An Improved PCR-RFLP Assay for Detection and Genotyping of Asymptomatic Giardia lamblia Infection in a Resource-Poor Setting

Laboratory workers, in resource-poor countries, still consider PCR detection of Giardia lamblia more costly and more time-consuming than the classical parasitological techniques. Based on 2 published primers, an in-house one-round touchdown PCR-RFLP assay was developed. The assay was validated with an internal amplification control included in reactions. Performance of the assay was assessed with DNA samples of various purities, 91 control fecal samples with various parasite load, and 472 samples of unknown results. Two cysts per reaction were enough for PCR detection by the assay with exhibited specificity (Sp) and sensitivity (Se) of 100% and 93%, respectively. Taking a published small subunit rRNA reference PCR test results (6%; 29/472) as a nominated gold standard, G. lamblia was identified in 5.9% (28/472), 5.2%, (25/472), and 3.6% (17/472) by PCR assay, RIDA® Quick Giardia antigen detection test (R-Biopharm, Darmstadt, Germany), and iodine-stained smear microscopy, respectively. The percent agreements (kappa values) of 99.7% (0.745), 98.9% (0.900), and 97.7% (0.981) were exhibited between the assay results and that of the reference PCR, immunoassay, and microscopy, respectively. Restriction digestion of the 28 Giardia-positive samples revealed genotype A pattern in 12 and genotype B profile in 16 samples. The PCR assay with the described format and exhibited performance has a great potential to be adopted in basic clinical laboratories as a detection tool for G. lamblia especially in asymptomatic infections. This potential is increased more in particular situations where identification of the parasite genotype represents a major requirement as in epidemiological studies and infection outbreaks.

Internal Amplification Control for a Cryptosporidium Diagnostic PCR: Construction and Clinical Evaluation

Various constituents in clinical specimens, particularly feces, can inhibit the PCR assay and lead to false-negative results. To ensure that negative results of a diagnostic PCR assay are true, it should be properly monitored by an inhibition control. In this study, a cloning vector harboring a modified target DNA sequence (approximately375 bp) was constructed to be used as a competitive internal amplification control (IAC) for a conventional PCR assay that detects approximately550 bp of the Cryptosporidium oocyst wall protein (COWP) gene sequence in human feces. Modification of the native PCR target was carried out using a new approach comprising inverse PCR and restriction digestion techniques. IAC was included in the assay, with the estimated optimum concentration of 1 fg per reaction, as duplex PCR. When applied on fecal samples spiked with variable oocysts counts, approximately2 oocysts were theoretically enough for detection. When applied on 25 Cryptosporidium-positive fecal samples of various infection intensities, both targets were clearly detected with minimal competition noticed in 2-3 samples. Importantly, both the analytical and the diagnostic sensitivities of the PCR assay were not altered with integration of IAC into the reactions. When tried on 180 randomly collected fecal samples, 159 were Cryptosporidium-negatives. Although the native target DNA was absent, the IAC amplicon was obviously detected on gel of all the Cryptosporidium-negative samples. These results imply that running of the diagnostic PCR, inspired with the previously developed DNA extraction protocol and the constructed IAC, represents a useful tool for Cryptosporidium detection in human feces.