Diagnosis and Identification of Blastocystis Subtypes in Primary School Children in Jakarta

Diagnosis and Identification of Blastocystis Subtypes in Primary School Children in Jakarta Abstract Blastocystis hominis is an enteric protozoan with many subtypes. It is frequently found in children and may cause chronic diarrhea. This study revealed Blastocystis subtypes among primary school children and comparison of molecular technique and culture method in Blastocystis diagnosis. A total of 141 stools were collected, examined microscopically, selected into the Blastocystis and negative parasite groups, for diagnostic comparison between culture and 18S rRNA polymerase chain reaction (PCR) methods. Positive PCR amplicons were subsequently sequenced for subtyping. The PCR results revealed 89%, 78%, 80% and 88% sensitivity, specificity and positive and negative predictive values, respectively, in comparison with the culture method (McNemar, p > 0.05). Sixteen PCR samples were successfully sequenced and resulted in three Blastocystis subtypes 1, 3 and 4. In conclusion, PCR was sensitive enough and can be used to exclude Blastocystis infection up to 88% of the cases. Subtypes 3 and 1 were the main subtypes found in apparently healthy school children in Jakarta. intestinal parasite, children, diagnostic, Blastocystis subtype INTRODUCTION Blastocystis hominis is an intestinal protozoan, infecting humans as well as animals such as mammals, amphibians, reptiles and arthropods. It is distributed widely in the world and transmitted via fecal-oral route through ingestion of cysts [1]. Blastocystis prevalence in children could reach 20–30% or even >50% as reported by Kurniawan and Pegelow [2, 3] in a survey among primary school children in North Jakarta and Sukaraja, respectively. The lowest prevalence of 0.5% was reported in Japan [4]. Blastocystis is often regarded as commensal or nonpathogenic and frequently found in epidemiological surveys. Some regarded Blastocystis as an opportunistic pathogen among the immune compromised while some epidemiological data support pathogenic characteristic of Blastocystis [5, 6]. The clinical symptoms of B. hominis infection may manifest as bloating, anorexia, abdominal pain, acute and chronic diarrhea or even ‘irritable bowel syndrome’. Chronic diarrhea in children will consequently disturb their growth and development particularly among those <5 years old [7]. Diagnosis of Blastocystis can be carried out microscopically; however, the result is subjective and depends on the technician’s experience and skill. The size and morphology of Blastocystis varies widely during developmental cycle; vacuolar form, cyst, amoeboid, granular, multivacuolar and avacuolar, which may mimic other intestinal protozoa or contaminants and subsequently cause misdiagnosis. A number of studies have reported other methods such as culture using ATCC Medium 1671, Locke Medium or Jones medium and polymerase chain reaction (PCR) to identify Blastocystis infection [8], and culture method was regarded as the gold standard for Blastocystis diagnosis [9]. As Blastocystis has a wide range of hosts, the genetic analysis of SSU rRNA from human, mammals, primates and birds reveals at least 17 subtypes of Blastocystis with subtype 3 (ST3) as the most common subtype found in human. Some subtypes (ST1-9) commonly seen in human can also be found in animals, suggesting potential zoonotic transmission [10]. This study aimed to compare the DNA detection by PCR and culture by Jones medium, determine the sensitivity and specificity of PCR to be used as diagnostic tool and to obtain the genetic profile of Blastocystis subtypes among the school children. Ethical clearance was obtained from the ethics committee, Faculty of Medicine, Universitas Indonesia and informed consents from the participants and/or the teachers. MATERIALS AND METHODS Study population A cross-sectional study was carried out among primary school children in Kampung Melayu, East Jakarta. The study area was densely populated and vulnerable to flood from Ciliwung river every year. As many as 141 students were recruited from two primary schools. Fecal samples were collected, transported and examined at Parasitology laboratory, Faculty of Medicine, Universitas Indonesia. The samples were divided into three parts: first part for direct smear examination, second part for culture and the third part were kept in −20°C until it was used for molecular analysis. Parasitology examination and culture Stools were examined directly with 0.2% Lugol solution under microscope to detect any intestinal parasites. To perform the diagnostic test comparison between culture method and molecular detection for B. hominis diagnosis, the stools were further screened for single infection of B. hominis (Blastocystis group) and no parasites (negative group): 18 samples in each group following sample size estimation formula for diagnostic test. Stools from the two groups were cultured in Jones medium supplemented with 10% horse serum [11], incubated at 37°C and kept for 72 h before reporting as positive or negative Blastocystis. Examination of culture was performed after 24 h incubation. DNA isolation and amplification Stool samples from the two groups (the Blastocystis and negative groups) were DNA extracted using QIAamp DNA Mini Kit following the procedure from the manufacturer (Qiagen, GmbH, cat no. 51306). The final isolated DNA was resuspended in 90 μl AE buffer and kept in −20°C. Extracted DNA was PCR amplified in 20 μl reaction using TopTaq Polymerase Master Mix Kit (Qiagen, GmbH, cat no. 200403), which contains Taq DNA Polymerase, PCR Buffer (with 1.5 mM MgCl2), 200 µM each dNTP, 10% BSA (Bovine Serum Albumin) to minimize the inhibitors effect and 2 μl DNA template with the following primers as previously described in [12]. Forward: RD5 5′-ATC-TGG-TTG-ATC-CTG-CCA-GT-3′ Reverse:. BhRDr 5′-GAG-CTT-TTT-AAC-TGC-AAC-AAC-G-3′ DNA amplification was performed by direct PCR targeting 18S rRNA gene. All PCR reactions were carried out on MJ Research PTC 200 thermocycler as follows: one cycle at 95 °C for 6 min followed by 30 cycles at 93 °C for 2 min, 65 °C for 2 min, 72°C for 2 min. The last cycle was extended for 10 min at 72 °C. The 600 bp PCR product was visualized on 2% agarose gel electrophoresis. DNA sequencing and subtype analysis To characterize genetic diversity of Blastocystis, all PCR products from Blastocystis positive samples were directly sequenced and chromatograms were validated using Chromas lite software (Technelysium Pty Ltd, Australia). Blasted sequences were multiple aligned with Blastocystis database retrieved from the GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi), using Mega 6 software (en.bio-soft.net/tree/MEGA.htm). Statistical analyses The data were statistically analyzed using Cat Maker® software [13], with p value ≤ 0.05 regarded as significant. Accuracy was calculated as specificity, sensitivity and positive and negative predictive values, with 95% confidence intervals (CI). RESULTS Prevalence of B. hominis As many as 141 samples were collected, and direct fecal smear examination revealed that 74 (52.5%) children were positive for intestinal parasites. Single infection of B. hominis was observed in 47 children (33.3%), mix infection of B. hominis in 11 children (7.8%) and 16 children (11.3%) infected with other intestinal parasites (Table 1). The mix infection of B. hominis can be with Giardia duodenalis, Entamoeba coli, Entamoeba histolytica or Trichuris trichiura. The overall prevalence of Blastocystis in the study population was 41.1% where 80% infection was present among the 1st–3rd year students. This result suggested high prevalence of parasitic infection among the primary school children in Kampung Melayu area, East Jakarta which was dominated by the protozoa B. hominis and G. duodenalis (Table 1). Table 1 Intestinal parasites profile among primary school children in East Jakarta Location  Single infection   Mixed infection   Negative  Total  B. hominis  G. duodenalis  E. coli  Ascaris lumbricoides  T. trichiura  Bh+Gd  Bh+Tt  Bh+Ec  Bh+Eh  School A  23  5  2  1  1  1  2  0  0  31  66  School B  24  6  1  0  0  3  0  4  1  36  75  Total  47  11  3  1  1  4  2  4  1  67  141  (%)  (33.3)  (7.8)  (2.1)  (0.7)  (0.7)  (2.8)  (1.4)  (2.8)  (0.7)  (47.5)  (100)  Location  Single infection   Mixed infection   Negative  Total  B. hominis  G. duodenalis  E. coli  Ascaris lumbricoides  T. trichiura  Bh+Gd  Bh+Tt  Bh+Ec  Bh+Eh  School A  23  5  2  1  1  1  2  0  0  31  66  School B  24  6  1  0  0  3  0  4  1  36  75  Total  47  11  3  1  1  4  2  4  1  67  141  (%)  (33.3)  (7.8)  (2.1)  (0.7)  (0.7)  (2.8)  (1.4)  (2.8)  (0.7)  (47.5)  (100)  Notes: Bh + Gd: B. hominis + G. duodenalis. Bh + Tt: B. hominis + T. trichiura. Bh + Ec: B. hominis + E. coli. Bh + Eh: B. hominis + E. histolytica. Table 1 Intestinal parasites profile among primary school children in East Jakarta Location  Single infection   Mixed infection   Negative  Total  B. hominis  G. duodenalis  E. coli  Ascaris lumbricoides  T. trichiura  Bh+Gd  Bh+Tt  Bh+Ec  Bh+Eh  School A  23  5  2  1  1  1  2  0  0  31  66  School B  24  6  1  0  0  3  0  4  1  36  75  Total  47  11  3  1  1  4  2  4  1  67  141  (%)  (33.3)  (7.8)  (2.1)  (0.7)  (0.7)  (2.8)  (1.4)  (2.8)  (0.7)  (47.5)  (100)  Location  Single infection   Mixed infection   Negative  Total  B. hominis  G. duodenalis  E. coli  Ascaris lumbricoides  T. trichiura  Bh+Gd  Bh+Tt  Bh+Ec  Bh+Eh  School A  23  5  2  1  1  1  2  0  0  31  66  School B  24  6  1  0  0  3  0  4  1  36  75  Total  47  11  3  1  1  4  2  4  1  67  141  (%)  (33.3)  (7.8)  (2.1)  (0.7)  (0.7)  (2.8)  (1.4)  (2.8)  (0.7)  (47.5)  (100)  Notes: Bh + Gd: B. hominis + G. duodenalis. Bh + Tt: B. hominis + T. trichiura. Bh + Ec: B. hominis + E. coli. Bh + Eh: B. hominis + E. histolytica. Comparison of PCR and culture methods in Blastocystis diagnosis The culture showed 100% agreement with direct examination result in both groups, the Blastocystis and negative groups. The PCR result showed 89% sensitivity (95% CI: 74–100%) and 78% specificity (95% CI: 59–97%), 80% positive predictive value and 88% negative predictive value in comparison with the culture method (Table 2). Statistical analysis showed no significant difference between those two methods in diagnosing Blastocystis infection (McNemar, p> 0.05). Table 2 Comparison between culture and PCR in Blastocystis diagnosis   Culture   Total  p value  Positive  Negative  PCR  Positive  16 (44.4%)  4 (11.1%)  20 (55.6%)    Negative  2 (5.6%)  14 (38.9%)  16 (44.4%)  0,687  Total  18 (50%)  18 (50%)  36 (100%)      Culture   Total  p value  Positive  Negative  PCR  Positive  16 (44.4%)  4 (11.1%)  20 (55.6%)    Negative  2 (5.6%)  14 (38.9%)  16 (44.4%)  0,687  Total  18 (50%)  18 (50%)  36 (100%)    Table 2 Comparison between culture and PCR in Blastocystis diagnosis   Culture   Total  p value  Positive  Negative  PCR  Positive  16 (44.4%)  4 (11.1%)  20 (55.6%)    Negative  2 (5.6%)  14 (38.9%)  16 (44.4%)  0,687  Total  18 (50%)  18 (50%)  36 (100%)      Culture   Total  p value  Positive  Negative  PCR  Positive  16 (44.4%)  4 (11.1%)  20 (55.6%)    Negative  2 (5.6%)  14 (38.9%)  16 (44.4%)  0,687  Total  18 (50%)  18 (50%)  36 (100%)    Blastocystis subtype analysis There were 16 of 18 samples positive for Blastocystis, which were successfully amplified targeting the 18S rRNA gene and sequenced. Sequences were deposited in Genbank with accession numbers JN 682513.1, KP 233714.1, KP 233717.1, KP 233722.1, KP 408437.1, KP 408442.1, KP 408449.1, KP 408450.1, KT 438687.1 and LT 222066.1. The result demonstrated five samples of subtype 1 (ST1), 10 samples of subtype 3 (ST3) and one of subtype 4 (ST4); ST3 was the major subtype (Figure 1). Only subtype 4 (ST4) found in this study perfectly matched the genbank sequence database. A phylogenetic tree of the Blastocystis isolates is displayed in Figure 1, using the neighbor-joining method with the bootstrap proportions (%) attached to the internal branches from 500 replicates. Fig. 1. View largeDownload slide Phylogenetic tree analysis of Blastocystis samples sequences on 18S rRNA. Fig. 1. View largeDownload slide Phylogenetic tree analysis of Blastocystis samples sequences on 18S rRNA. DISCUSSION This study showed a high prevalence of intestinal parasitic infection dominated by Blastocystis among the primary school children living in a densely populated area by Ciliwung river in East Jakarta, which always flooded every year during rainy season. This prevalence is similar to other studies performed among primary school children in Sumba, rural area in East Indonesia, North Jakarta and Sukaraja with the exception that higher soil-transmitted helminthiasis were found in the other studies [2, 3, 14]. Flood, which happened every year during the rainy season in that area, may become one factor that contributes to the high prevalence of Blastocystis apart from personal hygiene factor of the children, as noted in this study that 80% Blastocystis infection happened among younger students, aged 6–9 [15]. This urges the importance of continous health education and practice among the primary school students to prevent parasitic infections. Diagnosis of Blastocystis in this study used direct microscopic examination, culture and PCR on 18S rRNA gene. Culture and direct PCR methods from fresh stool samples were tested because direct smear examination has weakness in Blastocystis diagnosis because of diversity in morphology and size of Blastocystis, which may mimic other parasites or organisms or contaminant in the stool [8, 16]. This study did not show superiority of PCR against the culture method, which is in contrast to the statement from Stensvold et al. [16] that the sensitivity of culture is in 52–79% compared with DNA-based methods. Four samples that were negative by culture turned out to be positive by PCR, and two samples that were positive by culture turned out to be negative by PCR; however, statistical analysis did not show significant difference between those two methods. Several reasons may explain this finding. First, the DNA that was isolated from fresh stool may have less quality and quantity than those isolated from culture [5, 16]. Stensvold et al. [16] reported that PCR sensitivity will increase three times when using Blastocystis DNA isolated from culture than from the fresh stool. Secondly, the presence of inhibitors such as bile salts, polysaccharide complex in stool specimens will subsequently lower the DNA recovery or the amount of Blastocystis DNA was below detection level [5, 17]. However, with 88% negative predictive value, PCR can be used to exclude suspected Blastocystis infection up to 90%. The result of 16 of 18 Blastocystis positive stool samples, which were successfully amplified, sequenced and subtyped, yielded dominance of ST3 (67.95%), followed by ST1 (26.4%), ST2 (3.8%) and the least was ST4 (1.9%) in only one sample. Dominance of ST3 Blastocystis in human infection is in agreement with other previous reports in Europe, Africa, Asia and Australia, except in America, which was dominated by ST1 [1]. There have been several reports on Blastocystis subtypes from Indonesian population, which showed only subtypes 1, 2 and 3 in which ST3 predominated in the studies among migrant workers in Taiwan and Qatar [18, 19], while ST1 predominated among children in Sumba, East Nusa Tenggara [13]. ST4 is the rarest subtype among Asian people except Nepalese; however, it is quite prevalent and predominant among the Spanish, British and Australian [1, 10, 20] and was reported to be associated with acute diarrhea among Danish patients [21]. ST4 commonly infects rodents, marsupials, ratites and primate [22]. A study in Sumba, Indonesia by Yoshikawa et al. [14] showed that ST4 was observed only in wild rodents and none among the residents who live together with domestic and wild animals, and postulated the presence of host specificity and difficulty for zoonotic transmission, which is in contrast to other studies that suggested the role of rodents as reservoir host for zoonotic Blastocystis infection in human [5, 23, 24]. The finding of ST4 in our study showed that ST4 is an emerging Blastocystis subtype infecting human population in Asia, a minor subtype, which has been also observed among the school children in Malaysia and Turkey [25, 26]. The possibility of zoonotic transmission of ST4 from rat to human (the student) in our study cannot be avoided owing to the densely populated area where the student lives and rat is a common animal found in the surrounding area. Unlike ST3 and ST1, which can be found in symptomatic and asymptomatic infection, the presence of ST4 should raise concern and association to the possibility of pathology, which needs further study to clarify any association between particular subtype and clinical manifestation. In conclusion, high prevalence of Blastocystis infection is seen among the primary school children in East Jakarta, with ST3 as the most common subtype. PCR can be used to exclude up to 88% cases of suspected Blastocystis infection. Funding This work was supported by the Directorate of Higher Education, Republic of Indonesia through Universitas Indonesia. References 1 Alfellani MA, Stensvold CR, Vidal-Lapiedra A, et al.   Variable geographic distribution of Blastocystis subtypes and its potential implications. Acta Trop  2013; 126: 11– 8. Google Scholar CrossRef Search ADS PubMed  2 Kurniawan A. Intestinal parasitic infections among primary school children in North Jakarta: dominance of soil transmitted helminths and Blastocystis infections. Am J Trop Med Hyg  2012; 87: S37. 3 Pegelow K, Gross R, Pietrzik K, et al.   Parasitological and nutritional situation of school children in the Sukaraja district, West Java, Indonesia. Southeast Asian J Trop Med Public Health  1997; 28: 173– 90. Google Scholar PubMed  4 Yoshikawa H, Abe N, Iwasawa M, et al.   Genomic analysis of Blastocystis hominis strains isolated from two long-term health care facilities. J Clin Microbiol  2000; 38: 1324– 30. Google Scholar PubMed  5 Bart A, Wentink-Bonnema EM, Gilis H, et al.   Diagnosis and subtype analysis of Blastocystis sp. in 442 patients in a hospital setting in the Netherlands. BMC Infect Dis  2013; 13: 389. Google Scholar CrossRef Search ADS PubMed  6 Abdulsalam AM, Ithoi I, Al-Mekhlafi HM, et al.   Subtype distribution of Blastocystis isolates in Sebha, Libya. PloS One  2013; 8: e84372. Google Scholar CrossRef Search ADS PubMed  7 Brown KH. Diarrhea and malnutrition. J. Nutr  2003; 133: 328S– 32S. Google Scholar CrossRef Search ADS PubMed  8 Stensvold CR, Suresh GK, Tan KS, et al.   Terminology for Blastocystis subtypes–a consensus. Trends Parasitol  2007; 23: 93– 6. Google Scholar CrossRef Search ADS PubMed  9 Tan KS. New insights on classification, identification and clinical relevance of Blastocystis spp. Clin Microbiol Rev  2008; 21: 639– 65. Google Scholar CrossRef Search ADS PubMed  10 Popruk S, Udonsom R, Koompapong K, et al.   Subtype distribution of Blastocystis in Thai-Myanmar border, Thailand. Korean J Parasitol  2015; 53: 13– 9. Google Scholar CrossRef Search ADS PubMed  11 Leelayoova S, Taamasri P, Rangsin R, et al.   In-vitro cultivation: a sensitive method for detecting Blastocystis hominis. Ann Trop Med Parasitol  2002; 96: 803– 7. Google Scholar CrossRef Search ADS PubMed  12 Scicluna SM, Tawari B, Clark CG. DNA barcoding of Blastocystis. Protist  2006; 157: 77– 85. Google Scholar CrossRef Search ADS PubMed  13 CAT maker. http://www.cebm.net/catmaker-ebm-calculators (August 2015, date last accessed). 14 Yoshikawa H, Tokoro M, Nagamoto T, et al.   Molecular survey of Blastocystis sp. from humans and associated animals in an Indonesian community with poor hygiene. Parasitol Int  2016; 65: 780– 4. Google Scholar CrossRef Search ADS PubMed  15 Daryani A, Sharif M, Nasrolahei M, et al.   Epidemiological survey of the prevalence of intestinal parasites among school children in Sari, northern Iran. Trans R Soc Trop Med Hyg  2012; 106: 455– 9. Google Scholar CrossRef Search ADS PubMed  16 Stensvold R, Brillowska-Dabrowska A, Nielsen HV, et al.   Detection of Blastocystis hominis in unpreserved stool specimens by using polymerase chain reaction. J Parasitol  2006; 92: 1081– 7. Google Scholar CrossRef Search ADS PubMed  17 Monteiro L, Bonnemaison D, Vekris A, et al.   Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model. J Clin Microbiol  1997; 35: 995– 8. Google Scholar PubMed  18 Chen LH, Lee SL, Yang CC, et al.   Genetic variability of Blastocystis hominis in Indonesian immigrant workers. J Intern Med Taiwan  2014; 25: 199– 205. 19 Abu-Madi M, Aly M, Behnke JM, et al.   The distribution of Blastocystis subtypes in isolates from Qatar. Parasit Vectors  2015; 8: 465– 72. Google Scholar CrossRef Search ADS PubMed  20 Roberts T, Stark D, Harkness J, et al.   Update on the molecular epidemiology and diagnostic tools for Blastocystis sp. J Med Microb Diagn  2014; 3: 1. 21 Stensvold CR, Christiansen DB, Olsen KE, et al.   Blastocystis sp. subtype 4 is common in Danish Blastocystis-positive patients presenting with acute diarrhea. Am J Trop Med Hyg  2011; 84: 883– 5. Google Scholar CrossRef Search ADS PubMed  22 Alfellani MA, Taner-Mulla D, Jacob A, et al.   Genetic diversity of Blastocystis in livestock and zoo animals. Protist  2013; 164: 497– 509. Google Scholar CrossRef Search ADS PubMed  23 Silberman JD, Sogin ML, Leipe DD, et al.   Human parasite finds taxonomic home. Nature  1996; 380: 398. Google Scholar CrossRef Search ADS PubMed  24 Khoshnood S, Rafiei A, Saki J, et al.   Prevalence and genotype characterization of Blastocystis hominis among the Baghmalek people in Southwestern Iran in 2013-2014. Jundishapur J Microbiol  2015; 8: e23930. Google Scholar CrossRef Search ADS PubMed  25 Nithyamathi K, Chandramathi S, Kumar S. Predominance of Blastocystis sp. infection among school children in Peninsular Malaysia. PLoS One  2016; 11: e0136709. Google Scholar CrossRef Search ADS PubMed  26 Dogan N, Aydin M, Tuzemen NU, et al.   Subtype distribution of Blastocystis spp. isolated from children in Eskisehir, Turkey. Parasitol Int  2017; 66: 948– 51. Google Scholar CrossRef Search ADS PubMed  © The Author [2017]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Tropical Pediatrics Oxford University Press

Diagnosis and Identification of Blastocystis Subtypes in Primary School Children in Jakarta

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Abstract

Abstract Blastocystis hominis is an enteric protozoan with many subtypes. It is frequently found in children and may cause chronic diarrhea. This study revealed Blastocystis subtypes among primary school children and comparison of molecular technique and culture method in Blastocystis diagnosis. A total of 141 stools were collected, examined microscopically, selected into the Blastocystis and negative parasite groups, for diagnostic comparison between culture and 18S rRNA polymerase chain reaction (PCR) methods. Positive PCR amplicons were subsequently sequenced for subtyping. The PCR results revealed 89%, 78%, 80% and 88% sensitivity, specificity and positive and negative predictive values, respectively, in comparison with the culture method (McNemar, p > 0.05). Sixteen PCR samples were successfully sequenced and resulted in three Blastocystis subtypes 1, 3 and 4. In conclusion, PCR was sensitive enough and can be used to exclude Blastocystis infection up to 88% of the cases. Subtypes 3 and 1 were the main subtypes found in apparently healthy school children in Jakarta. intestinal parasite, children, diagnostic, Blastocystis subtype INTRODUCTION Blastocystis hominis is an intestinal protozoan, infecting humans as well as animals such as mammals, amphibians, reptiles and arthropods. It is distributed widely in the world and transmitted via fecal-oral route through ingestion of cysts [1]. Blastocystis prevalence in children could reach 20–30% or even >50% as reported by Kurniawan and Pegelow [2, 3] in a survey among primary school children in North Jakarta and Sukaraja, respectively. The lowest prevalence of 0.5% was reported in Japan [4]. Blastocystis is often regarded as commensal or nonpathogenic and frequently found in epidemiological surveys. Some regarded Blastocystis as an opportunistic pathogen among the immune compromised while some epidemiological data support pathogenic characteristic of Blastocystis [5, 6]. The clinical symptoms of B. hominis infection may manifest as bloating, anorexia, abdominal pain, acute and chronic diarrhea or even ‘irritable bowel syndrome’. Chronic diarrhea in children will consequently disturb their growth and development particularly among those <5 years old [7]. Diagnosis of Blastocystis can be carried out microscopically; however, the result is subjective and depends on the technician’s experience and skill. The size and morphology of Blastocystis varies widely during developmental cycle; vacuolar form, cyst, amoeboid, granular, multivacuolar and avacuolar, which may mimic other intestinal protozoa or contaminants and subsequently cause misdiagnosis. A number of studies have reported other methods such as culture using ATCC Medium 1671, Locke Medium or Jones medium and polymerase chain reaction (PCR) to identify Blastocystis infection [8], and culture method was regarded as the gold standard for Blastocystis diagnosis [9]. As Blastocystis has a wide range of hosts, the genetic analysis of SSU rRNA from human, mammals, primates and birds reveals at least 17 subtypes of Blastocystis with subtype 3 (ST3) as the most common subtype found in human. Some subtypes (ST1-9) commonly seen in human can also be found in animals, suggesting potential zoonotic transmission [10]. This study aimed to compare the DNA detection by PCR and culture by Jones medium, determine the sensitivity and specificity of PCR to be used as diagnostic tool and to obtain the genetic profile of Blastocystis subtypes among the school children. Ethical clearance was obtained from the ethics committee, Faculty of Medicine, Universitas Indonesia and informed consents from the participants and/or the teachers. MATERIALS AND METHODS Study population A cross-sectional study was carried out among primary school children in Kampung Melayu, East Jakarta. The study area was densely populated and vulnerable to flood from Ciliwung river every year. As many as 141 students were recruited from two primary schools. Fecal samples were collected, transported and examined at Parasitology laboratory, Faculty of Medicine, Universitas Indonesia. The samples were divided into three parts: first part for direct smear examination, second part for culture and the third part were kept in −20°C until it was used for molecular analysis. Parasitology examination and culture Stools were examined directly with 0.2% Lugol solution under microscope to detect any intestinal parasites. To perform the diagnostic test comparison between culture method and molecular detection for B. hominis diagnosis, the stools were further screened for single infection of B. hominis (Blastocystis group) and no parasites (negative group): 18 samples in each group following sample size estimation formula for diagnostic test. Stools from the two groups were cultured in Jones medium supplemented with 10% horse serum [11], incubated at 37°C and kept for 72 h before reporting as positive or negative Blastocystis. Examination of culture was performed after 24 h incubation. DNA isolation and amplification Stool samples from the two groups (the Blastocystis and negative groups) were DNA extracted using QIAamp DNA Mini Kit following the procedure from the manufacturer (Qiagen, GmbH, cat no. 51306). The final isolated DNA was resuspended in 90 μl AE buffer and kept in −20°C. Extracted DNA was PCR amplified in 20 μl reaction using TopTaq Polymerase Master Mix Kit (Qiagen, GmbH, cat no. 200403), which contains Taq DNA Polymerase, PCR Buffer (with 1.5 mM MgCl2), 200 µM each dNTP, 10% BSA (Bovine Serum Albumin) to minimize the inhibitors effect and 2 μl DNA template with the following primers as previously described in [12]. Forward: RD5 5′-ATC-TGG-TTG-ATC-CTG-CCA-GT-3′ Reverse:. BhRDr 5′-GAG-CTT-TTT-AAC-TGC-AAC-AAC-G-3′ DNA amplification was performed by direct PCR targeting 18S rRNA gene. All PCR reactions were carried out on MJ Research PTC 200 thermocycler as follows: one cycle at 95 °C for 6 min followed by 30 cycles at 93 °C for 2 min, 65 °C for 2 min, 72°C for 2 min. The last cycle was extended for 10 min at 72 °C. The 600 bp PCR product was visualized on 2% agarose gel electrophoresis. DNA sequencing and subtype analysis To characterize genetic diversity of Blastocystis, all PCR products from Blastocystis positive samples were directly sequenced and chromatograms were validated using Chromas lite software (Technelysium Pty Ltd, Australia). Blasted sequences were multiple aligned with Blastocystis database retrieved from the GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi), using Mega 6 software (en.bio-soft.net/tree/MEGA.htm). Statistical analyses The data were statistically analyzed using Cat Maker® software [13], with p value ≤ 0.05 regarded as significant. Accuracy was calculated as specificity, sensitivity and positive and negative predictive values, with 95% confidence intervals (CI). RESULTS Prevalence of B. hominis As many as 141 samples were collected, and direct fecal smear examination revealed that 74 (52.5%) children were positive for intestinal parasites. Single infection of B. hominis was observed in 47 children (33.3%), mix infection of B. hominis in 11 children (7.8%) and 16 children (11.3%) infected with other intestinal parasites (Table 1). The mix infection of B. hominis can be with Giardia duodenalis, Entamoeba coli, Entamoeba histolytica or Trichuris trichiura. The overall prevalence of Blastocystis in the study population was 41.1% where 80% infection was present among the 1st–3rd year students. This result suggested high prevalence of parasitic infection among the primary school children in Kampung Melayu area, East Jakarta which was dominated by the protozoa B. hominis and G. duodenalis (Table 1). Table 1 Intestinal parasites profile among primary school children in East Jakarta Location  Single infection   Mixed infection   Negative  Total  B. hominis  G. duodenalis  E. coli  Ascaris lumbricoides  T. trichiura  Bh+Gd  Bh+Tt  Bh+Ec  Bh+Eh  School A  23  5  2  1  1  1  2  0  0  31  66  School B  24  6  1  0  0  3  0  4  1  36  75  Total  47  11  3  1  1  4  2  4  1  67  141  (%)  (33.3)  (7.8)  (2.1)  (0.7)  (0.7)  (2.8)  (1.4)  (2.8)  (0.7)  (47.5)  (100)  Location  Single infection   Mixed infection   Negative  Total  B. hominis  G. duodenalis  E. coli  Ascaris lumbricoides  T. trichiura  Bh+Gd  Bh+Tt  Bh+Ec  Bh+Eh  School A  23  5  2  1  1  1  2  0  0  31  66  School B  24  6  1  0  0  3  0  4  1  36  75  Total  47  11  3  1  1  4  2  4  1  67  141  (%)  (33.3)  (7.8)  (2.1)  (0.7)  (0.7)  (2.8)  (1.4)  (2.8)  (0.7)  (47.5)  (100)  Notes: Bh + Gd: B. hominis + G. duodenalis. Bh + Tt: B. hominis + T. trichiura. Bh + Ec: B. hominis + E. coli. Bh + Eh: B. hominis + E. histolytica. Table 1 Intestinal parasites profile among primary school children in East Jakarta Location  Single infection   Mixed infection   Negative  Total  B. hominis  G. duodenalis  E. coli  Ascaris lumbricoides  T. trichiura  Bh+Gd  Bh+Tt  Bh+Ec  Bh+Eh  School A  23  5  2  1  1  1  2  0  0  31  66  School B  24  6  1  0  0  3  0  4  1  36  75  Total  47  11  3  1  1  4  2  4  1  67  141  (%)  (33.3)  (7.8)  (2.1)  (0.7)  (0.7)  (2.8)  (1.4)  (2.8)  (0.7)  (47.5)  (100)  Location  Single infection   Mixed infection   Negative  Total  B. hominis  G. duodenalis  E. coli  Ascaris lumbricoides  T. trichiura  Bh+Gd  Bh+Tt  Bh+Ec  Bh+Eh  School A  23  5  2  1  1  1  2  0  0  31  66  School B  24  6  1  0  0  3  0  4  1  36  75  Total  47  11  3  1  1  4  2  4  1  67  141  (%)  (33.3)  (7.8)  (2.1)  (0.7)  (0.7)  (2.8)  (1.4)  (2.8)  (0.7)  (47.5)  (100)  Notes: Bh + Gd: B. hominis + G. duodenalis. Bh + Tt: B. hominis + T. trichiura. Bh + Ec: B. hominis + E. coli. Bh + Eh: B. hominis + E. histolytica. Comparison of PCR and culture methods in Blastocystis diagnosis The culture showed 100% agreement with direct examination result in both groups, the Blastocystis and negative groups. The PCR result showed 89% sensitivity (95% CI: 74–100%) and 78% specificity (95% CI: 59–97%), 80% positive predictive value and 88% negative predictive value in comparison with the culture method (Table 2). Statistical analysis showed no significant difference between those two methods in diagnosing Blastocystis infection (McNemar, p> 0.05). Table 2 Comparison between culture and PCR in Blastocystis diagnosis   Culture   Total  p value  Positive  Negative  PCR  Positive  16 (44.4%)  4 (11.1%)  20 (55.6%)    Negative  2 (5.6%)  14 (38.9%)  16 (44.4%)  0,687  Total  18 (50%)  18 (50%)  36 (100%)      Culture   Total  p value  Positive  Negative  PCR  Positive  16 (44.4%)  4 (11.1%)  20 (55.6%)    Negative  2 (5.6%)  14 (38.9%)  16 (44.4%)  0,687  Total  18 (50%)  18 (50%)  36 (100%)    Table 2 Comparison between culture and PCR in Blastocystis diagnosis   Culture   Total  p value  Positive  Negative  PCR  Positive  16 (44.4%)  4 (11.1%)  20 (55.6%)    Negative  2 (5.6%)  14 (38.9%)  16 (44.4%)  0,687  Total  18 (50%)  18 (50%)  36 (100%)      Culture   Total  p value  Positive  Negative  PCR  Positive  16 (44.4%)  4 (11.1%)  20 (55.6%)    Negative  2 (5.6%)  14 (38.9%)  16 (44.4%)  0,687  Total  18 (50%)  18 (50%)  36 (100%)    Blastocystis subtype analysis There were 16 of 18 samples positive for Blastocystis, which were successfully amplified targeting the 18S rRNA gene and sequenced. Sequences were deposited in Genbank with accession numbers JN 682513.1, KP 233714.1, KP 233717.1, KP 233722.1, KP 408437.1, KP 408442.1, KP 408449.1, KP 408450.1, KT 438687.1 and LT 222066.1. The result demonstrated five samples of subtype 1 (ST1), 10 samples of subtype 3 (ST3) and one of subtype 4 (ST4); ST3 was the major subtype (Figure 1). Only subtype 4 (ST4) found in this study perfectly matched the genbank sequence database. A phylogenetic tree of the Blastocystis isolates is displayed in Figure 1, using the neighbor-joining method with the bootstrap proportions (%) attached to the internal branches from 500 replicates. Fig. 1. View largeDownload slide Phylogenetic tree analysis of Blastocystis samples sequences on 18S rRNA. Fig. 1. View largeDownload slide Phylogenetic tree analysis of Blastocystis samples sequences on 18S rRNA. DISCUSSION This study showed a high prevalence of intestinal parasitic infection dominated by Blastocystis among the primary school children living in a densely populated area by Ciliwung river in East Jakarta, which always flooded every year during rainy season. This prevalence is similar to other studies performed among primary school children in Sumba, rural area in East Indonesia, North Jakarta and Sukaraja with the exception that higher soil-transmitted helminthiasis were found in the other studies [2, 3, 14]. Flood, which happened every year during the rainy season in that area, may become one factor that contributes to the high prevalence of Blastocystis apart from personal hygiene factor of the children, as noted in this study that 80% Blastocystis infection happened among younger students, aged 6–9 [15]. This urges the importance of continous health education and practice among the primary school students to prevent parasitic infections. Diagnosis of Blastocystis in this study used direct microscopic examination, culture and PCR on 18S rRNA gene. Culture and direct PCR methods from fresh stool samples were tested because direct smear examination has weakness in Blastocystis diagnosis because of diversity in morphology and size of Blastocystis, which may mimic other parasites or organisms or contaminant in the stool [8, 16]. This study did not show superiority of PCR against the culture method, which is in contrast to the statement from Stensvold et al. [16] that the sensitivity of culture is in 52–79% compared with DNA-based methods. Four samples that were negative by culture turned out to be positive by PCR, and two samples that were positive by culture turned out to be negative by PCR; however, statistical analysis did not show significant difference between those two methods. Several reasons may explain this finding. First, the DNA that was isolated from fresh stool may have less quality and quantity than those isolated from culture [5, 16]. Stensvold et al. [16] reported that PCR sensitivity will increase three times when using Blastocystis DNA isolated from culture than from the fresh stool. Secondly, the presence of inhibitors such as bile salts, polysaccharide complex in stool specimens will subsequently lower the DNA recovery or the amount of Blastocystis DNA was below detection level [5, 17]. However, with 88% negative predictive value, PCR can be used to exclude suspected Blastocystis infection up to 90%. The result of 16 of 18 Blastocystis positive stool samples, which were successfully amplified, sequenced and subtyped, yielded dominance of ST3 (67.95%), followed by ST1 (26.4%), ST2 (3.8%) and the least was ST4 (1.9%) in only one sample. Dominance of ST3 Blastocystis in human infection is in agreement with other previous reports in Europe, Africa, Asia and Australia, except in America, which was dominated by ST1 [1]. There have been several reports on Blastocystis subtypes from Indonesian population, which showed only subtypes 1, 2 and 3 in which ST3 predominated in the studies among migrant workers in Taiwan and Qatar [18, 19], while ST1 predominated among children in Sumba, East Nusa Tenggara [13]. ST4 is the rarest subtype among Asian people except Nepalese; however, it is quite prevalent and predominant among the Spanish, British and Australian [1, 10, 20] and was reported to be associated with acute diarrhea among Danish patients [21]. ST4 commonly infects rodents, marsupials, ratites and primate [22]. A study in Sumba, Indonesia by Yoshikawa et al. [14] showed that ST4 was observed only in wild rodents and none among the residents who live together with domestic and wild animals, and postulated the presence of host specificity and difficulty for zoonotic transmission, which is in contrast to other studies that suggested the role of rodents as reservoir host for zoonotic Blastocystis infection in human [5, 23, 24]. The finding of ST4 in our study showed that ST4 is an emerging Blastocystis subtype infecting human population in Asia, a minor subtype, which has been also observed among the school children in Malaysia and Turkey [25, 26]. The possibility of zoonotic transmission of ST4 from rat to human (the student) in our study cannot be avoided owing to the densely populated area where the student lives and rat is a common animal found in the surrounding area. Unlike ST3 and ST1, which can be found in symptomatic and asymptomatic infection, the presence of ST4 should raise concern and association to the possibility of pathology, which needs further study to clarify any association between particular subtype and clinical manifestation. In conclusion, high prevalence of Blastocystis infection is seen among the primary school children in East Jakarta, with ST3 as the most common subtype. PCR can be used to exclude up to 88% cases of suspected Blastocystis infection. Funding This work was supported by the Directorate of Higher Education, Republic of Indonesia through Universitas Indonesia. References 1 Alfellani MA, Stensvold CR, Vidal-Lapiedra A, et al.   Variable geographic distribution of Blastocystis subtypes and its potential implications. Acta Trop  2013; 126: 11– 8. Google Scholar CrossRef Search ADS PubMed  2 Kurniawan A. 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Google Scholar CrossRef Search ADS PubMed  7 Brown KH. Diarrhea and malnutrition. J. Nutr  2003; 133: 328S– 32S. Google Scholar CrossRef Search ADS PubMed  8 Stensvold CR, Suresh GK, Tan KS, et al.   Terminology for Blastocystis subtypes–a consensus. Trends Parasitol  2007; 23: 93– 6. Google Scholar CrossRef Search ADS PubMed  9 Tan KS. New insights on classification, identification and clinical relevance of Blastocystis spp. Clin Microbiol Rev  2008; 21: 639– 65. Google Scholar CrossRef Search ADS PubMed  10 Popruk S, Udonsom R, Koompapong K, et al.   Subtype distribution of Blastocystis in Thai-Myanmar border, Thailand. Korean J Parasitol  2015; 53: 13– 9. Google Scholar CrossRef Search ADS PubMed  11 Leelayoova S, Taamasri P, Rangsin R, et al.   In-vitro cultivation: a sensitive method for detecting Blastocystis hominis. Ann Trop Med Parasitol  2002; 96: 803– 7. Google Scholar CrossRef Search ADS PubMed  12 Scicluna SM, Tawari B, Clark CG. DNA barcoding of Blastocystis. 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Parasitol Int  2017; 66: 948– 51. Google Scholar CrossRef Search ADS PubMed  © The Author [2017]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Journal of Tropical PediatricsOxford University Press

Published: Aug 31, 2017

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