Risk factors analysis for neglected human rickettsioses in rural communities in Nan province, Thailand: A community-based observational study along a landscape gradient.

In this study, we estimated exposure for Scrub typhus (STG), Typhus (TG) and Spotted fever groups (SFG) rickettsia using serology at a fine scale (a whole sub-district administration level) of local communities in Nan Province, Thailand. Geographical characteristics of the sub-district were divided into two landscape types: lowland agricultural area in an urbanized setting (lowland-urbanized area) and upland agricultural area located close to a protected area of National Park (upland-forested area). This provided an ideal contrast between the two landscapes with low and high levels of human-altered habitats to study in differences in disease ecology. In total, 824 serum samples of participants residing in the eight villages were tested by screening IgG ELISA, and subsequently confirmed by the gold standard IgG Immunofluorescent Assay (IFA). STG and TG IgG positivity were highest with seroprevalence of 9.8% and 9.0%, respectively; whereas SFG positivity was lower at 6.9%. Inhabitants from the villages located in upland-forested area demonstrated significantly higher STG exposure, compared to those villages in the lowland-urbanized area (chi-square = 51.97, p < 0.0001). In contrast, TG exposure was significantly higher in those villagers living in lowland-urbanized area (chi-square = 28.26, p < 0.0001). In addition to the effect of landscape types, generalized linear model (GLM) analysis identified socio-demographic parameters, i.e., gender, occupation, age, educational level, domestic animal ownership (dog, cattle and poultry) as influential factors to explain the level of rickettsial exposure (antibody titers) in the communities. Our findings raise the public health awareness of rickettsiosis as a cause of undiagnosed febrile illness in the communities.

Introduction

Human rickettsioses are neglected emerging and re-emerging diseases caused by a group of obligate intracellular bacteria in the Order Rickettsiales (e.g., Rickettsia and Orientia). Rickettsial pathogens causing disease in humans can be divided into the three main groups, known as scrub typhus (STG), typhus (TG) and spotted fever groups (SFG) [1]. These zoonotic bacteria are transmitted to humans and animals after bites by arthropod vectors, including ticks, mites, fleas or lice. Co-occurrences of rickettsial species among arthropod vectors, domestic animals and humans as well as increasing opportunities for interactions at the human-animal interface are of importance in zoonotic transmission cycles [2-4]. These vector-borne diseases have increasingly been recognized as one of the common causes of febrile illness in Southeast Asia, in addition to dengue, leptospirosis and malaria [1,5,6].

Scrub typhus is a chigger-borne rickettsiosis caused by intracellular bacteria in the genus Orientia, including Orientia tsutsugamushi (reported primarily in Asia-Pacific region), Orientia chuto (in the Arabian Peninsula and African countries) and Orientia sp. (Candidatus Orientia chiloensis, a potential new species from Chile) [7-11]. Larval stage of mites in the family Trombiculidae, known as “chiggers”, are the main vector of the disease. Previous studies have suggested that human scrub typhus is more common in rural or sub-urban areas [12,13] with the majority of disease in farmers, military soldiers and jungle trekkers [7,14,15]. Typhus group (TG) mainly involves murine typhus (also known as endemic typhus) infections caused by Rickettsia typhi, a flea-borne rickettsiosis with worldwide distribution, notably in tropical coastal regions [16,17]; and Rickettsia prowazekii which causes epidemic typhus, a louse-borne rickettsiosis in the areas of poor socioeconomic status and high prevalence of human body louse infestation [18]. Transmission of typhus group rickettsioses generally occur when the bacteria in the feces of arthropod vectors are rubbed into the bite wound or the vectors themselves are crushed and broke in to the skin [19]. For the spotted fever group (SFG), there are more than 25 rickettsial species and related strains distributed globally, and known to cause disease in human [20], including Rickettsia africae, Rickettsia asiatica, Rickettsia australis, Rickettsia conorii, Rickettsia heilongjiangensis, Rickettsia helvetica, Rickettsia honei, Rickettsia japonica, Rickettsia parkeri, Rickettsia raoultii, Rickettsia rickettsia, Rickettsia sibirica and Rickettsia tamurae. These SFG rickettsia are mainly harbored by ticks, and transmitted to human or other animal hosts via their bites. Other ectoparasites such as fleas and mites are also involved as the vectors of several SFG rickettsia including the flea-borne Rickettsia felis and the mite-borne Rickettsia akari [2,21,22].

Diagnosis of these pathogens relies on molecular techniques such as PCR-based amplification to demonstrate the presence of the infectious agent, as well as serological methods as a surrogate including enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assays (IFA) [23]. Serological investigations allow epidemiologists to investigate the history of rickettsial exposure and estimation of seroprevalence by detecting the occurrence of preexisting levels of antibodies in a population [24-26]. IFA detection of IgM and/or IgG is recognized as a gold standard for rickettsial serological diagnosis, particularly for STG, however there remains uncertainty as to the most suitable diagnostic cut-off for each geographic location [23,27]. ELISA-based diagnostics give acceptable levels of sensitivity and specificity for STG, SFG and TG antibody detection [23,28,29] as well as having increased diagnostic throughput and less subjectivity [29,30]. However, serological results are normally confined to the serogroup level. Although generally, there are cross-reactions within the serogroups, they occur less so between serogroups with the exception that cross-reactions between SFG and TG antibodies are recognised to a limited extent [1,31].

In Thailand, previous studies have indicated that all three rickettsial groups exist and are a cause for public health concern [2,5,32-34]. In terms of human febrile illness, scrub typhus is the most prevalent rickettsial pathogen which has markedly increased during the last two decades although this may be due to increased awareness and access to improved diagnostics. During 2003–2018, there were approximately 100,000 cases reported to the National Disease Surveillance system (Ministry of Public Health, Thailand). The disease is reported nation-wide, with the majority of the cases from the northern region accounting approximately half (53%) of the total reported cases per year [34]. Murine typhus is also recognized a rickettsiosis in Thailand. Case reports of R. typhi infection have been documented in several provinces, either in municipalities, rural communities or border areas [5,35-38]. Epidemiological data regarding SFG rickettiosis is rather more scarce compared to scrub typhus and murine typhus. In 1994, three human SFG rickettsiosis cases from Chiang Mai Province were confirmed by serology, and documented as the first report in Thailand [39]. The first human case of R. felis infection in Thailand (and in Asia) was reported at the Thai-Myanmar border in Kanchanaburi Province (32). Thai tick typhus Rickettsia TT-118 (also known as R. honei) was identified from a patient in urban area of Bangkok who had history of camping at a National Park prior to the onset of fever [40]. In addition, Rickettsia spp. closely related to R. japonica and R. helvetica have also reported to infect humans in Thailand [41-43].

Most of the case reports and epidemiological studies in Thailand were recognised in the context of passive surveillance or retrospective studies from stored clinical samples [44], while active surveillance data of human rickettsiosis is still limited. Here, within the research framework of FutureHealthSEA project (Predictive scenarios of health in Southeast Asia: linking land use and climate changes to infectious diseases, ANR-17-CE35-0003), we performed screening for human rickettsiosis at the sub-district administration level of local communities in Nan Province, northern Thailand. The objectives of the study were: (1) to estimate level of past exposure to rickettsial pathogens using serological diagnosis approach; and (2) to determine potential factors, such as socio-demographic, zoonotic potential and environmental parameters influencing rickettsial exposures in a local scale setting.

Materials and methods

Ethics statement

All procedures involving human samples and data collection were reviewed and approved by the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University (document no. MUTM 2018-035-01). Signed formal consent form was obtained from all the participants and also from the parent/guardian of the child participants.

Study population and individual demographic information

In 2018, we conducted a cross-sectional study at the local communities, all of the 8 villages in Saen Thong Sub-district, Tha Wang Pha District, Nan Province (latitude 19.133 and longitude 100.7680). Geographical distribution of the 8 villages is shown in Fig 1. In terms of geographical characteristics, the sub-district can be roughly divided into the two landscape types: (1) lowland agricultural area in an urbanized setting (Lowland-urbanized area), closed to Tha Wang Pha city on the east; and (2) upland agricultural area closed to the forested area of Nantaburi National Park (Upland-forested area) on the west.

Fig 1

Distribution maps demonstrating the level of rickettsiosis exposures in participants from the eight villages in Saen Thong Subdistrict, Nan Province.

Distribution and level of (A) STG, (B) TG and (C) SFG individual exposures from each village. Green circles indicate negativity. Red circles show positive exposures, and size of the circles indicate level of IgG IFA positivity at a diagnostic titer cut-off of 1:100.

The target population of this study was randomly selected with the following inclusion criteria: both males and females; age over 9 years old; recently and permanently residing in the study area; able to adequately respond to the questions and voluntary participation in the research project. Individual data regarding participants’ personal information was collected including age group (children at 9–17 years old; young adult at 18–35 years old; adult at 36–60 years old; and elderly at >60 years old), gender (male and female) and occupation (farmer or non-farmer). Information on companion pets and livestock ownership, which included numbers of dogs, cats, poultry, pigs and cattle, were also obtained to determine potential zoonotic association. Every household that the participants’ resided were geo-tagged to collect GPS coordinates information for further spatial case mapping.

Blood sample collection and serological diagnosis of rickettsial past exposure

Staff from the local primary health care unit (PCU) made an appointment with the village chiefs for preparation of participant recruitment for blood collection. Blood samples were collected via venipuncture (5ml) in non-anticoagulant tubes (VACUETTE TUBE Serum Clot Activator, Greiner Bio-One, Austria), and then centrifuged at 2,795 xg for 5 min within the same day of collection to separate serum samples in sterile 1.5 ml collection tubes (Axygen, US). The serum samples were frozen in -80°C prior to laboratory investigations.

Due to the large number of samples, sera were screened for the presence of IgG antibodies at 1:100 dilution using STG, TG or SFG IgG ELISA using diagnostic cut off with a high sensitivity of 0.50 nett OD. ELISA-positive samples were titrated in the “gold standard” IFA to determine the endpoint antibody titer. The MORU in-house STG IgG ELISA used specific antigens of O. tsutsugamushi Karp, Kato, Gilliam, and TA716 strains to detect scrub typhus IgG antibodies [45]. For the TG ELISA, R. typhi Wilmington strain and for SFG ELISA, R. conorii and R. honei antigens were applied using the same methodology. Consequently, we performed IgG IFA to produce quantitative results, with the full methodology described elsewhere [46]. Briefly, sera were serially 2-fold diluted from 1:100 to 1:25,600 and the endpoint was determined as the highest titer displaying specific fluorescence. Titer >1:100 was considered positive for the presence of IgG antibodies.

Statistical analysis

Estimation of STG, TG and SFG by village, landscape type and personal attributes were performed using descriptive statistics, i.e., seroprevalence with 95% confidence interval. A Venn diagram was created using “ggplot2” package [47] in R freeware [48] to illustrate number and proportion of participants exposed with STG, TG, SFG and co-exposures. Seropositive cases were geographically mapped to reveal distribution patterns of STG, TG and SFG using “tmap” [49] and “raster” [50] packages in R freeware [48].

Generalized linear model (GLM) was performed using R freeware [48] to determine socio-demographic and environmental factors explaining the level of STG, TG and SFG exposures (titers). Similar initial models were applied to the three rickettsial groups, comprising with the following variables: Landscape type, Gender, Occupation, Age, Educational level, as well as discrete variables for population numbers of dogs, cats, pigs, poultry and cattle. Poisson distribution was selected for model fitting after checking data distribution using “fitdistrplus” package [51]. GLM with Akaike’s information criterion corrected for sample size (AICc) was performed with negative binomial distribution using “glmulti” package [52]. The best models for each rickettsial group were selected for further discussion based on low AICc and high Akaike’s weight (Wr) scores [53]. To identify the degree of multicollinearity among explicative variables, the variance inflation factor (VIF) was computed using “car” package [54]. In addition, quality of the selected models was assessed with goodness-of-fit and power analysis tests using “sjstats” [55] and “lmSupport” [56] packages, respectively.

Results and discussion

Socio-demographic information of Saen Thong subdistrict

In total, 824 sera from the eight villages were subjected to serological testing yielding a participation rate of 19.8% (total registered population of 4,145 in the subdistrict). Of the total population registered in the system, it is likely that a proportion of the population (particularly those working age) did not live in the area because they moved to work in other cities during the study which may have artificially lowered the participation rate. Serum samples were collected from participants who lived in 524 households covering almost a half (48.3%) of the total number of eligible households (1,083 registered households in total). Adults were the dominant age group (477 samples, 57.8%), followed by the elderly (250 samples, 30.3%), children (45 samples, 5.4%), young adults (37 samples, 4.5%), however 15 samples (1.8%) were not able to assigned an age group category as this information was not recorded during the interview. The majority of the participants were farmers (601 samples, 72.9%), with non-farmers accounting for 21.9% (181 samples) and unknown occupation for 42 samples (5.1%). We were unable to assign their occupation as this information was not recorded during the interview (more details in S1 Table).

Over a half of the participants (483 individuals, 58.6%) kept or raised animals including companion pets and farm livestock at home, whereas 341 individuals (41.3%) responded as having no animals. Among these, poultry (mainly backyard chickens) were the most popular (375 people, 45.5%) followed by 185 people (22.4%) responded to have dogs, 57 people (6.9%) cats, 14 people (1.7%) cattle and 6 people (0.7%) raised pigs at home.

Rickettsiosis past exposure in Saen Thong subdistrict: Associations of environmental and socio-demographic factors

Rickettsial exposure was noted in the Saen Thong subdistrict with seroprevalence of STG, TG and SFG of 9.8%, 9.0% and 6.9%, respectively (Table 1). Comparing the results of STG seroprevalence presented here to previous publications, the study population in Nan Province demonstrated a similar rate of exposure. This agreed with the review data of Bonell et al. (2017) that Orientia tsutsugamushi infection endemic in several Asian countries (i.e., Bangladesh, Indonesia, Lao PDR, Malaysia, Papua New Guinea and Sri Lanka), with seroprevalence ranging from 9.3%-27.9% [44]. Compared with previous studies in Thailand, the STG seroprevalence in Nan province was slightly higher than a previous reported among the populations in the three previously studied provinces of Thailand (4.2%), i.e., Chiang Rai, Khon Kaen and Nakhon Phanom provinces [33], however it should be noted that the study used a different antibody cut-off titer (≥ 1:128 compared with ≥1:100 in this study to detect IgG using IFA) to define positivity. In addition, our result demonstrated much lower STG positivity rate than the population in the western provinces, i.e., 62.5% Ratchaburi, 64.6% Petchaburi and 49.1% Kanchanaburi province [57], however it is important to recognise that a lower diagnostic cut-off value was used for interpretation (IgG using IFA, ≥1:50 compared with ≥1:100 in our study). In terms of murine typhus, seroprevalence of TG in the present study was slightly higher (9.0%) than the study in Chiang Rai, Khon Kaen and Nakhon Phanom provinces (4.2%), [33]; and similar rate of exposure with a previous study (8.0%) in suburban Bangkok which used the indirect immunoperoxidase assay with a diagnostic cut-off of ≥1:50 [58]. For SFG, our result in Nan province demonstrated higher seroprevalence (6.9%) than that from a previous study in Chiangrai, Khon Kaen and Nakhon Phanom provinces (0.8%), [33]. In contrast, another seropositivity study reported much higher SFG exposures in Chiang Rai (33%) and Tak provinces (27.3%), but the study used different assays (SFGR Enzyme Immunoassay IgG Antibody Kit), [59]. Therefore, it is important to consider the serological diagnostic methods and selection of the diagnostic cut-off values when comparing exposure rates, otherwise this has the potential to cause analysis bias and misinterpretation.

Table 1

Seroprevalence of human exposure to rickettsial infections, scrub typhus group (STG), typhus group (TG) and spotted fever group (SFG) in rural communities of Nan Province, Thailand.

Category	n	Scrub typhus group (STG)		Typhus group (TG)		Spotted fever group (SFG)
		Number positive	Seroprevalence[95% CI]	Number positive	Seroprevalence[95% CI]	Number positive	Seroprevalence[95% CI]
Village
Lowland agricultural landscape
No.1 –Ban Na Noon	161	2	1.2 [0.2–4.5]	39	24.2 [17.9–31.6]	16	9.9 [6.1–15.7]
No.2 –Ban Na Sai	113	3	2.7 [0.7–7.4]	9	8.0 [4.1–14.4]	4	3.5 [1.2–8.7]
No.3 –Ban Pho	98	4	4.1 [1.4–10.1]	7	7.1 [3.4–14.1]	6	6.1 [2.7–12.6]
No.8 –Ban Hae	71	0	-	9	12.7 [6.5–22.4]	4	5.6 [1.9–13.8]
Upland forested landscape
No.4 –Ban Huak	141	13	9.2 [5.2–15.1]	3	2.1 [0.6–6.2]	11	7.8 [4.1–13.4]
No.5 –Ban Nam Krai	64	8	12.5 [5.8–23.2]	1	1.6 [0.1–8.3]	4	6.3 [2.2–15.3]
No.6 –Ban Huay Muang	122	32	26.2 [19.1–34.8]	5	4.1 [1.6–9.3]	4	3.3 [1.1–8.1]
No.7 –Ban Santisuk	54	19	35.2 [22.9–49.1]	1	1.9 [0.1–9.8]	8	14.8 [6.9–26.7]
Landscape type
Lowland agricultural area	443	9	2.0 [1.0–3.8]	64	14.4 [11.3–18.0]	30	6.8 [4.7–9.6]
Upland forested area	381	72	18.9 [15.2–23.2]	10	2.6 [1.4–4.8]	27	7.1 [4.8–10.2]
Gender
Male	352	56	15.9 [12.3–20.1]	31	8.8 [6.2–12.3]	27	7.7 [5.2–10.9]
Female	469	25	5.3 [3.6–7.7]	43	9.2 [6.8–12.1]	29	6.2 [4.3–8.7]
Age group
Children (9–17)	45	1	2.2 [0.1–11.8]	1	2.2 [0.1–11.8]	1	2.2 [0.1–11.8]
Young adult (18–35)	37	0	-	2	5.4 [0.9–18.5]	4	10.8 [3.8–25.4]
Adult (36–60)	477	39	8.2 [5.9–11.0]	44	9.2 [6.9–12.1]	33	6.9 [4.9–9.6]
Elderly (>60)	250	40	16.0 [11.8–21.1]	27	10.8 [7.3–15.3]	18	7.2 [4.5–11.1]
Occupation
Farmer	601	63	10.5 [8.2–13.2]	59	9.8 [7.6–12.4]	39	6.5 [4.7–8.8]
Non-farmer	181	14	7.7 [4.6–12.6]	13	7.2 [4.0–12.1]	15	8.3 [4.8–13.2]
Total	824	81	9.8 [7.9–12.1]	74	9.0 [7.2–11.1]	57	6.9 [5.3–8.8]

There was evidence of multiple positivity with the three rickettsial serogroups in a small proportion of participants. However, no individual demonstrated multiple positivity with all three rickettsial groups. Multiple positivity was occurred in 10 (5.2%), 6 (3.1%) and 3 (1.6%) cases between STG-SFG, TG-SFG and STG-TG, respectively (Fig 2). However, it is recognised that serological cross-reactivity among SFG and TG [60] may occur, which could account for the dual-positivity between these antigenic groups.

Fig 2

Venn diagram represents number of participants exposed with STG, TG, SFG and co-exposures.

Relative proportions of the rickettsiosis exposures are provided in parentheses.

GLM analysis revealed that environmental factors, including landscape type and socio-demographic parameters, such as gender, occupation, age, educational level, and number of domestic animals in possession (dog, cattle and poultry) was significantly associated with higher rickettsial antibody levels in the communities (Table 2). Inhabitants from the villages located in upland-forested area (Village 4, 5, 6 and 7) demonstrated significantly higher STG seroprevalence (9.2% to 35.2%) and higher level of STG exposure (titer), compared to those villages (Village 1, 2, 3 and 8) in the lowland-urbanized area (none to 4.1%), (chi-square = 51.97, p < 0.0001), (Figs 1A and 3). Individuals located in upland-forested areas (increased habitat complexity and biodiversity) would have higher rates of exposures to chigger mites resulting in higher STG seroprevalence than those who live in urban environments (7,34). The reverse trend was observed for TG exposure, where the villages in the lowland-urbanized area demonstrated significantly higher seroprevalence (7.1% to 24.2%) and higher TG titer level compared to those villages in the upland-forested area (1.6% to 4.1%), (chi-square = 28.26, p < 0.0001), (Figs 1B and 4). Individuals located in urban environments may have potentially higher rates of exposure to fleas (the vector for murine typhus) parasitizing on rats and companion pets [61,62] resulting in higher TG seroprevalence compared with those individuals that are located in more forested or non-urban environments. These trends are in accordance with the study of scrub typhus and murine typhus cases in Lao PDR [12] and Malaysia [63]. We found no significant effect of landscape type neither on SFG seroprevalence nor antibody titer level of SFG exposure. Distribution maps of STG-, TG- and SFG-positive individuals from each village are provided in S1–S3 Figs.

Table 2

GLM best model selections after 1,050 total computed models based on AICc to determine socio-demographic and environmental factors explaining level of STG, TG and SFG past exposures in the study area.

Similar initial models are applied comprising with the following variables: Landscape type, Gender, Occupation, Age, Educational level, Dog number, Cat number, Pig number, Poultry number and Cattle number (with Poisson distribution after checking data distribution using “fitdistrplus” package in R freeware). Analysis of deviance (type II test) significant level for each variable is indicated as following: *p = 0.05–0.01; **p = 0.01–0.001 and ***p < 0.001. VIF = Variance inflation factor; Wr = Akaike’s weights.

Dependent variable	Explicative variable	Estimate	Standard Error	p-value	VIF	AICc	Wr	Model power	Goodness of fit (R2)
STG titers	Landscape type (upland forested area)***	1.212	0.013	< 0.0001	1.184	9625.156	0.109	1.000	0.999
	Gender (male)***	0.778	0.010	< 0.0001	1.029
	Occupation (non-farmer)	-0.019	0.012	0.107	1.045
	Age***	0.035	0.001	< 0.0001	1.397
	Educational level***	-0.839	0.009	< 0.0001	1.412
	Dog number***	0.197	0.004	< 0.0001	1.161
TG titers	Landscape type (upland forested area)***	-1.771	0.031	< 0.0001	1.364	7561.437	0.048	1.000	0.989
	Occupation (non-farmer)**	0.107	0.025	< 0.0001	1.022
	Age***	0.023	0.001	< 0.0001	1.270
	Educational level***	-0.195	0.017	< 0.0001	1.286
	Cattle number***	0.087	0.004	< 0.0001	1.019
	Dog number***	0.327	0.011	< 0.0001	1.311
SFG titers	Occupation (non-farmer)***	0.138	0.026	< 0.0001	1.059	7640.394	0.101	1.000	0.994
	Age***	0.006	0.0001	< 0.0001	1.112
	Educational level***	-0.312	0.016	< 0.0001	1.276
	Poultry number***	0.006	0.0001	< 0.0001	1.327

Fig 3

Effect plots of explicative variables after best model fitting with GLM to estimate level of STG exposures in inhabitants of Saen Thong Subdistrict, Nan Province.

Marks on the X-axis represent individual observations.

Fig 4

Effect plots of explicative variables after best model fitting with GLM to estimate level of TG exposures in inhabitants of Saen Thong Subdistrict, Nan Province.

Marks on the X-axis represent individual observations.

Male participants demonstrated significantly higher STG seroprevalence (15.9%) when compared with females (5.3%), (chi-square = 19.47, p < 0.0001), (see Table 1). This may be explained by the need for males to enter into the forest for hunting or animal tending purposes. For TG and SFG positivity, no clear difference was observed between the two genders. Surprisingly, there was no discernible difference in rickettsial seroprevalence between farmer and non-farmer participants. One would normally associate farming with higher levels of scrub typhus seroprevalence, however it would depend where the farming was performed for example, whether the farming was performed close or remote from forested areas. In terms of educational level, the rickettsial exposures were found to be significantly more common among those with lower levels of education compared to those with higher education which had been previously identified in Lao PDR [12].

Domestic animals as influencing factor of rickettsiosis exposure in Saen Thong subdistrict

In terms of the human-animal interface, our results demonstrate a significant link between number of domestic animals owned and the level of human rickettsial exposures. Higher levels of STG, SFG and TG exposures were positively correlated with number of dogs, poultry and both dogs and cattle, respectively (Figs 3–5). Domestic animals can act as natural vertebrate reservoir hosts for Rickettsia spp. in Southeast Asia [2], together with the close contact between people, domestic animals and associated ectoparasites (e.g., ticks, mites, lice and fleas). These bring emerging or remerging zoonoses closer to humans.

Fig 5

Effect plots of explicative variables after best model fitting with GLM to estimate level of SFG exposures in inhabitants of Saen Thong Subdistrict, Nan Province.

Marks on the X-axis represent individual observations.

Dogs are one of the prime examples of this phenomenon as they have long been associated with humans for approximately 23,000 years ago, being the first domesticated species on earth [64]. Using a data science approach and network analysis, Morand et al. (2020), [3] revealed that domestic dogs (Canis lupus familiaris) were the most important species sharing a great number of potential zoonotic rickettsial species with human (e.g., R. conorii, R. felis, R. rickettsii, R. typhi, O. tsutsugamushi, Ehrlichia canis and Neorickettsia sennetsu) when compared with other animals. Recent publications have confirmed the role of domestic dogs as important reservoirs for Rickettsia felis [4,65]. The study, Ng-Nguyen et al. (2020), [4] demonstrated that dogs have ability to sustain R. felis for a long period being asymptomatic during the course of infection; and they can also act as biological vehicles passing the rickettsial bacteria to uninfected fleas (Ctenocephalides felis) through horizontal transmission. In Sri Lanka, all the three rickettsiosis groups were reported in dogs with a high exposure rates (seroprevalence) at 49% [66]. The information presented here, demonstrates that dogs may play a very important roles in epidemiology and transmission cycle of human rickettsioses. For surveillance and disease ecology aspects, dogs could be used as a sentinel animal to identify high disease burden foci to mitigate risk of rickettsiosis outbreaks in the future.

In addition to dogs, the numbers of cattle and poultry were also positively correlated with levels of human TG and SFG exposures, respectively. Normally, evidence of R. typhi exposure in companion pets (e.g., dogs and cats) or in wildlife (e.g., rodents and opossums) translates to increased risk of human infections [22]. To our knowledge, cattle has never been linked directly with transmission cycle of R. typhi. However, previous publications revealed potential occurrences of flea infestation on cattle; e.g., Ctenocephalides felis felis was found on buffalo calves in India [67] and on dairy calves in Australia [68]; Pulex irritans infested on cow and sheep in Iran [69]. In terms of poultry, several ectoparasite species such as biting lice (e.g., Lipeurus caponis, Goniodes dissimilis, G. gallinae, Menacanthus stramineus and Menopon gallinae), fleas (e.g., Echidnophaga gallinacea), ticks (e.g., Haemaphysalis wellingtoni) and mites (e.g., Ornithonyssus bursa, Dermanyssus gallinae, Megninia cubitalis and Pterolichus obtusus) parasitize domestic chickens in Thailand [70,71], and might be play a vectorial role for SFG rickettsial transmission. However, the study of Rickettsia-associated poultry is very limited in Southeast Asia. In the other parts of the world, information regarding rickettsial infections either in poultry or in ectoparasites associated with avian species have been documented but only in a limited manner. Antibodies against Rickettsia spp. (i.e., R. rickettsiii, R. parkeri and/or R. bellii) were detected in domestic chickens from Brazil [72]. Rickettsia sp. (closely related to R. africae) were detected in flea specimens, E. gallinacea collected from domestic chickens in Madagascar [73]. SFG rickettsiae (e.g., R. aeschlimannii, R. africae, R. helvetica, R. massiliae, R. monacensis and R. slovaca) were isolated from ticks of migratory birds in European countries [74-76]. Furthermore, R. helvetica and R. monacensis were detected in ticks removed from birds in Taiwan, alongside with other tick-borne pathogens such as Borrelia turdi, Anaplasma sp. and Ehrlichia sp [77]. These results highlight a prominent relationship among avian hosts and their ectoparasites in carrying and maintaining SFG rickettsiae in nature.

Further studies on ectoparasite burden in dogs, cattle, poultry and wild rodents as potential reservoirs of several ectoparasites of public health and veterinary importance. These studies should be combined with investigations of rickettsial infections in the animal hosts and their associated arthropod vectors in contrasting ecologies to better determine the role of these animals in zoonotic cycle of rickettsial transmission.

Demographic data of each village in Saen Thong Sub-district, Tha Wang Pha District, Nan Province, Thailand.

(DOCX)

Click here for additional data file.

Mapping of scrub typhus group (STG) seropositive cases by villages in Saenthong subdistrict, Nan Province.

Green and red circles indicate sero-negativity and sero-positivity of STG, respectively. Size of the red circles indicate level of STG positivity (IFA titers).

(TIF)

Click here for additional data file.

Mapping of typhus group (TG) seropositive cases by villages in Saenthong subdistrict, Nan Province.

Green and red circles indicate sero-negativity and sero-positivity of TG, respectively. Size of the red circles indicate level of TG positivity (IFA titers).

(TIF)

Click here for additional data file.

Mapping of spotted fever group (SFG) seropositive cases by villages in Saenthong subdistrict, Nan Province.

Green and red circles indicate sero-negativity and sero-positivity of SFG, respectively. Size of the red circles indicate level of SFG positivity (IFA titers).

(TIF)

Click here for additional data file.

8 Nov 2021

Dear Dr. Kittipong Chaisiri,

Thank you very much for submitting your manuscript "Risk factors analysis for neglected human rickettsioses in rural communities in Nan province, Thailand: A community-based observational study along a landscape gradient" 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.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

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

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Wen-Ping Guo

Associate Editor

PLOS Neglected Tropical Diseases

Nam-Hyuk Cho

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: Methods appear to be fine. I do not have the statistical expertise to critique the methods used, but they seem to be ok at a superficial level.

Reviewer #2: Methodology is well described and objectives are clear. Statistical analysis is correct. No ethical issues

Reviewer #3: Materials and Methods; Pg 7; Line 21 (Blood sample collection)

Please specify the details of the tubes used to collect serum or give the relevant reference

You could change it like this if needed:

Blood samples were collected and processed for rickettsial serology as described previously/elsewhere

--------------------

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: Results are valid

Reviewer #2: The analysis planned and presented matched each other

Results clearly and comprehensively presented

Quality of the images and tables is as per the standard requirements

Reviewer #3: Pg 10; lines 4 & 6, you have mentioned age and occupation could not be assigned in 1.8% and 5.1% respectively. Kindly give a reason/reasons for this. If data was not collected/ or was unclear, you can omit this statement

Pg 10; Line 7-11; Regarding individual interviewing ------ keeping these animals at home, respectively. Sentence is not clear (complicated) please re-write in simple words, so that it is easy to understand.

Pg 10 Line14-15: Simplify this sentence

--------------------

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 appear to be valid and based on the data.

Reviewer #2: Conclusions are supported by the data

Authors have explained the usefulness of the study with reference to Public health

Few limitations could have been added like need for exploring the burden of rodent and ecto parasites in the two contrasting settings

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: The standard of written English is not up to publication level. Suggest review by a native English speaker.

Introduction, line 14. The mites that appear to transmit Orientia sp in Chile are not of the Trombiculidae family.

Material & Methods, line 10/11. What is meant by "no communication problem" ? autism ? poor mobile phone coverage ?

I was surprised that only 6 sera (3.1%) showed cross-reactions between TG-SFG in IF serology. In my lab we see a much higher level of cross-reaction.

p. 12 line 1/2 "(non-exposure to 4.1%)" What does this mean ?

Table 1. Data is clear but why does village # 8 come after # 3 rather than after # 7 ? It looks untidy.

Reviewer #2: Cut-off titres for ST IgG IFA for epidemiological purposes as opined by few authors may be added.

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: An interesting study and well performed with clear data.

Reviewer #2: The study is well planned and meticulously carried out

Reviewer #3: Authors have conducted a study which expands the body of knowledge regarding the important factors contributing to rickettsial infections in Asia and Thailand in particular. As the methodology is robust, the results and the conclusions derived are reliable.

I have a few minor suggestions which are appended as comments.

Another suggestion is that the language and grammar though adequate in most places needs more attention

Example: Pg12; Line 24 (going to Pg13; Line 1): Lacking of education is recognized as one of poverty indices.

--------------------

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Reviewer #1: No

Reviewer #2: Yes: SELVARAJ STEPHEN

Reviewer #3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

21 Dec 2021

Submitted filename: Responses to reviewers_MS_Chaisiri et al 2021_final.docx

Click here for additional data file.

3 Feb 2022

Dear Dr. Chaisiri,

Thank you very much for submitting your manuscript "Risk factors analysis for neglected human rickettsioses in rural communities in Nan province, Thailand: A community-based observational study along a landscape gradient" 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.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

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

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Wen-Ping Guo

Associate Editor

PLOS Neglected Tropical Diseases

Nam-Hyuk Cho

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: line 9 of p7 states "age over 9 years" but line 12 states "children 7-9 years old". Which statement is correct ? Currently the 2 statements are inconsistent.

line 22 of p7 states "rpm". This is an inappropriate representation of centrifugal force as it can vary with the diameter of the centrifuge rotor. It should always be expressed as " x g ".

Reviewer #2: - Yes the objectives of the study is clearly stated in the testable hypothesis

- Study design is appropriately addressed to the stated objectives

- Sample size is sufficient to ensure adequate power to address the hypothesis being tested

- Statistical analysis used to support conclusions were correct

- Are there concerns about ethical or regulatory requirements being met? No

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: OK

Reviewer #2: - Does the analysis presented match the analysis plan? - Yes

-Are the results clearly and completely presented? - Yes

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

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: OK

Reviewer #2: -Are the conclusions supported by the data presented? - Yes

-Are the limitations of analysis clearly described? - Yes

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

-Is public health relevance addressed? - Yes

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: two minor changes only required

Reviewer #2: Accept

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: OK

Reviewer #2: May be accepted for publication

Reviewer #3: (No Response)

--------------------

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

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

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

Reviewer #1: No

Reviewer #2: Yes: SELVARAJ STEPHEN

Reviewer #3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

9 Feb 2022

Submitted filename: Response to the reviewers_resub2.docx

Click here for additional data file.

12 Feb 2022

Dear Dr. Chaisiri,

We are pleased to inform you that your manuscript 'Risk factors analysis for neglected human rickettsioses in rural communities in Nan province, Thailand: A community-based observational study along a landscape gradient' 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.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

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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,

Wen-Ping Guo

Associate Editor

PLOS Neglected Tropical Diseases

Nam-Hyuk Cho

Deputy Editor

PLOS Neglected Tropical Diseases

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

18 Mar 2022

Dear Dr. Chaisiri,

We are delighted to inform you that your manuscript, "Risk factors analysis for neglected human rickettsioses in rural communities in Nan province, Thailand: A community-based observational study along a landscape gradient," 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