2.9.1 Microscopy
Diagnosis of Blastocystis infection is usually based on microscopic observation of the various forms (mainly vacuolar form) of the parasite in faecal samples. The most common approaches for detection of Blastocystis sp. consist of direct smear (Kukoschke et al., 1990; Zman & Khan, 1994), permanent-stained smears (Windsor et al., 2002; Termmathurapoj et al., 2004), concentration techniques, such as formalin-ethyl acetate concentration technique (Suresh & Smith, 2004), and in vitro culture (Kukoschke et al., 1990; Leelayoova et al., 2002; Termmathurapoj et al., 2004).
The examination of direct wet smears either unstained or stained with Lugol‘s iodine is convenient and inexpensive yet it frequently leads to false negative or false positive results (Stenzel & Boreham, 1996). Concentration methods such as formalin- ethyl acetate concentration technique generally appear unsuitable for laboratory identification of Blastocystis sp. because of extremely poor sensitivity (Suresh & Smith, 2004; Stensvold et al., 2006; Stensvold et al., 2007a).
Permanent smears staining appeared to be the procedure of choice for light microscopic diagnosis of Blastocystis sp. (Stenzel & Boreham, 1996). Suitable staining methods include trichome and iron-haematoxylin, Giemsa stain, Fields stain, and modified Ziehl-Neelsen stain are used. Studies comparing unstained smears and permanent-stained smears have shown the latter to be more sensitive and specific (Windsor et al., 2002; Termmathurapoj et al., 2004; Stensvold et al., 2007a). Multiple stool specimens should be examined because the parasite may exhibit irregular shedding (Vennila et al., 1999; Suresh et al., 2009b).
2.9.2 Laboratory Culture
In vitro cultivation methods have been used to enhance detection of Blastocystis sp.
Polyxenic or monoxenic of Blastocystis sp. cultures generally grow well in Jones‘s (Jones, 1946) or Boeck and Drbohlav‘s (Boeck & Drbohlav, 1925) inspissated egg medium supplemented with horse serum. Jones‘ medium is the medium of choice in studies involving culture to identify the parasite in patient samples (Leelayoova et al., 2002; Suresh & Smith, 2004; Termmathurapoj et al., 2004; Stensvold et al., 2006;
Stensvold et al., 2007a; Wong et al., 2008). Short-term xenic in vitro culture (XIVC) increases the sensitivity of detection compared to that of direct faecal smears or permanent-stained smears (Leelayoova et al., 2002; Termmathurapoj et al., 2004;
Stensvold et al., 2007a). XIVC was also superior to formalin-ethyl acetate concentration technique (FECT) in term of sensitivity (Suresh & Smith, 2004; Stensvold et al., 2007a;
Stensvold & Nielsen, 2012). However, in case of mixed infections with concurrent Blastocystis subtypes, XIVC can be a source of underestimation as this method may favour the preferential growth of certain subtype over others (Parkar et al., 2007; Yan et al., 2007), although Stensvold et al. (2007a) indicated that XIVC has very little or no impact on the detection of mixed infections.
Blastocystis sp. was axenically grown in variety of media such as Iscove‘s modified Dulbecco‘s medium, minimal essential medium, or biphasic inspissated egg slant overlaid with Locke‘s solution (Zierdt & Swan, 1981; Ho et al., 1993). Axenic cultures of Blastocystis isolates are important for molecular and biochemical studies, however, the process is generally laborious, time consuming, and the successful elimination of microbial contaminants is not guaranteed.
2.9.3 Serology
Blastocystis infection results in a serologic response that can be detected by enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody (IFA) (Zierdt &
Nagy, 1993; Zierdt et al., 1995; Hussain et al., 1997; Kaneda et al., 2000; Mahmoud &
Saleh, 2003). Currently, serological diagnostic tests have not routinely been used for diagnosis of this infection. Furthermore, conflicting results obtained using these serological methods, which highlighted that Blastocystis sp. antigenic, and hence genetic diversity must be taken into consideration in the development of any serological test. In addition, development of monoclonal antibodies specific for Blastocystis subtypes would be helpful in detection the parasite from stool specimens (Tan et al., 2010).
2.9.4 Molecular techniques
Several molecular PCR-based techniques have been developed for identification of Blastocystis sp. in faecal samples. Subtype-specific diagnostic primers, also referred to as STS (sequence-tagged site primers), is a commonly used method. This approach was developed by Yoshikawa et al. (1998, 2000, 2004b), which amplified seven distinct subtypes: ST1 to ST7. Sequence-tagged site-PCR (STS-PCR) has provided information on the distribution of different subtypes among human and animal populations and offered the possibility to detection the mixed-subtype infections. However, this
approach allows only the amplification of seven subtypes of 13 known subtypes.
Besides, its use in large-scale epidemiological studies can be costly and time-consuming as several PCR analyses have to be carried out for each isolate (Stensvold et al., 2009c).
Blastocystis isolates can also be characterized by PCR of SSU rDNA gene followed by RFLP analysis. Major limitations of this approach are the lack of standardization of the PCR conditions, choice of primers, mutations at restriction sites, and the difficulty in interpreting RFLP profiles from mixed infections (Tan, 2008). Another method consists of amplification of a SSU rDNA gene followed by either direct sequencing of the PCR product or cloning and sequencing of a number of arbitrarily selected clones (Stensvold et al., 2007a; Rivera, 2008; Souppart et al., 2009; Meloni et al., 2011). However, these subtyping methods may lead to underestimation the prevalence of mixed infections with more than one Blastocystis subtype (Meloni et al., 2012). In comparative studies of methods for detection of Blastocystis infection, PCR–based methods coupled with short-term axenic in vitro culture were more efficient than the other approaches including the formalin-ether concentration technique (FECT) and permanent trichrome smears (Stensvold et al., 2007a).
Recently, a real-time PCR assay (qPCR) has been developed for detection and quantification of Blastocystis sp. isolated directly from human stool samples (Poirier et al., 2011). The assay targets a partial sequence of the Blastocystis small ribosomal subunit (SSU) rRNA gene, allowing subtyping of Blastocystis isolates by direct sequencing of qPCR products. The qPCR assay was able to detect Blastocystis sp. in 27 samples out of 186 samples examined with detection limits 102 Blastocystis cells per gm of stools, and identified six different subtypes (ST1, ST2, ST3, ST4, ST6, and ST7) by direct sequencing of qPCR products. The approach showed high sensitivity compared to direct-light microscopy and xenic in vitro culture. The qPCR assay can be easily applied to large epidemiological studies, with rapid results compared to xenic cultures. It also
might be useful for evaluating the clinical relevance of Blastocystis subtypes and their role in acute or chronic digestive disorders.
2.10 TREATMENT
The need to treat Blastocystis sp. infection remains a complicated issue, and this is due to the controversial pathogenesis of the organism and the apparent self-limiting nature of symptoms. However, a number of antimicrobial agents have been used for treatment of Blastocystis infection. Metronidazole is considered the drug of choice as it appears to be the most effective drug for Blastocystis chemotherapy (Stensvold et al., 2010), despite some evidence for treatment failures possibly due to drug resistance (Moghaddam et al., 2005). It is possible that the variations in drug susceptibility of Blastocystis sp. are due to variations in genotypes of this parasite. Cotrimoxazole (TMP/SMX) is the most common alternative to metronidazole, especially in patients who may not be able to tolerate or do not respond to treatment with metronidazole (Ok et al., 1999). Additional drugs, such as iodoquinol, tinidazole, nitazoxanide, emetine, pentamidine, iodochlorhydroxyquin, and furazolidone, have been used for treatment of Blastocystis infection and have shown variable efficacy in eradicating the infection (Coyle et al., 2012).