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 Table of Contents  
ORIGINAL ARTICLES
Year : 2018  |  Volume : 8  |  Issue : 1  |  Page : 33-40  

Detection of Blastocystis in clinical stool specimens using three different methods and morphological examination in Jones' medium


1 Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India
2 Department of Neonatology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India
3 Department of Medicine, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India

Date of Web Publication28-May-2018

Correspondence Address:
Subhash Chandra Parija
Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry - 605 006
India
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DOI: 10.4103/tp.TP_4_18

PMID: 29930905

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   Abstract 


Background: Blastocystis was identified almost a century ago, yet its biology and pathogenicity status in humans is obscure. Studies on Blastocystis in India are scanty and are mostly microscopy based. This study compared three detection modalities to determine their efficiency in the identification of Blastocystis in human feces.
Materials and Methods: A total of 279 stool samples were screened using microscopy, culture (Jones' medium), and polymerase chain reaction (PCR)-based methods. Among the three, PCR is considered the gold standard test for detection of Blastocystis, as it helps to authenticate the sensitivity, specificity, and kappa agreement obtained by the other two tests. The morphological features of Blastocystis were recorded at 24, 48, and 72 h. After positive morphological identification, ten samples were cultured on Löwenstein–Jensen (LJ) medium and Locke's egg slant medium.
Results: The sensitivity and specificity determined on the basis of microscopy were 36.2% and 99.4%, respectively. On the other hand, Jones' medium showed 67.6% sensitivity and 100% specificity. Further, we documented various morphological and reproductive features of Blastocystis using various staining techniques on cultures positive in Jones' medium. In addition, we also found that LJ medium was not equally efficacious as Jones' medium in assisting the growth of Blastocystis.
Conclusions: Although molecular diagnosis is a necessary tool for understanding the true epidemiology of Blastocystis, in laboratories devoid of molecular detection facilities, stool microscopy in conjunction with stool culture on Jones' medium could serve as the best alternative tool for the detection of Blastocystis.

Keywords: Blastocystis, India, Jones' medium, polymerase chain reaction, sensitivity, specificity


How to cite this article:
Padukone S, Mandal J, Rajkumari N, Bhat BV, Swaminathan RP, Parija SC. Detection of Blastocystis in clinical stool specimens using three different methods and morphological examination in Jones' medium. Trop Parasitol 2018;8:33-40

How to cite this URL:
Padukone S, Mandal J, Rajkumari N, Bhat BV, Swaminathan RP, Parija SC. Detection of Blastocystis in clinical stool specimens using three different methods and morphological examination in Jones' medium. Trop Parasitol [serial online] 2018 [cited 2018 Dec 14];8:33-40. Available from: http://www.tropicalparasitology.org/text.asp?2018/8/1/33/233336




   Introduction Top


Blastocystis, a unicellular stramenopile, inhabits the large intestine of humans and many other hosts. The pathogenic potential of Blastocystis in humans is debatable.[1] Many studies in the past decade have shown an association of Blastocystis with various enteric manifestations, as well as other extraintestinal complications.[2],[3]Blastocystis is cosmopolitan in occurrence, with a global prevalence ranging from 0.5% to 62%.[1] In fact, a study from Senegal reported 100% prevalence among schoolchildren.[4] It is one of the most common enteric parasites found in human stool samples in most of the developing countries. This could be attributable to the poor hygiene practices involved.[5] Currently, on the basis of the ribosomal lineages (small subunit [SSU] ribosomal RNA gene analyses), different Blastocystis species are designated as various subtypes (STs). The ST system is mainly based on the consensus developed by Stensvold et al. in 2007.[6] Till date, nine STs are known to infect humans, of which ST1, ST2, ST3, and ST4 together make up 90% of all Blastocystis STs reported in humans. Infection by Blastocystis can lead to an asymptomatic carrier state, or it may cause a varying degree of nonspecific symptoms such as abdominal pain, flatulence, nausea, vomiting, anorexia, weight loss, and acute or chronic diarrhea.[7],[8]

In India, data on Blastocystis are meager and chiefly derived from direct stool microscopy. Previous studies were based on microscopic techniques and have shown lesser prevalence rate ranging from 3% to 8%.[9],[10] However, a recent molecular-based study in a healthy population from the western part of India has reported 27% prevalence of Blastocystis.[11] Another study from the northern part of India reported the prevalence of Blastocystis to be 33.34% in patients with irritable bowel syndrome, which was much higher than the prevalence among the healthy individuals (15%) recruited in the same study.[12]

Blastocystis is highly polymorphic in appearance, thus complicating morphological diagnosis by microscopy. Hence, various alternative detection modalities such as culture and molecular methods are being employed for the identification of Blastocystis.[13],[14],[15] Among the molecular detection methods, real-time polymerase chain reaction (PCR) assay is considered to be a more sensitive method for detecting Blastocystis. However, sequencing of the PCR amplicon is mandatory to discriminate among the STs of Blastocystis.[16],[17] It has been reported that certain primer sets could not amplify all the STs of Blastocystis.[13],[18] On the other hand, the primers suggested by Scicluna et al. could do so and therefore, are the most preferred for subtyping. These primers can amplify the highly variable barcoding region of 18S SSU rDNA of all the relevant human Blastocystis STs.[19] Molecular detection studies on Blastocystis from India have also used the same barcoding primer set to determine its prevalence.[11],[12] Hence, in this study, the same barcoding primers have been used for detecting Blastocystis in DNA extracted directly from human stool samples.

In our study, we have employed microscopy, culture in Jones' medium, and PCR for the detection of Blastocystis from human stool specimens. The primary objective of the study was to compare the sensitivity and specificity obtained by microscopy and culture against a conventional PCR technique. In addition, the study also sought to explore the different morphological forms of Blastocystis in culture during various days of incubation. Besides, we have also attempted to determine the applicability of Locke's egg (LE) medium (NIH modification of Boeck and Drbohlav's medium) and Löwenstein–Jensen (LJ) medium for the growth of Blastocystis present in human stool samples.


   Materials and Methods Top


Stool specimens

A total of 279 stool samples were collected from the participants in the study after obtaining a signed informed consent. The study was conducted between June 2014 and August 2016. Approval was obtained from the Institute's Ethics Committee at JIPMER. Stool samples were examined macroscopically and microscopically at the Department of Microbiology, JIPMER, Puducherry, India. Further, the obtained stool samples were apportioned into three parts for use in microscopy, culture, and PCR techniques. For molecular analysis, the samples were stored at −80°C without any added preservatives.

Microscopy

Stool wet mount preparation was prepared using saline, iodine, and lactophenol cotton blue (LPCB) mount. Further, trichrome (Wheatley modified) staining was performed to detect Blastocystis and other possible parasitic infestations. During microscopy, to avoid ambiguity in diagnosing the polymorphic Blastocystis, only the presence of vacuolar form in two or more fields of stool wet mount preparation was considered as positive identification.

Culture

Following microscopy, approximately 50–100 mg of fresh stool samples were subjected toin vitro propagation in Jones' medium and incubated at 37°C. The growth of Blastocystis was confirmed by microscopic observation of culture at 24, 48, and 72 h of incubation. In positive cultures, the distinct morphological and reproductive stages of Blastocystis were recorded using various stains such as methylene blue, carbol fuchsin, Lugol's iodine, LPCB, and 4',6-diamidino-2-phenylindole (DAPI) fluorescent dyes.

Thereafter, ten microscopically positive stool samples were cultured on LE and LJ media. For LJ medium inoculation, we dissolved 50–100 mg of stool sample in 0.5–1 ml of normal saline, and these semi-solid stool samples were slowly poured on the slant surface of the medium. All the ten samples were simultaneously inoculated on three different media and incubated at 37°C for 48–72 h.

DNA extraction

DNA was extracted from the stool samples using QIAmp DNA Stool Mini Kit. The quality and quantity of the obtained DNA were evaluated either by running on 0.8% agarose gel or by ultraviolet absorbance using NanoDrop 2000C (Thermo Fisher, US). DNA was stored at −80°C until further molecular analysis.

Polymerase chain reaction

Blastocystis Primer Set 1 polymerase chain reaction and sequencing of representative samples

For PCR analysis, we used primers (Primer Set 1) suggested by Scicluna et al. (forward: RD5 5'-ATCGCCACTTCTCCAAT-3' and reverse: BhRDr 5'-GAGCTTTTTAACTGCAACAACG-3'), which amplifies the 600 bp product of 18S SSU rDNA of Blastocystis [Figure 1]. The PCR results obtained by Primer Set 1 were compared with the results obtained by microscopy and culture.[19] PCR amplification conditions consisted of 30 cycles of 1 min each at 95°C, 60°C, and 72°C, with a final elongation at 7 min. The PCR products were visualized by running on 1.5% agarose gel. Six representative PCR-positive amplicons (600 bp) were sequenced bidirectionally. Furthermore, CLUSTALW-aligned sequences were compared with the GenBank sequences using NCBI BLASTN server to confirm whether the amplified products belonged to Blastocystis ribosomal DNA.
Figure 1: Representative gel picture of Blastocystis-specific PCR using the Primer Set 1: L- 100 bp ladder, NC: Negative control, PC: Positive control and S1 to S7 – Stool DNA samples

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Blastocystis Primer Set 2–confirmatory polymerase chain reaction

All samples determined to be positive by Primer Set 1 were subjected to a confirmatory PCR by Primer Set 2 (forward: 5'-GGAGGTAGTGACAATAAATC-3' and reverse: 5'-ACTAGGAATTCCTCGTTCATG-3'), which amplifies 1112 bp of 18S SSUrDNA of Blastocystis.[20] For this PCR, the following conditions were used: initial denaturation at 94°C for 5 min followed by 94°C for 1 min, 49°C annealing for 1 min, 72°C 2-min elongation, and final extension for 7 min. PCR products were run on 1.3% agarose gel and visualized in gel documentation system (BIO-RAD).

Eukaryotic-specific polymerase chain reaction

All negative samples were screened by eukaryotic-specific PCR primers targeting a 140-bp fragment of the 18S ribosomal DNA (forward: 18SEUDIR 5′-TCTGCCCTATCAACTTTCGATGG-3′ and reverse: 18SEUINV 5′-TAATTTGCGCGCCTGCTG-3′). PCR products were visualized after running on 2% agarose gel, and it was considered as internal process control PCR.[21],[22] Samples that showed a negative identification by eukaryotic-specific PCR were again subjected to DNA extraction to eliminate false-negative results due to inhibitory substances in the PCR process.

All PCR reactions were conducted in 20-μl volumes. This volume was a mixture of Amplicon III Red Taq master mix (2X) (Tris HCL pH 8.5 (NH4)2 SO4, 4 mM MgCl2, 0.2% Tween 20, 0.4 mM dNTPs, and 0.2 units/μl amplicon Taq DNA polymerase), primers (0.4 μM), and 2 μl of DNA.

Statistical analysis

The PCR technique using Primer Set 1 is considered as the gold standard tool for the detection of Blastocystis. The sensitivity, specificity, positive predictive value, and negative predictive value of microscopy and culture techniques were calculated using MEDCALC ® online statistical software (https://www.medcalc.org/calc/diagnostic_test.php). The kappa agreement between the two tests was evaluated using the GraphPad online statistical software (https://graphpad.com/quickcalcs/kappa1/).


   Results Top


A total of 279 stool samples were collected from 153 males and 126 females, with ages ranging from 2 months to 81 years. All stool samples were screened for the presence of Blastocystis by microscopy, culture, and conventional PCR. Using microscopic techniques, 39 (13.98%) samples were determined as positive. On the other hand, Jones' medium assisted the growth of Blastocystis in 71 (25.45%) samples, which were determined as positive samples. However, PCR-based methods helped to determine the most number of positive samples (37.63%). On further confirmation, one sample, determined positive by microscopy, was found to be negative on the basis of culture and PCR techniques that the negative result was considered as a previous false-positive result. On the other hand, two samples, determined positive by culture, were found to be negative by PCR and microscopy techniques. However, the DNA of these samples could not be amplified in internal process control PCR, indicating PCR inhibition. Hence, these two samples were again subjected to DNA extraction, and DNA extracted from Blastocystis was successfully amplified using Primer Sets 1 and 2.

Subsequently, all the 105 PCR-positive samples amplified by Primer Set 1 were subjected to a confirmatory PCR using Primer Set 2. Primer Set 2 was able to amplify all the 105 positive samples. Further, the sequencing results of the six representative PCR-positive amplicons of Primer Set 1 confirmed that the PCR-amplified product (600 bp) belonged to Blastocystis SSU rDNA. Hence, all the 105 samples amplified using Primer Set 1 were considered as true positive. Sensitivity and specificity of the samples identified by microscopy in comparison to PCR methods were recorded as 36.19% and 99.43%, respectively. The kappa agreement on comparing the two tests was found to be 0.407 at 95% confidence interval [Table 1]. The culture method showed 67.62% sensitivity and 100.00% specificity when compared with PCR method. The kappa agreement on comparing these two tests was found to be 0.723 at 95% confidence interval [Table 2].
Table 1: Two-way contingency table to compare microscopy with polymerase chain reaction method

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Table 2: Two-way contingency table to compare culture with polymerase chain reaction method

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Coinfection with other parasites was recorded among the 105 PCR-positive samples. The highest rate of coexistence was found with Entamoeba spp. complex (n = 12), followed by Giardia spp., Strongyloides, Ascaris, and hookworms [Table 3].
Table 3: Microscopic results showing co-existence of other intestinal parasites among Blastocystis polymerase chain reaction-positive samples

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In the microscopy technique, all cultures were observed under ×20 and ×40 objectives of a light microscope at intervals of 24, 48, and 72 h [Figure 2]. During microscopic observation, we recorded all major forms of Blastocystis using various staining techniques [Figure 3]. The vacuolar forms were the most common forms present in 24- and 48-h cultures; in addition, these forms continued to exist even after 74 h of incubation. Usually, 3rd day cultures tend to possess a higher number of the granular form along with a rare presence of cystic forms. Variations in the size of Blastocystis are quite evident in most of the cultures; the vacuolar form shows a huge size variation ranging from 5 to 150 μm [Figure 4]. Using DAPI fluorescent dye, we observed multiple nuclei in the periphery of the cell [Figure 5]. We also recorded various reproductive modes of Blastocystis such as binary fission, budding, multiple budding, and schizogony-like reproduction [Figure 6]. Cystic forms of Blastocystis were seen in 72-h cultures or older cultures. Ameboid forms were observed only in two of the cultures, and it was a rare event [Figure 3]. In few cultures, motile Chilomastix mesnili were seen after 24 h of incubation [Figure 7], and one of these cultures was also positive for Blastocystis.
Figure 2: Appearance of Blastocystis grown in Jones' medium under ×200 (a) and ×400 (b)

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Figure 3: Four major morphological forms of Blastocystis in Jones' medium: (a) unstained wet mount preparation showing vacuolar form with peripheral nuclei (arrow head), (b) vacuolar form with translucent cell membrane and without prominent nucleus, (c) granular form unstained wet mount, (d) granular form stained with methylene blue, (e-g) cyst-like form unstained, in iodine stain, and lactophenol cotton blue stain, respectively (arrow head showing cyst formation inside degenerating cell), (h and i) Ameboid form of Blastocystis consisting of pseudopodia (arrowheads). Scale bar: 20 μm

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Figure 4: Size variation in Blastocystis. Scale bar: 20 μm

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Figure 5: 4',6-diamidino-2-phenylindole-stained Blastocystis culture showing peripheral nuclei (arrowheads)

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Figure 6: Different reproductive forms observed during in vitro culture in Jones' medium (a) A process of binary fission showing segregation of cytoplasmic and nuclear content, (b and c) multiple budding, (d) schizogony-like reproduction (arrowhead)

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Figure 7: Chilomastix mesnili grown in early culture (arrowheads)

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The ten microscopically positive fresh stool samples were cultured on LE medium and LJ medium, which assisted the growth of Blastocystis. LE medium facilitated the growth of Blastocystis in all ten microscopically positive samples. This is in concordance with the growth on Jones' medium. However, LJ medium was able to support the growth of Blastocystis in only two samples.


   Discussion Top


In this study, a total of 279 stool samples were screened by microscopy, culture, and PCR methods for the detection of Blastocystis in human stool specimens. The highest rate of positivity was recorded by PCR method. All the PCR-positive samples were subjected to another round of PCR to rule out any nonspecific amplification that might have occurred with the Primer Set 1. We also validated the PCR results by sequencing six representative positive PCR amplicons of Primer Set 1. The reasons for selecting Primer Set 1 as the main primer for detection and Primer Set 2 as a confirmatory primer were that most of the published Blastocystis diagnostic primers have been reported to amplify several other intestinal parasites or some of the primers could not amplify particular STs of Blastocystis.[13],[23] On the other hand, Primer Set 1 could amplify all the nine STs of Blastocystis relevant to human infection; sequencing of this PCR product is the most preferred and accepted method for subtyping of Blastocystis. Primer Set 2 was used to confirm the results obtained by Primer Set 1. Primer Set 2 is more specific, targeting a larger portion of Blastocystis ribosomal DNA, and it does not amplify any other intestinal parasitic DNA.[23] The Primer Set 2 is mainly used for subtyping of Blastocystis by PCR- RFLP analysis.[20] In microscopy, only the presence of a vacuolar form of Blastocystis was considered as positive to avoid perplexity in identification. However, the occurrence of one false-positive microscopic result illustrates the difficulties in microscopy-based detection, especially when the laboratory personnel are untrained or novices in the identification of Blastocystis. Stool microscopy is a widely used parasitological laboratory method, which is useful in diagnosing many intestinal parasites. However, in the case of Blastocystis, we found the microscopy technique to have low sensitivity, as the method failed to identify more than 50% of Blastocystis. The lower sensitivity of the microscopy technique could be because of lesser parasite load in the stool or because of our inclusion criteria considered, that is, only the presence of vacuolar form was considered as positive identification. Stool culture in Jones' medium augments the Blastocystis detection rate. The increase in the rate of detection using culture was attributable to the quick processing of fresh stool samples and the presence of persistent cystic stage of Blastocystis. The triple feces technique could have increased the detection rate of Blastocystis, both by microscopy and culture. Considering PCR as a gold standard detection method, microscopy showed sensitivity of <50%, and the strength of agreement between the tests was “moderate.” On the other hand, culture methods displayed 67.62% sensitivity and 100.00% specificity, with “good” strength of agreement between the tests. Hence, Jones' medium could be a better alternative detection modality for the detection of Blastocystis in resource-poor settings, where expensive PCR setups are unavailable.

The findings of our study are in consensus with previous reports that molecular detection modalities are superior over culture methods for the detection of Blastocystis from human stool samples.[12],[13],[15],[24] Conversely, there are few studies suggesting the usefulness of culture methods over PCR.[25],[26] The microscopy-based studies from India have shown a low prevalence of Blastocystis,[9],[10],[27] but molecular detection modalities have revealed a dramatic increase in the frequency of detection of Blastocystis.[11],[12] This study is the first molecular-based report from the southeastern part of India, revealing a higher detection rate of Blastocystis (37.63%) in the stool samples collected from participants attending a tertiary care hospital.

During culture examination, we were able to observe various morphological and reproductive forms of Blastocystis. Since the generation time for Blastocystis is 12–14 h, the vacuolar form of Blastocystis can be seen even just after 24 h of incubation, often with dividing cells. The granular forms start appearing with the depletion of the nutrients in Jones' medium. Rarely, we also encountered ameboid forms, which were usually present in the cultures of patients with intestinal manifestations. However, the identification of ameboid forms was obscure due to its sluggish motility. The cystic forms were smaller and frequent in old cultures. These findings from light microscopic examination were in consensus with the earlier reports.[28],[29] In Blastocystis, binary fission and budding are the well-accepted modes of reproduction. In addition to this, we also saw schizogony-like reproduction, as mentioned in earlier literatures.[28],[30] As light microscopy resolution is not sufficient to unveil the minor aspects of reproduction, the results should not be interpreted in excess of what was noted.[31] DAPI, a nuclear fluorescent dye, demonstrated the presence of peripherally located multiple nuclei. C. mesnili, which grew in Jones' medium, vanished upon prolonged incubation, and it did not interfere with the growth of Blastocystis.

A study from India employed LJ medium for culturing Blastocystis from stool.[27] In our laboratory, LJ and LE media are used routinely. Hence, out of curiosity, we considered growing ten microscopically positive fresh stool samples in both LJ and LE media to check the possibility of using these media as an alternative option in our setup. We found that LE medium and Jones' medium were equally effective in assisting the growth of Blastocystis, whereas in LJ medium, only two cultures were positive for the growth of Blastocystis. In culture, the facultative anaerobic bacteria present in stool are responsible for creating an anaerobic environment, which in turn assists the growth of Blastocystis. On the contrary, LJ medium contains malachite green, which inhibits most of the gut bacteria. The absence of anaerobiosis could be one of the reasons for the discrepant prevalence information of Blastocystis reported in a previous study that used LJ medium for the detection of Blastocystis.[27] Hence, LJ medium may not serve as a routine culture medium for the detection of Blastocystis in a diagnostic laboratory. Although LE medium was found to be equally efficient to Jones' medium in facilitating Blastocystis growth, the procedure for the LE medium preparation requires more time and additional sterile precautions. Hence, the use of Jones' medium is more advisable, as it is cost-effective in a resource-poor setting. However, in Jones' medium, avoiding contamination of the basal medium and usage of horse serum are matters of concern that should be taken care.


   Conclusions Top


In comparison with stool microscopy andin vitro culture, PCR method is an excellent detection tool for the identification of Blastocystis in human stool samples. Thus, prevalence studies should be based on the molecular detection methods to unveil the true prevalence of Blastocystis. Microscopy tends to miss out more than half of the Blastocystis infections. If three consecutive fecal sample screening techniques are employed, both microscopy and culture will pick up a higher number of Blastocystis infections. Hence, routine stool microscopy along with the xenic culture in Jones' medium could serve as an alternative detection tool for the identification of Blastocystis in resource-poor diagnostic laboratory settings.

Acknowledgments

We sincerely thank Dr. Christen Rune Stensvold, Statens Serum Institut, Copenhagen S, Denmark, and Dr. Prashant Kumar Pandey, Universidad Nacional Agraria La Molina, Lima, Perú, for providing us with Blastocystis DNA positive control.

Financial support and sponsorship

This study was financially supported by Intramural Research Grant, JIPMER.

Conflicts of interest

There are no conflicts of interest.



 
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Windsor JJ, Stenzel DJ, Macfarlane L. Multiple reproductive processes in Blastocystis hominis. Trends Parasitol 2003;19:289-90.  Back to cited text no. 31
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