Incidence and distribution of human leptospirosis in the Western Cape Province, South Africa, (2010-2019): A retrospective study

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16 Leptospirosis is an emerging zoonosis of global importance. In South Africa, the 17 infection is an underreported public health concern, with limited information on its 18 incidence and distribution. This study aimed to determine the incidence of human 19 leptospirosis from 2010 to 2019 in Western Cape Province (WPC), and to compare 20 the incidence based on seasonal and demographic factors. 21

22 A retrospective study was conducted with data on leptospirosis diagnoses by sex, age, 23 season, and year in WCP obtained from the National Health Laboratory Services. With 24 the provincial population sizes as the denominator, the incidence of leptospirosis was 25 estimated and expressed as cases per 100,000 population. Negative binomial 26 regression was used to estimate the effect of sex, season, and year on the incidence 27 of leptospirosis. 28

29 A total of 254 cases of leptospirosis were reported between 2010 and 2019, with the 30 highest number of cases being in 2015. The annual incidence ranged between 0.15 31 and 0.66/100,000 populati on with a 10 -year average incidence of 0.40/100,000 32 population. The incidence was higher among males than in females (0.55 vs. 0.25/ 33 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 3 100,000 population; incidence rate ratio (IRR) 2.2, 95% CI: 1.66,3.03). The 18-44 age 34 cohort and had the highest average in cidence (0.56/100,000 population), while the 35 ≤17age cohort had the lowest incidence (0.07/100,000 population). The 18 -44 (IRR 36 8.0, 95% CI: 4.65,15.15) and ≥45 (IRR 7.4, 95% CI: 4.17,14.17) age cohorts were 37 more at risk of infection compared to ≤17age cohor t. The average incidence of the 38 infection was similar among seasons and there was no significant association between 39 season and incidence of leptospirosis. 40

41 The results highlight that leptospirosis is an important zoonosis within the province 42 disproportionately affecting males and the productive age demographic groups. These 43 findings should enhance targeted prevention and provoke further investigation on the 44 importance of environmental and socioeconomic factors on leptospirosis burden. 45 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 4 Author Summary 46 Leptospirosis is an emerging zoonotic bacterial disease of global importance. Despite 47 its wide distribution, the disease is largely underestimated because its clinical 48 manifestations mimic certain commonly known febrile illnesses such as mal aria, 49 influenza, hepatitis, and yellow fever. Leptospirosis burden in South Africa has been 50 suggested to be moderately high however studies on the burden of the infection are 51 lacking. This study sought to determine the incidence and trends of leptospirosis in the 52 Western Cape Province, South Africa between 2010 and 2019. Overall, leptospirosis 53 average incidence was 0.4 cases per 100,000 population (ranging from 0.15 to 0.66 54 cases per 100,000 population). Leptospirosis incidence was highest among male 55 individuals and among those who were aged 18 -years and above, however, the 56 incidence did not differ by seasons. These estimates highlight that leptospirosis is an 57 important zoonotic disease within the province and potentially disproportionately 58 affecting males and productive age demographic groups. Therefore, this indicates the 59 need for an all-encompassing One Health Approach to obtain all relevant information 60 concerning leptospirosis distribution and risk factors in South Africa and in Africa at 61 large to effectively enhance leptospirosis prevention strategies. 62 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 5 1. Introduction 63 Leptospirosis is an emerging, global zoonosis with an estimated 1.03 million cases 64 and 58,900 deaths occurring annually [1–5]. The disease is caused by pathogenic 65 spirochetes Leptospira species currently reported to comprise 66 different species with 66 more than 300 serovars [6]. The infection in humans is associated with varying clinical 67 manifestations ranging from a mild self -limiting febrile illness to severe illness 68 characterized by dysfunction of multiple organs such as liver, kidneys, lungs, and the 69 brain [7] potentially leading to pulmonary hemorrhagic syndrome and acute kidney 70 injury due to Weil’s disease [5,8–10] . In addition, leptospirosis presents with 71 symptoms that can mimic commonly known infections that cause febrile illnesses such 72 as malaria, influenza, hepatitis, yellow fever, and viral hemorrhagic diseases among 73 others [11,12]. Consequently, the infection ha s often been underdiagnosed and 74 underreported [11]. Despite the life -threatening nature of leptospirosis, there is little 75 published data on morbidity associated with the infection [4,5,13] contributing to its 76 neglected status. 77 The risk of leptospirosis infection among humans occurs either through direct 78 exposure to urine or aborted tissues of wild or domestic reservoir animals such as 79 rodents and livestock/pets [4,14], or through indirect exposure such as contact with 80 contaminated water, soil, and food [3,14]. In South Africa, a recent prison outbreak of 81 leptospirosis was associated with exposure of inmates to rat urine in an overcrowded 82 prison. Due to the predominant modes of transmission, leptospirosis is regarded as 83 an occupational hazard affectin g mainly farmers, sewer workers, veterinarians, and 84 military personnel [11,15–18]. However, the transmission patterns are changing 85 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 6 because there are increasing reports of leptospirosis in the general population. These 86 cases are due to recreational exposu res such as camping, kayaking, adventure 87 travelling, hiking, cave exploration and other activities done in infected water 88 [11,16,18,19]. Due to the diverse avenues through which humans can be exposed to 89 the leptospires, occurrence of leptospirosis within the general population requires 90 consideration [7,20]. 91 Globally, tropical regions as well as resource limited settings are reported to have a 92 higher burden of leptospirosis compared to temperate regions [5]. However, in Africa, 93 few countries have data and reports on human leptospirosis [15,20] and research 94 studies are scarce [2,5]. A previously published systematic review, of peer reviewed 95 studies conducted in Africa, reported a prevalence of acute human leptospirosis 96 ranging from 2.3% to 19.8% among hospital patients presenting with febrile illness and 97 an estimated total of 750,000 cases per annum [15]. It was further concluded in this 98 study that the morbidity of human leptospirosis in Africa is likely to be high relative to 99 other global regions [15]. The need of more studies on the occurrence of leptospirosis 100 in Africa was emphasized in another systematic review by De Vries et.al [20] to reliably 101 understand the extent of the problem. 102 The incidence of human leptospirosis in South Africa has be en suggested to be 103 moderately high within the population [21–23]. Findings about the continued 104 circulation of highly pathogenic Leptospira spp. among rodents suggests that the 105 infection maybe an important underreported public health concern in the country 106 [21,22]. A study conducted between 2009 and 2011 reported a seroprevalence that 107 ranged from 9% to12.5% among all clinical samples sent t o the NHLS for IgM ELISA 108 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 7 testing from all over the country [23] . Environmental conditions in Western Cape South 109 Africa, (the coastal, and temperate conditions with wet winters and warmer summers) 110 have been suggested to favor transmission of pathogenic leptospira species L. 111 borgpetersenii and L. interrogans traditionally associated with rats [4,21,22]. The most 112 recent retrospective study conducted between 2005 -2015, focused on patients 113 presenting at a tertiary referral hospital in Western Cape Province (WCP), and a 114 seroprevalence of 20% was reported [24]. 115 Studies outside Africa have highlighted the influence of season, gender and age on 116 the distribution and occurrence of human leptospirosis [25–27]. Such associations 117 have scarcely been studied in Africa despite changes in climatic factors, demographic 118 shifts, urbanization, and globalization [2,11,20,28–30]. The risk conferred by rodent 119 infestation and overcrowding have been demonstrated in a recent leptospirosis 120 outbreak that occurred in a prison in WCP, South Africa. [22]. Despite this information, 121 studies describing leptospirosis occurrence in relation to demographic and seasonal 122 factors are lacking in WCP. 123 To address these knowledge gaps, a retrospective study on human leptospirosis 124 cases in the WCP, South Africa was conducted using data generated from diagnostic 125 tests done in a public health care setting in Cape Town, South Africa. The study aimed 126 at determining the incidence and trends of human leptospirosis over a 10-year period 127 (2010-2019), with respect to year and seasonality and to describe the demographic 128 characteristics of leptospirosis cases. 129 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 8 2. Methods 130 2.1 Study setting 131 WCP is located on the south -western coast of South Africa and is bordered by the 132 Northern Cape and Eastern Cape Provinces. WCP has a population of approximately 133 seven million inhabitants [31], three quarters of whom use public -sector healthcare 134 services [32]. The province is approximately 129,462 km2 and has a population density 135 of 45 inhabitants per Km 2 [31]. Administratively, the province is divided into one 136 metropolitan municipality (City of Cape Town) and five district municipalities (Central 137 Karoo, Garden Route, Overberg, West Coast and Cape Winelands) [31]. The province 138 has a diverse climate but is dominated by a Mediterranean climate with a cool, wet 139 winter and a warm, dry summer. The inland daily maximum temperatures range from 140 20 ◦C in winter to 32 ◦C in summer and the mean annual rainfall is less than 380 mm. 141 2.2 Study design and study population 142 A retrospective study was conducted by using data provided by the data warehouse 143 of the National Heath Laboratory Services (NHLS), on all human leptospirosis tests 144 conducted at public healthcare facilities in the WCP, South Africa. The NHLS does 145 serological testing on all serum samples of patients who are clinically suspected to be 146 having a leptospirosis infection. Specimens from suspected patients are collected from 147 different public healthcare facilities within the province and sent to a central NHLS 148 laboratory in Cape Town. In this analysis, data included are from patients who had 149 laboratory confirmed leptospirosis between 1st January 2010 and 31st December 2019. 150 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 9 2.3 Data collection 151 Human leptospirosis cases were considered for all ELISA IgM serological tests that 152 were positive for Leptospirosis during the study period (2010 to 2019). ELISA IgM 153 testing is the serological test conducted on a ll samples submitted to NHLS for 154 leptospirosis screening and is sensitive in detecting new onset of illness [11]. Patient 155 data extracted included the patient’s age, sex, year, date of test (year, month, and 156 day) and the name of the health facility that submitted the specimen for testing. 157 The population sizes based on year, sex, and age in WCP for the 10-year period were 158 extracted from the Western Cape Department of Health population circular H102 of 159 2020 [33]. This data was used as the denominator when calculating the incidence of 160 leptospirosis infection. 161 2.4 Statistical analysis 162 All human leptospirosis cases were tabulated according to sex, age, season, and year 163 of occurrence (2010-2019) and their frequency and proportions were calculated. The 164 cases were not grouped based on their geographical location (district municipality) 165 because the patient address information was not part of received data. Furthermore, 166 the location of the health facility that submitted the specimen could not be assumed to 167 be the same as the patient’s location because of the possibility of patients seeking 168 healthcare or being hospitalized in a facility outside of their actual district of residence. 169 The date when the test was done was used to assign the case to a particular year and 170 season. Season was categorized as Summer (1 st December-28/29th February), 171 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 10 Autumn/Fall (1 st March-31st May), Winter (1 st June-31st August), and Spring (1 st 172 September-30th November). The variable year was c onceptualized as a consecutive 173 12-month period from 1st January to 31st December, thus there were ten categories for 174 year (2010 to 2019). 175 The incidence proportion of human leptospirosis for each year (2010 -2019) was 176 estimated and expressed as leptospirosi s cases per 100,000 population. The 177 incidence proportion by sex and age group as well as for each season of the year was 178 also estimated. The Kruskal-Wallis Rank Sum Test was used to compare the average 179 annual incidence between seasons of the year, age grou ps and between years. The 180 Wilcoxon Rank Sum Exact Test was used to compare the average annual incidence 181 between sex demographics. The negative binomial regression was used to estimate 182 the effect of sex, gender, year of occurrence and season of the year on the incidence 183 of human leptospirosis over the study period. This is a suitable regression model to 184 use instead of the traditional Poisson regression in situations where modelling involves 185 a count variable that is over-dispersed (the mean is less than the variance). The results 186 were presented as incidence rate ratios (IRR) with 95% confidence intervals (CI). The 187 cut-off value for statistical significance was 0.05. All statistical analysis was conducted 188 using R software version 1.2.5033. 189 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 11 2.5 Ethical considerations 190 Ethical approval was obtained from the University of Cape Town Human Research 191 Ethics Committee (UCT -HREC) (reference number: HREC REF: 303/2021). The 192 Western Cape Provincial approval was granted by Groote Schuur Hospital Research 193 Ethics Committee. The approval to use data from NHLS database was given by the 194 NHLS Academic Affairs and Research office (reference number: PR2118953). 195 3. Results 196 3.1 Distribution of leptospirosis cases in WCP, 2010-2019 197 During the 10 -year study period (2010-2019), a total of 254 cases of human 198 leptospirosis were recorded by the National Health Laboratory Services (NHLS) 199 (Table 1Error! Reference source not found. ) for persons in the Western Cape 200 Province, South Africa. Most of the cases reported had their specimen submitted from 201 healthcare facilities located within the City of Cape Town Metropolitan (S2 Table 1). 202 The highest number of cases was recorded in 2015 (42 cases, 16.5%) and lowest in 203 2012 (9 cases, 3.5%). When grouped by month of occurrence, the month of March (30 204 cases) had the highest number of cases over the 10-year period while September (16 205 cases) had the lowest number of the cases (Fig 1). When grouped into seasons, more 206 cases were observed in fall season (75 cases), followed by summer, spring, and winter 207 (64, 62, 53 cases respectively) (Table 1). More males were infected than females, with 208 the males accounting for 173 (68.1%) cases compared to 81 (31.9%) cases who were 209 females, and the overall male to female ratio was 2.136:1 (Table 1). The median age 210 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 12 of the observed cases was 37.0 (Inter Quantile Range (IQR) 28.0 -48.0) years, with a 211 mean age of 38.12 years. Overall, 163 cases (64.2 %) were in the 18–44-year-old age 212 group, 78 cases (30.7%) were in ≥ 45-year-old age group while 13 cases (5.1%) were 213 in the ≤17-year-old age group over the 10-year study period (Table 1). 214 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 13 Table 1: Distribution of leptospirosis cases by age, sex, season, and year of 215 occurrence in Western Cape Province South Africa (2010-2019) 216 217 Variables All Cases (n=254) n (%) Age (years) median (IQR) 37.0 (28.0,48.0) Age group ≤17 13 (5.1) 18-44 163 (64.2) ≥ 45 78 (30.7) Sex Male 173 (68.1) Female 81 (31.9) Season of the Year Summer 64 (25.2) Fall 75 (29.5) Winter 53 (20.9) Spring 62 (24.4) Year of occurrence 2010 34 (13.4) 2011 19 (7.5) 2012 9 (3.5) 2013 12 (4.7) 2014 28 (11.0) 2015 42 (16.5) 2016 37 (14.6) 2017 22 (8.7) 2018 31 (12.2) 2019 20 (7.9) IQR: Inter Quantile Range; n: frequency. 218 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 14 Fig 1: Monthly distribution of leptospirosis cases in Western Cape Province over 219 a 10-year period (2010-2019) 220 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 15 3.2 Incidence of leptospirosis in the WCP, 2010-2019 221 The incidence of leptospirosis fluctuated widely across the 10 years. The annual 222 incidence ranged from 0.15 to 0.66 cases per 100,000 population, with an average 223 annual incidence of 0.40 cases per 100,000 population. The annual number of cases 224 and the incidence are plotted against the year of occurrence to show the trends across 225 the years (Fig 2). The incidence followed a downward trend between 2010 (0.59 cases 226 per 100,000 population) and 2012 (0.15 cases per 100,000 population). However, 227 there was an increase in the incidence from 2013 (0.20 cases per 100,000 population) 228 which peaked in 2015 (0.66 cases per 100,000 population). This was followed by a 229 gradual decrease in the incidence between 2015 and 2019 (0.66 -0.27 cases per 230 100,000 population) except for year 2018 where the annual incidence was higher than 231 the one observed in 2017. 232 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 16 Fig 2: The number of cases and incidence of leptospirosis per year (2010 to 233 2019) in Western Cape Province, South Africa. 234 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 17 Throughout the study period, the incidence was highest among males compared to 235 females ( Fig 3), with the average incidence among males being 0.55 cases per 236 100,000 population and 0.25 cases per 100,000 population among females (p -237 values=0.004). During the entire study period, the incidence was highest in the 18-44-238 year-old and ≥ 45 years-old age groups compared to the ≤17-year-old age group (Fig 239 4 and Error! Reference source not found. ). The average incidence differed 240 substantially between the age groups with the highest average incidence being among 241 the 18-44-year-old age group (0.56 cases per 100,000 population) over the 10 -year 242 period, followed by ≥ 45 years-old age group (0.49 cases per 100,000 population) and 243 the ≤17 -year-old age group had the low est incidence (0.07 cases per 100,000 244 population) (Error! Reference source not found.). 245 The results indicate that on average, the annual incidence was higher during th e fall 246 season (0.12 cases per 100,000 population) and lowest in the winter season (0.08 247 cases per 100,000 population) over the 10 -year period ( Table 2). However, the 248 observed difference in the average incidence of leptospirosis between the seasons of 249 the year was not statistically significant. 250 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 18 Fig 3: Incidence of leptospirosis in males and females from 2010 to 2019 in 251 Western Cape Province. 252 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 19 Fig 4: Incidence of leptospirosis by age group from 2010 to 2019 in Western 253 Cape Province, South Africa. 254 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 20 Table 2: Seasonal incidence of leptospirosis for the years 2010-2019, in Western 255 Cape Province, South Africa. 256 Year Summer Fall Winter Spring IP/100,000 (95% CI) IP/100,000 (95% CI) IP/100,000 (95% CI) IP/100,000 (95% CI) 2010 0.10 (0.02,0.19) 0.28 (0.14,0.41) 0.12 (0.03,0.21) 0.09(0.01,0.16) 2011 0.12 (0.03,0.21) 0.03 (-0.01,0.08) 0.07 (0.001,0.13) 0.10 (0.02,0.18) 2012 0.07 (0.001,0.13) 0.03 (-0.01,0.08) 0.02 (-0.02,0.05) 0.03 (-0.01,0.08) 2013 0.05 (-0.01,0.10) 0.05 (-0.01,0.10) 0.07 (0.001,0.13) 0.03 (-0.01,0.08) 2014 0.02 (-0.01,0.05) 0.10 (0.02,0.17) 0.14 (0.05,0.24) 0.19 (0.08,0.30) 2015 0.20 (0.09,0.31) 0.17 (0.07,0.27) 0.16 (0.06,0.25) 0.13 (0.04,0.21) 2016 0.18 (0.08,0.29) 0.21 (0.10,0.33) 0.03 (-0.01,0.07) 0.14 (0.05,0.23) 2017 0.12 (0.03,0.18) 0.09 (0.02,0.16) 0.08 (0.01,0.14) 0.06 (0.001,0.12) 2018 0.12 (0.04,0.20) 0.15 (0.06,0.24) 0.09 (0.02,0.16) 0.10 (0.03,0.18) 2019 0.04(-0.01,0.09) 0.07 (0.01,0.14) 0.07 (0.01,0.14) 0.10 (0.03,0.18) Average incidence 0.10 (0.03,0.18) 0.12 (0.04,0.20) 0.08 (0.02,0.15) 0.10 (0.02,0.17) IP: incidence proportion; CI: confidence interval 257 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 21 3.3 Effect of sex, age, year, and season on leptospirosis 258 incidence in WCP, 2010-2019 259 Table 3 shows the results from the Multivariable Negative Binomial Regression 260 (MNBR) model that was run to identify factors associated with the incidence of 261 leptospirosis during the 10-year period. The incidence rate of leptospirosis among the 262 male demographic was 2.2 (95% CI: 1.66,3.03) times higher than among females 263 during the entire study period. The model results indicated that on average the 264 incidence rate of lepto spirosis among 18 -44 and ≥ 45 age groups was 8.0 (95% CI: 265 4.65,15.15) and 7.4 (95% CI: 4.17,14.17) times higher than the incidence rate among 266 ≤17 years age group. The incidence was substantially lower in 2012 (IRR 0.25; 95% 267 CI 0.11-0.54), 2013 (IRR 0.34; 9 5% CI 0.16 -0.68), 2017 (IRR 0.53; 0.28 -0.98) and 268 2019 (IRR 0.47; 95% CI 0.24-0.87) compared to the incidence in 2010. From the model 269 results, the incidence rate in fall, summer and spring seasons was 1.4 (95% CI: 270 0.95,2.15), 1.2 (95% CI: 0.78,1.81) and 1.2 (95% CI: 0.77,1.78) times higher than what 271 was observed in winter season, however the observed difference was not statistically 272 significant. 273 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 22 Table 3: MNBR analysis results between year, season, sex, and age group with 274 incidence of leptospirosis 275 Variables IRR 95% CI P.value Year of occurrence 2010 Ref _ _ 2011 0.543 (0.281,1.026) 0.061 2012 0.25 (0.106,0.536) 0.001 2013 0.337 (0.157,0.684) 0.003 2014 0.73 (0.402,1.314) 0.294 2015 1.033 (0.597,1.796) 0.907 2016 0.956 (0.546,1.677) 0.874 2017 0.53 (0.282,0.98) 0.046 2018 0.748 (0.418,1.334) 0.323 2019 0.466 0.244,0.872 0.019 Season of the year Winter Ref _ _ Spring 1.166 (0.765,1.782) 0.475 Summer 1.184 (0.778,1.808) 0.431 Fall/autumn 1.422 0.947,2.147 0.091 Sex Female Ref _ _ Male 2.239 (1.664,3.034) <0.001 Age group ≤17 Ref _ _ 18-44 8.045 (4.654,15.147) <0.001 ≥45 7.391 (4.167,14.169) <0.001 IRR: incidence rate ratio; CI: confidence interval; ref: reference category; MNBR: 276 Multivariable Negative Binomial Regression. 277 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 23 4. Discussion 278 This study provides a first description of the incidence of human leptospirosis in the 279 Western Cape Province (WCP), South Africa across a 10-year period. Here we report 280 an average incidence of 0.40 leptospirosis cases per 100,000 population, ranging from 281 0.15 in 2012 to 0.66 in 2015 cases per 100,000. There was no overall increase in the 282 annual incidence of leptospirosis, however the incidence fluctuated widely across the 283 10-year period. The incidence was significantly higher in males compared to female. 284 The 18 -44-year-old and ≥ 45 years -old age groups had a higher incide nce of the 285 infection compared to the ≤17 -year-old age group. The incidence did not vary based 286 on season over the 10-year period. 287 The annual incidence observed during this 10-year period lies within the range of the 288 incidence that has been reported globally to be occurring in temperate regions (0.1-1 289 cases per 100,000 population) [34]. The cold wet winters and warm dry summers 290 might allow transmission and survival of pathogenic Leptospira species [21]. WCP is 291 known for its harsh drought, and different ty pes of floods such as river floods, flash 292 floods and these occur in each year at varying intensities [35]. Annual fluctuation in 293 such environmental conditions could partly explain the observed wide fluctuation in 294 the annual incidence of leptospirosis bec ause increase in incidence is normally 295 reported after occurrence of extreme weather events such as heavy rains and high 296 temperatures [29,34,36]. In addition, rodent infestation, increased prevalence of 297 infected rodents and overcrowded settings were repor ted as risk factors for human 298 leptospirosis outbreak in a one of the prisons in WCP in 2015 [22]. This outbreak could 299 have contributed to the observed increase in the incidence in 2015. In a study 300 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 24 conducted after this outbreak, Naidoo et al. further highlighted the importance of 301 continued circulation of pathogenic leptospira spp among rodents in maintaining the 302 status of leptospirosis transmission particularly in informal settlements [22]. 303 Previous studies have indicated a significant relationship between leptospirosis 304 incidence and seasonality [34,36,37]. In Western Cape, seasonality was not 305 associated with the incidence of leptospirosis between 2010 and 2019, however the 306 incidence was slightly higher in fall and summer as compared to winter and spring. 307 This is consistent with some reports from other countries in temperate regions [34,38]. 308 Studies on correlation between leptospirosis incidence and seasonality require 309 consideration of other var iables such as rodent seasonal population, temperature, 310 rainfall, and other climate related parameters to be included in the analysis [37]. This 311 could help in understanding the importance of the interaction of different environmental 312 and climatic factors in determining the incidence of leptospirosis in resource limited 313 settings. 314 The results showed that the overall incidence was significantly higher in males as 315 compared to females (0.55 cases vs 0.25 cases per 100,000 population) during the 316 study period. S imilar results have been reported in both seroprevalence studies in 317 Africa and in incidence studies conducted using surveillance data globally 318 [26,27,34,38,39]. The higher incidence among males has been largely attributed to 319 occupational or environmental exposure, whereby males engage in activities that may 320 put them at higher risk of contracting the infection [36,40]. Epidemiological studies 321 collecting data on occupation, underlying medical conditions, and place of settlement 322 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 25 could aid in categorically establishing risk groups hence guiding policies on targeted 323 prevention of leptospirosis. 324 The incidence of leptospirosis was highest among those within the 18 -44 and ≥ 45 -325 year-old age groups compared to those below ≤17 years of age. These findings are in 326 line with a systematic review by Costa et al. that reported the highest incidence to be 327 occurring among adult males aged 20 -49 years [5]. The observed results also 328 correspond with other studies, where incidence has been shown to be higher among 329 the active adult population [34,38]. People belonging to the 18-44 and ≥ 45 age groups 330 would theoretically have an increased environmental exposure compared to those 331 belonging to ≤17 age group, thus increasing their risk of being infected. 332 The data presented h ere are the first results to inform on the incidence trends and 333 distribution of leptospirosis in WCP. The data covers a 10 -year period, hence could 334 help to guide designing of preventive strategies. However, there are some study 335

that need to be considered. Firstly, the study utilized data collected from 336 passive surveillance and from a public health laboratory setting excluding data from 337 private laboratories, hence there is a great potential for underestimation of the real 338 occurrence of leptospiro sis in WCP. Some bias may still be inherent in the analysis 339 due to ambiguity in clinical presentation; the latter contributes to underestimation of 340 actual incidence. Secondly, the analysis does not provide information on the 341 geographical distribution of the cases within the province as such information was not 342 included in provided data. Consideration of the geographical distribution of the 343 incidence would have given insights into which specific districts are at risk, hence 344 informing targeted interventions. 345 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 26 Findings from this analysis highlight the continued circulation of leptospirosis infection 346 within WCP with age and sex being significant risk factors for infection. However, there 347 is need of a good knowledge about the epidemiology of leptospirosis (such a s the 348 geographical and seasonal patterns, the specific risk populations, circulating 349 Leptospira strains and the importance of reservoir animals) [13] within the province to 350 improve prevention strategies, prediction, and detection of leptospirosis burden and 351 outbreaks. For instance, future studies focusing on the impact of heavy rainfall, floods, 352 seasonal fluctuation in weather-related factors, occupation, geographical location, and 353 rodent dynamic parameters on leptospirosis incidence could help address th e 354 knowledge gaps on the actual burden of this emerging zoonosis. 355 In conclusion, the incidence of leptospirosis in WCP fluctuated within the range that 356 has been reported to be occurring in temperate regions. The incidence was strongly 357 related to sex and age; however, it did not differ across the seasons of the year. These 358

show that leptospirosis is an important zoonosis within the province and 359 potentially disproportionately affecting males and the productive age demographic 360 groups. The findings, t herefore, can guide targeted intervention strategies within the 361 province to decrease the burden of human leptospirosis. 362 . CC-BY-NC 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.22268774doi: medRxiv preprint 27 5. Acknowledgement 363 Special thanks to the National Health Laboratory Services (NHLS), South Africa for 364 providing all the necessary data on leptospirosis tests conducted in Western Cape 365 Province. 366 . 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