Simultaneous detection of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum in fecal samples by using multiplex real-time PCR

Entamoeba histolytica, Giardia lamblia, and Cryptosporidium are three of the most important diarrhea-causing parasitic protozoa. For many years, microscopic examination of stool samples has been considered to be the "gold standard" for diagnosis of E. histolytica, G. lamblia, and C. parvum infections. Recently, more specific and sensitive alternative methods (PCR, enzyme-linked immunosorbent assay, and direct fluorescent-antibody assay) have been introduced for all three of these parasitic infections. However, the incorporation in a routine diagnostic laboratory of these parasite-specific methods for diagnosis of each of the respective infections is time-consuming and increases the costs of a stool examination. Therefore, a multiplex real-time PCR assay was developed for the simultaneous detection of E. histolytica, G. lamblia, and C. parvum in stool samples. The multiplex PCR also included an internal control to determine efficiency of the PCR and detect inhibition in the sample. The assay was performed on species-specific DNA controls and a range of well-defined stool samples, and it achieved 100 percent specificity and sensitivity. The use of this assay in a diagnostic laboratory would provide sensitive and specific diagnosis of the main parasitic diarrheal infections and could improve patient management and infection control.     

Prevalence of Dientamoeba fragilis,Giardia duodenalis,Entamoeba histolytica/dispar,and Cryptosporidium spp in Da Nang,Vietnam, detected by a multiplex real‐time PCR

We surveyed the prevalence of Dientamoeba fragilis, Giardia duodenalis, Entamoeba histolytica, Entamoeba dispar, and Cryptosporidium spp in individuals with and without gastrointestinal (GI) symptoms residing in and around Da Nang city, Vietnam. Fecal samples were collected from children (n = 100) and adults (n = 80) with GI symptoms and from healthy individuals (n = 88) reporting no GI symptoms. Parasite detection was performed by multiplex real‐time PCR. Overall, except for G. duodenalis, we found a low prevalence (<5%) of D. fragilis and E. dispar and no detection of E. histolytica and C. spp in all participants with GI symptoms. Specifically for D. fragilis this contrasts with findings in European populations of children with GI symptoms showing prevalence up to 73%. Moreover, our results indicate that the prevalence of G. duodenalis is higher in patients with GI symptoms compared to asymptomatic individuals and this difference is most obvious in young patients.     

Evaluation of the Roche LightMix Gastro parasites multiplex PCR assay detecting Giardia duodenalis,Entamoeba histolytica,cryptosporidia, Dientamoeba fragilis,and Blastocystis hominis

ObjectivesMultiplex PCR assays offer highly sensitive and specific tools for the detection of enteric pathogens. This prospective study aimed at comparing the novel Roche LightMix Modular Assay Gastro Parasites (LMAGP) detecting Giardia duodenalis, Entamoeba histolytica, Cryptosporidium spp., Blastocystis hominis, and Dientamoeba fragilis with routine laboratory procedures.MethodsStool specimens (n = 1062 from 1009 patients) were consecutively examined by LMAGP, R-Biopharm Ridascreen enzyme immunoassays (EIAs) detecting G. duodenalis or E. histolytica/dispar, and microscopy of wet mounts. Discrepant results were analysed by in-house PCR.ResultsD. fragilis or B. hominis were detected by LMAGP in 131 (14.4%) and 179 (19.9%; 16 samples positive by microscopy; p < 0.0001) of 909 samples, respectively. Of 918 samples analysed for Cryptosporidium spp., six were positive by LMAGP (three could be confirmed by Kinyoun staining and one by in-house PCR). G. duodenalis was detected by LMAGP, EIA, or microscopy in 20, 16, or 9 of 1039 stool samples, respectively; all four samples missed by EIA were confirmed by in-house PCR. In total, 938 stool samples were analysed for E. histolytica/dispar. Nine of ten EIA-positive samples were negative by LMAGP but positive by in-house PCR for E. dispar. One E. histolytica infection (positive by both LMAGP and in-house PCR) was missed by EIA and microscopy. Parasites only detected by microscopy included Enterobius vermicularis eggs (n = 3) and apathogenic amoebae (n = 27).ConclusionsThe data call for routine use of multiplex PCR assays for the detection of enteric protozoan parasites in laboratory diagnostics.     

Evaluation of three diagnostic methods, including real-time PCR, for detection of Dientamoeba fragilis in stool specimens

Dientamoeba fragilis is a protozoan parasite of humans that infects the mucosa of the large intestine and is associated with gastrointestinal disease. We developed a 5' nuclease (TaqMan)-based real-time PCR assay, targeting the small subunit rRNA gene, for the detection of D. fragilis in human stool specimens and compared its sensitivity and specificity to conventional PCR and microscopic examination by a traditional modified iron-hematoxylin staining procedure. Real-time PCR exhibited 100% sensitivity and specificity.     

Comparison of microscopy, two xenic culture techniques, conventional and real-time PCR for the detection of Dientamoeba fragilis in clinical stool samples

Dientamoeba fragilis is a pathogenic protozoan parasite that is notoriously difficult to diagnose. The aim of this study was to determine the gold standard for laboratory detection of D. fragilis. A total of 650 human faecal samples were included in the study. All specimens underwent the following: microscopy using a permanent stain (modified iron-haematoxylin), culture using a modified Boeck and Drbohlav’s medium (MBD) and TYGM-9, a conventional polymerase chain reaction (PCR) and a real-time PCR (RT-PCR). The overall prevalence of D. fragilis in the study population was 5.4% (35/650). RT-PCR detected 35 isolates, conventional PCR detected 15 isolates, MBD culture detected 14 isolates, TYGM-9 detected ten isolates, while microscopy detected 12 isolates. RT-PCR detected an additional 15 positive samples compared to the other diagnostic methods, all of which were confirmed by sequencing. When all methods were compared to each other, RT-PCR showed a sensitivity and specificity of 100 and 100%, conventional PCR 42.9 and 100%, MBD culture 40 and 100%, TYGM-9 culture 28.6 and 100%, and microscopy 34.3 and 99%, respectively. These results show that RT-PCR is the diagnostic method of choice for the detection of D. fragilis in clinical samples and, as such, should be considered as the gold standard for diagnosis.     

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

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.     

Evaluation of an immunochromatographic dip strip test for simultaneous detection of Cryptosporidium spp, Giardia duodenalis, and Entamoeba histolytica antigens in human faecal samples

Immunochromatographic (IC) tests may play an important role in the future diagnosis of parasitic diseases because of their speed and simplicity of use. A recently developed test to detect Cryptosporidium spp, Giardia duodenalis and Entamoeba histolytica was evaluated. Microscopy and PCR were the "gold standard" reference techniques and the results of this IC test were compared with those obtained with ELISA and IC single test for the three parasites. One hundred sixty stool samples were assayed. Using microscopy, 22 samples were diagnosed as positive for Cryptosporidium spp., 31 for Giardia duodenalis, 41 for Entamoeba histolytica/dispar, and 68 had a negative diagnosis for the three parasites. Results of IC tests show sensitivities of 70-72% for Cryptosporidium, 90-97% for Giardia and 62.5% for Entamoeba histolytica. Specificities were of 93.6-94.9%, >99% and 96.1%, respectively. In all diagnoses, agreement with microscopy and PCR was over 90%, except in the triple test and microscopy in E. histolytica detection that was 76.3%, due to the inability of microscopy to differentiate E. histolytica from nonpathogenic species such as E. dispar or E. moshkovskii. The triple stool immunoassays provide adequate sensitivities and specificities for use in outbreak situations, for screening proposals and for massive assays in endemic areas where a large number of samples must be analysed or as complementary test for individual diagnosis.     

Evaluation of the Triage Micro Parasite Panel for detection of Giardia lamblia, Entamoeba histolytica/Entamoeba dispar, and Cryptosporidium parvum in patient stool specimens

A study comparing the Triage Micro Parasite Panel (Biosite Diagnostics, Inc., San Diego, Calif.) to conventional O&P examination (O&P) was performed using patient fecal specimens. Five hundred twenty-three stool samples were compared. Nineteen specimens were found to be positive by Triage, and 29 were found to be positive by O&P. Seven specimens were positive for Giardia lamblia, four were positive for Entamoeba histolytica/E. dispar, and three were positive for Cryptosporidium parvum as determined by both methods. There was one false positive by Triage (C. parvum) and four false negatives by O&P (two G. lamblia, one E. histolytica/E. dispar, and one C. parvum). The Triage test accurately detected all 18 specimens that contained one of the three organisms that it was designed to detect. The Triage test is a rapid, easy-to-use enzyme immunoassay for the detection of G. lamblia, E. histolytica/E. dispar, and C. parvum in fresh or fresh-frozen fecal specimens. These data suggest that the Triage test can be used as a screen for the immediate testing of stool specimens for these three pathogenic parasites. If Triage test results are negative, O&P can be performed if parasitic infections other than G. lamblia, E. histolytica/E. dispar, or C. parvum are suspected.     

Evaluation of seven commercial antigen detection tests for Giardia and Cryptosporidium in stool samples

Stool samples from patients with abdominal symptoms were used to evaluate different copro-diagnostic assays for the detection of Giardia and Cryptosporidium. Results from microscopical examination following conventional stool concentration and direct fluorescent-antibody methods were compared with various commercially available immunochromatographic and enzyme immunoassays. Of 220 samples, 45 were positive for Giardia and 17 for Cryptosporidium. For Giardia, the sensitivities obtained by Ridascreen Giardia, Rida Quick Giardia, Rida Quick Combi and Giardia-Strip were 82%, 80%, 80% and 44%, respectively. For Cryptosporidium, the sensitivities obtained by Rida Quick Cryptosporidium, Ridascreen Cryptosporidium, Rida Quick Combi and Cryptosporidium-Strip were 88%, 82%, 82% and 75%, respectively. The specificity of all tests was > or = 98%. Other intestinal parasites were present in 68 samples, but cross-reactions with other protozoan or helminthic parasites were not observed. Overall, the copro-antigen assays were less time-consuming and easier to perform, but were less sensitive than conventional microscopical methods. Thus, these tests might be a useful addition to, but not a substitute for microscopical methods in the diagnosis of travel-associated giardiasis and cryptosporidiosis.     

Multisite performance evaluation of an enzyme-linked immunosorbent assay for detection of Giardia, Cryptosporidium, and Entamoeba histolytica antigens in human stool

A novel fecal antigen detection assay for fresh and frozen human samples that detects but does not differentiate Giardia spp, Cryptosporidium spp, and Entamoeba histolytica, the Tri-Combo parasite screen, was compared to three established enzyme-linked immunosorbent assays (ELISAs) at three international sites. It exhibited 97.9% sensitivity and 97.0% specificity, with positive and negative predictive values of 93.4% and 99.1%, respectively. The Tri-Combo test proved a reliable means to limit the use of individual parasite ELISAs to positive samples.     

Evaluation of a new multiplex PCR assay (ParaGENIE G-Amoeba Real-Time PCR kit) targeting Giardia intestinalis,Entamoeba histolytica and Entamoeba dispar/Entamoeba moshkovskii from stool specimens: evidence for the limited performances of microscopy-based approach for amoeba species identification

ObjectivesBesides the potential to identify a wide variety of gastrointestinal parasites, microscopy remains the reference standard in clinical microbiology for amoeba species identification and, especially when coupled with adhesin detection, to discriminate the pathogenic Entamoeba histolytica from its sister but non-pathogenic species Entamoeba dispar/Entamoeba moshkovskii. However, this approach is time-consuming, requires a high-level of expertise that can be jeopardized considering the low prevalence of gastrointestinal parasites in non-endemic countries. Here, we evaluated the CE-IVD-marked multiplex PCR (ParaGENIE G-Amoeba, Ademtech) targeting E. histolytica and E. dispar/E. moshkovskii and Giardia intestinalis.MethodsThis evaluation was performed blindly on a reference panel of 172 clinical stool samples collected prospectively from 12 laboratories and analysed using a standardized protocol relying on microscopy (and adhesin detection by ELISA for the detection of E. histolytica) including G. intestinalis (n = 37), various amoeba species (n = 55) including E. dispar (n = 15), E. histolytica (n = 5), as well as 17 other gastrointestinal parasites (n = 80), and negative samples (n = 37).ResultsThis new multiplex PCR assay offers fast and reliable results with appropriate sensitivity and specificity for the detection of G. intestinalis and E. dispar/E. moshkovskii from stools (89.7%/96.9% and 95%/100%, respectively). Detection rate and specificity were greatly improved by the PCR assay, highlighting several samples misidentified by microscopy, including false-negative and false-positive results for both E. dispar/E. moshkovskii and E. histolytica.ConclusionGiven the clinical relevance of amoeba species identification, microbiologists should be aware of the limitations of using an algorithm relying on microscopy coupled with adhesin detection by ELISA.     

Real-time PCR for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in fecal samples

A closed-tube, real-time PCR assay was developed for sensitive and specific detection and differentiation of the two closely related intestinal protozoan parasites Entamoeba histolytica and Entamoeba dispar directly from human feces. The assay is performed with the LightCycler system using fluorescence-labeled detection probes and primers amplifying a 310-bp fragment from the high-copy-number, ribosomal DNA-containing ameba episome. The assay was able to detect as little as 0.1 parasite per g of feces. The two pairs of primers used were specific for the respective ameba species, and results were not influenced by the presence of other Entamoeba species even when present in exceeding amounts. PCR was evaluated using several hundred stool samples from areas of amebiasis endemicity in Vietnam and South Africa, and results were compared with those of microscopy and ameba culture. PCR was found to be significantly more sensitive than microscopy or culture, as all samples positive by microscopy and 22 out of 25 (88%) samples positive by culture were also positive by PCR, but PCR revealed a considerable number of additional E. histolytica- or E. dispar-positive samples. Compared to culture and subsequent ameba differentiation by isoenzyme analysis, PCR was 100% specific for each of the two Entamoeba species. Interestingly, the comparison with PCR revealed that culture, in particular, underestimates E. histolytica infections. Given the high sensitivity and specificity of the developed PCR assay, the inability of microscopy to distinguish between the two ameba species, and the time it takes to culture and subsequently differentiate entamoebae by isoenzyme analysis, this assay is more suitable than microscopy or culture to correctly diagnose intestinal E. histolytica or E. dispar infection.     

Blastocystis spp., Cryptosporidium spp., and Entamoeba histolytica exhibit similar symptomatic and epidemiological patterns in healthcare-seeking patients in Karachi

In this study, we collected data on the incidence of enteric parasites in healthcare-seeking individuals along with their symptoms to quantify the potential roles of factors such as age, sex, and seasonality in infection. We performed analysis to identify factors which could help differentiate parasitic infection from other causes of gastrointestinal illness in a community. The size of the patient population (n?=?339), patient selection methodology, collection methods, and statistical analysis followed approaches from similar studies in core clinical journals. Ethical approval was obtained from the University of Karachi’s Ethical Review Board. Fecal specimens (n?=?339) submitted by symptomatic patients were collected from two clinical laboratories, along with information about the patients’ age, sex, and symptoms. We found that symptoms of fever, vomiting, and constipation were 100 % predictive of finding a parasitic infection, while diarrhea was 88 % predictive of a parasitic infection. Gastrointestinal parasite-positive patients reported diarrhea (~60 %), vomiting (~30 %), fever (~25 %) and constipation (~25 %), while parasite-negative patients exhibited a symptomatic profile without fever, vomiting, and constipation. The distribution of symptoms in parasite-positive patients remained relatively invariant regardless of the parasite identified. Blastocystis spp.-mono-infected patients reported a similar profile to patients positive for Entamoeba histolytica/Entamoeba dispar and Cryptosporidium spp. Most parasitic infections exhibited a strong seasonal pattern, with a peak incidence in summer months. Infection by Blastocystis spp. was the most prevalent, and it was the only infection mathematically correlated to rainfall by Pearson’s method. We observed no increase in healthcare-seeking behavior following a stressful community event, namely, the attempted assassination of Benazir Bhutto in Karachi. The data suggest that parasitological testing would produce a high yield of positive results when performed on healthcare-seeking patients in Karachi in 2007 with symptoms of fever, vomiting, or constipation and a low yield when performed on patients noting only abdominal pain. Parasitological testing also produces a higher yield on patients seen in summer months.     

A medium available in Czechoslovakia for the culture of Entamoeba histolytica and Giardia intestinalis

From nutritive media produced in the CSSR a modification of Diamond's medium TYI-S-33 was prepared which ensures the long-term axenic cultivation of Entamoeba histolytica and Giardia intestinalis. Details of the cultivation technique are also presented.     

Detection and differentiation of Cryptosporidium spp. in human clinical samples by use of real-time PCR

Real-time PCR has the potential to streamline detection and identification of Cryptosporidium spp. in human clinical samples. In the present article, we report the first such assay to allow not only detection and differentiation of the most common human pathogens, Cryptosporidium hominis and Cryptosporidium parvum, but also simultaneous amplification of a region of the small subunit (SSU) rRNA gene, permitting direct sequence analysis to identify any Cryptosporidium species. An internal control is incorporated to identify the presence of PCR inhibitors. Analytical sensitivity was determined to be as low as 200 oocysts per gram of feces processed, equivalent to 2 oocysts per PCR. The C. hominis and C. parvum PCRs specifically detected only species/genotypes in their respective target clades. Diagnostic sensitivity and specificity, evaluated against a widely used conventional nested SSU rRNA gene PCR as a nominated gold standard using a panel of 258 (151 positive and 107 negative) samples, were 100% and 99.1%, respectively. The assay agreed with PCR-restriction fragment length polymorphism analysis of the Cryptosporidium oocyst wall protein gene for 134 of 136 (98.5%) samples tested prospectively and typed two additional isolates. The real-time PCR assay was sensitive, specific, and reproducible and significantly improved laboratory work flow and turnaround times.     

Detection and genotyping of Entamoeba histolytica, Entamoeba dispar, Giardia lamblia, and Cryptosporidium parvum by oligonucleotide microarray

Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum are the most frequently identified protozoan parasites causing waterborne disease outbreaks. The morbidity and mortality associated with these intestinal parasitic infections warrant the development of rapid and accurate detection and genotyping methods to aid public health efforts aimed at preventing and controlling outbreaks. In this study, we describe the development of an oligonucleotide microarray capable of detecting and discriminating between E. histolytica, Entamoeba dispar, G. lamblia assemblages A and B, and C. parvum types 1 and 2 in a single assay. Unique hybridization patterns for each selected protozoan were generated by amplifying six to eight diagnostic sequences/organism by multiplex PCR; fluorescent labeling of the amplicons via primer extension; and subsequent hybridization to a set of genus-, species-, and subtype-specific covalently immobilized oligonucleotide probes. The profile-based specificity of this methodology not only permitted for the unequivocal identification of the six targeted species and subtypes, but also demonstrated its potential in identifying related species such as Cryptosporidium meleagridis and Cryptosporidium muris. In addition, sensitivity assays demonstrated lower detection limits of five trophozoites of G. lamblia. Taken together, the specificity and sensitivity of the microarray-based approach suggest that this methodology may provide a promising tool to detect and genotype protozoa from clinical and environmental samples.     

Evaluation of an antigencapture enzyme immunoassay for detection ofEntamoeba histolytica in stool samples

In order to identify the prevalence ofEntamoeba histolytica in tourists with diarrhoea returning from countries of the developing world, sensitivity and specificity of a commercially available enzyme immunoassay (EIA) kit for the detection ofEntamoeba histolytica coproantigen in stool were evaluated. Five hundred seventy-seven specimens from 469 patients were examined by microscopy and EIA. Sixty-two specimens from 49 patients were considered positive forEntamoeba histolytica. Compared with microscopic examination of stool samples, the EIA was found to be slightly more sensitive (90.3% vs. 87.1%) and was 97.7% specific forEntamoeba histolytica.     

Simple differential detection of Entamoeba histolytica and Entamoeba dispar in fresh stool specimens by sodium acetate-acetic acid-formalin concentration and PCR.

Amoebiasis is caused by two distinct species, a pathogenic form (Entamoeba histolytica) and a nonpathogenic form (Entamoeba dispar), which are morphologically identical. Although the distinction between these two species is of great clinical importance, the methods developed for this purpose either are very time-consuming or involve laborious procedures for isolation of the DNA. We report here a simple PCR method starting with fresh stool specimen that allows for the sensitive and reliable distinction between E. histolytica and E. dispar. After initial concentration by the sodium acetate-acetic acid-formalin (SAF) method and digestion with proteinase K, a 0.88-kb sequence of the multicopy 16S rRNA gene served as a target for PCR amplification. The method starting with unpreserved specimens proved to be very sensitive and was not influenced by the quick exposure to SAF fixative during the initial concentration step. However, storage in SAF fixative prior to testing resulted in a decreased sensitivity within 2 days. The detection limit of the method was as low as one copy of the 16S rRNA gene. No cross-reactivity was observed with other common intestinal protozoa. Mixed infections involving both E. histolytica and E. dispar could easily be detected at a ratio of 1:10,000 by agarose gel electrophoresis or a DNA hybridization immunoassay.