Transmission of Enterocytozoon bieneusi genotype a in a Thai orphanage

Am. J. Trop. Med. Hyg., 73(1), 2005, pp. 104–107 Copyright © 2005 by The American Society of Tropical Medicine and Hygiene

TRANSMISSION OF ENTEROCYTOZOON BIENEUSI GENOTYPE A IN A THAI ORPHANAGE SAOVANEE LEELAYOOVA, ITTISAK SUBRUNGRUANG, RAM RANGSIN, PORNTIP CHAVALITSHEWINKOON-PETMITR, JEERAPAN WORAPONG, TAWEE NAAGLOR, AND MATHIRUT MUNGTHIN* Department of Parasitology, and Department of Military and Community Medicine, Phramongkutklao College of Medicine, Bangkok, Thailand; Department of Protozoology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Center for Biotechnology and Department of Biotechnology, Institute of Science and Technology for Research and Development, Salaya, Nakornpratom, Thailand

Abstract. A cross-sectional study of Enterocytozoon bieneusi infection in children who lived in an orphanage in Bangkok, Thailand was conducted in April 2003. Two hundred ninety stool specimens were collected and examined under light microscopy after staining with gram-chromotrope. Confirmation of E. bieneusi was done using transmission electron microscopy. Of 290 samples, 12 (4.1%) were positive for E. bieneusi. Genotypic characterization of 10 E. bieneusi showed that all were genotype A, which might indicate the same source of infection. Multivariate analysis showed that orphans who were 12–23 months old, girls, and living in one particular house were independently associated with E. bieneusi infection. Our study suggests that E. bieneusi infection in this orphanage might be transmitted person to person. April 2003. The study was reviewed and approved by the Ethical Committee of the Medical Department of the Royal Thai Army. Stool specimens collected from 290 orphans were stained with gram-chromotrope as previously described13 and examined by light microscopy (100× objective) for E. bieneusi. Confirmation of infection with E. bieneusi was performed using electron microscopy.14 Genotypic characterization of E. bieneusi was determined by polymorphic sites on the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene. This region was amplified by a polymerase chain reaction (PCR) using primers MSP3 (5⬘-GGAATTCACACCGCCCGTC(A/G) (C/ T)TAT-3⬘) and MSP4B (5⬘-CCAAGCTTATGCTTAAGTCCAGGGAG-3⬘) as previously described.15 The specific primer pair amplifies 508-basepair fragment of E. bieneusi containing 122 basepairs of the 3⬘ end of the small subunit rRNA gene, 243 basepairs of the ITS, and 143 basepairs of the 5⬘ region of the large subunit rRNA gene. The PCR products were sent to Bioservice Unit in Bangkok, Thailand for DNA sequencing using a 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). Nucleotide sequences were determined with Sequencer program (Gene Codes Corporation, Inc., Ann Arbor, MI). The genotype of E. bieneusi from each specimen was confirmed by the homology of the sequenced PCR product to the published sequence in GenBank. To determine the risk factors of E. bieneusi infection, standardized questionnaires concerning demographic data were used in this study. There were 10 small houses in this orphanage. Orphans positive for human immunodeficiency virus (HIV) lived in the same house, while HIV-negative orphans lived separately from the others. The child care providers of each house were asked to complete the questionnaires for each child. Significant differences among groups were assessed using the chi-square test with a 95% confidence interval (CI). Univariate analysis was performed using Epi-Info version 6.04b (Centers for Disease Control and Prevention, Atlanta, GA). Odds ratios with 95% CIs and P values were calculated to compare outcomes among study groups. Multivariate logistic regression using SPSS for Windows version 9.6 (SPSS, Inc., Chicago, IL) was performed for multivariate analysis to assess the independent association of the risk factors and E. bieneusi infections.

INTRODUCTION Intestinal microsporidiosis, an opportunistic infection that causes chronic diarrhea in patients with acquired immunodeficiency syndrome (AIDS), is caused by two species of microsporidia (Enterocytozoon bieneusi and Encephalitozoon intestinalis).1,2 These obligate intracellular eukaryotes are now considered as fungi by molecular phylogenic analysis.3 Enterocytozoon bieneusi is the most common microsporidial infection found in humans.4,5 It also causes diarrhea in other immunosuppressive conditions 6 and immunocompetent hosts.7,8 The epidemiology of these infections is still unclear in many aspects. It is assumed that these organisms are transmitted via the fecal-oral route since their spores are excreted via stool. A few studies showed the evidence supporting the role of person-to-person transmission in intestinal microsporidiosis.9,10 Thus, sources of the infection are apparently from those who excrete E. bieneusi spores both with and without diarrhea. Asymptomatic E. bieneusi infections were reported in a few studies,11,12 and could be one of the major sources of infections. Our surveys in a Thai orphanage located in Bangkok, Thailand found that E. bieneusi infection is common in this population (Mungthin M, unpublished data). Although these children usually had no gastrointestinal symptoms, monitoring of their infections will provide more epidemiologic information. In the present study, we conducted a cross-sectional study of E. bieneusi infection in these orphans to determine the prevalence and associated risk factors. In addition, genotypic characterization of E. bieneusi isolates from these orphans was also performed. MATERIALS AND METHODS A cross-sectional study of intestinal microsporidiosis was conducted in Thai children who lived in an orphanage (Phyathai Babies’ Home) located in Bangkok, Thailand in * Address correspondence to Mathirut Mungthin, Department of Parasitology, Phramongkutklao College of Medicine, 315 Ratchawithi Road, Ratchathewi, Bangkok, 10400, Thailand. E-mail: [email protected]

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E. BIENEUSI GENOTYPE A INFECTION IN A THAI ORPHANAGE

RESULTS Of the 290 stool specimens collected from the orphans, E. bieneusi spores were detected in 12 specimens using gramchromotrope staining. All these specimens were then confirmed by transmission electron microscopy. Unfortunately only 10 specimens had sufficient material for PCR amplification to determine the genotypic characterization of E. bieneusi isolated from these orphans. The PCR amplification of the 10 specimens gave the expected PCR product of 508 basepairs. The ITS of these specimens indicated that all 10 isolates were E. bieneusi type A (GenBank No. AY357195AY397204). The characteristics of the orphans enrolled in the study are shown in Table 1. One HIV-positive child and 11 HIVnegative children between 14 and 33 months of age were positive for E. bieneusi. Significant differences were observed in the prevalence of E. bieneusi infections among different age groups and houses. The prevalence of E. bieneusi infections among HIV-positive and HIV-negative orphans was 2.6% (95% CI ⳱ 0.1–15.1%) and 4.4% (95% CI ⳱ 2.3– 7.9%), respectively. None of the infected orphans had gastrointestinal symptoms during the surveys. TABLE 1 Characteristics of 290 children in the orphanage*

Characteristics

Age group (months) 0–11 12–23 24–35 36–47 ⱖ 48 Sex Male Female House No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 No. 9 No. 10 Nutritional status High (normal/overweight) Normal Low (normal/underweight) HIV-1 status Positive Negative Duration of stay (months) 0–11 12–23 24–35 36–47 ⱖ 48 Age at admission (months) 0–11 ⱖ 12 Total

No. positive for Enterocytozoon bieneusi (%)

Total (%)

P

90 (31.0) 114 (39.3) 38 (13.1) 20 (6.9) 28 (9.7)

0.02

198 (68.3) 92 (31.7)

0.06

3 (8.8)

31 (10.7) 32 (11.0) 13 (4.5) 39 (13.4) 33 (11.4) 23 (7.9) 28 (9.7) 29 (10) 28 (9.7) 34 (11.7)

0.01

5 (3.4)

8 (3.0) 147 (54.9)

7 (6.2)

113 (42.2)

0.46

1 (2.6) 11 (4.4)

39 (13.4) 251 (86.6)

1.00

5 (3.2) 7 (7.7)

158 (54.5) 91 (31.4) 16 (5.5) 14 (4.8) 11 (3.8)

0.28

10 (8.8) 2 (5.3)

5 (2.5) 7 (7.6)

1 (2.6) 1 (3.0) 2 (8.7) 5 (17.2)

10 (4.4) 2 (3.2) 12 (4.1)

* HIV-1 ⳱ human immunodeficiency virus type 1.

228 (78.6) 62 (21.4) 290 (100)

0.51

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Table 2 shows the univariate and multivariate analysis of risk factors and E. bieneusi infection. Univariate analysis showed that orphans who were 12–23 months old and lived in house no. 8 were 8.4 and 7.6 times at greater risk, respectively, of acquiring an infection with this organism. Multivariate logistic regression analysis showed that orphans who were 12– 23 months old, girls, and living in house No. 8 were independently associated with E. bieneusi infections. Orphans 12–23 months old were 5.6 times more likely to acquire an E. bieneusi infection than other age groups. Girls living in this orphanage had a five times higher risk of infection than boys. Moreover, orphans who lived in house no. 8 had a 5.4 times greater risk of acquiring an infection with this organism. There was no significant association between nutritional status, HIV status, duration of stay, and age at admission. DISCUSSION In Thailand, intestinal microsporidiosis in HIV-positive children has been reported with a range of 10.8–25.3%.16,17 Thus, it should be considered as one of the common opportunistic infections in HIV-positive children in Thailand. This infection is not uncommon in HIV-negative children. A recent study in Thailand showed that 13 of 87 HIV-negative children who had acute diarrhea excreted E. bieneusi.17 Asymptomatic infections with E. bieneusi have also been reported in both HIV-positive and HIV-negative patients.11,12,18 In the present study, we showed that 4.1% of the orphans without diarrhea excreted E. bieneusi. The prevalence of infection with E. bieneusi in these orphans was also in the same range as in our previous report.11 Data from these studies suggested that infection with E. bieneusi is rather common in children. Among 12 E. bieneusi-positive orphans, one orphan was infected with HIV and seven orphans were underweight. Infection with HIV and being underweight were not associated with E. bieneusi infection in this population. Most of the cases of symptomatic intestinal microsporidiosis were AIDS patients with very low CD4+ cell counts.4,5 Thus, asymptomatic infection in these children should be explained by their immune status. The high prevalence of the infection among children 12–23 months old in this study was parallel to that in recent reports from Uganda18 and Thailand.17 This might be explained by the fact that their behavior is related to more active movement and more independent eating habits compared with those in the first year of age. Additionally, children in this age group still have poor toilet training and poor food-handling hygiene. The transmission via the fecal-oral route might explain the higher prevalence of the infection among orphans in the second year of age than among the infants in this facility. Although, our study was a cross-sectional evaluation, the data might indicate the possibility of self-limited E. bieneusi infection since the prevalence of the disease decreased after the second year of age. It is also possible that E. bieneusi persisted but was shed less consistently or at levels below detection in the older children. An analysis of the ITS sequence of rDNA in E. bieneusi showed the polymorphisms that have been used for genotypic characterization. Although a variation of genotypes has been shown in other studies,19–21 we found no variation of the genotype in this population. Compared with other study populations in Thailand, we found a wide variation of E. bieneusi genotypes in Thai HIV-positive patients (Subrungru-

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LEELAYOOVA AND OTHERS

TABLE 2 Univariate and multivariate analysis of risk factors associated with Enterocytozoon bieneusi infection*

Characteristics

Age group (months) Others 12–23 Sex Male Female House Others No. 8 Nutritional status Overweight /High normal/normal Low normal/underweight HIV-1 status Positive Negative Duration of stay (months) Others 12–23 Age at admission (months) 0–11 ⱖ 12

Prevalence of E. bieneusi (%)

Total (%) (n ⳱ 290)

Crude odds ratio (95% CI)

2 (1.1) 10 (8.8)

176 (60.7) 114 (39.3)

1 8.4 (1.8–38.9)

5 (2.5) 7 (7.6)

198 (68.3) 92 (31.7)

1 3.2 (0.9–10.3)

0.054

1 5.0 (1.3–18.5)

0.016

7 (2.7) 5 (17.2)

261 (90) 29 (10)

1 7.6 (2.2–25.6)

0.01

1 5.4 (1.3–22.4)

0.021

5 (3.2)

155 (57.8)

7 (6.2)

113 (42.2)

1 1.9 (0.6–6.4)

0.254

1 (2.6) 11 (4.4)

39 (13.4) 251 (86.6)

1 0.6 (0.1–4.5)

0.6

5 (2.5) 7 (7.7)

199 (68.6) 91 (31.4)

10 (4.4) 2 (3.2)

228 (78.6) 62 (21.4)

1 3.2 (0.9–10.5) 1 0.3 (0.1–1.0)

P

0.07

Adjusted odds ratio† (95% CI)

1 5.6 (1.0–29.4)

P

0.041

0.051 0.051

* CI ⳱ confidence interval; HIV-1 ⳱ human immunodeficiency virus type 1. † Adjusted for age group, sex, and house.

ang I, unpublished data). This might indicate that these children acquired the infection from the same source. Several studies have shown that different genotypes of E. bieneusi are prevalent in different populations. The occurrence of genotype A, as shown in this study, is also predominant in HIVpositive Peruvians with diarrhea.21 Major sources of the infections are apparently those both with and without diarrhea who excrete E. bieneusi spores. Thus, it is possible that E. bieneusi has been directly passed from one individual to another. A few studies provide evidence to support the role of person-to-person transmission in intestinal microsporidiosis. A case-control study in HIVpositive patients showed that male homosexuals had a greater risk of intestinal microsporidiosis.9 This finding suggested sexual transmission in the male homosexual group. Personto-person transmission was also indicated in the study of E. bieneusi infection in HIV-positive patients in Zimbabwe.10 Those with a history of contact with diarrhea patients had a 1.9 times greater risk of getting the infection. In this study, the risk of E. bieneusi infection was independently greater in those orphans who lived in house no. 8. This house was designated to house newly admitted children for two weeks. If they remained healthy, they were then relocated to other houses. Children who needed medical care were also temporarily transferred to this house. Thus, these newly admitted children could be directly exposed to a source of infection. Our study also showed a significantly higher risk of acquiring the infection in this house. Multivariate analysis also showed that girls who lived here had a significantly higher risk of being infected than boys. Child care workers indicated that girls had a greater risk of being infected because they were more active and social than boys. The evidence from this study suggests that infection with E. bieneusi in this orphanage is transmitted from person to person. Although these children were asymptomatic, they could be an unsuspected source of this infection for those who are

susceptible. In an institution such as an orphanage where infection with E. bieneusi could spread easily, universal precautions should be taken since there are no specific prevention and control strategies for this infection. Received October 11, 2004. Accepted for publication November 25, 2004. Acknowledgments: We gratefully thank the director and childcare workers of the orphanage for their support of this study. Financial support: This work was supported by the Phramongkutklao Research Fund and the Thailand-Tropical Diseases Research Program (T-2), ID 02-2-ARI-24-007. Authors’ addresses: Saovanee Leelayoova, Tawee Naaglor, and Mathirut Mungthin, Department of Parasitology, Phramongkutklao College of Medicine, 315 Ratchawithi Road, Ratchathewi, Bangkok, 10400, Thailand, Telephone and Fax: 66-2-354-7761, E-mail: [email protected]. Ittisak Subrungruang and Porntip Chavalitshewinkoon-Petmitr, Department of Protozoology, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400 Thailand. Ram Rangsin, Department of Military and Community Medicine, Phramongkutklao College of Medicine, 315 Ratchawithi Road, Ratchathewi, Bangkok 10400, Thailand. Jeerapan Worapong, Center for Biotechnology and Department of Biotechnology, Institute of Science and Technology for Research and Development, Salaya, Nakornpratom 73170, Thailand.

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