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Fulltext Amoebic forms of Blastocystis spp. - evidence for a pathogenic role

Rajamanikam A, Govind SK

Parasit Vectors, 2013;6(1):295.
PMID: 24499467 DOI: 10.1186/1756-3305-6-295

Blastocystis spp. are one of the most prevalent parasites isolated from patients suffering from diarrhea, flatulence, constipation and vomiting. It's pathogenicity and pathophysiology remains controversial to date. Protease activity and amoebic forms have been reported previously in symptomatic isolates but there has been no conclusive evidence provided to correlate the protease activity and any specific life cycle stage of the parasite thus far.
Modified fields' stain: ideal to differentiate Dientamoeba fragilis and Blastocystis sp

Ragavan AD, Govind SK

Parasitol Res, 2015 Mar;114(3):1163-6.
PMID: 25614298 DOI: 10.1007/s00436-014-4296-8

Dientamoeba fragilis, a trichomonad parasite is usually found in the gastrointestinal tract of human, and it is known to be the cause for gastrointestinal disease. The parasite is globally distributed and mostly found in rural and urban areas. The parasite is found in humans and nonhuman primates such as the macaques, baboons, and gorillas. Often, the parasite is confused with another largely found organism in stools called Blastocystis sp. especially when seen directly under light microscopy on culture samples containing both parasites. Both sometimes are seen with two nuclei with sizes tending to be similar which complicates identification. Stools were collected fresh from nine previously diagnosed persons infected with D. fragilis who also were found to be positive for Blastocystis sp. Samples were then cultured in Loeffler's medium and were stained with Giemsa, iron hematoxylin, and modified Fields' (MF) stain, respectively. D. fragilis was differentiated from Blastocystis sp. when stained with MF stain by the presence of a thinner outer membrane with clearly demarcated nuclei in the center of the cell whilst Blastocystis sp. had a darker and thicker stained outer membrane with the presence of two nuclei. The staining contrast was more evident with modified Fields' stain when compared with the other two. The simplicity in preparing the stain as well as the speed of the staining procedure make MF stain an ideal alternate. The modified Fields' stain is faster and easier to prepare when compared to the other two stains. MF stain provides a better contrast differentiating the two organisms and therefore provides a more reliable diagnostic method to precisely identify one from the other especially when cultures show mixed infections.
Establishing a protocol for water sample processing for the detection of Blastocystis sp

Lee IL, Tan TC, Govind SK

Exp Parasitol, 2019 Mar;198:105-110.
PMID: 30695704 DOI: 10.1016/j.exppara.2019.01.007

This study was aimed at establishing a protocol for water sample processing for the detection of Blastocystis sp. using distilled water spiked with Blastocystis sp. cysts. The study established a protocol involving eight technical aspects, namely, storage temperature, storage duration, minimum water sample volume, optimum relative centrifugal force, centrifugation duration, minimum number of cyst for inoculation in Jones' medium and turn-around-time for the detection of vacuolar forms of Blastocystis sp. Results showed a minimum of 1.0 L water sample should be collected and processed on the same day. Otherwise, it should be stored at 4 °C and processed within 3 days. Water sample should be centrifuged at 1400×g for 10 min. For the isolation of Blastocystis sp. cysts, parasite pellet could be layered on top of Ficoll-Paque™ PLUS, centrifuged at 1400×g for 20 min and washed twice using 0.9% saline with centrifugation at 1400×g for 10 min. A minimum of 1 × 105 cysts could then be inoculated in Jones' medium supplement with 10% horse serum, incubated at 37 °C and examined for any presence of vacuolar forms of Blastocystis sp. after 3 days of inoculation. A protocol for water sample processing for the detection of Blastocystis sp. has successfully been established. The protocol was validated using 106 various water samples. This protocol will be very useful in determining the extent of Blastocystis sp. contamination in water sources in order to identify the seriousness of contamination.
In vitro and in vivo thermal stress induces proliferation of Blastocystis sp

Thergarajan G, Govind SK, Bhassu S

Parasitol Res, 2018 Jan;117(1):177-187.
PMID: 29188368 DOI: 10.1007/s00436-017-5688-3

Blastocystis sp. is known to be the most commonly found intestinal protozoan parasite in human fecal surveys and has been incriminated to cause diarrhea and abdominal bloating. Binary fission has been widely accepted as the plausible mode of reproduction for this parasite. The present study demonstrates that subjecting the parasites in vitro to higher temperature shows the proliferation of parasite numbers in cultures. Transmission electron microscopy was used to compare the morphology of Blastocystis sp. subtype 3 isolated from a dengue patient having high fever (in vivo thermal stress) and Blastocystis sp. 3 maintained at 41 °C (in vitro thermal stress) and 37 °C (control). Fluorescence stains like acridine orange (AO) and 4',6'-diamino-2-phenylindole (DAPI) were used to demonstrate the viability and nuclear content of the parasite for both the in vitro and in vivo thermal stress groups of parasites. Blastocystis sp. at 37 °C was found to be mostly vacuolar whereas the in vitro thermal stressed isolates at 41 °C were granular with electron dense material seen to protect the granules within the central body. Parasites of the in vivo thermal stressed group showed similar ultrastructure as the in vitro ones. AO and DAPI staining provided evidence that these granules are viable which develop into progenies of Blastocystis sp. These granular forms were then observed to rupture and release progenies from the mother cells whilst the peripheral cytoplasmic walls were seen to degrade. Upon exposure to high temperature both in vitro and in vivo, Blastocystis sp. in cultures show higher number of granular forms seen to be protected by the electron dense material within the central body possibly acting as a protective mechanism. This is possibly to ensure the ability to survive for the granules to be developed as viable progenies for release into the host system.
Fulltext Phenotypic variation in Blastocystis sp. ST3

Ragavan ND, Govind SK, Chye TT, Mahadeva S

Parasit Vectors, 2014;7:404.
PMID: 25174569 DOI: 10.1186/1756-3305-7-404

Blastocystis, is one of the most common human intestinal protozoan, which has many conflicting reports on its pathogenic role. Gut conditions which obviously varies in asymptomatic individuals, symptomatic and irritable bowel syndrome (IBS) patients in terms of gut flora, pH, osmotic pressure and water potentials could play an important role in its pathogenicity. The present study is the first study to investigate phenotypic characteristics of Blastocystis sp. ST3 isolated from asymptomatic, symptomatic and IBS isolates.
Fulltext Blastocystis sp.: waterborne zoonotic organism, a possibility?

Lee LI, Chye TT, Karmacharya BM, Govind SK

Parasit Vectors, 2012;5:130.
PMID: 22741573 DOI: 10.1186/1756-3305-5-130

Blastocystis sp. is a common intestinal parasite found in faecal sample surveys. Several studies have implicated human-to-human, zoonotic and waterborne transmissions by Blastocystis sp. However, there has been no study providing evidence interlinking these three transmissions in a community. We have previously shown a high prevalence of Blastocystis sp. subtype 4 amongst village dwellers in Bahunipati, Nepal, and the present study extends the observation to assess if the same subtype of Blastocystis sp. occurs in animals they rear and rivers they frequent.
Factors that influence the shedding of Blastocystis cysts in an irritable bowel syndrome (IBS) patient--an evidence-based case study

Ragavan ND, Govind SK, Chye TT, Mahadeva S

Parasitol Res, 2015 Aug;114(8):2999-3005.
PMID: 25982571 DOI: 10.1007/s00436-015-4502-3

Blastocystis is one of the most common gut parasites found in the intestinal tract of humans and animals. We have previously reported the irregular shedding of Blastocystis cysts in stools from infected patients. In the present study, we assess the factors influencing shedding patterns from a Blastocystis ST3-infected IBS patient. The stools samples were voluntarily submitted for examination for a period of 30 days from Blastocystis ST3-infected IBS patient. A questionnaire on the factors that could influence the shedding pattern of the cysts was designed to assess the following information: (a) the frequency of frequenting the toilet in a day, (b) the timing of frequenting the toilet, (c) the stool forms, (d) the type of mood the patient was in when frequenting the toilet and (e) food intake. A total of 79 stool samples were collected for 30 days. The highest number of cysts recorded when the patient visited the toilet three times a day was 22.2 × 10(6) cysts/g. Frequenting the toilet between 6 a.m. to 11.59 a.m. showed the highest number of cysts, i.e. 21.7 × 10(6) cysts/g. Semi-solid forms showed the highest cyst count, i.e. 2.00 × 10(6) cysts/g. Irregular shedding of cysts was seen in 10 out of 30 days where the widest range recorded on day 17 was between 0 to 1.2 × 10(6) cysts/g. The average daily cyst count on days of emotional fluctuations was from 0 to 5.13 × 10(6) cysts/g. In conclusion, the study confirms that there are factors influencing shedding patterns of Blastocystis, and these have important implications when it comes to diagnosis and transmission of the parasite.
Blastocystis sp. reduces the efficacy of 5-fluorouracil as a colorectal cancer chemotherapeutic treatment

Kumarasamy V, Kuppusamy UR, Jayalakshmi P, Govind SK

Exp Parasitol, 2023 Aug;251:108564.
PMID: 37308003 DOI: 10.1016/j.exppara.2023.108564

Blastocystis is an enteric protozoan parasite with extensive genetic variation and unclear pathogenicity. It is commonly associated with gastrointestinal symptoms such as nausea, diarrhea, vomiting and abdominal pain in immunocompromised individuals. In this study, we explored the in vitro and in vivo effects of Blastocystis on the activity of a commonly used CRC chemotherapeutic agent, 5-FU. The cellular and molecular effects of solubilized antigen of Blastocystis in the presence of 5-FU were investigated using HCT116, human CRC cell line and CCD 18-Co, normal human colon fibroblast cells. For the in vivo study, 30 male Wistar rats were divided into six groups, as follows; Control Group: oral administration of 0.3 ml Jones' medium, Group A: rats injected with azoxymethane (AOM), Group A-30FU: Rats injected with AOM and administered 30 mg/kg 5-FU, Group B-A-30FU: rats inoculated with Blastocystis cysts, injected with AOM and administered 30 mg/kg 5-FU, Group A-60FU: rats injected with AOM and administered 60 mg/kg 5-FU and Group B-A-60FU: rats inoculated with Blastocystis cysts, injected with AOM and administered 60 mg/kg 5-FU. The in vitro study revealed that the inhibitory potency of 5-FU at 8 μM and 10 μM was reduced from 57.7% to 31.6% (p
Fulltext High lipid storage in vacoular forms of subtype 6 Blastocystis sp. in ostrich

Chandrasekaran H, Govind SK, Panchadcharam C, Bathmanaban P, Raman K, Thergarajan G

Parasit Vectors, 2014;7:469.
PMID: 25358755 DOI: 10.1186/s13071-014-0469-7

Blastocystis sp., a widely prevalent intestinal protozoan parasite is found in a wide range of animals, including humans. The possibility of zoonotic transmission to human from birds especially ostriches led us to investigate on the cross infectivity of Blastocystis sp. isolated from the ostrich feces as well as the phenotypic and subtype characteristics. There is a need to investigate this especially with the rising number of ostrich farms due to the growing global ostrich industry.
Prevalence and subtypes of Blastocystis among migrant workers from different working sectors in Peninsular Malaysia

Sahimin N, Meor Termizi FH, Rajamanikam A, Mohd Nazri NA, Govind SK, Mohd Zain SN

Parasitol Res, 2020 Oct;119(10):3555-3558.
PMID: 32875397 DOI: 10.1007/s00436-020-06865-3

Blastocystis sp. is a common enteric parasite of humans and animals associated with inadequate sanitation and poor personal hygiene. Over the years, the Malaysian thriving economy has been facilitated largely by migrant workers from developing countries, and there is concern that diseases endemic to their countries may be imported. Therefore, this study aimed to determine the current status of Blastocystis infection as well as subtypes (STs) from fecal samples among migrant workers in Selangor and Kuala Lumpur, Malaysia. Overall, almost a third of the study cohort (30.9%; n = 68/220) screened were infected with Blastocystis sp. predominantly with ST3 (54.5%; n = 12), followed by ST1 (36.4%; n = 8) and ST2 (9.1%; n = 2). Infection levels was almost similar among the different sectors; manufacturing (32.8%), domestic service (32.3%), and food service (27.3%) with common symptoms for infection included stomach and abdominal pain or discomfort and diarrhea (48.5%; n = 33). None of the socio-demographic risk factors evaluated were significant. Therefore, this study warrants continuous monitoring as well as understanding the impact of transmission among the migrant community with the local population especially those involved in food service sector.
Fulltext Diagnostic implications of miRNAs in Liquid Biopsy for Oral Squamous Cell Carcinoma (OSCC): Clinical validity and interpretation

Jayaraj R, Kumaraswamy C, Raymond G, Ravishankar Ram M, Govind SK, Chandramoorthy HC, et al.

Oral Oncol, 2020 10;109:104634.
PMID: 32171663 DOI: 10.1016/j.oraloncology.2020.104634
Fulltext Clinical validity of clinicopathological and prognostic significance of circulating tumor cells in head and neck squamous cell carcinoma

Jayaraj R, Shetty S, Kumaraswamy C, Raymond G, Ram M R, Govind SK, et al.

Oral Oncol, 2020 10;109:104727.
PMID: 32327312 DOI: 10.1016/j.oraloncology.2020.104727
Fulltext Clinical approaches to interpreting the findings of systematic review and meta-analysis of the effectiveness of probiotics in the prevention and treatment of Cancer Therapy-Induced Oral Mucositis (CTIOM)

Jayaraj R, Kumaraswamy C, Shetty S, Raymond G, Ram M R, Govind SK, et al.

Oral Oncol, 2020 05;104:104622.
PMID: 32115371 DOI: 10.1016/j.oraloncology.2020.104622
Fulltext Clinical validity and conceptual interpretation of systematic review and meta-analysis on elective neck dissection (END) versus observation for early-stage oral squamous cell carcinoma (OSCC)

Jayaraj R, Shetty S, Kumaraswamy C, Raymond G, Ram M R, Govind SK, et al.

Oral Oncol, 2020 10;109:104764.
PMID: 32402654 DOI: 10.1016/j.oraloncology.2020.104764
Fulltext Gut bacteria influence Blastocystis sp. phenotypes and may trigger pathogenicity

Rajamanikam A, Isa MNM, Samudi C, Devaraj S, Govind SK

PLoS Negl Trop Dis, 2023 Mar;17(3):e0011170.
PMID: 36989208 DOI: 10.1371/journal.pntd.0011170

Whilst the influence of intestinal microbiota has been shown in many diseases such as irritable bowel syndrome, colorectal cancer, and aging, investigations are still scarce on its role in altering the nature of other infective organisms. Here we studied the association and interaction of Blastocystis sp. and human intestinal microbiota. In this study, we investigated the gut microbiome of Blastocystis sp.-free and Blastocystis sp. ST3-infected individuals who are symptomatic and asymptomatic. We tested if the expression of phenotype and pathogenic characteristics of Blastocystis sp. ST3 was influenced by the alteration of its accompanying microbiota. Blastocystis sp. ST3 infection alters bacterial composition. Its presence in asymptomatic individuals showed a significant effect on microbial richness compared to symptomatic ones. Inferred metagenomic findings suggest that colonization of Blastocystis sp. ST3 could contribute to the alteration of microbial functions. For the first time, we demonstrate the influence of bacteria on Blastocystis sp. pathogenicity. When Blastocystis sp. isolated from a symptomatic individual was co-cultured with bacterial suspension of Blastocystis sp. from an asymptomatic individual, the parasite demonstrated increased growth and reduced potential pathogenic expressions. This study also reveals that Blastocystis sp. infection could influence microbial functions without much effect on the microbiota diversity itself. Our results also demonstrate evidence on the influential role of gut microbiota in altering the characteristics of the parasite, which becomes the basis for the contradictory findings on the parasite's pathogenic role seen across different studies. Our study provides evidence that asymptomatic Blastocystis sp. in a human gut can be triggered to show pathogenic characteristics when influenced by the intestinal microbiota.
Fulltext Clinical Theragnostic Potential of Diverse miRNA Expressions in Prostate Cancer: A Systematic Review and Meta-Analysis

Jayaraj R, Raymond G, Krishnan S, Tzou KS, Baxi S, Ram MR, et al.

Cancers (Basel), 2020 May 09;12(5).
PMID: 32397507 DOI: 10.3390/cancers12051199

Background: Prostate cancer (PrC) is the second-most frequent cancer in men, its incidence is emerging globally and is the fifth leading cause of death worldwide. While diagnosis and prognosis of PrC have been studied well, the associated therapeutic biomarkers have not yet been investigated comprehensively. This systematic review and meta-analysis aim to evaluate the theragnostic effects of microRNA expressions on chemoresistance in prostate cancer and to analyse the utility of miRNAs as clinical theragnostic biomarkers. Methods: A systematic literature search for studies reporting miRNA expressions and their role in chemoresistance in PrC published until 2018 was collected from bibliographic databases. The evaluation of data was performed as per PRISMA guidelines for systematic review and meta-analysis. Meta-analysis was performed using a random-effects model using Comprehensive Meta-Analysis (CMA) software. Heterogeneity between studies was analysed using Cochran's Q test, I2 and the Tau statistic. Quality assessment of the studies was performed using the Newcastle-Ottawa Scale (NOS) for the methodological assessment of cohort studies. Publication bias was assessed using Egger's bias indicator test, Orwin and classic fail-safe N test, Begg and Mazumdar rank collection test, and Duval and Tweedie's trim and fill methods. Findings: Out of 2909 studies retrieved, 79 studies were shortlisted and reviewed. A total of 17 studies met our eligibility criteria, from which 779 PrC patients and 17 chemotherapy drugs were examined, including docetaxel and paclitaxel. The majority of the drug regulatory genes reported were involved in cell survival, angiogenesis and cell proliferation pathways. We studied 42 miRNAs across all studies, out of which two miRNAs were found to be influencing chemosensitivity, while 21 were involved in chemoresistance. However, the remaining 19 miRNAs did not appear to have any theragnostic effects. Besides, the prognostic impact of the miRNAs was evaluated and had a pooled HR value of 1.960 with 95% CI (1.377-2.791). Interpretation: The observation of the current study depicts the significance of miRNA expression as a theragnostic biomarker in medical oncology. This review suggests the involvement of specific miRNAs as predictors of chemoresistance and sensitivity in PrC. Hence, the current systematic review and meta-analysis provide insight on the use of miRNA as PrC biomarkers, which can be harnessed as molecular candidates for therapeutic targeting.
Fulltext Establishing associated risk factors, including fungal and parasitic infections among Malaysians living with schizophrenia

Franklin F, Rajamanikam A, Phang WK, Raju CS, Gill JS, Francis B, et al.

Sci Rep, 2024 Jan 03;14(1):385.
PMID: 38172146 DOI: 10.1038/s41598-023-50299-7

The aetiology of schizophrenia is multifactorial, and the identification of its risk factors are scarce and highly variable. A cross-sectional study was conducted to investigate the risk factors associated with schizophrenia among Malaysian sub-population. A total of 120 individuals diagnosed with schizophrenia (SZ) and 180 non-schizophrenic (NS) individuals participated in a questionnaire-based survey. Data of complete questionnaire responses obtained from 91 SZ and 120 NS participants were used in statistical analyses. Stool samples were obtained from the participants and screened for gut parasites and fungi using conventional polymerase chain reaction (PCR). The median age were 46 years (interquartile range (IQR) 37 to 60 years) and 35 years (IQR 24 to 47.75 years) for SZ and NS respectively. Multivariable binary logistic regression showed that the factors associated with increased risk of SZ were age, sex, unemployment, presence of other chronic ailment, smoking, and high dairy consumption per week. These factors, except sex, were positively associated with the severity of SZ. Breastfed at infancy as well as vitamin and supplement consumption showed a protective effect against SZ. After data clean-up, fungal or parasitic infections were found in 98% (39/42). of SZ participants and 6.1% (3/49) of NS participants. Our findings identified non-modifiable risk factors (age and sex) and modifiable lifestyle-related risk factors (unemployment, presence of other chronic ailment, smoking, and high dairy consumption per week) associated with SZ and implicate the need for medical attention in preventing fungal and parasitic infections in SZ.
Fulltext Research funding impact and priority setting - advancing universal access and quality healthcare research in Malaysia

Fun WH, Sararaks S, Tan EH, Tang KF, Chong DWQ, Low LL, et al.

BMC Health Serv Res, 2019 Apr 24;19(1):248.
PMID: 31018843 DOI: 10.1186/s12913-019-4072-7

BACKGROUND: Health Research Priority Setting (HRPS) in the Ministry of Health (MOH) Malaysia was initiated more than a decade ago to drive effort toward research for informed decision and policy-making. This study assessed the impact of funded prioritised research and identified research gaps to inform future priority setting initiatives for universal access and quality healthcare in Malaysia.
METHODS: Research impact of universal access and quality healthcare projects funded by the National Institutes of Health Malaysia were assessed based on the modified Payback Framework, addressing categories of informing policy, knowledge production, and benefits to health and health sector. For the HRPS process, the Child Health and Nutrition Research Initiative methodology was adapted and adopted, with the incorporation of stakeholder values using weights and monetary allocation survey. Workshop discussions and interviews with stakeholders and research groups were conducted to identify research gaps, with the use of conceptual frameworks to guide the search.
RESULTS: Seventeen ongoing and 50 completed projects were identified for research funding impact analysis. Overall, research fund allocation differed from stakeholders' expectation. For research impact, 48 out of 50 completed projects (96.0%) contributed to some form of policy-making efforts. Almost all completed projects resulted in outputs that contributed to knowledge production and were expected to lead to health and health sector benefits. The HRPS process led to the identification of research priority areas that stemmed from ongoing and new issues identified for universal access and quality healthcare.
CONCLUSION: The concerted efforts of evaluation of research funding impact, prioritisation, dissemination and policy-maker involvement were valuable for optimal health research resource utilisation in a resource constrained developing country. Embedding impact evaluation into a priority setting process and funding research based on national needs could facilitate health research investment to reach its potential.

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