does not seem correct. This may severely impact the study.

On line 128 it also states that a probable case was defined as a patient with a typical ulcer (who determines what is a typical ulcer) or plaque, ascertained by a medical officer in the study team during the study period. However, the study period was only 2 weeks, so how can this refer to the part of the study that led to the line list (which to my understanding is based on retrospective file review).

In addition, why are only ulcers or plaques mentioned as probable CL, while Sang already stated in 1993 that lesions were rarely ulcerated, and Toroitich also said that many lesions were in fact nodular.

-Why was no laboratory confirmation done? Logistical challenges are mentioned, these should be specified.

-For the record review, how experienced were the staff in the health facilities in diagnosing CL?

- How experienced was the medical officer

Field work

It states that a lab scientist was there during the field visit, but if no lab confirmation was done, what did this person actually do?

Matching

Why was matching done by neighbourhood done? This does not make sense when factors related to the outdoor environment are one of the main interests of the study. By matching by neighbourhood, neighbourhood itself (micro-environment) cannot be studied properly as a risk factor in that way. �I think the third level of factors related to outdoor environment cannot be properly studied if neighbours are chosen as controls (unless the houses are very far apart, which should then be adequately explained).

Because no direct neighbours of ‘CL cases’ could be taken as controls, how can having an immediate neighbour with CL be studied as a risk factor?

Why matching when multivariable regression is done in analysis to deal with confounding anyway?

Sample size

The manuscript states that OpenEpi was used to calculate the sample size. However, OpenEpi does not seem to have an option for calculating sample size for matched case control studies. When I redo the sample size calculation in open epi using unmatched case control I get 180, 167 or 189, not 174. It should be clearly indicated how they reached 174. After trying to repeat the sample size calulcation it seems that a sample size calculation for matched case control assuming correlation is 0.15 was used. However, this is not explained clearly, and if 0.15 correlation was used, this should be explained.

Sample size uses assumption that mosquito net use was present in 31% of controls. In fact this was <20%. I think this may lead to your study being underpowered, as when I retrospectively calculate the sample size using the 18% proportion controls using a mosquito net, the sample size is 306, 279 or 317, which is almost double of the current sample size.

Ethics:

This study was not reviewed by any ethical review body. The reason given is that the study was conducted as part of an MOH led public health response to an acute event. However, to my understanding even research carried out in emergency response (e.g. in Ebola research) requires (expedited) ethical review. The researchers should elaborate more on the reason and regulations as to why the study was not reviewed by any ethical board. Just because permission was given by the health authorities does not automatically mean the study is carried out according to the ethical principles of medical research. For instance regarding the consent procedures (see below).

The manuscript states that oral consent was obtained. However, there is no mention of any proof of this consent (e.g. recording of oral consent, or ideally written consent). It is also not stated clearly why for this study oral (non-recorded) consent suffices. I have my doubts about this, and therefore the reasons for these matters should be explicitly explained.

In addition, the informed consent should be explained in more detail (especially since it was not approved by any IRB), was the consent procedure organised in an appropriate and complete manner? Did the patient receive a participant information sheet describing the research and giving the contact information of the PI?

Measures were taken to assure confidentiality. Please specify which methods.

The manuscript states that no personal identifying information was collected or analysed. However, in line 133 it states that name, sex, age, date seen at the facility, residence, signs and symptoms, diagnosis and treatment given were all among collected data. This would definitely qualify as personal identifying information.

Why use the 3 steps in multivariate? Why not skip the analysis per level?

Results

It is mentioned that one death was suspected due to CL. This is strange as CL is normally not deadly, and therefore this should be elaborated on.

Line 203: IQR should give the 25th and 75th percentile, and not just one value)

Line 218-219 should be left out, as matching means that the age is very similar

Line 220: states that distribution of other social and demographic variables studied among cases and controls was comparable. However, occupation was significantly different between the two groups.

Line 227: mean duration of illness was two years .

228: it says all cases had typical ulcers, but it does not make sense to describe a feature that was used to select the population. If typical ulcers were the way the population was selected, of course all patients will have typical ulcers.

The number of lesions should be mentioned. If the big majority of cases had both a typical ulcer but also a papule or nodule, does this mean they had multiple lesions with different presentations or one lesion with both an ulcerative and indurated presentation?

Line 232: why mention nasal stuffiness? This seems random, unless you want to link it MCL.

Line 238-244. If patients were close neighbours how can there be a difference in sighting rock hyraxes around the house or in living close to the forest? Could this be purely due to bias?

252: here 2 decimals are given, throughout the manuscript it should be consistent, either 1 or two decimals for percentages and CI.

Line 257: certain activities were not analysed because they could potentially have a close link with forest visits. However they should be analysed in multivariable which can check whether they are independent risk factors.

Are the factors displayed in table 3 the only ones that were assessed? Or did a selection take place and only certain ones are displayed? This should be described.

Table 2 and 3 can be merged into one, as there is significant overlap.

Line 264: should bivariate analysis not be multivariate here?

The results from the risk factor analysis can be shortened. The essence should be which factors were risk factors in the bivariate, and how many of them were independent risk factors after multivariate analysis?

Discussion

I feel the burden of CL has not been sufficiently highlighted. At least, more background should be given that can help quantify the burden in relation to the study area.

Limitations regarding recall bias and the case definition should be mentioned.

Line 310: this seems to be over interpretation of the findings, this study does not allow saying anything about indoor or outdoor transmission being observed.

The CL outbreak of 2016 is never mentioned again. Was this actually an outbreak?

In line 355 it mentioned there may be accelerated transmission. A reference from 1993 is given. This indicates that CL is not a new thing here and therefore points in the other direction in fact.

Line 369: it is not clear whether the statement of low levels of education is about the cases in the case control study, the hospital records or in the population comprising both cases and controls. In addition the 90.8% that is mentioned to have less than primary education can not be found in any of the tables, which mention 94.8% of cases having primary education or less (not less than primary education).

Line 375: patients above 35 are mentioned being ‘older individuals.’ This statement is related to immunosuppression of old age. This is not appropriate, as only 10% of patients were above 60 in fact.

Line 386: states the most common presentation of cases was a skin ulcer, if this was the way the cases were selected do not use it to describe the presenting feature of CL.

The limitations of the case control study should be elaborated on, as there are quite a few (see comments from methods section).

Conclusions

404: no real grounds for this statement. What is large?

407: study does not provide real evidence on transmission, just on risk factors

414: what is meant by ‘there is need to institute surveillance for CL’?

References

Please correct the mistakes here, I found a few

-ref 9: should be Sang D, Ashford RW, Njeru WK etc.

-ref 10: authors name is Toroitich AK (not Itor Toroitich)

-ref 11: third author’s first rather than last name is written in full

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Mohammed Musid Alkulaibi

Reviewer #4: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

31 Oct 2019

Dear Editor,

The following are the responses to comments raised by reviewers on my manuscript “Cases of Cutaneous Leishmaniasis in a peri-urban settlement in Kenya, 2016" (#PONE-D-19-19202):

Editor comments

1. Please check all PLOS ONE style requirements available via https://journals.plos.org/plosone/s/submission-guidelines before submitting the revised version. Finally, please consider a careful language revision for your manuscript

These have been addressed. I have revised sections of the manuscript in line with the suggested changes from the reviewers. I have aligned the document to PLS ONE style requirements.

2. Additional comments (journal requirements):

a. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming

This has been addressed

b. Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information. Moreover, please include more details on how the questionnaire was pre-tested, and whether it was validated

The study questionnaire has been included as supporting information S2

c. Please correct your reference to "p=0.000" to "p<0.001" or as similarly appropriate, as p values cannot equal zero

These changes have been made in the revised manuscript.

d. Data availability and sharing:

There is no ethical or legal restriction against sharing data. The minimal anonymized dataset for both the records review and case-control study have been shared in as supporting information file (“S5_Cutaneos_Leishmaniasis_Data”)

Reviewer #1:

1. The efforts of the authors are appreciated. However, there is a grave concern about the cases recruited in the study. Suspected cases, with the definition authors provided, must not be included in the study

‘Suspected cases’ were only included in the review of hospital records. Only probable cases were included in the case control study. Probable cases were defined as residents with typical ulcers (with typical raised edges and depressed center) or plaques (a circumscribed palpable or visible lesion made up of multiple coalescent nodules or papules) as was ascertained by an experienced medical officer in the study team.

Reviewer #2:

2. The time period the clinical reports that were reviewed covered 2010-2016. This is clearly indicated in the paper text but should be indicated in the initial paper Summary Section as well.

This has been addressed (See revised manuscript abstract section)

3. Ethical review of the study was not required as this was a MOH response to an acute situation. It seems however, that the appropriate measures were taken to assure patient protections (confidentiality and consent). Was MOH number (approval) given to the Study—might this be cited?

MOH approval was given for the outbreak response, see the approval letter in the supplementary materials.

4. The authors state that cases are higher in June-December period. However, this is really only the case for 2014. This may be more a case of continuous transmission. It is not clear why there is a peak for June 2010 and then little disease reported subsequently. In 2011, 2012 and even 2013- disease appears continuous.

This has been addressed. Continuous transmission was seen throughout the period with occasional peaks in June 2010 and December 2014

5. In reporting factors relevant to disease—some are positively related (forest time, neighbors with disease) while others are negatively associated (protective- crop growing). These are not really distinguished in the tables—although the text is clear. It would be useful to make this clearer in the Tables presented.

I have revised Table 3 to show protective factors in a separate category

6. It is unclear to this reviewer that some of the hypotheses concerning the epidemiological features of the leishmaniasis outbreak are reasonable. 1) The authors suggest (Discussion Section) that as many lesions are on the nose and mouth that mucocutaneous leishmaniasis may be occurring. This would be unusual for the species likely transmitting in the area- which is likely (based on the reservoir hosts implicated), L. tropica. Actually a report in 1994 (Trans R Soc Trop Med Hyg. 1994 . 88:35-7; note this reference (9) has been miscited in the Reference Section) from this area indicated that this species is in circulation in this area of Kenya. No mention is made of this point though other findings of this paper were mentioned in the text. MCL is not straightforward (as the authors appear to realize) and these really sound more like these lesions are where the insect bite occurred and not really a dissemination/metastasis from a cutaneous site to mucosal tissue. L. tropica (to date) is most commonly associated with CL rather than MCL.

After carefully reviewing available references and discussion with subject matter expert, this statement has been revised to read “Mouth and nostril ulcers were observed among some of the cases, possibly because such areas of the body are also exposed to insect bites. Ordinarily, mucosal lesions are typical of muco-cutaneous leishmaniasis (MCL), however, MCL would be highly unlikely to occur in this region given that the agent (L. tropica) that is known to be present in this area is mainly associated with CL”- See text in the revised manuscript

7. The authors (Discussion) mention that there is a higher susceptibility at the ends of the age spectrum – older and younger. They suggest that the susceptibilities of these populations might reflect impaired immune responses associated with these groups. However, this does not match their data. The lower immune responses of children are generally associated with infants and the group here with higher level of infection was the >5-15 year olds and not the <5 year old group. In fact the highest incidence of cases (48% of total) is in this group. Few cases appear in the >60 years group. This group (5-15 years) would likely be more mobile and in and out of the nearby forests. Certainly, the data available could be used to examine this point. However, immune impairment does not seem reasonable.

Indeed, our data does show that more cases of CL were younger. Some literature explains that prevalence of CL increases with age up to about 15 years after immunity is acquired following childhood infections in endemic areas (Reithinger et al 2003). However, it is also likely that as children grow and become more mobile, they get exposed many times when they visit forest around their homes. We have therefore factored in these explanations in the revised discussion section of our manuscript.

8. In Table 4, it is indicated that one of the “interior” factors for disease is “Living in a house with cracked walls”. This sounds like the barrier to the outside is broken. Would this not reflect the exterior conditions then? In these cases, were the houses closer to the forests (another disease factor) as well? It would be useful to assess this in terms of identifying intervenable control measures.

Cracked walls had visible lines, sometimes with crevices but not broken apart, therefore, these constituted ‘indoor factors’. Most of these walls were made of mud/earthen and rocks. These cracks provided daytime hiding places for the sand-fly after blood meal.

9. Minor Points:

a. On the Title Page- it is not clear what the symbol & represents

‘’ is an ampersand symbol, and is used to represent the 2nd set of equal contributors in the authors list. The symbol is recommended in the PLOS ONE formatting guidelines https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

b. Line 114- Leishmania donovani, the d should not be capitalized; line 115, the same for the c in L. chagasi. The Word program is annoying in that it does this- capitalizes letters after periods (.)

Corrected

c. Map in Figure 1 is useful but could be improved with the addition of specific geographical landmarks (City, river).

Corrected (Fig. 1)

Reviewer #3:

1. The study is within the scope of the journal considered about one of the neglected diseases in the world. The title should be matched the objective, methodology and conclusion, it better to be as. “Risk factors of cutaneous leishmaniasis in a pre-urban settlement in Kenia, 2016”

I have revised the title to read “Burden and Risk Factors of Cutaneous Leishmaniasis in a peri-urban settlement in Kenya, 2016” in line with the major objectives of the study. This study identified and characterized cases of cutaneous leishmaniasis in Gilgil through a review of records and active case search in the community to establish the burden of cutaneous leishmaniasis and explored individual, indoor and outdoor factors that may promote spread of the disease.

2. Abstract should not include any abbreviations and not exceed more than 300 wards. So the abbreviation “CL” should be dropped from the conclusion section of the abstract and the abstract wards (389) should be decreased to match the journal criteria.

The abstract has been revised for conciseness and word count updated

3. Really, the study has a clear design of a case control study type however, all the cases of the study group either suspected or probably cases of cutaneous leishmaniasis. All cases diagnosed clinically so in conclusion section, it is better to use “there is evidence” rather than “there is strong evidence”

Agreed. This has been rectified

4. Second:

a. The pronoun “we” was used several time through the paper, it is better to avoid using the subjective pronouns and it should be dropped from the text.

b. In the line number 93, the abbreviation “CL” was not previously defined and should be written as cutaneous leishmaniasis rather than the short form “CL”.

c. In the line number 81, “intermediate hosted” it is better to be “reservoir” rather than intermediate.

d. In the line number 182, drop the wards “at the” from the sentence

e. In the line number 228, the ward “while” it is better to be dropped.

f. In the line number 262, it is better to add word “Kenia” at the end of the title of table 3.

g. In the line number 290, it is better to add the time of the study “2016” at the end of the title of the table 4.

h. In the line, number 337, it is better to uses “reservoir mammals” rather than “infected wild rodents”

i. In the line number 474, the references 15 should be corrected by 17

j. In the line number 481, the reference 18 should be corrected number 20

Comments a-j have been addressed and updated in the revised manuscript

5. The map should be clearer illustrating all valleys, villages and cities which were mentioned through the paper.

This has been corrected. See new map fig 1

Reviewer #4:

1. General: Abbreviations should be used consistently. CL and cutaneous leishmaniasis are used throughout the manuscript (e.g. in full in line 293). After the first time (line 70?) using the full name the abbreviation should be used.

This has been corrected throughout the document

2. Introduction: Line 93, there are several vectors for CL, which one is meant here?

The species have been clarified and a reference shown

3. Methods: To make what was done easier to understand the paper should consequently discuss the hospital review first (and separately) and the case control study after

The discussion has been re-organized for flow. We first discuss the records review and then present the risk factor analysis. However, some overlapping issues between these two parts of the study have been highlighted under the risk factors for cutaneous leishmaniasis

4. Study population: As a whole, the selection of the study population should be more clearly described. Specifically, the relationship between the line list made based on the hospital records to the case control study. From the 255 only 41 were included as cases. How were the 41 selected? What happened to the rest? Could there have been a bias in selecting these 41? Also for the controls, did all the controls agree to participate in the study? How was the house to house survey used to detect the additional 18 cases carried out? What was the strategy? What were the exact criteria to define cases in the house to house survey.

This has been addressed. 41 probable cases were selected from the outbreak line-list because they could be reached during the study period. Identification of additional cases trough door-to-door case search was a respondent-driven sampling process, whereby study staff working together with village guides would be guided by villagers to identify additional cases. Out of the controls that were approached, 5 were dropped on account of not meeting the criteria for controls. This has been updated in the text and in fig. 2

5. There is mention of suspected, probable or confirmed cases, and that all are included as cases. However, is this correct? How many were actually suspected, probably and confirmed?

Only probable cases were included in the case-control study. The outbreak line-list had a total of 255 suspected and probable cases, out of which 59 probable cases were enrolled in the case control study. No laboratory confirmation was done. See text and updated Fig. 2

6. Were there any patients with missing data? Did this lead to any exclusions?

Patients whose data (e.g. clinical information, physical address, contact information) were missing from the line-list were excluded from enrollment. This has been updated

7. Definition of cases and control: I have a big issue with the definition of cases, as I fear the definition of a CL case is too broad, and therefore the cases contain false positives. Patients with ‘dermatitis’, ‘skin infection’, or ‘skin wound’ now all seem to be recorded as cases, which does not seem correct. This may severely impact the study.

All entries of ‘skin ulcer’, ‘skin wound’, ‘plaque’, ‘dermatitis’, ‘skin infection’ or ‘cutaneous leishmaniasis’ as captured in hospital registers were only included in the outbreak line-list as ‘suspected cases of CL’. For cases that were included in the case-control study, medical officers in the study team had to first ascertain if their skin lesion was a typical CL lesion (ulcer with typical raised edges and depressed center or a skin plaque-a circumscribed palpable or visible lesion made up of multiple coalescent nodules or papules).

8. On line 128 it also states that a probable case was defined as a patient with a typical ulcer (who determines what is a typical ulcer) or plaque, ascertained by a medical officer in the study team during the study period. However, the study period was only 2 weeks, so how can this refer to the part of the study that led to the line list (which to my understanding is based on retrospective file review). In addition, why are only ulcers or plaques mentioned as probable CL, while Sang already stated in 1993 that lesions were rarely ulcerated, and Toroitich also said that many lesions were in fact nodular.

Medical officers in the study team, working together with village locators (community health volunteers and village chiefs) and some of the already recruited cases, identified cases for inclusion in the study during door-to-door visits in a respondent-driven sampling process. These cases were identified on the basis of typical skin lesions (ulcer with raised edges and depressed center or a skin plaque-a circumscribed palpable or visible lesion made up of multiple coalescent nodules or papules) and were updated in the outbreak line-list as ‘probable cases’. The definition of plaques used included skin lesions that were coalescent ‘nodular’ or ‘papular’. This information has been revised in the revised manuscript for clarity.

9. Why was no laboratory confirmation done? Logistical challenges are mentioned, these should be specified. Field work: It states that a lab scientist was there during the field visit, but if no lab confirmation was done, what did this person actually do?

Laboratory confirmation was not done during the outbreak because laboratory supplies needed had not been delivered by the time of the outbreak investigation (sample collection supplies, leishmania skin test supplies and laboratory reagents for tissue staining). Despite having the laboratory scientist in the team, we were not able to conduct any skin tests or collect any samples (skin scrapings/aspirates/biopsies) from the identified cases owing to lack of the necessary supplies.

10. For the record review, how experienced were the staff in the health facilities in diagnosing CL? How experienced was the medical officer

The study did not gauge the experience of hospital staff (medical doctors, nurses and physician assistants) in diagnosing CL. However, these staff would ordinarily clinically evaluate and capture details of patients with skin lesions in hospital records either as ‘skin ulcer’, ‘skin wound’, ‘plaque’, ‘dermatitis’, ‘skin infection’ or a diagnosis ‘cutaneous leishmaniasis’ in instances when laboratory diagnosis was possible. During review of these hospital records, we abstracted this information to create the outbreak line-list and further updated the line-list by examination of the lesions for patients that could be reached during the 2 weeks of the outbreak response. The two medical officers in the study team had been working in the study area for at least 5 years and had been diagnosing and referring patients with CL for treatment at a specialist clinic located at the referral hospital in Nakuru town.

11. Matching: Why was matching by neighborhood done? This does not make sense when factors related to the outdoor environment are one of the main interests of the study. By matching by neighbourhood, neighbourhood itself (micro-environment) cannot be studied properly as a risk factor in that way. �I think the third level of factors related to outdoor environment cannot be properly studied if neighbors are chosen as controls (unless the houses are very far apart, which should then be adequately explained). Because no direct neighbors of ‘CL cases’ could be taken as controls, how can having an immediate neighbor with CL be studied as a risk factor? Why matching when multivariable regression is done in analysis to deal with confounding anyway?

The study area is relatively sparsely populated (Population density of 148 persons per sq. Km), with most households located beyond 150-500 meters apart, sometimes separated by thickets, valleys, or large farms (See description of the study site in the revised manuscript). To get eligible controls, the study team would skip between 2 and 5 households in a chosen direction before approaching a potential control for recruitment. Therefore, many times the controls would land in the next village, far from the location of the case and would not necessarily share the same micro-environment.

Matching by age-group and neighborhood at design level of this study does not necessarily control for all confounding, hence the need for us to still conduct a multivariate regression to control for any residual confounding. On analysis of matched case-control studies, Neil Pearce recommends that matching alone does not eliminate confounding, and may still require controlling of the matching factors in the analysis (Neil Pearce- Analysis of matched case-control studies, BMJ 2016 available from https://www.bmj.com/content/bmj/352/bmj.i969.full.pdf )

12. Sample size: The manuscript states that OpenEpi was used to calculate the sample size. However, OpenEpi does not seem to have an option for calculating sample size for matched case control studies. When I redo the sample size calculation in open epi using unmatched case control I get 180, 167 or 189, not 174. It should be clearly indicated how they reached 174. After trying to repeat the sample size calculation it seems that a sample size calculation for matched case control assuming correlation is 0.15 was used. However, this is not explained clearly, and if 0.15 correlation was used, this should be explained. Sample size uses assumption that mosquito net use was present in 31% of controls. In fact this was <20%. I think this may lead to your study being underpowered, as when I retrospectively calculate the sample size using the 18% proportion controls using a mosquito net, the sample size is 306, 279 or 317, which is almost double of the current sample size.

The numbers of eligible cases present in the study area during the study period (58 cases) largely determined the sample size. A minimum sample size of 174 (2 controls per case) was used, giving the study had at least 80% power at the 5% significance level and able to detect an odds ratio (OR) of 0.3 for an exposure present in 31% of controls. We used OpenEpi to calculate sample size for unmatched case-control studies. This information has been updated in the revised manuscript

13. Ethics: This study was not reviewed by any ethical review body. The reason given is that the study was conducted as part of an MOH led public health response to an acute event. However, to my understanding even research carried out in emergency response (e.g. in Ebola research) requires (expedited) ethical review. The researchers should elaborate more on the reason and regulations as to why the study was not reviewed by any ethical board. Just because permission was given by the health authorities does not automatically mean the study is carried out according to the ethical principles of medical research. For instance, regarding the consent procedures (see below). The manuscript states that oral consent was obtained. However, there is no mention of any proof of this consent (e.g. recording of oral consent, or ideally written consent). It is also not stated clearly why for this study oral (non-recorded) consent suffices. I have my doubts about this, and therefore the reasons for these matters should be explicitly explained. In addition, the informed consent should be explained in more detail (especially since it was not approved by any IRB), was the consent procedure organized in an appropriate and complete manner? Did the patient receive a participant information sheet describing the research and giving the contact information of the PI?

This study was approved by Ministry of Health (MOH) in Kenya and was conducted as part of public health response to an acute event and as such was not reviewed by an ethical review body. Oral consent was obtained from the case-control study subjects and documented in the study questionnaires as indicated in the revised manuscript. Study information was provided in form of written leaflets given to all study participants and printed brochures displayed at strategic locations in health facilities. The records review and active community case finding were conducted as part of routine surveillance and response activities by the MOH. In instances of acute events (such as outbreaks), the practice is that outbreak investigation, response and regulatory oversight is offered by the government Ministry of Health. However, approvals from various levels of administration (e.g. county and subcounty levels) are still mandatory and were sought by the response team. All the data collected was anonymized by dropping all personal identifiers (e.g. name, inpatient/outpatient number, phone contact) before analysis.

14. Measures were taken to assure confidentiality. Please specify which methods.

Measures taken to assure confidentiality of the information provided during these interviews included conducting interviews in private place convenient for respondents, secure storage of paper questionnaires in lockable cabinets accessible only with authorization of the PI and password protection of electronic databases. These have been updated in the revised manuscript.

15. The manuscript states that no personal identifying information was collected or analysed. However, in line 133 it states that name, sex, age, date seen at the facility, residence, signs and symptoms, diagnosis and treatment given were all among collected data. This would definitely qualify as personal identifying information.

Patient data captured in health facility outbreak line-list but was de-identified by dropping all the personal identifying information (patient name, IP/OP number, phone contact) before analysis.

16. Why use the 3 steps in multivariate? Why not skip the analysis per level?

Grouping of the risk factors into three categories was necessary since a number of the factors being explored were potentially inter-related and their parameters would likely vary at more than one level (i.e. multilevel). For example, individual factors would affect a person’s exposure to and vulnerability to sand-fly bites, indoor factors would affect the abundance of sandflies in the house and/or vulnerability to bites while outdoor factors would affect abundance and likelihood of bites when the individual is outside the residence. An individual (with his factors) would ordinarily be nested within an indoor or outdoor environment that has a mix of related factors, some of which are mutually exclusive in this study e.g. examining the relationship between age (individual factor), spending time within the house (indoor factor and is likely associated with individual’s age) and spending time outside the residence (outdoor factor that is related to age and is mutually exclusive of spending most time indoors) gives an example of how mutual exclusivity of the factors would affect the levels of analysis.

17. Results:

a. It is mentioned that one death was suspected due to CL. This is strange as CL is normally not deadly, and therefore this should be elaborated on.

The death was reported in a 67-year old woman who had undergone weeks of treatment for CL

b. Line 203: IQR should give the 25th and 75th percentile, and not just one value)

c. Line 218-219 should be left out, as matching means that the age is very similar

d. Line 220: states that distribution of other social and demographic variables studied among cases and controls was comparable. However, occupation was significantly different between the two groups.

e. Line 227: mean duration of illness was two years .

f. Line 228: it says all cases had typical ulcers, but it does not make sense to describe a feature that was used to select the population. If typical ulcers were the way the population was selected, of course all patients will have typical ulcers.

b-f have been addressed in the revised manuscript

g. The number of lesions should be mentioned. If the big majority of cases had both a typical ulcer but also a papule or nodule, does this mean they had multiple lesions with different presentations or one lesion with both an ulcerative and indurated presentation?

The majority of cases had multiple lesions with different skin presentations, mainly ulcerative and nodular lesions. This statement has been revised

h. Line 232: why mention nasal stuffiness? This seems random, unless you want to link it MCL.

Nasal stuffiness was observed in 2 patients that had severely ulcerative lesions affecting the peri-nasal area.

i. Line 238-244. If patients were close neighbors how can there be a difference in sighting rock hyraxes around the house or in living close to the forest? Could this be purely due to bias?

Rock hyraxes are territorial animals, live is specific rocky habitat and do not forage far (>50 meters) from their residence. In the study area, the population density is low and houses are located up to 200 meters from each other. I therefore believe that sighting rock hyraxes around the residence is a valid question for this study.

j. 252: here 2 decimals are given, throughout the manuscript it should be consistent, either 1 or two decimals for percentages and CI.

This has been addressed

k. Line 257: certain activities were not analysed because they could potentially have a close link with forest visits. However, they should be analyzed in multivariable which can check whether they are independent risk factors.

I would still be hesitant to analyze each of these factors separately in the multivariate model since the underlying denominator seems to be visiting the forest (which was identified as an independent risk factor for CL in the regression model). It would be complex to interpret the aORs for each of these activities separately, when put in the same model with ‘forest visits’

l. Are the factors displayed in table 3 the only ones that were assessed? Or did a selection take place and only certain ones are displayed? This should be described.

We reported on all the factors that were assessed except for the activities such as charcoal burning, hunting, herding, stone masonry and mining that were included among the occupations involving forest visits. It has been explained that these factors had significantly large ORs but were not reported here due to possible close link between each of these occupations with forest visits (see revised manuscript with explanation)

m. Table 2 and 3 can be merged into one, as there is significant overlap.

The two tables communicate different information from our analysis. Table 2 shows the overall demographic characteristics of the study respondents while table 3 presents the results of the bivariate analysis for the factors that were assessed. The two tables were presented separately for simplicity and clarity of the two messages

n. Line 264: should bivariate analysis not be multivariate here?

Corrected

o. The results from the risk factor analysis can be shortened. The essence should be which factors were risk factors in the bivariate, and how many of them were independent risk factors after multivariate analysis?

This section has been revised

18. Discussion:

a. I feel the burden of CL has not been sufficiently highlighted. At least, more background should be given that can help quantify the burden in relation to the study area.

This has been addressed by addition of more background information about the study area and revision of first paragraph of the discussion.

b. Limitations regarding recall bias and the case definition should be mentioned.

c. Line 310: this seems to be over interpretation of the findings, this study does not allow saying anything about indoor or outdoor transmission being observed.

b and c have been revised

d. The CL outbreak of 2016 is never mentioned again. Was this actually an outbreak? In line 355 it mentioned there may be accelerated transmission. A reference from 1993 is given. This indicates that CL is not a new thing here and therefore points in the other direction in fact.

Indeed, CL has been known to be present in this geographical area since the early 1990’s as seen from the work done by Rosemary Sang (1993) and Toroitich (1995). In our investigation, we found a continuous transmission pattern of cases between 2010-2016 without any obvious peaks that would characterize an outbreak. We therefore concluded that this was a case of accelerated transmission owing to existence of multiple factors of spread (indoor and outdoor) in the area.

e. Line 369: it is not clear whether the statement of low levels of education is about the cases in the case control study, the hospital records or in the population comprising both cases and controls. In addition the 90.8% that is mentioned to have less than primary education can not be found in any of the tables, which mention 94.8% of cases having primary education or less (not less than primary education).

This has been reviewed. The correct percentage for primary level of education and below is 90.8%

f. Line 375: patients above 35 are mentioned being ‘older individuals.’ This statement is related to immunosuppression of old age. This is not appropriate, as only 10% of patients were above 60 in fact.

This has been corrected. ‘Older individuals’ has been dropped from this statement

g. Line 386: states the most common presentation of cases was a skin ulcer, if this was the way the cases were selected do not use it to describe the presenting feature of CL.

h. The limitations of the case control study should be elaborated on, as there are quite a few (see comments from methods section).

g and h have been revised

19. Conclusions:

a. 404: no real grounds for this statement. What is large?

b. 407: study does not provide real evidence on transmission, just on risk factors

c. 414: what is meant by ‘there is need to institute surveillance for CL’?

I have revised this section in line with the revisions in the rest of the manuscript

20. References: Please correct the mistakes here, I found a few

a. -ref 9: should be Sang D, Ashford RW, Njeru WK etc.

b. -ref 10: authors name is Toroitich AK (not Itor Toroitich)

c. -ref 11: third author’s first rather than last name is written in full

These have been addressed

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

2 Dec 2019

**********

**********

**********

**********

**********

**********

3 Dec 2019

REVIEWER #1.

1. Abstract

“Of the 255 suspected cases of cutaneous leishmaniasis identified, females constituted 56% 28 (142/255) and the median age was 7 years (IQR 7-21). Cases were clustered in two locations: Gitare (28.6%, 73/255) and Kambi-Turkana (14%, 36/255) and a continuous transmission 30 pattern throughout the period was depicted”

The authors mentioned 255 cases. Then, they mention 73 case in Gitare and 36 case in Kambi giving a total of 109 case! This is really confusing!

Response: This statement has been revised to read “Cases occurred in clusters with up to 43% of cases originating from Gitare (73/255) and Kambi-Turkana (36/255) villages and a continuous transmission pattern throughout the period was depicted”

2. Methodology

Sample recruiting is very confusing and I was not satisfied with the explanation given to my previous concerns. In line number 141, the authors mentioned 172 cases. This number (172) was never mentioned again!

They recruited 41 cases from previous medical records assuming that those cases are CL. The medical record did not mention the diagnosis clearly and authors inferred from the description of lesions that those patients had CL. If the medical personnel who treated the patients when they visited the hospital could make the diagnosis, how authors could do so! Recruiting samples is very weak and hence the conclusions cannot be reliable, unfortunately.

Response: The section outlining recruitment of cases and controls and sample size estimation has been re-organized for clarity and flow. The sample size was determined by the number of eligible cases present in the study area during the study period. A sample size of 58 patients and 116 controls (2 controls per case) was therefore adopted to give the study at least 80% power at the 5% significance level and able to detect an odds ratio (OR) of 0.3 for an exposure present in 31% of controls. All the 41 cases that were selected from the outbreak line-list were first ascertained to be probable CL on the basis of typical skin lesions (a skin ulcer or a plaque) by experienced medical officers who were part of the study team.

REVIEWER #3.

1. Thank you for the revised revision, however, other comment need to be addressed

In the references section.

a. In the line 477, the reference number should be corrected to be 10 rather than 11

b. In the line 501, the reference number should be corrected to be 19 rather than 15

c. In the line 508, the reference number should be corrected to be 22 rather than 18

Response: The numbering has been corrected in the reference section of the revised manuscript

REVIEWER #3.

The manuscript has improved significantly and the link between the hospital based study and case control study is clearer now. The limitations have also been made more clear. There are still a few things I would like to point out

1. The CL abbreviation is still not done correctly (e.g. line 64, line 70, line 216)

2. Be consistent in formatting e.g. rift valley (92), Rift Valley (85), Sub-county (98) or sub county (100)

3. Typo's : line 98 (the study 2x), line 443-444 ("would be in theory be effective but would not be practical, (102) larva should be 'lava'

Response: These typos have been corrected

4. The statement on line 68-69 is from WHO I think (not from the review so change reference) and is also incorrect, an estimated 2 million new infections with leishmania (including 0.5 mil for VL) occur yearly, of which 1.5 million are for CL

Response: This reference has been corrected

5. The sample size is still not clear to me based on the new version of the manuscript and the reply to the comments. It seems all cases that could be found were included, and then a power calculation was done afterwards, rather then a sample size calculation beforehand, which would have guided when to stop recruiting patients. If this is the case I would phrase it more like below for clarity

"Based on the 58 patients and 116 controls (2 controls per case) , the study had 80% power to detect an odds ratio of 0.3 (using the 5% significance level for mosquito nets which was estimated to be present in 31% of controls."

Response: This section has been re-written for clarity and flow. The sample size was determined by the numbers of eligible cases present in the study area during the study period. A sample size of 58 patients and 116 controls (2 controls per case) was adopted to give the study at least 80% power at the 5% significance level and able to detect an odds ratio (OR) of 0.3 for an exposure present in 31% of controls. The exposure chosen was use of mosquito nets.

All other comments and typographical errors have been corrected

Submitted filename: Response to Reviewers_PLOSONE.doc

Click here for additional data file.

5 Dec 2019

PONE-D-19-19202R2

Burden and risk factors of Cutaneous Leishmaniasis in a peri-urban settlement in Kenya, 2016

PLOS ONE

Dear Dr Ngere,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Jan 19 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Abdallah M. Samy, PhD

Academic Editor

PLOS ONE

Comments:

- The authors should provide the figures in 300 dpi as per Journal regulations; all figures looks unsuitable as it currently stands.

- Table 3: :"Net use by the individual", do you mean the bednet?

- Please carefully revise the language of your manuscript in all sections and tables.

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

17 Dec 2019

- The authors should provide the figures in 300 dpi as per Journal regulations; all figures looks unsuitable as it currently stands.

This has been addressed. The figures have been edited to the right format and uploaded via the PACE digital diagnostic tool for compliance to journal requirements

- Table 3: :"Net use by the individual", do you mean the bednet?

This table has been edited to read ‘individual using bed net’

- Please carefully revise the language of your manuscript in all sections and tables. Other variable names in the table have been revised accordingly

We have revised the language and flow of the entire manuscript, including the tables.

All other comments and typographical errors have also been corrected

Submitted filename: Response to Reviewers_PLOSONE.doc

Click here for additional data file.

27 Dec 2019

Burden and risk factors of cutaneous leishmaniasis in a peri-urban settlement in Kenya, 2016

PONE-D-19-19202R3

Dear Dr. Ngere,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Abdallah M. Samy, PhD

Academic Editor

PLOS ONE

31 Dec 2019

PONE-D-19-19202R3

Burden and risk factors of cutaneous leishmaniasis in a peri-urban settlement in Kenya, 2016

Dear Dr. Ngere:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Abdallah M. Samy

Academic Editor

PLOS ONE

Table 1

Demographic characteristics of suspected cases of cutaneous leishmaniasis based on review of records from nine health facilities in Gilgil, 2010–2016 (n = 255).

Cases by age group	Female	(%)	Male	(%)	Total	(%)
<5 years	51	(60.0)	34	(40.0)	85	(33.3)
5–15 years	38	(43.7)	49	(56.3)	87	(34.1)
16–34 years	27	(61.4)	17	(38.6)	44	(17.3)
35–59 years	9	(81.8)	2	(18.2)	11	(4.3)
60+ years	1	(20.0)	4	(80.0)	5	(2.0)
Age not recorded a	12	(52.2)	11	(47.2)	23	(9.0)
Total	138	(54.1)	117	(45.9)	255	(100.0)
Cases by village of residence
Gitare	36	(49.3)	37	(50.7)	73	(28.6)
Kambi Turkana	21	(58.3)	15	(41.7)	36	(14.1)
Oljorai	9	(50.0)	9	(50.0)	18	(7.1)
Kongasis	11	(68.8)	5	(31.3)	16	(6.3)
Diatomite	8	(66.7)	4	(33.3)	12	(4.7)
Other Villages	59	(57.8)	43	(42.2)	102	(40.0)
Total	142	(55.7)	113	(44.3)	255	(100.0)

Age variable not captured in hospital records

Table 2

Demographic characteristics of cases and controls in Gilgil, Kenya 2016.

Variable	Cases		Controls		p
	(n = 58)	%	(n = 116)	%
Sex
Male	32	55.2	57	49.1	0.451
Female	26	44.8	59	50.9
Age Group
under 5 years	7	12.1	12	10.3
5–15	26	44.8	47	40.5
16–34	6	10.3	24	20.7
35–59	13	22.4	24	20.7
60+	6	10.3	9	7.8	0.553
Level of education
Primary level and below	55	94.8	103	88.8
Secondary level or more	3	5.2	13	11.2	0.194
Occupation
In school	20	34.5	51	44.0
Involving forest visit b	20	34.5	12	10.3
Farming	17	29.3	49	42.2
Other occupation c	1	1.7	4	3.5	0.002

Occupation involving Forest visit- included those who were engaged in charcoal burning, herding, hunting, stone masonry and mining.

Other occupation- Included children out of school, housewives, those engaged in business and casual labourers.

Table 4

Final model showing adjusted odds ratios and 95% confidence intervals for factors related to cutaneous leishmaniasis in Gilgil-Kenya, 2016.

Variable	Adjusted odds Ratio	95% CI	P-value
Occupation involves forest visit	3.4	1.1–10.7	0.038
Living in a house with cracked walls	5.5	1.6–19.3	0.008
Cultivated crop farm near residence	0.1	0.0–0.5	0.007
Presence of a nearby forest or thicket	5.8	1.7–19.6	0.005
Immediate neighbor with ulcerating disease	5.3	1.8–15.7	0.003
Sharing residence with household member with typical skin lesions	26.7	5.2–135.8	<0.001
5 or fewer regular household members	2.8	1.1–7.1	0.029