Use of multiplex real-time PCR for detection of common diarrhea causing protozoan parasites in Egypt

Abstract

Diarrhea is an important cause of morbidity and mortality, worldwide. Giardia intestinalis, Cryptosporidium spp., and Entamoeba histolytica are the most common diarrhea-causing parasitic protozoa. Diagnosis of these parasites is usually performed by microscopy. However, microscopy lacks sensitivity and specificity. Replacing microscopy with more sensitive and specific nucleic acid based methods is hampered by the higher costs, in particular in developing countries. Multiplexing the detection of more than one parasite in a single test by real-time polymerase chain reaction (PCR) has been found to be very effective and would decrease the cost of the test. In the present study, stool samples collected from 396 Egyptian patients complaining of diarrhea along with 202 faecal samples from healthy controls were examined microscopically by direct smear method and after concentration using formol-ethyl acetate. Frozen portions of the same samples were tested by multiplex real-time for simultaneous detection of E. histolytica, G. intestinalis, and Cryptosporidium spp. The results indicate that among diarrheal patients in Egypt G. intestinalis is the most common protozoan parasite, with prevalence rates of 30.5 and 37.1 %, depending on the method used (microscopy vs. multiplex real-time PCR). Cryptosporidium spp. was detected in 1 % of the diarrheal patients by microscopy and in 3 % by real-time PCR. While E. histolytica/dispar was detected in 10.8 % by microscopy, less than one fifth of them (2 %) were found true positive for Entamoeba dispar by real-time PCR. E. histolytica DNA was not detected in any of the diarrheal patients. In comparison with multiplex real-time PCR, microscopy exhibited many false positive and negative cases with the three parasites giving sensitivities and specificities of 100 and 91 % for E. histolytica/dispar, 57.8 and 85.5 % for G. intestinalis, and 33.3 and 100 % for Cryptosporidium spp.

Molecular Testing for Clinical Diagnosis and Epidemiological Investigations of Intestinal Parasitic Infections

“…g Abbreviations: RFLP, restriction fragment length polymorphism; LNA, locked nucleic acid; SSU, small subunit; ITS, internal transcribed spacer; cox1, mitochondrial cytochrome c oxidase subunit I gene; Clp, cathepsin L-like cysteine peptidase; HDP1, T. saginata-specific repetitive sequence; HDP2, cestode-specific sequence. 87,104,107,162,414). However, parasites that are not included as targets in the multiplex PCR will not be detected, which is one of the arguments that is frequently raised against the use of molecular diagnostics in parasitology.…”

“…Recently, novel sequence information was used to identify new assemblage A-and B-specific loci. Two assemblage-specific PCRs based on these loci showed an excellent performance when used on DNAs extracted from feces (86,87). The molecular epidemiology of Giardia and genotyping methods have recently been reviewed (88,89).…”

“…While partial SSU rRNA gene sequences are available for all species of Cryptosporidium known to infect humans, surprisingly, only a fraction of these species are currently represented by complete SSU rDNA sequences in GenBank (Table 4). COWP sequences are readily available, but for some other loci, for instance, the DnaJ-like protein used in widely used diagnostic assays (27,79,87,104,107,158,(160)(161)(162), sequence data are not available for most species other than C. parvum and C. hominis, and other Cryptosporidium species may go undetected by this assay. Because there is little genetic variation across Cryptosporidium SSU rRNA genes, the design of primers/probes targeting the entire genus is relatively straightforward, but there are limited targets for designing species-specific primers/probes.…”

Molecular diagnosis of infectious diarrhea: focus on enteric protozoa

“…PCR-based methods typically offer improved sensitivity and specificity for enteric protozoa relative to microscopy and Molecular diagnosis of infectious diarrhea: focus on enteric protozoa Review informahealthcare.com antigen detection in clinical samples [51][52][53][54]. PCR-based assays can also distinguish closely related species and differentiate between pathogenic and non-pathogenic strains of the same species.…”

Intestinal Amebae

“…[83][84][85][86] It is also commercially available in kit format (Fast-Track Diagnostics Ltd, Malta). Nazeer and colleagues 87 added E dispar to the panel of diarrhea-causing pathogens and developed a new tetraplex TaqMan real-time PCR. This PCR may prove helpful in finding any association of E dispar with the diarrhea or dysentery in the infected individuals.…”