Diagnosis of Giardia infections by PCR-based methods in children of an endemic area

Abstract

The present study was designed to estimate the prevalence of Giardia infection in preschool-and school-aged children living in an endemic area. Fecal samples from 573 children were processed by zinc sulfate centrifugal flotation, centrifugal sedimentation (using a commercial device for fecal concentration-TF-Test kit®) and polymerase chain reaction (PCR)-based methods. Of the stool samples assessed, 277 (48.3%) were positive for intestinal parasites and/or commensal protozoa. Centrifugal flotation presented the highest diagnostic sensitivity for Giardia infections. The kappa index revealed that both coproparasitological techniques closely agreed on the Giardia diagnosis (86%) versus satisfactory (72%) and poor (35%) concordances for commensal protozoan and helminth infections, respectively. Concerning Giardia molecular diagnosis, from the 71 microscopy-positive samples, specific amplification of gdh and tpi fragments was noted in 68 (95.7%) and 64 (90%) samples, respectively. Amplification of gdh and tpi genes was observed, respectively, in 95.7% and 90% of microscopy-positive Giardia samples. For 144 microscopynegative samples, gdh and tpi gene amplification products were obtained from 8.3% and 35.9% samples, respectively. The agreement between these genes was about 40%. The centrifuge-flotation based method was the most suitable means of Giardia diagnosis assessed in the present study by combining accuracy and low cost.

Coproscopy and molecular screening for detection of intestinal protozoa

“…The prevalence values obtained in the current study after analyzing different subjects are in close agreement, with 65.2% vs 7.6% for the molecular and microscopy tools, respectively. Similar results in detecting intestinal protozoa have been reported elsewhere in Brazil, Australia and Denmark [ 17 – 19 ], although the difference between the two methodologies was not as disparate as our experience [ 20 ].…”

“…False negatives, especially in the diagnosis of pathogenic agents, have significant consequences for both treatment and rapid containment of disease in affected subjects and in populations. They may be due to the presence of inhibitors in faeces resulting in non-amplification of the targeted gene fragments [ 17 ], and this appears to be perhaps the most important complication, limiting the efficiency of PCR-based techniques. It is suggested that the frequency of occurrence of PCR inhibitors in human faeces is higher in the adult populations compared to that in pediatric populations [ 23 ].…”

“…DNA extraction by kits using “spin columns” can reduce the concentration of PCR inhibitors, as well as enabling greater efficiency in obtaining the DNA sample. However, some inhibitors can be co-eluted with the DNA [ 17 ]. A more general and widely applied method is the dilution of the sample or the extracted nucleic acid, which will automatically result in a dilution of the PCR inhibitors.…”

Application of a Multiplex Quantitative PCR to Assess Prevalence and Intensity Of Intestinal Parasite Infections in a Controlled Clinical Trial

“…For STH diagnostics they have been shown to be more sensitive than microscopy, particularly at low infection intensities [ 11 , 30 ]. For detection of protozoal infections, PCR-based techniques have shown significantly higher sensitivity compared to microscopy and/or immunodiagnostic techniques [ 31 – 37 ]. Adapting PCR assays to multiplex real-time platforms enables simultaneous detection of multiple parasites, thereby minimizing reagent costs and processing time [ 12 , 30 , 38 ].…”

Epidemiology of Giardia duodenalis infection in ruminant livestock and children in the Ismailia province of Egypt: insights by genetic characterization

“…This technique suffers from low sensitivity in comparison to improved methods based on immunodiagnostic fecal antigen detection or polymerase chain reaction (PCR) [ 7 - 9 ]. The latter assays are thus more useful for epidemiological studies [ 5 , 9 , 10 ], in particular in combination with genetic characterization of the parasites [ 6 ]. It is well established that G. duodenalis represents a species complex composed of at least eight genetic groups (assemblages A to H) that are distinguishable based on genetic polymorphisms in, e.g., the triosephosphate isomerase ( tpi ), glutamate dehydrogenase ( gdh ), beta-giardin ( bg ) and small-subunit rDNA (ssu-rDNA) genes [ 6 , 11 - 15 ].…”