Damien Stark

Laboratory Diagnostic Techniques for Entamoeba Species

SUMMARY The genus Entamoeba contains many species, six of which (Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba polecki, Entamoeba coli, and Entamoeba hartmanni) reside in the human intestinal lumen. Entamoeba histolytica is the causative agent of amebiasis and is considered a leading parasitic cause of death worldwide in humans. Although recent studies highlight the recovery of E. dispar and E. moshkovskii from patients with gastrointestinal symptoms, there is still no convincing evidence of a causal link between the presence of these two species and the symptoms of the host. New approaches to the identification of E. histolytica are based on detection of E. histolytica-specific antigen and DNA in stool and other clinical samples. Several molecular diagnostic tests, including conventional and real-time PCR, have been developed for the detection and differentiation of E. histolytica, E. dispar, and E. moshkovskii in clinical samples. The purpose of this review is to discuss different methods that exist for the identification of E. histolytica, E. dispar, and E. moshkovskii which are available to the clinical diagnostic laboratory. To address the need for a specific diagnostic test for amebiasis, a substantial amount of work has been carried out over the last decade in different parts of the world. The molecular diagnostic tests are increasingly being used for both clinical and research purposes. In order to minimize undue treatment of individuals infected with other species of Entamoeba such as E. dispar and E. moshkovskii, efforts have been made for specific diagnosis of E. histolytica infection and not to treat based simply on the microscopic examination of Entamoeba species in the stool. The incorporation of many new technologies into the diagnostic laboratory will lead to a better understanding of the public health problem and measures to control the disease.

Methicillin-Resistant Staphylococcus aureus (MRSA) Detection: Comparison of Two Molecular Methods (IDI-MRSA PCR Assay and GenoType MRSA Direct PCR Assay) with Three Selective MRSA Agars (MRSA ID, MRSASelect, and CHROMagar MRSA) for Use with Infection-Control Swabs

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is an increasing problem. Rapid detection of MRSA-colonized patients has the potential to limit spread of the organism. We evaluated the sensitivities and specificities of MRSA detection by two molecular methods (IDI-MRSA PCR assay and GenoType MRSA Direct PCR assay) and three selective MRSA agars (MRSA ID, MRSASelect, and CHROMagar MRSA), using 205 (101 nasal, 52 groin, and 52 axillary samples) samples from consecutive known MRSA-infected and/or -colonized patients. All detection methods had higher MRSA detection rates for nasal swabs than for axillary and groin swabs. Detection of MRSA by IDI-MRSA was the most sensitive method, independent of the site (94% for nasal samples, 80% for nonnasal samples, and 90% overall). The sensitivities of the GenoType MRSA Direct assay and the MRSA ID, MRSASelect, and CHROMagar MRSA agars with nasal swabs were 70%, 72%, 68%, and 75%, respectively. All detection methods had high specificities (95 to 99%), independent of the swab site. Extended incubation for a further 24 h with selective MRSA agars increased the detection of MRSA, with a corresponding decline in specificity secondary to a significant increase in false-positive results. There was a noticeable difference in test performance of the GenoType MRSA Direct assay in detection of MRSA (28/38 samples [74%]) compared with detection of nonmultiresistant MRSA (17/31 samples [55%]) (susceptible to two or more non-β-lactam antibiotics). This was not observed with selective MRSA agar plates or IDI-MRSA. Although it is more expensive, in addition to rapid turnaround times of 2 to 4 h, IDI-MRSA offers greater detection of MRSA colonization, independent of the swab site, than do conventional selective agars and GenoType MRSA Direct.

PCR detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in stool samples from Sydney, Australia.

ABSTRACT This study investigated the presence of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in stool samples from a patient population in Sydney, Australia. Stool samples were tested by microscopy and PCR. Five patients were found with E. histolytica infections, while E. dispar and E. moshkovskii were observed in 63 (70.8%) and 55 (61.8%) patients, respectively, by PCR. This is the first study in Australia using molecular techniques to determine the presence of E. histolytica, E. dispar, and E. moshkovskii.

A review of Dientamoeba fragilis carriage in humans: Several reasons why this organism should be considered in the diagnosis of gastrointestinal illness

Dientamoeba fragilis is a protozoan that inhabits the human gut. It is approximately 100 years since Dientamoeba’s discovery and first description when it was described as a rare and harmless commensal. Since then it has struggled to gain recognition as a pathogen despite the evidence supporting its pathogenicity. Dientamoeba remains neglected, probably due to the misconceptions that it is uncommon and non-pathogenic. Usually, carriage of Dientamoeba is associated with symptoms such as abdominal pain and diarrhea. Moreover, antimicrobial therapy followed by resolution of symptoms coincides with the eradication of Dientamoeba. This manuscript reviews the scientific literature relating to Dientamoeba’s prevalence and pathogenicity. While much of the evidence supporting its pathogenicity is only circumstantial, it is apparent that most researchers agree that Dientamoeba is pathogenic. Therefore, in symptomatic patients who harbor Dientamoeba and no other pathogen, Dientamoeba should be considered as the etiological agent and treated as such.

Blastocystis infection is associated with irritable bowel syndrome in a Mexican patient population

In recent times, some common “non-pathogenic” parasites, such as Blastocystis and Dientamoeba fragilis, have been associated to the aetiology of irritable bowel syndrome (IBS), while host pro-inflammatory cytokine gene polymorphisms might have a role in the pathophysiology of the disease. Therefore, Blastocystis subtypes (ST), D. fragilis and gene promoter single nucleotide polymorphisms of interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α) in IBS patients and controls were studied. After giving written consent, 45 patients with symptoms of IBS according to the Rome III criteria and 45 controls were enrolled. DNA was extracted from peripheral blood for SNP analysis at position -174 for IL-6 as well as -238 and -308 for TNF-α. Blastocystis was more common in the IBS group (p = 0.043). Interestingly, D. fragilis was found more frequently in the control group (p = 0.002); Blastocystis ST1 and 3 were most frequent in both groups. Haploview analysis revealed linkage disequilibrium in TNF-α (p < 0.0001); however, none of the SNPs for IL-6 and TNF-α were found to be significantly related with IBS. The clinical and molecular approaches undertaken for the first time in Latin American IBS patients demonstrated an association with Blastocystis that supports a pathogenic role of this parasite in IBS Furthermore, co-infections with ST1 and ST3 were frequent; thus, the genetic diversity proposed within ST polymorphisms does not rule out that particular strains might be associated with disease. In addition, our results do not support a major contribution of IL-6 and TNF-α gene polymorphisms in the susceptibility to IBS.

Comparison of Stool Antigen Detection Kits to PCR for Diagnosis of Amebiasis

ABSTRACT The present study was conducted to compare two stool antigen detection kits with PCR for the diagnosis of Entamoeba histolytica infections by using fecal specimens submitted to the Department of Microbiology at St. Vincents Hospital, Sydney, and the Institute of Medical and Veterinary Science, Adelaide, Australia. A total of 279 stool samples containing the E complex (E. histolytica, Entamoeba dispar, and Entamoeba moshkovskii) were included in this study. The stool specimens were tested by using two commercially produced enzyme immunoassays (the Entamoeba CELISA PATH and TechLab E. histolytica II kits) to detect antigens of E. histolytica. DNA was extracted from all of the samples with a Qiagen DNA stool mini kit (Qiagen, Hilden, Germany), and a PCR targeting the small-subunit ribosomal DNA was performed on all of the samples. When PCR was used as a reference standard, the CELISA PATH kit showed 28% sensitivity and 100% specificity. The TechLab ELISA (enzyme-linked immunosorbent assay) kit did not prove to be useful in detecting E. histolytica, as it failed to identify any of the E. histolytica samples which were positive by PCR. With the TechLab kit, cross-reactivity was observed for three specimens, one of which was positive for both E. dispar and E. moshkovskii while the other two samples contained E. moshkovskii. Quantitative assessment of the PCR and ELISA results obtained showed that the ELISA kits were 1,000 to 10,000 times less sensitive, and our results show that the CELISA PATH kit and the TechLab ELISA are not useful for the detection of E. histolytica in stool samples from patients in geographical regions where this parasite is not endemic.

Prospective Study of the Prevalence, Genotyping, and Clinical Relevance of Dientamoeba fragilis Infections in an Australian Population

ABSTRACT A prospective study was conducted over a 30-month period, in which fecal specimens from 6,750 patients were submitted to the Department of Microbiology at St. Vincents Hospital, Sydney, Australia. Trophozoites of Dientamoeba fragilis were detected in 60 (0.9%) patients by permanent staining, and confirmation was performed by PCR. Gastrointestinal symptoms were present in all patients, with diarrhea and abdominal pain the most common symptoms. Thirty-two percent of patients presented with chronic symptoms. The average age of infected patients was 39.8 years. No correlation was found between D. fragilis and Enterobius vermicularis, a proposed vector of transmission for D. fragilis. The genetic diversity of 50 D. fragilis isolates was examined by PCR, and the PCR products were analyzed for the presence of restriction fragment length polymorphisms. These results showed no variation in the small-subunit rRNA gene and demonstrated a single genotype for all Australian isolates. This study shows the potential pathogenic properties of D. fragilis and the need for all laboratories to routinely test for this organism.

Entamoeba moshkovskii infections in Sydney, Australia

Entamoeba moshkovskii is considered to be a free-living ameba, which is morphologically similar, but biochemically and genetically different, to Entamoeba histolytica and Entamoeba dispar. However, recent studies have suggested that E. moshkovskii may be a “potential” pathogen, with infections giving rise to diarrhea and other intestinal disorders. Microscopy and polymerase chain reaction (PCR) targeting the 18S ribosomal (r) DNA was performed on fecal samples collected from patients presenting with diarrhea and other gastrointestinal disorders (test group), as well as fecal samples collected from healthy controls (control group). Of the 110 patients microscopically positive for the Entamoeba species, 55/110 (50%) samples were positive for E. moshkovskii in the test group of patients presenting with diarrhea. E. moshkovskii was the only pathogen detected (including bacteria or viruses) in 3/55 (5.5%) of the test group of patients presenting with diarrhea and abdominal pain. The DNA of E. moshkovskii was not detected in the fecal samples collected from the healthy controls. These results suggest that E. moshkovskii may not simply be a commensal of the human gastrointestinal tract and provides evidence for E. moshkovskii as a “potential” pathogen in the case of diarrhea and other gastrointestinal disorders. Further studies are needed to determine the role of E. moshkovskii in causing diarrheal diseases in our population.

Comparison of microscopy, culture, and conventional polymerase chain reaction for detection of blastocystis sp. in clinical stool samples.

We tested 513 stool samples from patients in Sydney, Australia for Blastocystis by using five diagnostic techniques: microscopy of a permanently stained smear using a modified iron-hematoxylin stain, two xenic culture systems (a modified Boeck and Drbohlavs medium and tryptone, yeast extract, glucose, methionine-9 medium), and two published conventional polymerase chain reaction methods specific for the small subunit ribosomal DNA. Ninety-eight (19%) samples were positive for Blastocystis in one or more of the diagnostic techniques. The PCR 2 method was the most sensitive at detecting Blastocystis with a sensitivity of 94%, and the least sensitive was microscopy of the permanent stain (48%). Subtype 3 was the most predominant subtype (present in 43% of samples assigned to this group). This study highlights the low sensitivity of microscopy when used as the sole diagnostic modality for detection of Blastocystis sp.

Update on the pathogenic potential and treatment options for Blastocystis sp

Although Blastocystis is one of the most common enteric parasites, there is still much controversy surrounding the pathogenicity and potential treatment options for this parasite. In this review we look at the evidence supporting Blastocystis as an intestinal pathogen as shown by numerous case studies and several in vivo studies and the evidence against. We describe the chronic nature of some infections and show the role of Blastocystis in immunocompromised patients and the relationship between irritable bowel syndrome and Blastocystis infection. There have been several studies that have suggested that pathogenicity may be subtype related. Metronidazole is the most widely accepted treatment for Blastocystis but several cases of treatment failure and resistance have been described. Other treatment options which have been suggested include paromomycin and trimethroprim- sulfamethoxazole.