Cryptosporidium spp., Giardia lamblia and Entamoeba histolytica are the most frequently identified enteric protozoa in water-borne disease outbreaks. Many PCR assays, with satisfactory results in terms of sensitivities and specificities, have been developed for detection of the aforementioned three protozoa in faecal specimens but the majority of these assays have a limited usage in the clinical laboratories due to being more costly and more time-consuming than the conventional diagnostic methods. \ud Based on published oligonucleotide primers, three individual uniplex PCR assays were developed, properly optimised and subsequently combined into a conventional multiplex PCR format to screen for the three protozoa in the same stool specimen. The multiplex PCR assay was clinically validated with 185 control and 212 randomly selected stool samples. The assay was optimised with DNA directly retrieved from stool samples using a modified QIAamp® DNA Stool Mini Kit (Qiagen) DNA extraction protocol subsequent amplification using single-round well-controlled PCR protocol. \ud Like the individual PCRs, the multiplex PCR assay detected genomic DNA from control isolates matching 12, 12 and four copies of the Cryptosporidium, G. lamblia and E. histolytica genomes, respectively. Similarly, ~100 (oo)cysts per 200μl stool were successfully identified by the multiplex and the matching uniplex-PCR assays as detection limits. The diagnostic sensitivity, specificity, negative predictive value and positive predictive value of the multiplex and the individual PCR assays were comparable and equal to 97 %, 100 %, 95 % and 100 %, respectively. Furthermore, by nominating three nested PCRs as 'gold standards', the multiplex PCR demonstrated specificity and sensitivity exceeding that achieved by the combined copro-antigen immunoassay adopted for Cryptosporidium/Giardia diagnosis at the Clinical Microbiology laboratory, Leicester Royal Infirmary, University Hospital of Leicester. \ud In conclusion, the newly developed multiplex PCR was demonstrated to be a simple, cost-effective, adequately sensitive and highly specific assay. An assay with this broad-spectrum format has a great potential to be adopted as a routine test in diagnostic laboratories especially those in resource-poor countries where parasitic protozoal infections are endemic
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