• 1

    Stensvold CR, Suresh GK, Tan KSW, Thompson RCA, Traub RJ, Viscogliosi E, Yoshikawa H, Clark CG, 2007. Terminology for Blastocystis subtypes—a consensus. Trends Parasitol 23 :93–96.

    • Search Google Scholar
    • Export Citation
  • 2

    Stensvold CR, Alfellani MA, Nørskov-Lauritsen S, Prip K, Maddox C, Nielsen HV, Clark CG, 2009. Subtype distribution of Blastocystis isolates from synanthropic and zoo animals and identification of a new subtype. Int J Parasitol 39 :473–479.

    • Search Google Scholar
    • Export Citation
  • 3

    Stensvold CR, Nielsen HV, Smith HV, 2009. Pursuing the clinical impact of Blastocystis—diagnostic limitations. Trends Parasitol 25 :23–29.

    • Search Google Scholar
    • Export Citation
  • 4

    Clark CG, 2000. Cryptic genetic variation in parasitic protozoa. J Med Microbiol 49 :489–491.

  • 5

    Amin OM, 2000. Seasonal prevalence of intestinal parasites in the United States during 2000. Am J Trop Med Hyg 66 :799–803.

  • 6

    Noor Azian MY, San YM, Gan CC, Yusri MY, Nurulsyamzawaty Y, Zuhaizam AH, Maslawaty MH, Norparina I, Vythilingam I, 2007. Prevalence of intestinal protozoa in an aborigine community in Pahang, Malaysia. Trop Biomed 24 :55–62.

    • Search Google Scholar
    • Export Citation
  • 7

    Stensvold CR, Arendrup MC, Mølbak K, Nielsen HV, 2007. The prevalence of Dientamoeba fragilis in patients with suspected enteroparasitic disease in a metropolitan area in Denmark. Clin Microbiol Infect 13 :839–842.

    • Search Google Scholar
    • Export Citation
  • 8

    Suresh K, Ng GC, Ramachandran NP, Ho LC, Yap EH, Singh M, 1993. In vitro encystment and experimental infections of Blastocystis hominis. Parasitol Res 79 :456–460.

    • Search Google Scholar
    • Export Citation
  • 9

    Moe KT, Singh M, Howe J, Ho LC, Tan SW, Chen XQ, Ng GC, Yap EH, 1997. Experimental Blastocystis hominis infection in laboratory mice. Parasitol Res 83 :319–325.

    • Search Google Scholar
    • Export Citation
  • 10

    Hussein EM, Hussein AM, Eida MM, Atwa MM, 2008. Pathophysiological variability of different genotypes of human Blastocystis hominis Egyptian isolates in experimentally infected rats. Parasitol Res 102 :853–860.

    • Search Google Scholar
    • Export Citation
  • 11

    Leelayoova S, Rangsin R, Taamasri P, Naaglor T, Thathaisong U, Mungthin M, 2004. Evidence of waterborne transmission of Blastocystis hominis. Am J Trop Med Hyg 70 :658–662.

    • Search Google Scholar
    • Export Citation
  • 12

    Suresh K, Smith HV, Tan TC, 2005. Viable Blastocystis cysts in Scottish and Malaysian sewage samples. Appl Environ Microbiol 71 :5619–5620.

    • Search Google Scholar
    • Export Citation
  • 13

    Mehlhorn H, 1988. Blastocystis hominis, Brumpt 1912: are there different stages or species? Parasitol Res 74 :393–395.

  • 14

    Stenzel DJ, Boreham PFL, 1991. A cyst-like stage of Blastocystis hominis. Int J Parasitol 21 :613–615.

  • 15

    Zaman V, Howe J, Ng M, 1995. Ultrastructure of Blastocystis hominis cysts. Parasitol Res 81 :465–469.

  • 16

    Zaman V, 1996. The diagnosis of Blastocystis hominis cysts in human feces. J Infect 33 :15–16.

  • 17

    Suresh K, Smith H, 2004. Comparison of methods for detecting Blastocystis hominis. Eur J Clin Microbiol Infect Dis 23 :509–511.

  • 18

    Yoshikawa H, Yoshida K, Nakajima A, Yamanari K, Iwatani S, Kimata I, 2004. Fecal-oral transmission of the cyst form of Blastocystis hominis in rats. Parasitol Res 94 :391–396.

    • Search Google Scholar
    • Export Citation
  • 19

    Zaman V, Howe J, Ng M, 1997. Variation in the cyst morphology of Blastocystis hominis. Parasitol Res 83 :306–308.

  • 20

    Stenzel DJ, Lee MG, Boreham PFL, 1997. Morphological differences in Blastocystis cysts—an indication of different species? Parasitol Res 83 :452–457.

    • Search Google Scholar
    • Export Citation
  • 21

    Moe KT, Singh M, Howe J, Ho LC, Tan SW, Chen XQ, Yap EH, 1999. Development of Blastocystis hominis cysts into vacuolar forms in vitro. Parasitol Res 85 :103–108.

    • Search Google Scholar
    • Export Citation
  • 22

    Jones WR, 1946. The experimental infection of rats with Entamoeba histolytica; with a method for evaluating the anti-amoebic properties of new compounds. Ann Trop Med Parasitol 40 :130–140.

    • Search Google Scholar
    • Export Citation
  • 23

    Stensvold R, Brillowska-Dabrowska A, Nielsen HV, Arendrup MC, 2006. Detection of Blastocystis hominis in unpreserved stool specimens by using polymerase chain reaction. J Parasitol 92 :1081–1087.

    • Search Google Scholar
    • Export Citation
  • 24

    Stensvold CR, Arendrup MC, Jespersgaard C, Mølbak K, Nielsen HV, 2007. Detecting Blastocystis using parasitologic and DNA-based methods: a comparative study. Diagn Microbiol Infect Dis 59 :303–307.

    • Search Google Scholar
    • Export Citation
  • 25

    Henriksen SA, Pohlenz JF, 1981. Staining of cryptosporidia by a modified Ziehl-Neelsen technique. Acta Vet Scand 22 :594–596.

  • 26

    Hall TA, 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41 :95–98.

    • Search Google Scholar
    • Export Citation
  • 27

    Kumar S, Tamura K, Nei M, 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5 :150–163.

    • Search Google Scholar
    • Export Citation
  • 28

    Noël C, Dufernez F, Gerbod D, Edcomb VP, Delgado-Viscogliosi P, Ho LC, Singh M, Wintjens R, Sogin ML, Capron M, Pierce R, Zenner L, Viscogliosi E, 2005. Molecular phylogenies of Blastocystis isolates from different hosts: implications for genetic diversity, identification of species and zoonosis. J Clin Microbiol 43 :348–355.

    • Search Google Scholar
    • Export Citation
  • 29

    Badsberg JH, 2001. A guide to CoCo. J Stat Softw 6. Available at: http://www.jstatsoft.org/v06/i04.

  • 30

    Vennilla GD, Suresh Kumar G, Khairul Anuar A, Rajah S, Saminathan R, Sivanandan S, Ramakrishnan K, 1999. Irregular shedding of Blastocystis hominis. Parasitol Res 85 :162–164.

    • Search Google Scholar
    • Export Citation
  • 31

    Zaman V, Zaki M, Manzoor M, Howe J, Ng M, 1999. Postcystic development of Blastocystis hominis. Parasitol Res 85 :437–440.

  • 32

    Stensvold CR, Arendrup MC, Nielsen HV, Bada A, Thorsen S, 2008. Symptomatic infection with Blastocystis sp. subtype 8 successfully treated with trimethoprim-sulfamethoxazole. Ann Trop Med Parasitol 102 :271–274.

    • Search Google Scholar
    • Export Citation

 

 

 

 

Subtype Analysis of Blastocystis Isolates from Blastocystis Cyst Excreting Patients

View More View Less
  • 1 Department of Bacteriology, Mycology and Parasitology; Department of Quality Control, Statens Serum Institut, Copenhagen, Denmark

To determine the distribution of Blastocystis sp. subtypes from Blastocystis cyst excreters, 1,000 fecal samples from patients suspected of enteroparasitic disease were scored for stool consistency, submitted to xenic in vitro culture (XIVC), formol ethyl acetate concentration (FECT) with subsequent isopycnic centrifugation, and polymerase chain reaction (PCR) with subtype (ST) analysis. Blastocystis was significantly more prevalent in specimens from patients with travel-associated diarrhea (15.6%) than those with persistent diarrhea (8.3%) (P = 0.005). Overall, 115 (11.5%) and 35 (3.5%) specimens were positive by XIVC and FECT, respectively. Blastocystis cysts were detected in 33 (28.7%) of the XIVC-positive specimens. A positive FECT result was associated with ST3 (P = 0.05). The presence of Blastocystis in general or Blastocystis cysts was independent of stool consistency, and no particular ST was significantly associated with cyst identification. In view of these data, the present study indicates that Blastocystis cyst formation is independent of Blastocystis sp. subtype and gastrointestinal transit time.

INTRODUCTION

Blastocystis is a ubiquitous enteric protistan parasite of humans and a wide range of synanthropic and wild animals.13 It is perhaps the most frequently isolated protist in human fecal samples with a high prevalence in both developing and developed countries.47

Despite this, the clinical significance of Blastocystis remains poorly investigated, and other aspects regarding transmission and epidemiology are only partly uncovered. Direct, fecal-oral transmission of Blastocystis is probable, however, water-borne transmission may play a role as well.812 The life cycle of Blastocystis is similarly not completely settled but may involve some or all of the following stages: Vacuolar, avacuolar, multivacuolar, amoeboid, granular, and cyst stages.3 It is also possible that some of the stages develop only in vitro, e.g., in laboratory cultures.

The inconspicuous cyst stage was only recently discovered and described and can be detected in feces from Blastocystis-positive individuals after isopycnic centrifugation of fresh feces or fecal concentrates using Ficoll-Paque density gradients. 1317 However, in a recent study, only about 20% of Blastocystis-positive individuals were shown to excrete cysts. 17 It has been suggested that the cyst stage is the transmissible stage; however, it is not known whether, and in what ways, some or all of the other forms are transmissible. 9,12,13,18 Cysts may be seen with or without a membraneous layer, and reports on cyst sizes and morphology vary. 16,1921

The Blastocystis genus includes isolates that display extensive genetic diversity, and it was recently proposed that all Blastocystis isolates from humans, mammals, and birds be assigned to one of nine subtypes (STs) (ST1 through ST9); lately, a novel subtype (ST10) was shown to be hosted by primates and mammals.13

In this study we aimed to 1) determine the prevalence of Blastocystis among patients suffering primarily from persistent and travel-associated diarrhea by xenic in vitro culture (XIVC) and formol ethyl acetate concentration (FECT), 2) establish whether the consistency of stools is associated to Blastocystis carriage in general or Blastocystis sp. subtype, 3) determine the proportion of Blastocystis cyst excreters among Blastocystis-positive patients, and 4) establish whether cyst excretion is associated to Blastocystis sp. subtype.

MATERIALS AND METHODS

Specimens, parasitologic examination, and cyst identification.

One thousand fresh stool specimens submitted to the Laboratory of Parasitology, Statens Serum Institut, Copenhagen, by 799 patients with suspected enteroparasitic disease were randomly selected and analyzed over a period of 6 months (November 2007–May 2008). Specimens were submitted to Blastocystis-specific XIVC using Jones’ medium and evaluated for the presence of Blastocystis after 48 hours. 22,23 From each specimen, 250 mg of feces was stored at −20°C for subsequent DNA extraction. Finally, the remainder of each of the specimens was processed by an in-house FECT. 24 Ziehl-Neelsen staining was done to enable the detection of sporozoa. 25 Fecal concentrates obtained by FECT were evaluated for ova, (oo)cysts, and larvae, and fecal concentrates from XIVC-positive patients were submitted to Ficoll-Paque centrifugation and examined for the presence of Blastocystis cysts. 17 Briefly, fecal concentrates obtained by the FECT were mixed with 1 mL of distilled water and layered on 10 mL of Ficoll-Paque (Phamacia, Uppsala, Sweden) column and centrifuged at 2,000 × g for 20 minutes. Ficoll-Paque concentrated cysts were aspirated into a clean test tube and re-centrifuged in 20 mL of distilled water at 300 × g for 20 minutes to remove excess Ficoll-Paque. Pellet was resuspended and examined for the presence of Blastocystis cysts by phase contrast microscopy at ×600 magnification. Quantification of cysts was not performed.

The stool consistency (SC) of each fresh fecal specimen was determined and scored as follows: SC1 = solid, SC2 = semi-solid, SC3 = loose, and SC4 = watery. Records also included information on whether a specimen was submitted because of persistent diarrhea (R193), travel-associated diarrhea (R194), diarrhea in an immunocompromised patient (R195), diarrhea in an institutionalized patient (R196), or whether the sample was submitted to ordinary parasitologic examination (R071). Demographic data such as age and gender were collected.

PCR and ST analysis.

The DNA was extracted directly from feces from samples positive by XIVC or FECT using the QIAGEN Stool Mini Kit (QIAGEN, Hilden, Germany), and from each DNA sample 2 μL were used as template in a 25 μL PCR using primers described by Stensvold and others. 23,24 Inhibition controls were not incorporated. The PCR products were gel-purified using the UltraClean GelSPin DNA Purification Sample kit (SANBIO, Uden, The Netherlands) and dideoxy sequenced in one direction using the BHCRseq3 primer as the sequencing primer. 24 For ST identification, sequence chromatograms were analyzed, aligned, and manually interpreted using the software program BioEdit Sequence Alignment Editor. 26 Distance-based analysis was conducted with MEGA 3.1, and trees constructed using the neighbor-joining algorithm with the Kimura 2-parameter model; reference sequences available in GenBank were used as reference sequences, Proteromonas lacertae (U37108) was used as the out-group, and a ST was assigned to a given sequence based on the reference sequences it clustered with. 27,28 Blastocystis sp. subtype terminology according to a previous study was used.1

Statistical analysis included testing for independence by Pearson’s χ2 test using the software package CoCo, 29 however, where specifically indicated Fisher’s exact test was used.

RESULTS

Of the 1,000 specimens submitted by the 799 patients, 132 specimens were submitted to ordinary parasitologic examination, 411 specimens were from individuals with persistent diarrhea, 436 from patients with travel-associated diarrhea, 11 from immunocompromised patients, and 10 specimens were from institutionalized patients (Table 1). A total of 115 (11.5%) of the specimens were positive for Blastocystis by XIVC, whereas 35 (3.5%) were positive by FECT, 27 of which were positive by XIVC. Of the 115 specimens, 13 were also positive for other parasites (Giardia duodenalis, Entamoeba spp., and Endolimax nana). The 115 positive specimens were from a total of 87 patients, and thus the overall prevalence of Blastocystis was 10.9%. Sixty-eight specimens (59.1%) were from patients with travel-associated diarrhea, and the proportion of Blastocystis-positive specimens was significantly higher among the patients examined for travel-associated diarrhea (R194) than among patients from other categories (P = 0.005).

Of the XIVC-positive specimens, 65.2% were characterized as loose (SC3) (Table 2). However, the association between SC and the presence of Blastocystis as determined by XIVC was non-significant (P = 0.13), and there was no significant association between SC and the presence of cysts of Blastocystis (P = 0.34). In total, 33 specimens were positive for cysts of Blastocystis, and the proportion of XIVC-positive specimens positive for cysts was 28.7%. Of the cyst-positive specimens 78.8% were scored as loose. Mostly “naked” cysts were detected, attaining sizes of 3–4 μm and appearing more rounded than ovoid.

Of 115 XIVC-positive specimens, 99 Blastocystis isolates from 74 patients were successfully PCR amplified and sequenced (Table 3). Overall, ST3 was the most prevalent subtype (36.5%), whereas ST1, ST4, and ST2 were seen in 20.3%, 16.2%, and 14.9% of the patients, respectively. The ST6 and ST8 were each seen in one case and in both cases FECT results were negative. Mixed ST infection was detected in 7 samples from 6 patients (8.1%). The association between ST and SC was non-significant (P = 0.21). In cases of single ST infections where nucleotide sequences were obtained from multiple samples from the same patient, no sequence variation was detected.

Six of 8 samples positive by FECT and negative by XIVC were shown to be positive for Blastocystis by PCR, and sequencing revealed that the STs amplified in these samples were ST3 (3 samples), ST2 (2 samples), and ST1 (1 sample).

Although there was a tendency toward the FECT being less sensitive with regard to the detection of ST3 (Table 4), a positive FECT result was independent of ST (P = 0.07); however, when FECT results for ST3 were compared with pooled results for ST1, ST2, ST3, the trend toward positive FECT results depending on subtype became stronger (P = 0.05, Fisher’s exact test). The presence of cysts was non-significantly associated with ST (P = 0.20).

Among patients with travel-associated diarrhea ST3 predominated, whereas ST1 was slightly predominant among patients with persistent diarrhea (Table 5). Eighty percent of ST2 isolates were from patients with travel-associated diarrhea. However, because of small numbers and the fact that 13 of the PCR-positive patients contributed with more than one specimen, this finding should be interpreted with caution.

Analysis of demographic data did not reveal any significant association between age group or gender and Blastocystis sp. subtype (data not shown).

DISCUSSION

Studies seeking to combine phenotypical and molecular analyses of Blastocystis are still few, but necessary for the understanding of the clinical significance and epidemiology of the parasite.

The majority of the specimens analyzed in this study were from patients with travel-associated diarrhea (43.6%) and persistent diarrhea (41.1%), and the prevalence of Blastocystis among patients with travel-associated diarrhea was significantly higher than among patients with chronic diarrhea. Previous data from a similar Danish study saw no such association; however, being based on the analysis of 1,000 samples, the present study should be considered noticeably weightier than the previous one, in which only 117 samples from 103 patients were analyzed.7

Cysts were detected in 28.7% of the samples positive by XIVC compared with 20.5% reported by Suresh and Smith. 17 The fact that only a fraction of the stools were positive for cysts might be explained by the possibility that cysts are shed only intermittently. 30 Because the amount of feces per specimen analyzed could not be standardized, it cannot be ruled out that the probability of detecting cysts by isopycnic centrifugation of fecal concentrates could be proportional to the amount of material analyzed.

Interestingly, the data do not support the hypothesis that cysts are more prevalent in solid or semi-solid stools than watery stools, which indicates that the formation and presence of cysts in stools might be independent of gastrointestinal transit time, unless encystation (also) occurs outside the intestine, i.e., in the environment, resulting from physical and chemical conditions. Recently, it was shown that the relative proportion of FECT-positive samples from ST3-positive individuals was significantly lower than that of FECT-positive samples from individuals harboring other STs. 24 It was speculated that ST3-positive individuals were more prone to cyst excretion than individuals infected with other subtypes, which could be the reason why a significant proportion of ST3-positive samples was negative by conventional microscopy of formol ethyl acetate concentrates. Furthermore, in this study the relative sensitivity of the FECT with regard to the detection of ST3 isolates was low compared with other STs. However, the fact that the percentage of cyst positives was similar to the percentage of XIVC positives for each ST suggests that the amount of cyst production is also similar between STs and because no association between the detection of cysts and ST could be established, this issue remains unresolved.

Blastocystis cysts have been shown to develop into vacuolar stages in vitro within 24 hours. 12,21 It is therefore possible and plausible that the vacuolar stages often detected in Blastocystis cultures arise from the cyst stage, despite the absence of gastric acid and intestinal enzymes. 31 The present data, however, suggest that cysts are present in less than 30% of the XIVC-positive specimens. Therefore, it might be speculated that non-cyst life cycle stages present in fresh fecal material (e.g., vacuolar forms) may (re-)develop into vacuolar forms.

The distribution of subtypes detected in the present study resembles the one reported in similar studies.2 Hence, ST3 was the most prevalent subtype, followed in prevalence by ST1, ST4, and ST2. Two possibly zoonotic STs were detected: ST6 was detected in one case from a patient with solid stools, whereas ST8 was detected in another case from a patient with loose stools.

To the authors’ knowledge, this study is the first large-scale study to date seeking to unravel any potential correlation between SC and Blastocystis carriage in a situation where the presence of co-infecting parasites is negligible. Although there was no significant association between SC and Blastocystis carriage in general or any particular Blastocystis sp. subtype, it appeared that all seven specimens characterized by mixed subtype infections (MSI) were scored as loose (SC3). Moreover, there was a tendency toward ST4 isolates being less associated with loose or watery stools than other subtypes.

The majority (80%) of the 15 ST2 isolates were seen in patients with travel-associated diarrhea, whereas, for instance, only 35% of the 20 ST1 isolates were from such patients (Table 5). So far, potential differences in the geographic distribution of the different subtypes have been scarcely investigated. It is, however, difficult to draw conclusions on this based on the present data, because patients also with persistent diarrhea may have been exposed to travel previously and it is likely that Blastocystis infections can persist for months, maybe even years, if left untreated. 32

Although the XIVC was negative in 8 FECT-positive cases, the present study confirms findings from a recent study, where the FECT was found to be significantly less sensitive in diagnosing Blastocystis infections.24 In this study, the sensitivity of the FECT was only approximately 23% of that of XIVC. Likewise, Suresh and Smith 17 reported that none of the 1,000 specimens were positive by FECT, whereas 39 samples were positive by XIVC. Therefore, the present data reinforce the significant differences in the sensitivity of various diagnostic methods.

Table 1

Relationship between Blastocystis-positive specimens by xenic in vitro culture (XIVC) or formol ethyl acetate concentration technique (FECT) and test category

Table 1
Table 2

Relationship between stool consistency (SC) and the presence of Blastocystis as determined by xenic in vitro culture (XIVC) and Blastocystis cysts as determined by isopycnic concentration of fecal concentrates obtained by formol ethyl acetate concentration (FECT)

Table 2
Table 3

Relationship between stool consistency (SC) and the presence of Blastocystis sp. subtypes (ST) as determined by polymerase chain reaction (PCR) and sequencing of isolates detected by xenic in vitro culture (XIVC)

Table 3
Table 4

Number of Blastocystis sp. subtypes (ST) isolated by xenic in vitro culture (XIVC), formol ethyl acetate concentration (FECT), and isopycnic centrifugation of FECT concentrates (cyst detection)

Table 4
Table 5

Relationship between Blastocystis sp. subtype (ST) and test category (see text for details)

Table 5

*

Address correspondence to Christen R. Stensvold, Department of Bacteriology, Mycology and Parasitology, Building 43, Room no. 114, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark. E-mail: RUN@ssi.dk

Authors’ addresses: Betangbeh A. Rene, Christen R. Stensvold, and Henrik V. Nielsen, Department of Bacteriology, Mycology and Parasitology, Building 43, Room no. 114, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark. Jens H. Badsberg, Department of Quality Control, Building 207, Room no. 307, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark.

Acknowledgments: We thank Jørgen Grønvold and Per Moestrup Jensen for kind supervision. Lis Lykke Wassmann and her colleagues at the Laboratory of Parasitology, Statens Serum Institut, are thanked for excellent technical assistance. Graham Clark is thanked for a critical revision of the manuscript.

Financial support: No financial support was received for the study.

Disclosure: The present study represents the work undertaken by Betangbeh Ategwa Rene in order to obtain a MSc degree in Parasitology at the University of Copenhagen.

REFERENCES

  • 1

    Stensvold CR, Suresh GK, Tan KSW, Thompson RCA, Traub RJ, Viscogliosi E, Yoshikawa H, Clark CG, 2007. Terminology for Blastocystis subtypes—a consensus. Trends Parasitol 23 :93–96.

    • Search Google Scholar
    • Export Citation
  • 2

    Stensvold CR, Alfellani MA, Nørskov-Lauritsen S, Prip K, Maddox C, Nielsen HV, Clark CG, 2009. Subtype distribution of Blastocystis isolates from synanthropic and zoo animals and identification of a new subtype. Int J Parasitol 39 :473–479.

    • Search Google Scholar
    • Export Citation
  • 3

    Stensvold CR, Nielsen HV, Smith HV, 2009. Pursuing the clinical impact of Blastocystis—diagnostic limitations. Trends Parasitol 25 :23–29.

    • Search Google Scholar
    • Export Citation
  • 4

    Clark CG, 2000. Cryptic genetic variation in parasitic protozoa. J Med Microbiol 49 :489–491.

  • 5

    Amin OM, 2000. Seasonal prevalence of intestinal parasites in the United States during 2000. Am J Trop Med Hyg 66 :799–803.

  • 6

    Noor Azian MY, San YM, Gan CC, Yusri MY, Nurulsyamzawaty Y, Zuhaizam AH, Maslawaty MH, Norparina I, Vythilingam I, 2007. Prevalence of intestinal protozoa in an aborigine community in Pahang, Malaysia. Trop Biomed 24 :55–62.

    • Search Google Scholar
    • Export Citation
  • 7

    Stensvold CR, Arendrup MC, Mølbak K, Nielsen HV, 2007. The prevalence of Dientamoeba fragilis in patients with suspected enteroparasitic disease in a metropolitan area in Denmark. Clin Microbiol Infect 13 :839–842.

    • Search Google Scholar
    • Export Citation
  • 8

    Suresh K, Ng GC, Ramachandran NP, Ho LC, Yap EH, Singh M, 1993. In vitro encystment and experimental infections of Blastocystis hominis. Parasitol Res 79 :456–460.

    • Search Google Scholar
    • Export Citation
  • 9

    Moe KT, Singh M, Howe J, Ho LC, Tan SW, Chen XQ, Ng GC, Yap EH, 1997. Experimental Blastocystis hominis infection in laboratory mice. Parasitol Res 83 :319–325.

    • Search Google Scholar
    • Export Citation
  • 10

    Hussein EM, Hussein AM, Eida MM, Atwa MM, 2008. Pathophysiological variability of different genotypes of human Blastocystis hominis Egyptian isolates in experimentally infected rats. Parasitol Res 102 :853–860.

    • Search Google Scholar
    • Export Citation
  • 11

    Leelayoova S, Rangsin R, Taamasri P, Naaglor T, Thathaisong U, Mungthin M, 2004. Evidence of waterborne transmission of Blastocystis hominis. Am J Trop Med Hyg 70 :658–662.

    • Search Google Scholar
    • Export Citation
  • 12

    Suresh K, Smith HV, Tan TC, 2005. Viable Blastocystis cysts in Scottish and Malaysian sewage samples. Appl Environ Microbiol 71 :5619–5620.

    • Search Google Scholar
    • Export Citation
  • 13

    Mehlhorn H, 1988. Blastocystis hominis, Brumpt 1912: are there different stages or species? Parasitol Res 74 :393–395.

  • 14

    Stenzel DJ, Boreham PFL, 1991. A cyst-like stage of Blastocystis hominis. Int J Parasitol 21 :613–615.

  • 15

    Zaman V, Howe J, Ng M, 1995. Ultrastructure of Blastocystis hominis cysts. Parasitol Res 81 :465–469.

  • 16

    Zaman V, 1996. The diagnosis of Blastocystis hominis cysts in human feces. J Infect 33 :15–16.

  • 17

    Suresh K, Smith H, 2004. Comparison of methods for detecting Blastocystis hominis. Eur J Clin Microbiol Infect Dis 23 :509–511.

  • 18

    Yoshikawa H, Yoshida K, Nakajima A, Yamanari K, Iwatani S, Kimata I, 2004. Fecal-oral transmission of the cyst form of Blastocystis hominis in rats. Parasitol Res 94 :391–396.

    • Search Google Scholar
    • Export Citation
  • 19

    Zaman V, Howe J, Ng M, 1997. Variation in the cyst morphology of Blastocystis hominis. Parasitol Res 83 :306–308.

  • 20

    Stenzel DJ, Lee MG, Boreham PFL, 1997. Morphological differences in Blastocystis cysts—an indication of different species? Parasitol Res 83 :452–457.

    • Search Google Scholar
    • Export Citation
  • 21

    Moe KT, Singh M, Howe J, Ho LC, Tan SW, Chen XQ, Yap EH, 1999. Development of Blastocystis hominis cysts into vacuolar forms in vitro. Parasitol Res 85 :103–108.

    • Search Google Scholar
    • Export Citation
  • 22

    Jones WR, 1946. The experimental infection of rats with Entamoeba histolytica; with a method for evaluating the anti-amoebic properties of new compounds. Ann Trop Med Parasitol 40 :130–140.

    • Search Google Scholar
    • Export Citation
  • 23

    Stensvold R, Brillowska-Dabrowska A, Nielsen HV, Arendrup MC, 2006. Detection of Blastocystis hominis in unpreserved stool specimens by using polymerase chain reaction. J Parasitol 92 :1081–1087.

    • Search Google Scholar
    • Export Citation
  • 24

    Stensvold CR, Arendrup MC, Jespersgaard C, Mølbak K, Nielsen HV, 2007. Detecting Blastocystis using parasitologic and DNA-based methods: a comparative study. Diagn Microbiol Infect Dis 59 :303–307.

    • Search Google Scholar
    • Export Citation
  • 25

    Henriksen SA, Pohlenz JF, 1981. Staining of cryptosporidia by a modified Ziehl-Neelsen technique. Acta Vet Scand 22 :594–596.

  • 26

    Hall TA, 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41 :95–98.

    • Search Google Scholar
    • Export Citation
  • 27

    Kumar S, Tamura K, Nei M, 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5 :150–163.

    • Search Google Scholar
    • Export Citation
  • 28

    Noël C, Dufernez F, Gerbod D, Edcomb VP, Delgado-Viscogliosi P, Ho LC, Singh M, Wintjens R, Sogin ML, Capron M, Pierce R, Zenner L, Viscogliosi E, 2005. Molecular phylogenies of Blastocystis isolates from different hosts: implications for genetic diversity, identification of species and zoonosis. J Clin Microbiol 43 :348–355.

    • Search Google Scholar
    • Export Citation
  • 29

    Badsberg JH, 2001. A guide to CoCo. J Stat Softw 6. Available at: http://www.jstatsoft.org/v06/i04.

  • 30

    Vennilla GD, Suresh Kumar G, Khairul Anuar A, Rajah S, Saminathan R, Sivanandan S, Ramakrishnan K, 1999. Irregular shedding of Blastocystis hominis. Parasitol Res 85 :162–164.

    • Search Google Scholar
    • Export Citation
  • 31

    Zaman V, Zaki M, Manzoor M, Howe J, Ng M, 1999. Postcystic development of Blastocystis hominis. Parasitol Res 85 :437–440.

  • 32

    Stensvold CR, Arendrup MC, Nielsen HV, Bada A, Thorsen S, 2008. Symptomatic infection with Blastocystis sp. subtype 8 successfully treated with trimethoprim-sulfamethoxazole. Ann Trop Med Parasitol 102 :271–274.

    • Search Google Scholar
    • Export Citation

Author Notes

Reprint requests: Christen R. Stensvold, Department of Bacteriology, Mycology and Parasitology, Building 43, Room no. 114, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark, Tel: +45 3268 3604, Fax: +45 3268 3033, E-mail: RUN@ssi.dk.
Save