High Prevalence of Tropheryma whipplei in Lao Kindergarten Children

Background Tropheryma whipplei is a bacterium commonly found in feces of young children in Africa, but with no data from Asia. We estimated the prevalence of T. whipplei carriage in feces of children in Lao PDR (Laos). Methods/Principal Findings Using specific quantitative real-time PCR, followed by genotyping for each positive specimen, we estimated the prevalence of T. whipplei in 113 feces from 106 children in Vientiane, the Lao PDR (Laos). T. whipplei was detected in 48% (51/106) of children. Those aged ≤4 years were significantly less frequently positive (17/52, 33%) than older children (34/54, 63%; p< 0.001). Positive samples were genotyped. Eight genotypes were detected including 7 specific to Laos. Genotype 2, previously detected in Europe, was circulating (21% of positive children) in 2 kindergartens (Chompet and Akad). Genotypes 136 and 138 were specific to Chompet (21% and 15.8%, respectively) whereas genotype 139 was specific to Akad (10.55%). Conclusions/Significance T. whipplei is a widely distributed bacterium, highly prevalent in feces of healthy children in Laos. Further research is needed to identify the public health significance of this finding.


Introduction
Tropheryma whipplei is a common bacterium found in human feces; its prevalence depends mainly on age, exposure to saliva and/or human feces, and geographical area [1][2][3][4][5][6][7][8]. The prevalence in feces is estimated to be between 1 and 11% of European adults, but reaches 75% in children <4 years old in rural Senegal and 38% in relatives of those with Whipple's disease or chronic carriers of the bacterium in France [1,3,5,7]. T. whipplei carriage was estimated to be 12.9% in homeless people in France and between 12 to 26% among European sewerage workers [1,6,9]. In France, this bacterium was also detected in the feces of 15% of children aged 2 to 4 years with gastroenteritis but not in control subjects of the same age [8].
T. whipplei is viable in culture on Specific Axenic Medium (SAM) using the feces of patients with Whipple's disease [10] and data strongly suggest that it is contagious [3,5,6,8,11,12]. Indeed, specific clones were observed with a high prevalence in children from the same city with gastroenteritis, among individuals from the same village in Senegal, among relatives of patients with Whipple's disease and chronic carriers and among homeless people living in the same temporary visitor centre [3,5,6,8,11].
Recent studies suggest that T. whipplei infection is probably contracted during childhood [12]. There is a high prevalence of T. whipplei in rural Senegalese children <4 years old, up to 75%, that decreases to 30% among the 5-10 years old [12]. Moreover, in Senegal for the majority of children <6 years old, but not for those >6 years old, the presence of an immune response against the bacterium is consistently associated with the presence of the bacterium in their feces. This suggests that primary infection occurs before 5 years of age.
Data about T. whipplei in Asia are few, although T. whipplei has been reported in the saliva of healthy Koreans [17][18][19][20]. We therefore sought to determine the prevalence of T. whipplei carriage and identify genotypes circulating among children in Laos.

Population recruitment and ethical statement
The initial objective of this study was to look for enterovirus carriage in Lao kindergarten children. It is only secondarily that the carriage of T. whipplei has been investigated in children. For this purpose, we analyzed the feces from healthy children collected between 2010 and 2012 in the framework of enterovirus study [21] in 3 kindergartens in Vientiane City, Lao PDR (Laos), located in 3 different villages (Sailom, Chompet, and Akad) (Fig. 1). Although children were present at the kindergarten the day of sampling and thus considered healthy, they were also examined for conjunctival suffusion, pharyngeal erythema, rhinitis or runny nose, diarrhea, vomiting, mouth ulcer, vesicle on hands, and vesicles on feet.
Fresh feces were collected in sterile containers from children aged 1-7 years whose guardians/parents gave informed written consent. After collection, each specimen was stored at+4°C up to 24 hours. Each specimen was vortexed in MEM medium (Minimum Essential Medium, Invitrogen, Carlsbad, CA, USA), that is a commonly used in Lao laboratory as viral transport medium as recommended by WHO, with glass beads for 1 minute. Then, after 30 minutes centrifugation at 3,000g at +4°C (adaptation of the protocol from the polio laboratory manual 2004 WHO/IVB/04.10 with the replacement of chloroform by antibiotics and fungizone), the supernatant was recovered and stored at -80°C for culture of enterovirus but the presence of antibiotics in the transport medium prevented the culture of T. whipplei. After enterovirus screening in Laos, 113 feces, from 106 children, were shipped on dry ice to virology laboratory in Marseille, France, for enterovirus confirmation (94/113, 83% were confirmed to be positive for enterovirus), all were positive for β-actin and included in the present study.
The study was granted ethical approval by the Lao National Ethics Committee for Health Research (331/NECHR) and by the Oxford Tropical Research Ethics Committee (OXTREC 020-10), with amendments from both committees for feces collection for T. whipplei.

Molecular assays
DNA was extracted from 100μl of each sample using Machery Nagel DNA Mini Kit (Machery Nagel Gmbh & Co., Düren, Germany) in accordance with the manufacturer's instructions. Quantitative real-time PCR (qPCR) was performed using CFX96TM Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) with the FAST qPCR Master mix No ROX (Eurogentec, Liege Science Park, Belgium).
Genotyping of T. whipplei was attempted for each positive specimen and performed as described [26]. Briefly, four highly variable genomic sequences (HVGSs) of T. whipplei were amplified and sequenced. The genotype was obtained after the analysis of each four HVGSs obtained from each specimen and compared with those available in both the GenBank database and our internal laboratory database in order to determine their corresponding genotype.

Statistical analysis
PASW statistics 17 software (SPSS, Chicago, IL, USA) was used for data analysis and nonparametric values were compared using χ2 or the Fisher's exact tests. Statistical significance was defined as p<0.05.
Among 51 Lao children T. whipplei positive, genotypes were obtained for 19 samples. We obtained 8 different genotypes. Genotype 2 has been detected in France and Germany but 7 were new genotypes described from Laos for the first time (genotypes 136, 137, 138, 139, 140,  141, and 142; Fig. 3). Genotype 2 was detected in 4/19 (21%) children, 3 were from Chompet and one from Akad kindergarten. New genotypes 136 and 138 were both detected in Chompet kindergarten in 4/19 (21%) and 3/19 (15.8%), respectively, whilst genotype 139 was identified in Akad kindergarten in 2/19 (10.5%). The new genotypes 140, 141, and 142 were each detected in one child in each kindergarten (Fig. 3). Only one genotype (137) occurred in all three kindergartens, but was only identified once in each. From the two children positive on two different occasions, we obtained successive genotypes from only one child; in whom the same genotype (138) was found after an 8 month interval.
Among the children included in this study, only 1 presented pharyngeal erythema and 15 rhinitis. All the other symptoms, including diarrhea, were not observed in any of the children. Among the 51 children positive for T. whipplei, 5 exhibited rhinitis (9.8%).

Discussion
We describe T. whipplei in the stools of apparently healthy children for the first time in Asia. The data are based on strict experimental procedures, including positive and negative controls to validate each assay. The positivity of the β-actin gene was checked and confirmed for each included sample [2] and each T. whipplei qPCR was systematically confirmed by the successful amplification of another specific DNA sequence. Overall, the prevalence of T. whipplei carriage in feces from Lao children aged <7 years is 48% [5]. The only symptom observed among children positive for T. whipplei was rhinitis (9.8%) but this symptom was also observed in 16.1% of the negative children. Thus, no link between the presence of rhinitis and that of the bacterium can be established.
Lao children were contaminated with T. whipplei significantly later than those of rural Senegal. In France T. whipplei DNA was not detected in feces of healthy children <6 years old [8].
As far as we aware no one has investigated T. whipplei fecal carriage in Asia, but it has been detected in Korea among 1.5% of healthy peoples' saliva, but not in any of 108 small-bowel biopsies in Malaysia or in any of ten marginal supragingival plaque samples from Chinese patients with gingivitis [17,18,20]. In rural Senegal, the first contact with the bacterium occurred mainly in children before 4 years of age, with the prevalence of T. whipplei in feces declining from 75% in those 4 years to 30.3% among those >4 years [5]. This trend was confirmed by a recent study performed in Gabon, with a high prevalence of T. whipplei in children <5 years old, up to 40%, with a lower prevalence (36.4%) among those aged 5-10 years and 12.6% among those of 11-20 years [27]. In contrast, in Laos we observed a significantly lower prevalence in children 4 years (33%) than in children >4 years (63%).
A current hypothesis is that T. whipplei is transmitted between humans according to hygiene conditions [5,7], through saliva (oral-oral transmission) if hygiene conditions are good, or feces (fecal-oral transmission) if hygiene poor [28]. T. whipplei has rarely been identified in environmental samples and humans appear to be the main reservoir and source of this bacterium in rural Senegal [7,12]. Poor sanitary conditions and close contacts between children from the earliest age could explain the difference in exposure to T. whipplei between Senegalese and Gabonese children versus Laotian children for whom contacts with other children are probably much less common before entering kindergarten [7,27]. In Senegal, households with high prevalence of T. whipplei carriage had also significantly less access to toilets, supporting the interhuman transmission of the bacterium being from human feces through hand transmission. In Laos, toilets were present at home for all included children and also at kindergarten. However, no information was available about the hand washing practice. Finally, close contact could play a role in transmission of T. whipplei as well as the lack of hand hygiene [6].
In Europe and Africa, 135 different genotypes have been identified from 359 people positive for T. whipplei. Eight genotypes of T. whipplei, including 7 newly described, were identified in feces from healthy Lao children. Previous work showed that T. whipplei genotypes found in Senegal and in Europe were different [2,[4][5][6]. Genotype 2 was detected in 4/19 (21%) Lao children, 3 were from Chompet and one from Akad kindergarten. This genotype had been previously detected in 12 out of 359 other people (3.34% versus 21%; p<0.001) in Europe, but not in Senegal. The presence of highly prevalent clones specific to some kindergartens, such as genotypes 136 and 138 in Chompet kindergarten, suggest that these clones are probably epidemic and support the contagiousness of T. whipplei. Furthermore, it is important to underline that currently no specific genotypes have been associated with disease versus asymptomatic carriage. Besides, a same genotype may be observed in acute infections, chronic infections and/or asymptomatic carriage [4]. Overall, T. whipplei genotyping is a useful tool to differentiate a relapse of Whipple's disease to a reinfection with another genotype but also to detect circulating or epidemic clones [3][4][5][6]8,26,29,30].
Chronic infections due to T. whipplei such as classic Whipple's disease or endocarditis have been scarcely reported in Asia and no such patients have been reported from Laos or adjoining North East Thailand [31]. To the best of our knowledge, only two Japanese patients and three individuals from India with Whipple's disease have been reported in the literature [31,32]. These data reinforces that although many people are exposed to T. whipplei, only few individuals develop chronic infection [4]. The prevalence of T. whipplei carriage is high in areas where poor sanitary conditions and close contacts are observed but T. whipplei seems to be an opportunistic bacterium that causes chronic infections probably linked to an, as yet unknown, specific immunological defect. Indeed, several dysregulated T-cell functions, a persistent deficiency in, or the absence of, a T. whipplei-specific T-helper cell-1 response have been observed in the patients with classic Whipple's disease [33]. Furthermore, the susceptibility of patients to Whipple's disease, with relapses and reinfections with new strains, along with a lack of, or a weak, serological response supports also a specific T. whipplei immunodeficiency [30,34]. Several observations support a genetic predisposition for Whipple's disease. Indeed, the majority of patients reported to date are Caucasian, mainly males [4,29,35,36]. It is also remarkable for a rare disease that six familial proven cases of Whipple's disease have been reported in the literature [15,36,37]. A higher frequency of the HLA alleles DRBÃ13 and DQB1Ã06 in patients with classic Whipple's disease has been observed [38]. All these data may explain why T. whipplei carriage is so high in Africa and Asia relative to Europe, whereas there are so few or no confirmed cases of chronic Whipple's disease in these populations.
The high prevalence of T. whipplei carriage in the feces of Lao children, confirms that this bacterium occurs in South East Asia and that infection occurs in childhood. Further research is needed to better understand the natural history, public health significance, and effect of T. whipplei on the health in Laos.