ABSTRACT
Low parasite density in chronic infection with S. stercoralis is a challenging diagnostic issue. Generally, molecular techniques don't depend on parasite viability while copro-culture or Baermann concentration method relies on the presence of living larvae in fecal samples. Therefore, evaluation of PCR-based methods is important to increase the detection rates in light or chronic infection. This study was designed to analyze the sensitivity of quantitative PCR [qPCR] and nested PCR [nPCR] regarding the minimum amount of S. stercoralis DNA template that can be reliably detected by each molecular technique. Strongyloides larvae were collected from cultured stool samples from suspected infected Egyptian children. After counting larvae present in a known volume under the microscope, DNA extraction was done and serial dilution of genomic materials was prepared. Then, qPCR and nPCR targeting the small subunit of the rRNA gene were performed. Regarding qPCR, the limit of detection was 0.0005 S.stercoralis larvae/microl, with crossing threshold [Ct] value ranged from 17.8 to 38.7 while, nPCR did not detect from [0.002 to 0.00012 S. stercoralis larvae/microl] with minimum limit 0.004 S. stercoralis larvae/microl. Real-time quantitative PCR is very sensitive technique that can detect very low genomic load up to about 10 [9.765] genome copies/reaction compared to nested PCR which started positivity from 78.125 genome copies/reaction. Therefore, qPCR is recommended to detect chronic strongyloidiasis especially in high-risk groups to prevent lif-etheratening spread of such infection
ABSTRACT
Toxoplasmosis caused by Toxoplasma gondii is one of the most prevalent parasitic diseases in human beings. Human toxoplasmosis can be associated with serious clinical manifestations, particularly in developing fetus. The aim of the current study was to identify the possible lineage type of Toxoplasma gondii, molecularly detected in placental samples of women whose pregnancies were spontaneously terminated in the first trimester. Preliminary detection of Toxoplasma genomic materials was done by a SYBR green qPCR technology. Subsequent identification of Toxoplasma strain was done for the positive samples using PCR- restriction fragment length polymorphism [RFLP] at the SAG2 loci of T. gondii using restriction enzymes HhaI and Sau3AI. Out of 72 tested samples, Toxoplasma B1 gene was detected in 9 cases. Toxoplasma genotypes I and II in addition to unknown type were identified in 4, 3 and 2 cases respectively, while type III was not detected in our samples, hence excluded as a leading cause of abortion in humans in our preliminary study. Nevertheless, it remains uncertain to what extent the genotype of the parasite directly contributes to the clinical severity of human toxoplasmosis. Certainly, advanced molecular techniques targeting different Toxoplasma strains are crucial for better understanding of human toxoplasmosis. For more elucidation, additional studies are recommended intended for genetic characterization of such serious parasitic infection using larger number of samples