Epidemiology of leptospirosis in Tanzania: A review of the current status, serogroup diversity and reservoirs.

BACKGROUND: Tanzania is among the tropical countries of Sub-Saharan Africa with the environmental conditions favorable for transmission of Leptospira. Leptospirosis is a neglected zoonotic disease, and although there are several published reports from Tanzania, the epidemiology, genetic diversity of Leptospira and its host range are poorly understood.
METHODS: We conducted a comprehensive review of human and animal leptospirosis within the 26 regions of the Tanzanian mainland. Literature searches for the review were conducted in PubMed and Google Scholar. We further manually identified studies from reference lists among retrieved studies from the preliminary search.
RESULTS: We identified thirty-four studies describing leptospirosis in humans (n = 16), animals (n = 14) and in both (n = 4). The number of studies varied significantly across regions. Most of the studies were conducted in Morogoro (n = 16) followed by Kilimanjaro (n = 9) and Tanga (n = 5). There were a range of study designs with cross-sectional prevalence studies (n = 18), studies on leptospirosis in febrile patients (n = 13), a case control study in cattle (n = 1) and studies identifying novel serovars (n = 2). The most utilized diagnostic tool was the microscopic agglutination test (MAT) which detected antibodies to 17 Leptospira serogroups in humans and animals. The Leptospira serogroups with the most diverse hosts were Icterohaemorrhagiae (n = 11), Grippotyphosa (n = 10), Sejroe (n = 10), Pomona (n = 9) and Ballum (n = 8). The reported prevalence of Leptospira antibodies in humans ranged from 0.3-29.9% and risk factors were associated with occupational animal contact. Many potential reservoir hosts were identified with the most common being rodents and cattle.
CONCLUSION: Leptospirosis is prevalent in humans and animals in Tanzania, although there is regional and host variation in the reports. Many regions do not have information about the disease in either humans or their animal reservoirs. More studies are required to understand human leptospirosis determinants and the role of livestock in leptospirosis transmission to humans for the development of appropriate control strategies.

Introduction

Leptospirosis is a serious infectious disease caused by spirochete bacteria in the genus Leptospira [1]. It is considered a re-emerging zoonosis widespread in tropical and sub-tropical regions, where there are limited surveillance and disease control measures [2]. Leptospirosis infections may be acute, subacute or chronic [1] and may result in severe health problems such as pulmonary haemorrhagic syndrome (PHS) [3], or renal and liver dysfunctions [2,4,5]. Leptospirosis often presents with varied symptoms that mimic those of several other unrelated febrile illnesses including dengue and malaria [6]. Therefore, leptospirosis is an important undifferentiated febrile illness that requires differential diagnosis [7].

The incidence of leptospirosis is poorly known and this may be partially attributed to inadequate data and surveillance [8]. In addition, there is a shortage of appropriate diagnostic facilities in developing countries, and clinicians may fail to recognize leptospirosis in febrile patients, consequently it remains underreported [2]. However, it is estimated that around the globe there are 1.03 million leptospirosis cases annually and 2.9 million Disability Adjusted Life Years (DALYs), where the majority of infections and burden are in low and middle-income countries (LMICs) [4,9].

Leptospires are mainly harboured in the renal tubule and excreted in the urine of accidental and maintenance hosts including cattle, rodents, pigs, dogs, sheep and goats [1,10]. Humans contract leptospirosis from contaminated environments, consumption or handling waste products from infected animals [1,11]. More than 250 serovars have been serotyped into 31 serogroups which can potentially cause leptospirosis in humans and animals worldwide [12,13]. Based on DNA hybridization techniques and phylogenetic analysis, 64 species have been recognized and rearranged into two clades (pathogenic “P” and saprophytic “S”) and two subclades in each clade (subclade P1 and P2 and subclades S1 and S2) [14]. There are 17 species classified in subclade P1 of which 8 can cause severe disease in humans and 21 species in subclade P2 that can cause mild disease, and the remaining species, considered non-pathogenic, are in clade S subclade S1 and S2 [14].

Leptospirosis in Tanzania was reported in the early 1990s [15]. The authors of that study aimed to determine seroprevalence in humans, domestic and wild animals based on the microscopic agglutination test (MAT). The seroprevalence of Leptospira antibodies was reported as 38% in dogs, 5.6% in cattle, 1.8% in rodents and 0.3% in humans [15]. Despite the low prevalence of Leptospira antibodies in humans, it was sufficient to indicate a public health concern and the need for control and prevention strategies. Several studies have been conducted since and leptospirosis has been reported in a range of species [11,16-18]. Two recent investigations estimated human leptospirosis incidence in Tanzania. The study populations involved were hospitalized patients with fever related symptoms. The disease incidence was estimated by the two studies to be 75-102/100,000 persons annually in 2007–2008 [19] and 11-18/100,000 persons annually in 2012–2014 [20]. Humans are at high risk of contracting leptospirosis based on the fact that multiple animal species harbour and transmit the disease including livestock and wildlife [11,18,21]. Although three decades have elapsed since the first detection of leptospirosis in Tanzania the epidemiology and the diversity of leptospiral serovars and their reservoirs are not well articulated. This review comprehensively examined the disease epidemiology and Leptospira diversity in Tanzania to inform stakeholders of any existing knowledge gaps and for appropriate management of the disease.

Methods

Search strategy

A thorough and comprehensive search of the literature was carried out to identify studies associated with human, domestic or wild animal leptospirosis and Leptospira in Tanzania. To retrieve all related information, a boolean operator (“OR” and “AND”) with a combination of keywords was set and both PubMed and Google Scholar electronic search engines were used to retrieve published papers, peer-reviewed articles, theses, case reports, posters and conference presentations. Retrieval of materials from PubMed and Google search engine was done on 24th May 2020. In the PubMed search engine search terms were: (‘human’ OR ‘people’ OR ‘domestic animals’ OR ‘bovine’ OR ‘cattle’ OR ‘pigs’ OR ‘porcine’ OR ‘rodent’ OR ‘rat’ OR ‘dogs’ OR ‘canine’ OR wildlife’ OR ‘wild animals’) AND (“leptospirosis” OR ‘Leptospira’ OR ‘Weils disease’ OR ‘Weils syndrome’ OR ‘Leptospira serovars’ OR ‘sokoine serovar’ OR ‘interrogans serovar’ OR ‘Icterohaemorrhagiae serovar’ OR ‘Hebdomadis serovar’) AND (‘Tanzania’ OR ‘Northern zone’ OR ‘Kilimanjaro’ OR ‘Morogoro’ OR ‘Rukwa’ OR ‘Katavi’ OR ‘Tanga’ OR ‘Kagera’ OR ‘Simiyu’ OR ‘Mara’ OR ‘Geita’ OR ‘Shinyanga’ OR ‘Songwe’ OR ‘Moshi’) AND (‘prevalence’ OR ‘epidemiology’ OR ‘risk factors’ OR ‘febrile illness’ OR ‘acute leptospirosis’); while in Google scholar ((‘Leptospira’ OR ‘leptospirosis’) AND Tanzania)) were the key search terms used.

Study selection

The search returned a large number of publications, and the contents were collated in Mendeley citation manager version 1.19.4. Additional papers were identified from reference lists of retrieved articles to find appropriate studies that might not have been identified during the preliminary search. All papers were checked for duplicates and removed in Mendeley software. In the subsequent stage, those papers remaining after cleaning were then screened dependent on their titles and relevant geographical study location. Consequently, the full content of those papers was further assessed as far as their significance and by considering the inclusion and exclusion criteria.

Criteria for study eligibility

Inclusion and exclusion criteria

In this review, all publications including published papers, theses, poster or conference presentations were included if the source contained primary data citing leptospirosis/ febrile illness in humans, domestic or wild animals. Theses and poster presentations were excluded if the data had been published in another peer-review journal. All texts written in English and focused on Tanzania as the geographical area of attention were eligible.

Results

At a preliminary search, a total of 3767 documents were retrieved from two database search engines and pooled into the Mendeley citation manager. Of those articles, 3720 were recovered from Google Scholar and 47 from PubMed. A further 13 papers were searched and added manually after being identified from reference lists among the retrieved articles to make 3780 papers in total. Then articles were checked for duplicates in Mendeley, 3465 articles remained and met the criteria for the initial stage of inclusion and exclusion after duplicate removal. The initial screening was based on the title of the article and relevant study location (i.e. Tanzania), 3395 articles were excluded in the review process due to failure to fulfil the inclusion criteria for the next stage of assessment. A large number of articles recovered from Google scholar were excluded as they did not report leptospirosis in Tanzania. These articles were detected by the search engine because Tanzania was mentioned in the text of the paper as the author had referenced a previous publication. The publications were most often reporting leptospirosis in another country. After the selected literature underwent full text screening, 32 published papers were identified with primary data describing Leptospira and leptospirosis from Tanzania in humans and various animal species. In addition, two papers were identified, which were published after the initial retrieval was conducted, and these have been included in the review [22,23]. The flow diagram Fig 1 describes the process of identifying studies for this review. A summary of each study is available in S1 Table including the year of research, study design, geographical location, target populations, diagnostics tests, and results for each study (n = 34).

Fig 1

Flow diagram indicating how articles were included in the review regarding leptospirosis in Tanzania.

Of the 34 studies identified, sixteen described Leptospira seropositivity or leptospirosis in humans, fourteen investigated animals and four focused on both humans and animals S1 Table. There was a range of study designs with more than fifty per cent of studies being prevalence studies (n = 18). Over thirty percent were targeted studies investigating Leptospira as a cause of illness in febrile patients (n = 13) or disease in animals (n = 1) and a small number identified novel serovars (n = 2).

Geographic distribution of Leptospira studies

The Tanzanian mainland comprises 26 regions that are divided into 6 zones which are as follows: Lake Zone (Mwanza, Kagera, Shinyanga, Geita, Mara and Simiyu), Western Zone (Katavi and Kigoma), Southern Highland Zone (Songwe, Rukwa, Ruvuma, Mbeya, Iringa and Njombe), Eastern Zone (Morogoro, Pwani, Dar es Salaam, Lindi and Mtwara), Central Zone (Dodoma, Singida and Tabora) and Northern Zone (Kilimanjaro, Manyara, Tanga and Arusha). The geographical distributions of the recovered studies are shown in Fig 2.

Fig 2

Geographical distribution of Leptospira studies reported from human, domestic and wild animals: Regions colored pink indicate areas with Leptospira studies from 1990s to date and regions colored blue indicate regions where no study was retrieved from the search engine.

This map was prepared using Simplemaps https://simplemaps.com/resources/svg-tz.

There was an unequal distribution in the Leptospira studies conducted across the country. Human or animal related studies were only conducted in 10 (38.5%) out of 26 regions of the Tanzanian mainland. The majority of the studies reporting Leptospira or leptospirosis were from Morogoro region (n = 16) followed by Kilimanjaro (n = 9) and Tanga (n = 5). Additional studies were conducted in Dar es Salaam (n = 3), Katavi (n = 2), Mwanza regions (n = 2), Kagera (n = 1), Arusha (n = 1), Singida (n = 1) and Mbeya (n = 1) Fig 2. Only one study was conducted in multiple regions [15]. In some regions such as Mbeya and Singida the research was conducted many years ago at the onset of the disease identification in the country.

Studies from Morogoro region (n = 16) were mostly cross-sectional studies in animals (n = 8), or humans and animals (n = 2) and among these the animals studied were: rodents, shrews, cattle, goats, sheep, pigs, dogs, cats, fish and bats. The other studies from Morogoro described leptospirosis in hospital patients (n = 4) or new serovars (n = 2). On the other hand, studies conducted in the Kilimanjaro (n = 9) region were hospital-based studies describing leptospirosis in humans (n = 7). There was one cross-sectional study in humans and animals (n = 1) and one study focused only on animals with the target animals being cattle, goats, sheep and rodents. Among the five studies in Tanga, there were four cross-sectional studies, including two animal studies, one human study, one study in both humans and animals, and one targeted study investigated clinical disease in animals. Of the studies in Dar es Salaam two were hospital based and one was a cross sectional study of animals and humans. The study in Arusha was hospital based and the studies in the remaining regions were cross sectional in humans (Katavi and Mwanza) and in both humans and animals (Kagera, Mbeya, Katavi, Mwanza and Singida).

Diagnostic approaches for detecting Leptospira or antibodies to Leptospira

Various diagnostic methods for leptospirosis were identified during the review S1 Table. These diagnostic techniques include microscopic agglutination test (MAT) (n = 28), culture and isolation (n = 7), cross agglutinin absorption test (CAAT)(n = 2), Eiken latex agglutination test (n = 1), enzyme linked immunosorbent assay (ELISA) (n = 1) and polymerase chain reaction (PCR) (n = 9). Despite the advancement of diagnostic technology, currently few studies use molecular typing [10,22,24] for characterising Leptospira sp. Most of the studies (n = 22) employed a single technique for leptospirosis detection. Microscopic agglutination test (MAT) was broadly utilized in 85% of the studies (n = 28) for leptospirosis diagnosis and in nine of these studies it was utilized in combination with other methods such as ELISA, culture, or PCR S1 Table. Recent studies used advanced diagnostics methods including either polymerase chain reaction, molecular typing or in combination (n = 9). For PCR, the studies used a variety of tissues such as kidney, culture isolate and blood sample for detection and the assays had different gene targets [10,22,23,25,26].

Leptospiral serogroups used in studies that utilized MAT

The studies utilizing the MAT test for detection of antibodies to Leptospira included a wide range of Leptospira serogroups Fig 3. In general, human studies tended to use a wider range of serogroups compared to studies from animals Fig 3 [20,27-29]. Serogroups commonly used in human studies included: Australis (n = 7), Ballum (n = 9), Canicola (n = 4), Grippotyphosa (n = 9), Hebdomadis (n = 7), Icterohaemorrhagiae (n = 10), Pomona (n = 6), Sejroe (n = 7), and Tarassovi (n = 4). Leptospira serogroup panels which have been widely used for animal studies have included: Australis (n = 7), Ballum (n = 12), Canicola (n = 7), Grippotyphosa (n = 7), Hebdomadis (n = 8), Icterohaemorrhagiae (n = 14), Pomona (n = 12) and Sejroe (n = 9). The serogroups investigated for each animal group were not always detected as indicated in Fig 3 and S1 Table.

Fig 3

Serogroups used in the Microscopic Agglutination Test (MAT) for detection of antibodies to Leptospira in humans and animals in Tanzania (1997–2019).

The coloured box indicates that samples were screened for these serogroups and the black outline indicates that the serogroup was detected (n = 28).

Predominant Leptospira serogroups detected in human and animals

Thirty (n = 30) studies were able to report and describe serogroup diversity out of those studies using MAT, CAAT and molecular typing diagnostic approaches. The review found 17 Leptospira serogroups reported from humans and across animal species in Tanzania. In the case of humans, the most detected serogroups were Icterohaemorrhagiae (n = 11), Grippotyphosa (n = 8), Australis (n = 8), Ballum (n = 7), Hebdomadis (n = 6) and Sejroe (n = 6). We only counted the MAT serogroup once for samples that were used by multiple studies [19-21,27,30]. The most prevalent serogroups in people were Sejroe, Icterohaemorrhagiae and Australis Tables 1 and S2. The serogroups detected in the highest proportion of hospital patients were Australis, Icterhaemorrhagiae and Djasiman Tables 1 and S2.

Table 1

Mean prevalence of antibodies to Leptospira serogroups in people in cross-sectional studies; in febrile patients; in rodents; in cattle in Tanzania in leptospirosis papers published 1997–2021.

Study type, number and references	Seroprevalence (%)
	Australis	Ballum	Djasiman	Grippotyphosa	Hebdomadis	Icterohaemorrhagiae	Sejroe	Tarassovi
Cross-sectional studies in people (n = 5) [15,26,31–33]	3.43	0.48	NT	1.58	1.50	5.38	9.35	1.00
Hospital based studies in febrile patients (n = 4) [21,27,29,34]	20.48	5.78	16.20	8.20	6.13	17.45	4.18	7.4
Cross sectional studies in rodents (n = 7) [11,15,31,35–38]	8.38	1.47	NT	2.07	0.28	7.29	0.37	NT
Cross sectional studies in cattle (n = 6) [11,15,39–41]	0.80	0.00	NT	4.80	5.10	4.25	15.94	15.10

NT—Not tested

The most predominant Leptospira serogroups being reported in different animals were Icterohaemorrhagiae in 11 different animals (cattle, rodents, shrew, dogs, goat, sheep, bats, buffalo, pigs, cats and fish), Grippotyphosa and Sejroe in 10 animals (cattle, rodents, shrew, dogs, goat, sheep, buffalo, lion, cats, pigs), Pomona in 9 animals (cattle, rodents, shrew, dogs, goat, sheep, pigs, cats and fish), and Ballum in 8 animals (rodents, dogs, goats, sheep, bats, pigs, cats, and fish). The study carried out in wildlife found zero leptospiral antibodies in zebras which may be due to the small number of samples tested [11]. The most prevalent serogroups in rodents were Australis and Icterohaemorrhagiae and in cattle the most prevalent serogroups were Sejroe and Tarassovi Table 1.

Leptospirosis and prevalence in humans

Leptospirosis in humans was reported by 20 eligible studies from 10 regions of Tanzania. Six of these studies were cross sectional studies investigating seroprevalence in the general population Table 2. The findings from two papers which conducted studies on people in Katavi from 2012–2014 are reported once [11,26]. From 1997–2019, a total of 209 out of 1546 tested samples were seropositive for antibodies against Leptospira spp. serogroups Table 2. The prevalence of antibodies to Leptospira varied depending on the study area, study design and interpretation of the results from 0.3% to 29.9%. Risk factors identified from 5 studies include occupational exposures such as contact with animals, animal waste and animal products Table 2.

Table 2

Summary of studies reporting leptospirosis and seroprevalence of antibodies to Leptospira in humans in Tanzania 1997–2019.

Reference	Year	Study area	N	Seroprevalence (%)	Acute leptospirosis (%)	Risk factors/exposure
[15]	1996	Morogoro, Dar es Salaam, Mbeya, Kilimanjaro, Tanga, Singida and Mwanza	375	0.3	ND	ND
[33]	2005	Tanga	199	15.1	ND	ND
[11]*[26]	2012–2013	Katavi	267	29.96	ND	Slaughtering and handling of bush meat
[32]	2017	Mwanza	250	10	ND	Abattoir workers and meat vendors
[31]	No date	Kagera	455	15.8	ND	Fishing and working in sugarcane plantation
[18]	1996–2006	Morogoro	506	ND	0.2 Patients	ND
			83	ND	3.6 Abattoir workers	ND
[30]	2007–2008	Kilimanjaro	831	ND	8.4	ND
[42]	2008	Dar es Salaam	1005	ND	0.47	ND
[34]	2013	Morogoro	370	ND	11.6	Heavy rain and presence of rodents in residential areas
[43]	2014	Morogoro	191	ND	2	ND
[23]	2013–2014	Dar es Salaam	519	ND	0.2	ND
[25]	2014	Morogoro	842	ND	3	ND
[21]	2012–2014	Kilimanjaro	1293	ND	1.9	Cleaning animal waste and rice farming
[29]	2016–2017	Arusha	104	ND	5.8	ND

ND: not described

*Studies used the same data

There were also 14 hospital-based studies examining acute leptospirosis. Seven papers reported findings from the same patients in Kilimanjaro from 2007–2008 and/or 2013–2014 [10,19-21,27,28,30]. We have only reported the acute cases from these seven studies as defined by the authors and reported in 2 papers [21,27]. From 1997–2019 there were 173 acute cases of leptospirosis identified from 5661 febrile patients. Additionally, one study reported Leptospira in the urine of abattoir workers (3/83) which is not included in this number [18].

Leptospirosis incidence was estimated by two systematic hospital based and health care utilization surveys from the Kilimanjaro region. There was a large difference in the incidence estimations between the two studies. One study was conducted between 2007–2008 with the calculated incidence of acute leptospirosis ranging from 75–102 per 100,000 people annually [19]. The other study reported a lower leptospirosis incidence of 11–18 cases per 100,000 people annually from 2012–2014 [20].

Animal leptospirosis and prevalence

Several leptospirosis studies have been carried out in various animal species in Tanzania, and in this review, a total of 18 studies met the inclusion criteria and were examined, 15 were cross sectional prevalence studies Table 3, 1 case control study [39] and 2 identified new serovars [44,45]. The total number of animals tested was 9090, though there were variations in the sample size and species between regions. The animals investigated were rodents (n = 10), shrews (n = 7), cattle (n = 8), goats (n = 3), pigs (n = 2), dogs (n = 2), bats (n = 2), sheep (n = 1), fish (n = 1), buffaloes (n = 1), lions (n = 1), zebra (n = 1). Eleven animal types were confirmed to have been exposed to Leptospira. These include rodents, shrews, cattle, goats, pigs, dogs, bats, sheep, fish, buffaloes, and lions Table 3. Among the animals studied, rodents (Aesthomys chrysophilus, Dasmys incomtus, Mastomys natalensis, Rattus rattus, Lemniscomys griselda, Lemniscomys rosalia and Gerbilliscus vicinus) were the most investigated followed by cattle in Tanzania. The prevalence of antibodies to Leptospira in cattle ranged from 5.6–51.0%, and in rodents from 1.8–25.8%. The presence of antibodies in serum samples was determined by MAT with recent studies adopting qPCR and molecular sequencing to confirm the infection from kidney samples for explorations of Leptospira serogroups diversity [10,22].

Table 3

Summary of studies reporting animals with leptospirosis in Tanzania 1997–2019.

Reference	Year	Study area	Animal species	N	Seroprevalence (%)	Leptospira detected by culture* or PCR** (%)
[15]	1996	Morogoro, Dar es salaam, Mbeya, Kilimanjaro, Tanga, Singida and Mwanza	Cattle	MAT n = 374	5.6
			Cattle	Culture n = 1021		0.7*
			Dogs	208	38
			Rodent	537	1.8
[18]	1996–2006	Morogoro	Giant pouch rats	285		8.4*
			Field rats	1382		0.6*
			Shrews	298		3.7*
			Goats	100	38
			Pigs	100	41
			Dogs	100	39
			Cats	64	14.1
			Small rodents	500	5
			Small rodents	90	16.9
			African giant rats	65	15.4
			Shrew	4	25
[17]	2003	Morogoro	Fish	48	54.2
[37]	No date	Morogoro	Rodent	20	0	0* & 5**
			Shrew	7	0	29* & 29**
[41]	2002–2004	Tanga	Cattle	51	51
[40]	2003–2004	Tanga	Cattle	655	30.3
[39]	2005	Tanga	Cattle	80	21.3
[46]	2007–2008	Morogoro	Pig	MATn = 385	4.4
				Culturen = 236		0.8*
[36]	2007–2008	Morogoro	Rodent and shrew	348	17.8
[11]	2012–2013	Katavi	Cattle	1103	30.37
			Goat	248	8.47
			Rodent	207	20.29
			Shrew	11	9.09
			Buffalo	38	28.95
			Lion	2	50
			Zebra	2	0
[16]	2013	Morogoro	Bat	36	19.4
[38]	2012–2013	Morogoro	Rodent	89	25.8
			Shrew	1	100
[10]	2013–2014	Kilimanjaro	Cattle	452		7**
			Goat	167		1.2**
			Sheep	89		1.1**
			Rodents	384		0**
[47]	2016–2017	Morogoro	Dogs	232	9.5
[35]	No date	Morogoro	Rodents	70	22.9
[31]	No date	Kagera	Shrew and rodent	24	16.7	0*

Discussion

This review gives an insight on Leptospira prevalence and exposure, leptospirosis and the predominant Leptospira serogroups and their diversity in human and animal populations in Tanzania. It is evident after a detailed review of the published literature that leptospirosis is a prevalent zoonosis in Tanzania and present in various hosts including humans, livestock, wild animals, and aquatic life. There is an uneven distribution of research studies with large regions having inadequate or no leptospirosis information. The presence of universities or research institutions in regions that were overrepresented may reflect a degree of bias in the study site selection. For example, Kilimanjaro Clinical Research Institute (KCRI) conducted several human leptospirosis studies in the northern part of Tanzania while the Sokoine University of Agriculture conducted predominantly animal studies in the Morogoro region.

Our findings show that human leptospirosis is an important zoonosis of public health impact in Tanzania. Leptospirosis is widespread and prevalence varies between different settings and different populations. The actual burden of leptospirosis in humans may be difficult to estimate due to the limited and uneven distribution of studies and disease underestimation in the country. However, this trend is not unique to Tanzania, with the majority of low and middle-income countries (LMICs) facing similar challenges of inadequate surveillance data and diagnostic facilities [2]. Similar reports of leptospirosis prevalence as identified in this review have been reported in neighbouring countries. A study conducted in Kenya reported an apparent seropositivity of 13.4% in slaughterhouse workers [48], and a study of non-pregnant women in Uganda found 35% seropositive [49].

There was a large difference between the incidence reported in 2007–2008 and 2012–2014 in Kilimanjaro. This may be due to differences in the population selected, sample size or there may be variation in the leptospirosis incidence dependant on unknown factors [19,20]. Human leptospirosis in Tanzania may result from complex interactions between humans, animal carriers (such as cattle, rodents, dogs and pigs), and environments that favour perpetuation of leptospires and disease transmission.

The serological approaches utilized by various studies identified a diversity of Leptospira serogroups circulating in humans and animals. The MAT test was used in the majority of studies. MAT is a widely used diagnostic reference method for many studies, though not accessible in many laboratories due to its cost. MAT testing has many limitations: high levels of detectable antibodies are needed for a positive result and usually do not occur before the fourth week after disease onset [50] and it is time consuming and labour intensive [51,52]. Despite these drawbacks, the MAT test remains the only gold standard serological test and is considered a reference diagnostic test for leptospirosis in many settings [6,53].

The review found a large variation in the serogroup panels and the definition of positivity used across the studies S1 Table. When establishing a diagnostic panel it is advisable to include locally circulating serogroups or if these are not known to include a wide panel of pathogenic serogroups [6]. A list of candidate Leptospira serovars for diagnosis of leptospirosis using MAT in the African region was recently published based on research conducted in Tanzania [18]. However, emergence of new serovars suggests widening the serovar panels [14].

Most serogroups detected in animal species in the reviewed studies were also reported in humans. The most prevalent serogroups detected in rodents were Australis and Icterohaemoraghiae and Sejroe in cattle. These were also the most prevalent serogroups detected in people. This suggests that rodents and cattle may be an important source of infection in these settings. However, it is difficult to demonstrate transmission between animals and humans in our review because of the variability in the serogroup panel and different study designs.

A variety of domestic and wild animals in eighteen studies provide evidence of leptospirosis infections in animal populations in Tanzania. The review suggests that the main animal reservoirs for human leptospirosis may vary across the country, with primarily cattle, rodents, pigs, and dogs playing significant roles in disease transmission to humans. Rodents are important reservoirs of pathogenic Leptospira in many settings [12]. This review identified 10 studies reporting evidence of Leptospira in rodents and a diverse range of serogroups were detected Fig 3. There were only two studies in which Leptospira was detected in the sampled rodents using culture and PCR [18,37]. The lack of evidence of Leptospira in rodents in other studies using culture and qPCR techniques may indicate a methodological problem or lack of infected animals [22]. This scenario has also been reported in other studies, though such studies were associated with a limited sample size [12]. There may be differences in the prevalence of Leptospira in rodents between regions and between rural and urban settings [54]. Inappropriate sampling technique, sample preservation and an inadequate number of micro-organisms or loss of bacteria during culture can lead to false negative results.

Among exposed animals, cattle had the highest seropositivity, though this varied depending on geographical area. Cattle may be potential reservoirs and sources of human infection in Tanzania, particularly in rural areas where the majority of residents are smallholder dairy farmers and pastoralists [55]. Cattle are an important maintenance host for serogroup Sejroe [1,56] and transmission to farm workers and slaughtermen has been documented [48,57]. Animal contact particularly occupational exposures was identified as a risk factor by the reviewed papers and this is likely to have an important role in the epidemiology of leptospirosis in people in Tanzania [11,21,31,32].

Conclusion and recommendation

This review provides a summary of important information on the prevalence and distribution of the predominant Leptospira serogroups in humans and animals in Tanzania. Our review suggests that more comprehensive leptospirosis studies are needed in rodents and livestock across different agro-ecological zones for a deeper understanding of the epidemiology and to understand the risks of human leptospirosis for better management and control of the disease. The role of livestock in disease transmission among the smallholder farmers and other risk factors for human leptospirosis should be well studied for future disease control plans.

In most studies conducted in Tanzania, the MAT is the only diagnostic test used widely for leptospirosis detection however MAT may be impractical in many clinical laboratories due to the cost and complexity [52]. An alternative tool, such as rapid diagnostic tests (RDTs), was proposed by a recent policy brief and may be appropriate in a clinical setting for routine screening of patients with non-malaria fever [58]. The performance of RDTs is variable and would need to be trialled before implementation [28,59]. Raising awareness among health providers and the community on leptospirosis is recommended as a vital strategy for disease control and prevention.

Summary of the papers included in this review of leptospirosis in Tanzania 1997–2019 including year of research, study design, geographical location, target populations, diagnostics tests, and results for each study.

(DOCX)

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A) Prevalence of antibodies to Leptospira serogroups in people in cross-sectional studies; B) in febrile patients; C) in rodents; D) in cattle in Tanzania in leptospirosis papers published 1997–2021.

(DOCX)

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7 Mar 2021

Dear Mr Motto,

Thank you very much for submitting your manuscript "Epidemiology of leptospirosis in Tanzania: A review of the current status, serovar diversity and reservoirs" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

Reviewer #1: Thank you for the opportunity to review your systematic review. It was exciting to see a summary of recent research and a pleasure to read. I have some suggestions about areas in which the manuscript could be improved.

MAJOR:

1. Search strategy - You seem to be missing a number of relevant studies. It is not clear from the published search strategy why this is the case. When I use your search terms in Google I find additional references and understanding how/why these were excluded is important. For example:

a. Allan KJ, Maze MJ, Galloway RL, Rubach MP, Biggs HM, Halliday JE, Cleaveland S, Saganda W, Lwezaula BF, Kazwala RR, Mmbaga BT. Molecular Detection and Typing of Pathogenic Leptospira in Febrile Patients and Phylogenetic Comparison with Leptospira Detected among Animals in Tanzania. The American Journal of Tropical Medicine and Hygiene. 2020 Oct;103(4):1427. - contains additional molecular data from Kilimanjaro Region

b. Maze MJ. The Impact of Leptospirosis in Northern Tanzania: A Thesis Submitted for the Degree of Doctor of Philosophy at the University of Otago, Dunedin, New Zealand (Doctoral dissertation, University of Otago). This thesis contains a chapter on leptospirosis in the Ngorongoro Conservation Area.

c. D'acremont V, Kilowoko M, Kyungu E, Philipina S, Sangu W, Kahama-Maro J, Lengeler C, Cherpillod P, Kaiser L, Genton B. Beyond malaria—causes of fever in outpatient Tanzanian children. New England Journal of Medicine. 2014 Feb 27;370(9):809-17. This study investigated leptospirosis among children with fever

d. Boillat-Blanco N, Mbarack Z, Samaka J, Mlaganile T, Kazimoto T, Mamin A, Genton B, Kaiser L, D'Acremont V. Causes of fever in Tanzanian adults attending outpatient clinics: a prospective cohort study. Clinical Microbiology and Infection. 2020 Sep 4. This study used PCR to identify leptospirosis.

2. Leptospira exposure (ie seropositivity) vs leptospirosis (ie the acute disease) - It is important to clearly differentiate between the two as they are very different things. The lack of clarity is evident in line 299 - the Biggs 2011 study found 8.8% of patients hospitalized with a fever had acute leptospirosis with an additional 33.3% (ie 42.1% in total) seropositive. I suggest that you need to review all your papers regarding human infection again and consider how they should be interpreted. The interpretation is of course complex because acute leptospirosis would be unexpected in a cross sectional survey, and the incidence can only really be assessed by investigating those with a compatible illness.

3. Differentiating absent from not investigated. When considering MAT results I think you need to distinguish between serogroups that are not identified because they were not included in a panel and those that were included but tested negative. The entire section around predominant serovars needs reconsideration. In addition, Figure 5 is (I think) potentially misleading. As an alternative I think you should consider whether to report the prevalence of seropositivity by serogroup.

MINOR

General - Please review the written English within the paper - for example 'biasness' (line163) and numerous examples of disagreement between pronoun and noun (singular vs plural) and missing prepositions. Apologies for bringing this up, but the errors are to the point that they affect reading.

INTRODUCTION:

1. There are some sentences (eg lines 82-83 about leptospirosis in Malaysia) and references (eg reference 5 the case report from Russia) that seemed of limited relevance to the topic. I suggest focusing comments and references towards Tanzania.

2. Lines 82-83 regarding modes of transmission fails to mention contact with a contaminated environment as a potential source of infection.

Methods:

1. Including the search date is important (see above).

2. The ILS meeting is probably the most important scientific meeting for identifying leptospirosis abstracts and as far as I am aware it does not put the submitted abstracts on PubMed or Google. Did you have a strategy for reviewing these?

Results:

In addition to the 2 major comments above,

1. Lines 246-247. The statement regarding the confirmed leptospirosis cases needs clarification. What is the definition? Where has the denominator come from? As above, a cross sectional seroprevalence study should not be expected to identify acute leptospirosis cases and shouldn't feature as part of a denominator.

2. Lines 256-263 - When considering the variation in estimated leptospirosis incidence you highlight study design, population sampling and study size, but neglect to consider variation in leptospirosis incidence. Is this not possible?

Discussion:

1. Line 291 - While I am sympathetic to the perspective that leptospirosis doesn't get enough attention, I think the finding does need some support from your data. You have identified 30 studies - most published within the last decade. How much attention does it deserve, and from whom?

2. Lines 294-296 - The discussion about sources of leptospirosis does not mention rodents. Several authors have identified seropositive rodents and Mgode et al have isolated Leptospira from rodents. Totally discounting them seems a little premature.

3. Incidence of human leptospirosis - perhaps an alternative explanation of the data is that the incidence of leptospirosis varies by location and over time. The assertion that it has increased (line 298) is not supported by the only 2 actual datapoints on acute leptospirosis incidence (Biggs 2013 and Maze 2016).

4. Lines 324-327 - These seem to say that there has been only one study of rodents and it was negative. Your review shows this to be false. Mgode et al have identified Leptospira in rodents. Further you cite a number of technical errors that may have accounted for it. What about the possibility that Leptospira weren't (often) present in rats in Kilimanjaro region at the time of the study? It is notable that Allan et al did not trap many (if any) Mastomys species, whereas Mgode cultured Leptospira from them. There is precedent (a paper from Thailand last year I think) for low prevalence of leptospira carriage among peri-urban Rattus species, but high prevalence among rural rice field rats (Losea species from memory).

5. The paragraph on serologic approaches could more concisely state that MAT does not distinguish between infecting serovars. I think this is what you are getting at, but it is not clear.

6. The conclusion around RDTs is appealing, but the challenge is that (in my opinion at any rate) there isn't a good available RDT. While I appreciate that there isn't a lot of published data on this yet (there was an abstract at the ILS in 2017 reporting poor performance of lepto IgM RDTs in Kilimanjaro Region) your data doesn't really support this statement.

REFERNCES

There are numerous errors (eg non italisized genus names) and a mish-mash of referencing styles and text (including all caps).

Reviewer #2: This is an interesting review on leptospirosis in Tanzania that could also be representing the situation in African region where this disease is highly neglected.

The search seem to have skewed to mainly open access journals that full articles were retrievable and some important hosts have not been fully considered.

Relevants comments can be found on respective sections and lines. Generally the work is worth and increases awareness and knowledge on this disease in the region.

ABSTRACT

Line 53: ...typhoid and brucellosis. should be typhoid, brucellosis and other diseases. Delete Despite in the next sentence in this line to start with "More than 250 pathogenic.....

Line 56: leptospira should be Leptospira

INTRODUCTION

Line 73: ....including Tanzania (10) where the disease.... This citation number 10 refers to Thailand not Tanzania.

Line 79: ......"incidences of 72-102 and 529 leptospiral cases....." a denominator for the 529 might be useful here.

Line 82-83: .The high leptospiral cases in Malaysia.......296 deaths in 5 years: this sentence is not clear what it refers needs rephrasing and linking it well with the rest of contents in this paragraph.

Line 87: .....non-pathogenic Leptospira (25, 26): Original references would fit better here, or add to these ones.

METHODOLOGY -

RESULTS:

Line 178: ....in humans (n=8) needs references. similarly, ...........two studies on animals (cattle, goat and rodents) - needs references as well.e.g. the two studies could be cited here.

Line 226: ......serovars serotyped: should be serovars reported.

Line 264: table 1: the locality named Moshi in column 3 should preferably be named as Moshi Kilimanjaro because the rest of the areas listed in this Table represent the regions while Moshi is an area in Kilimanjaro region.

Line 272: see comment on Moshi. should be Moshi Kilimanjaro.

Line 277: .....Lemniscomys Griselda should be Lemniscomys griselda. the species name is small letter.

Line 282: Table 2. omit DFM among the listed test - it is not a test but a tool for examining leptospires

Row number 19 column no.5 the 455 are humans not rodents and shrews. In this study (39) there were rodents and shrews but the 455 are humans.

For citation no. 59 (row no. 14 column 4 add MAT and culture.

This Table can be enriched by including information from a study by Mgode et al (2015) published in PLOSNTD which gives insights on leptospirosis situation in broad range of reservoir hosts including data on cattle, cats, goats, sheep, pigs, rodents and shrews. Another citation that reports genetic diversity of Leptospira isolates from Tanzania published by Ahmed et al (2006) could also add important information on Leptospira serovars reported from Tanzania which were described/identified using multilocus sequence typing. This paper is missing in this manuscript and has a large/ in-depth report of Leptospira isolates from Tanzania (DOI: 10.1186/1476-0711-5-28).

DISCUSSION:

Line 290.... it has not received sufficient attention.....This probably requires rephrasing because some work on leptospirosis in Tanzania has been used/cited in the National One Health strategic plan in Tanzania and East Africa community (specifically the work on leptospirosis in bats from Morogoro Tanzania, as well as publication on Leptospira serovar Sokoine from cattle. However, much would have been done given the amount of evidence that is available on leptospirosis and Leptospira from Tanzania. A policy brief on Leptospirosis in Tanzania is also in place (on researchgate) which highlights the situation of this disease in humans in Tanzania - probably contributes to the increasing awareness of non-malarial fevers.

Line 297: For the past 9, ... this is incomplete, 9 what? years?

Line 306: ...establish = established

Line 368: ..... a list of candidate Leptospira serovars for diagnosis of leptospirosis using MAT in African region has been published (Mgode et al. 2015) which is based on data obtained from use of local serovars vs imported serovars. Similarly, policy brief (2016) recommending introduction of rapid diagnostic kits for leptospirosis has been recommended for patients with non-malarial fevers (DOI: 10.13140/RG.2.2.13464.60165) could be relevant in this discussion line 373.

REFERENCES:

Line 460: citation no. 23: seem to be incomplete

Reviewer #3: This manuscript synthesises what is known about the epidemiology of leptospirosis through a literature review. There is some confusion throughout the manuscript in the use of prevalence (presence of pathogen) and seroprevalence (presence of antibodies to the pathogen). The subject area is important and relevant and justifies publication.

Line 14-15: "Leptospirosis is…" - Awkward sentence. Try rewording

Line 34-36: Previously stated that 8 studies were undertaken in Kiliminjaro but this adds to 10

Line 37: "Sejroe" not "Serjoe"

Line 41-43: State the range in prevalence to provide some detail on how large the variation is

Line 77: "subacute and long-lasting illness" - I'm not sure that these are the most appropriate references they just reference other studies which state this. Cite the initial studies where this was shown

Line 81-83: Not sure why this is relevant

Line 84: It is more than 250 serovars (as stated in abstract)

Line 86: It is many more than 21 species. See recent paper https://doi.org/10.1371/journal. pntd.0007270

line 169-178: When describing the number of studies focussing on each host type in each region these do not add up to the number of studies performed in each region (line 169-170)

line 181: "serological diagnostic techniques" - many of these examples (culture, PCR) are not serological techniques

line 184 -186: This doesn't make sense. All the tests are diagnostic tests and I'm not sure what "widely recognised" means

line 180-203: I don't think this figure is publication quality yet and I think it would be worth that providing references for each of the assays (perhaps as a supplementary table)

line 209: If some serovars were frequently used then please list which ones

line 218-219: provide a reference

line 219-221: This is for discussion not results

Figure 4: I couldn't make sense of this figure. It needs to be improved

Line 230-232: Confusing sentence. Not sure what the point is. Is it that lepto has been detected in every species except zebra or that it hasn't been detected in zebra because few animals were sampled?

Line 246: I think this is misleading. It give the impression that all 7129 cases were suspected leptospirosis and only 576 were confirmed

Line 262-263: The most likely possibility is that lepto incidence truly did decrease

Line 279: presence of leptospires is not detected by MAT. Only leptospire antibodies are detected by MAT

Line 297-298: "For the past 9.." something has been left out of this sentence

Line 306: "leptospirosis" not "Leptospirosis"

Line 306: "base level" - I am not clear that a base level prevalence is established in this study? To what does this refer?

Line 313: To what does n=16 refer?

Line 315: dogs aren't livestock

Line 327: Again. The most likely cause of this finding is that the rodents weren't infected. It isn't uncommon as you point out. There doesn't have to be a methodology problem.

Line 338-339: Risk of contamination of what? I'm not sure the author fully understands MAT

Line 339-340: "MAT cannot be considered as a gold standard test" - but it is considered the gold standard serological test unless the author wishes to propose another test as gold standard

Line 349-351: I think the discussion should include human exposure identified in clinical cases (fever) and human cases identified in surveillance (no fever). There are some serovars (Hardjo) that might not cause severe human disease.

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Reviewer #1: Thank you for the opportunity to review your systematic review. It was exciting to see a summary of recent research and a pleasure to read. I have some suggestions about areas in which the manuscript could be improved.

MAJOR:

1. Search strategy - You seem to be missing a number of relevant studies. It is not clear from the published search strategy why this is the case. When I use your search terms in Google I find additional references and understanding how/why these were excluded is important. For example:

a. Allan KJ, Maze MJ, Galloway RL, Rubach MP, Biggs HM, Halliday JE, Cleaveland S, Saganda W, Lwezaula BF, Kazwala RR, Mmbaga BT. Molecular Detection and Typing of Pathogenic Leptospira in Febrile Patients and Phylogenetic Comparison with Leptospira Detected among Animals in Tanzania. The American Journal of Tropical Medicine and Hygiene. 2020 Oct;103(4):1427. - contains additional molecular data from Kilimanjaro Region

b. Maze MJ. The Impact of Leptospirosis in Northern Tanzania: A Thesis Submitted for the Degree of Doctor of Philosophy at the University of Otago, Dunedin, New Zealand (Doctoral dissertation, University of Otago). This thesis contains a chapter on leptospirosis in the Ngorongoro Conservation Area.

c. D'acremont V, Kilowoko M, Kyungu E, Philipina S, Sangu W, Kahama-Maro J, Lengeler C, Cherpillod P, Kaiser L, Genton B. Beyond malaria—causes of fever in outpatient Tanzanian children. New England Journal of Medicine. 2014 Feb 27;370(9):809-17. This study investigated leptospirosis among children with fever

d. Boillat-Blanco N, Mbarack Z, Samaka J, Mlaganile T, Kazimoto T, Mamin A, Genton B, Kaiser L, D'Acremont V. Causes of fever in Tanzanian adults attending outpatient clinics: a prospective cohort study. Clinical Microbiology and Infection. 2020 Sep 4. This study used PCR to identify leptospirosis.

2. Leptospira exposure (ie seropositivity) vs leptospirosis (ie the acute disease) - It is important to clearly differentiate between the two as they are very different things. The lack of clarity is evident in line 299 - the Biggs 2011 study found 8.8% of patients hospitalized with a fever had acute leptospirosis with an additional 33.3% (ie 42.1% in total) seropositive. I suggest that you need to review all your papers regarding human infection again and consider how they should be interpreted. The interpretation is of course complex because acute leptospirosis would be unexpected in a cross sectional survey, and the incidence can only really be assessed by investigating those with a compatible illness.

3. Differentiating absent from not investigated. When considering MAT results I think you need to distinguish between serogroups that are not identified because they were not included in a panel and those that were included but tested negative. The entire section around predominant serovars needs reconsideration. In addition, Figure 5 is (I think) potentially misleading. As an alternative I think you should consider whether to report the prevalence of seropositivity by serogroup.

MINOR

General - Please review the written English within the paper - for example 'biasness' (line163) and numerous examples of disagreement between pronoun and noun (singular vs plural) and missing prepositions. Apologies for bringing this up, but the errors are to the point that they affect reading.

INTRODUCTION:

1. There are some sentences (eg lines 82-83 about leptospirosis in Malaysia) and references (eg reference 5 the case report from Russia) that seemed of limited relevance to the topic. I suggest focusing comments and references towards Tanzania.

2. Lines 82-83 regarding modes of transmission fails to mention contact with a contaminated environment as a potential source of infection.

Methods:

1. Including the search date is important (see above).

2. The ILS meeting is probably the most important scientific meeting for identifying leptospirosis abstracts and as far as I am aware it does not put the submitted abstracts on PubMed or Google. Did you have a strategy for reviewing these?

Results:

In addition to the 2 major comments above,

1. Lines 246-247. The statement regarding the confirmed leptospirosis cases needs clarification. What is the definition? Where has the denominator come from? As above, a cross sectional seroprevalence study should not be expected to identify acute leptospirosis cases and shouldn't feature as part of a denominator.

2. Lines 256-263 - When considering the variation in estimated leptospirosis incidence you highlight study design, population sampling and study size, but neglect to consider variation in leptospirosis incidence. Is this not possible?

Discussion:

1. Line 291 - While I am sympathetic to the perspective that leptospirosis doesn't get enough attention, I think the finding does need some support from your data. You have identified 30 studies - most published within the last decade. How much attention does it deserve, and from whom?

2. Lines 294-296 - The discussion about sources of leptospirosis does not mention rodents. Several authors have identified seropositive rodents and Mgode et al have isolated Leptospira from rodents. Totally discounting them seems a little premature.

3. Incidence of human leptospirosis - perhaps an alternative explanation of the data is that the incidence of leptospirosis varies by location and over time. The assertion that it has increased (line 298) is not supported by the only 2 actual datapoints on acute leptospirosis incidence (Biggs 2013 and Maze 2016).

4. Lines 324-327 - These seem to say that there has been only one study of rodents and it was negative. Your review shows this to be false. Mgode et al have identified Leptospira in rodents. Further you cite a number of technical errors that may have accounted for it. What about the possibility that Leptospira weren't (often) present in rats in Kilimanjaro region at the time of the study? It is notable that Allan et al did not trap many (if any) Mastomys species, whereas Mgode cultured Leptospira from them. There is precedent (a paper from Thailand last year I think) for low prevalence of leptospira carriage among peri-urban Rattus species, but high prevalence among rural rice field rats (Losea species from memory).

5. The paragraph on serologic approaches could more concisely state that MAT does not distinguish between infecting serovars. I think this is what you are getting at, but it is not clear.

6. The conclusion around RDTs is appealing, but the challenge is that (in my opinion at any rate) there isn't a good available RDT. While I appreciate that there isn't a lot of published data on this yet (there was an abstract at the ILS in 2017 reporting poor performance of lepto IgM RDTs in Kilimanjaro Region) your data doesn't really support this statement.

REFERNCES

There are numerous errors (eg non italisized genus names) and a mish-mash of referencing styles and text (including all caps).

Reviewer #2: This is an interesting review on leptospirosis in Tanzania that could also be representing the situation in African region where this disease is highly neglected.

The search seem to have skewed to mainly open access journals that full articles were retrievable and some important hosts have not been fully considered.

Relevants comments can be found on respective sections and lines. Generally the work is worth and increases awareness and knowledge on this disease in the region.

ABSTRACT

Line 53: ...typhoid and brucellosis. should be typhoid, brucellosis and other diseases. Delete Despite in the next sentence in this line to start with "More than 250 pathogenic.....

Line 56: leptospira should be Leptospira

INTRODUCTION

Line 73: ....including Tanzania (10) where the disease.... This citation number 10 refers to Thailand not Tanzania.

Line 79: ......"incidences of 72-102 and 529 leptospiral cases....." a denominator for the 529 might be useful here.

Line 82-83: .The high leptospiral cases in Malaysia.......296 deaths in 5 years: this sentence is not clear what it refers needs rephrasing and linking it well with the rest of contents in this paragraph.

Line 87: .....non-pathogenic Leptospira (25, 26): Original references would fit better here, or add to these ones.

METHODOLOGY -

RESULTS:

Line 178: ....in humans (n=8) needs references. similarly, ...........two studies on animals (cattle, goat and rodents) - needs references as well.e.g. the two studies could be cited here.

Line 226: ......serovars serotyped: should be serovars reported.

Line 264: table 1: the locality named Moshi in column 3 should preferably be named as Moshi Kilimanjaro because the rest of the areas listed in this Table represent the regions while Moshi is an area in Kilimanjaro region.

Line 272: see comment on Moshi. should be Moshi Kilimanjaro.

Line 277: .....Lemniscomys Griselda should be Lemniscomys griselda. the species name is small letter.

Line 282: Table 2. omit DFM among the listed test - it is not a test but a tool for examining leptospires

Row number 19 column no.5 the 455 are humans not rodents and shrews. In this study (39) there were rodents and shrews but the 455 are humans.

For citation no. 59 (row no. 14 column 4 add MAT and culture.

This Table can be enriched by including information from a study by Mgode et al (2015) published in PLOSNTD which gives insights on leptospirosis situation in broad range of reservoir hosts including data on cattle, cats, goats, sheep, pigs, rodents and shrews. Another citation that reports genetic diversity of Leptospira isolates from Tanzania published by Ahmed et al (2006) could also add important information on Leptospira serovars reported from Tanzania which were described/identified using multilocus sequence typing. This paper is missing in this manuscript and has a large/ in-depth report of Leptospira isolates from Tanzania (DOI: 10.1186/1476-0711-5-28).

DISCUSSION:

Line 290.... it has not received sufficient attention.....This probably requires rephrasing because some work on leptospirosis in Tanzania has been used/cited in the National One Health strategic plan in Tanzania and East Africa community (specifically the work on leptospirosis in bats from Morogoro Tanzania, as well as publication on Leptospira serovar Sokoine from cattle. However, much would have been done given the amount of evidence that is available on leptospirosis and Leptospira from Tanzania. A policy brief on Leptospirosis in Tanzania is also in place (on researchgate) which highlights the situation of this disease in humans in Tanzania - probably contributes to the increasing awareness of non-malarial fevers.

Line 297: For the past 9, ... this is incomplete, 9 what? years?

Line 306: ...establish = established

Line 368: ..... a list of candidate Leptospira serovars for diagnosis of leptospirosis using MAT in African region has been published (Mgode et al. 2015) which is based on data obtained from use of local serovars vs imported serovars. Similarly, policy brief (2016) recommending introduction of rapid diagnostic kits for leptospirosis has been recommended for patients with non-malarial fevers (DOI: 10.13140/RG.2.2.13464.60165) could be relevant in this discussion line 373.

REFERENCES:

Line 460: citation no. 23: seem to be incomplete

Reviewer #3: This manuscript synthesises what is known about the epidemiology of leptospirosis through a literature review. There is some confusion throughout the manuscript in the use of prevalence (presence of pathogen) and seroprevalence (presence of antibodies to the pathogen). The subject area is important and relevant and justifies publication.

Line 14-15: "Leptospirosis is…" - Awkward sentence. Try rewording

Line 34-36: Previously stated that 8 studies were undertaken in Kiliminjaro but this adds to 10

Line 37: "Sejroe" not "Serjoe"

Line 41-43: State the range in prevalence to provide some detail on how large the variation is

Line 77: "subacute and long-lasting illness" - I'm not sure that these are the most appropriate references they just reference other studies which state this. Cite the initial studies where this was shown

Line 81-83: Not sure why this is relevant

Line 84: It is more than 250 serovars (as stated in abstract)

Line 86: It is many more than 21 species. See recent paper https://doi.org/10.1371/journal. pntd.0007270

line 169-178: When describing the number of studies focussing on each host type in each region these do not add up to the number of studies performed in each region (line 169-170)

line 181: "serological diagnostic techniques" - many of these examples (culture, PCR) are not serological techniques

line 184 -186: This doesn't make sense. All the tests are diagnostic tests and I'm not sure what "widely recognised" means

line 180-203: I don't think this figure is publication quality yet and I think it would be worth that providing references for each of the assays (perhaps as a supplementary table)

line 209: If some serovars were frequently used then please list which ones

line 218-219: provide a reference

line 219-221: This is for discussion not results

Figure 4: I couldn't make sense of this figure. It needs to be improved

Line 230-232: Confusing sentence. Not sure what the point is. Is it that lepto has been detected in every species except zebra or that it hasn't been detected in zebra because few animals were sampled?

Line 246: I think this is misleading. It give the impression that all 7129 cases were suspected leptospirosis and only 576 were confirmed

Line 262-263: The most likely possibility is that lepto incidence truly did decrease

Line 279: presence of leptospires is not detected by MAT. Only leptospire antibodies are detected by MAT

Line 297-298: "For the past 9.." something has been left out of this sentence

Line 306: "leptospirosis" not "Leptospirosis"

Line 306: "base level" - I am not clear that a base level prevalence is established in this study? To what does this refer?

Line 313: To what does n=16 refer?

Line 315: dogs aren't livestock

Line 327: Again. The most likely cause of this finding is that the rodents weren't infected. It isn't uncommon as you point out. There doesn't have to be a methodology problem.

Line 338-339: Risk of contamination of what? I'm not sure the author fully understands MAT

Line 339-340: "MAT cannot be considered as a gold standard test" - but it is considered the gold standard serological test unless the author wishes to propose another test as gold standard

Line 349-351: I think the discussion should include human exposure identified in clinical cases (fever) and human cases identified in surveillance (no fever). There are some serovars (Hardjo) that might not cause severe human disease.

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16 Jul 2021

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

19 Aug 2021

Dear Mr Motto,

Thank you very much for submitting your manuscript "Epidemiology of leptospirosis in Tanzania: A review of the current status, serogroup diversity and reservoirs" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Reviewer #1: Thank you for the opportunity to review this revised manuscript. My concerns from the initial paper have for the most part been addressed. Having read the revised manuscript, I have a small number of additional questions/ comments.

Introduction:

Line 74: Including cattle, rodents and pigs - all true. Is there a role for dogs, sheep or goats?

Methods: Search strategy - I worry that the PubMed search strategy is too narrow. That it only identified 47 articles compared to 3720 identified in Google attests to that. It is perhaps not feasible to re-engineer your searches, but perhaps acknowledging the discrepancy between identified articles in Google vs PubMed and the potential to have missed articles that were not identified by the Google search would be appropriate.

Discussion - The claim of primacy isn't entirely true. What about Allan et al and De Vries et al who both conducted systematic reviews of the entire continent. Your review provides greater detail for Tanzania - but isn't the first to address the topic.

Reviewer #3: Dear authors

Thank you for your revisions. Please see below a few suggestions.

Line 81-83: Check this. There are 17 species in P1 and 21 in P2

Line 87: "small prevalence" should be "low prevalence"

Lines 178-184: These sentences are more discussion than results

Lines 208-209: Molecular typing is not a diagnostic technique. It is way of identifying genetic diversity

Line 215: Should be Figure 3

Figure 3: "Javonica" should be "Javanica"

Line 227: "serovars" should be "serogroups"

Line 278: Please be careful using incidence rather than prevalence. Incidence always includes a time period e.g. XX cases/ 100000 individuals/year. I don't think any of these animal studies describe incidence.

Lines313-314: Please include the references

Supplementary Table 1:

This table needs to be checked. I am unclear what the serogroup (serovar) column indicates. I have checked a few references and for reference 2 (Allan et al 2020) the serogroups refer to those identified as causing human infections whereas reference 4 (Biggs et al 2011) appears to show all the serogroups included in the MAT. Only 8 of these serogroups were identified in human cases. I notice that in later references it is shown whether serogroups were not detected. Perhaps this was an oversight for earlier references?

Under the PCR column some studies list DNA fingerprinting - this isn't PCR. There is also reference to TAC. I'm not sure what this is? Perhaps it should be defined in the footnotes? In addition, for some references e.g. 22 the results from hosts are shown in the PCR column.

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Associate Editor

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Melissa Caimano

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer #1: Thank you for the opportunity to review this revised manuscript. My concerns from the initial paper have for the most part been addressed. Having read the revised manuscript, I have a small number of additional questions/ comments.

Introduction:

Line 74: Including cattle, rodents and pigs - all true. Is there a role for dogs, sheep or goats?

Methods: Search strategy - I worry that the PubMed search strategy is too narrow. That it only identified 47 articles compared to 3720 identified in Google attests to that. It is perhaps not feasible to re-engineer your searches, but perhaps acknowledging the discrepancy between identified articles in Google vs PubMed and the potential to have missed articles that were not identified by the Google search would be appropriate.

Discussion - The claim of primacy isn't entirely true. What about Allan et al and De Vries et al who both conducted systematic reviews of the entire continent. Your review provides greater detail for Tanzania - but isn't the first to address the topic.

Reviewer #3: Dear authors

Thank you for your revisions. Please see below a few suggestions.

Line 81-83: Check this. There are 17 species in P1 and 21 in P2

Line 87: "small prevalence" should be "low prevalence"

Lines 178-184: These sentences are more discussion than results

Lines 208-209: Molecular typing is not a diagnostic technique. It is way of identifying genetic diversity

Line 215: Should be Figure 3

Figure 3: "Javonica" should be "Javanica"

Line 227: "serovars" should be "serogroups"

Line 278: Please be careful using incidence rather than prevalence. Incidence always includes a time period e.g. XX cases/ 100000 individuals/year. I don't think any of these animal studies describe incidence.

Lines313-314: Please include the references

Supplementary Table 1:

This table needs to be checked. I am unclear what the serogroup (serovar) column indicates. I have checked a few references and for reference 2 (Allan et al 2020) the serogroups refer to those identified as causing human infections whereas reference 4 (Biggs et al 2011) appears to show all the serogroups included in the MAT. Only 8 of these serogroups were identified in human cases. I notice that in later references it is shown whether serogroups were not detected. Perhaps this was an oversight for earlier references?

Under the PCR column some studies list DNA fingerprinting - this isn't PCR. There is also reference to TAC. I'm not sure what this is? Perhaps it should be defined in the footnotes? In addition, for some references e.g. 22 the results from hosts are shown in the PCR column.

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References

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13 Sep 2021

Submitted filename: Response to reviewers.docx

Click here for additional data file.

16 Oct 2021

Dear Mr Motto,

We are pleased to inform you that your manuscript 'Epidemiology of leptospirosis in Tanzania: A review of the current status, serogroup diversity and reservoirs' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Alan J A McBride, Ph.D.

Associate Editor

PLOS Neglected Tropical Diseases

Melissa Caimano

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: THe authors have addressed my previous comments and I have no additional concerns.

Reviewer #3: (No Response)

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Previous comments have been addressed, and I have no additional comments

Reviewer #3: (No Response)

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Conclusions are appropriate to the data presented. My previous queries have been addressed and I have no further comments.

Reviewer #3: (No Response)

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Nil

Reviewer #3: (No Response)

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: All critiques have been addressed. I have no further comments.

Reviewer #3: Very minor comment - In the abstract MAT is defined as "Microagglutination test". It should be "Microscopic Agglutination Test" as defined in the rest of the manuscript.

**********

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Reviewer #1: No

Reviewer #3: Yes: Mark Moseley

11 Nov 2021

Dear Mr Motto,

We are delighted to inform you that your manuscript, "Epidemiology of leptospirosis in Tanzania: A review of the current status, serogroup diversity and reservoirs," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases