High Seroprevalence Against Typhus Group and Spotted Fever Group Rickettsiae in Rural Indigenous Populations of Peninsular Malaysia

Introduction

Rickettsioses of the typhus group (TG) and spotted fever group (SFG) are vector-borne emerging zoonotic bacterial infections worldwide. Whereas Rickettsia typhi, a member of the TG rickettsiae, is transmitted to humans by fleas from a rodent reservoir, SFG rickettsiae are transmitted by ticks from various mammals. A growing number of rickettsial species are discovered with importance for human disease (Parola et al. 2013). Particularly in tropical regions, rickettsial infections play a larger role than previously thought.

Initial studies on these pathogens and their respective clinical diseases in Malaysia were represented by a few investigations in humans, vectors, and wildlife in the 1960s–1980s (Marchette 1966, Brown et al. 1977, 1984). The first and only isolation of a rickettsial species in Malaysia so far, R. typhi, succeeded in 1932 from a febrile patient in the capital city Kuala Lumpur. In this context, the corresponding human disease, murine (endemic or “urban”) typhus, was described in Malaysia for the first time (Lewthwaite and Savoor 1936). The detection of rickettsial DNA (R. typhi, Rickettsia raoultii-like organism, and Rickettsia felis-like organism) from febrile patients in the country has only recently been successful in a case series from Kuala Lumpur (Kho et al. 2016). Until today, the rickettsial species responsible for the spotted fevers occurring after an infective tick bite in humans is unknown in Malaysia, but, based on scanty molecular data from tick vectors feeding on snakes and domesticated animals, might be similar to R. raoultii, Rickettsia tamurae, and Rickettsia heilongjiangensis (Kho et al. 2015, 2017), or, based on geography, Rickettsia honei (Parola et al. 2013).

Several seroprevalence studies with febrile and healthy patients have been performed in the country, showing high infection rates with R. typhi and members of the SFG rickettsiae in rural and urban areas (Brown et al. 1984, Tee et al. 1999, Sekhar and Devi 2000, Tay et al. 2002a, b, Tay et al. 2003). However, rickettsioses in indigenous people (Orang Asli) have very scarcely been investigated so far in Malaysia (Tay et al. 2000, Kho et al. 2017). Here, we conducted a seroprevalence study among healthy indigenous people from peninsular Malaysia for TG and SFG rickettsioses in several rural and peri-urban settlements.

Materials and Methods

A total of 544 sera from Orang Asli who were believed to be healthy and were afebrile from nine settlements in different geographical areas of peninsular Malaysia were obtained after venipuncture (2014–2015). The nine study sites were located in five different federal states and had different geographical characteristics (Table 1). The Orang Asli communities at Kuala Betis, Pos Betau, Pos Iskandar, and Kampung Layau are under a resettlement village scheme, whereas the Orang Asli communities at Sungai Bumbun, Sungai Miak, Gurney, Kampung Pangsun, and Sungai Perak lived in small traditional villages. The study cohort encompassed 406 children (<13 years of age), 20 adolescents (13–17 years of age), and 118 adults (≥18 years of age) with an overall male:female ratio of 0.8:1. Their age ranged from 1 to 80 years, with a median of 11 (interquartile range 9–13) years. The study was approved by the Human Ethics Committee of the University of Malaya (Reference no.: 638.36). Sera were analyzed by in-house immunofluorescence assays (IFA) for IgG antibodies against TG and SFG rickettsiae. TG rickettsial IFA was performed using R. typhi strain Wilmington, and SFG rickettsial IFA was conducted using R. honei strain RB^T, both with reference values of <1:80. The strains were grown in L929 mouse fibroblast cell culture.

Results

The overall seroprevalence of rickettsiosis in indigenous people was 48.5% (264/544), with 27.9% seroprevalence against TG rickettsiae (152/544) and 20.6% seroprevalence against SFG rickettsiae (112/544). In a total of 7.9% of the study participants, antibodies against both rickettsial groups could be found in the same individual (43/544). There were no gender differences in seropositivity against TG or SFG rickettsiae. The highest rate of seropositivity against TG rickettsiae was noted in the age group of 1–4 years, whereas the highest rates of seropositivity against SFG rickettsiae was noted in the age groups of 1–4 years and >18 years (Table 2). In the various settlements, seroprevalences against TG rickettsiae ranged from 18.8% to 54.8% (median 26.3%), whereas seroprevalences against SFG rickettsiae ranged from 0% to 38.7% (median 19.6%). Simultaneous seropositivity in single individuals against TG and SFG rickettsiae ranged from 0% to 29% (median 10.5%). The highest seroprevalence against TG and SFG rickettsiae with nearly 54.8% and nearly 38.7%, respectively, was found in indigenous people living in Sungai Miak. Sungai Miak was also the settlement with the highest simultaneous seropositivity in single individuals against TG and SFG rickettsiae (29%). Except for Sungai Perak and Gurney, TG rickettsioses seroprevalence in indigenous people was higher than SFG rickettsioses seroprevalence. No antibodies against SFG rickettsiae could be found in individuals from Sungai Bumbun. There were no differences in the seroprevalence distribution between villages under the resettlement scheme and villages that were traditional (Fig. 1).

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FIG. 1.

Map of the study sites in peninsular Malaysia and seroprevalence against SFGR and TGR in indigenous people. Borders with Thailand (north) and Singapore (south) are shown. Rickettsia honei immunofluorescence tests were used for testing of seropositivity against SFGR and Rickettsia typhi immunofluorescence tests were used for the serology of TGR. A high seroprevalence against both rickettsial groups is evident, except for the indigenous people of the Sungai Bumbun settlement in whom no antibodies against SFGR could be demonstrated. TGR, typhus group rickettsiae; SFGR, spotted fever group rickettsiae.

Discussion

Due to the basic lifestyle of many Orang Asli in rural areas (houses without piped water, electricity, or sanitary toilets), indigenous people are likely the population most exposed to vectors of rickettsial pathogens in peninsular Malaysia. In our study comprising 544 healthy Orang Asli, we show high past infection rates as reflected by a high seroprevalence against TG (27.9%) and SFG (20.6%) rickettsiae in these populations.

In an older report of a group of 18 febrile indigenous people from peninsular Malaysia who had been included in a larger study with febrile patients from rural regions, antibodies against TG and SFG rickettsiae were shown in roughly similar dimensions (22.2% and 27.8%, respectively; Tay et al. 2000) to our findings. A breakdown by geographical location and ecology of the settlements for the indigenous group had not been performed, however. In a recent investigation encompassing 102 healthy indigenous people from six rural villages, a 50% seroprevalence against SFG rickettsiae was shown (Kho et al. 2017). However, the seropositivity against R. typhi in the indigenous population had not been investigated and ecological properties of the different villages had not been analyzed. In our study of several hundred healthy indigenous individuals, we have analyzed the seroprevalences in indigenous people according to geography and ecology of the nine settlements against both TG and SFG rickettsiae. While our results of TG and SFG seroprevalences were comparable with the older study with only a few indigenous people (Tay et al. 2000), the SFG seroprevalences of a recent investigation with more Orang Asli participants (Kho et al. 2017) were more than twice as high as in our study, which might be due to geographical and/or ecological factors that had not been investigated. Except for individuals from the Sungai Bumbun settlement in our study, who did not exhibit antibodies against SFG rickettsiae, and participants from Sungai Miak, who had the highest seroprevalences against TG and especially against SFG rickettsiae, the seroprevalences against the two rickettsial groups were strikingly comparable in people from all other settlements in our study. Sungai Miak, a settlement surrounded by plantations like many other settlements in our investigation, is located in secondary hill land forest, an ecological setting that might favor tick populations as vectors for SFG rickettsiae. Rickettsial strains closely related to R. raoultii have been described in Rhipicephalus sanguineus and Haemaphysalis spp. ticks infesting peri-domestic animals in rural Malaysian villages (Kho et al. 2017). Sungai Bumbun, in the vicinity of Kuala Lumpur, is also surrounded by oil palm plantations but is located at the coast line. This setting might favor rat populations as reservoir for R. typhi, and in concordance with our observation, R. typhi infections are believed to occur more often in tropical coastal environments (Walter et al. 2012). However, only 16 study participants came from Sungai Bumbun, and thus, these data might not be representative.

In contrast to our study with indigenous people, the seropositivity against R. typhi in healthy nonindigenous individuals from rural regions of Malaysia was much lower (3%; Tee et al. 1999) but had reached 45% after a local outbreak of murine typhus in a village (Brown et al. 1977). Our findings thus likely reflect past outbreaks in the indigenous communities or, due to the living conditions of the Orang Asli, increased contact to flea vectors and rat reservoir hosts. Also in contrast to our investigation, a very high SFG rickettsial seroprevalence of 57.3% was seen in healthy nonindigenous individuals from a rural area (Tee et al. 1999). This prevalence was found in rubber estate workers and likely reflects a special working situation.

Our study shows gender-independent seroprevalences against both groups of rickettsiae, but high past infection rates in the youngest study participants and in adults >18 years of age. While an increasing seroprevalence with increasing age is an expected finding, the high past infection rate in the youngest (1–4 years of age) to TG and SFG rickettsiae deserves further attention. A high exposition to the arthropod vectors in the children's living environment that is often frequented by cats, dogs, and likely rats might be an explanation. As a limitation of our study, serological cross-reactions exist between both the TG and the SFG rickettsiae with R. felis, a member of the transitional group of rickettsiae. Thus, as R. felis infections were not analyzed in our study with further serological assays, some past infections in the Orang Asli may have been due to R. felis instead of TG or SFG rickettsiae.

Conclusions

Our study shows high past infection rates against rickettsiae in indigenous populations in rural Malaysia. Future studies are needed to understand the ecological and geographical differences, and thus, the risk factors and preventive measures for TG and SFG rickettsioses in these areas. This is especially important for young children and adults who are highly exposed to the transmitting vectors.