Qualitative and Quantitative Detection of Chlamydophila pneumoniae DNA in Cerebrospinal Fluid from Multiple Sclerosis Patients and Controls

A standardized molecular test for the detection of Chlamydophila pneumoniae DNA in cerebrospinal fluid (CSF) would assist the further assessment of the association of C. pneumoniae with multiple sclerosis (MS). We developed and validated a qualitative colorimetric microtiter plate-based PCR assay (PCR-EIA) and a real-time quantitative PCR assay (TaqMan) for detection of C. pneumoniae DNA in CSF specimens from MS patients and controls. Compared to a touchdown nested-PCR assay, the sensitivity, specificity, and concordance of the PCR-EIA assay were 88.5%, 93.2%, and 90.5%, respectively, on a total of 137 CSF specimens. PCR-EIA presented a significantly higher sensitivity in MS patients (p = 0.008) and a higher specificity in other neurological diseases (p = 0.018). Test reproducibility of the PCR-EIA assay was statistically related to the volumes of extract DNA included in the test (p = 0.033); a high volume, which was equivalent to 100 µl of CSF per reaction, yielded a concordance of 96.8% between two medical technologists running the test at different times. The TaqMan quantitative PCR assay detected 26 of 63 (41.3%) of positive CSF specimens that tested positive by both PCR-EIA and nested-PCR qualitative assays. None of the CSF specimens that were negative by the two qualitative PCR methods were detected by the TaqMan quantitative PCR. The PCR-EIA assay detected a minimum of 25 copies/ml C. pneumoniae DNA in plasmid-spiked CSF, which was at least 10 times more sensitive than TaqMan. These data indicated that the PCR-EIA assay possessed a sensitivity that was equal to the nested-PCR procedures for the detection of C. pneumoniae DNA in CSF. The TaqMan system may not be sensitive enough for diagnostic purposes due to the low C. pneumoniae copies existing in the majority of CSF specimens from MS patients

Lack of specificity of a single positive 50-kDa band in the electroimmunotransfer blot (EITB) assay for cysticercosis

ABSTRACTDiagnosis of the parasitic infection cysticercosis is usually confirmed by serological assays. The electroimmunotransfer blot (EITB) for cysticercosis is a sensitive and specific assay, which uses six glycoprotein antigens on a strip to detect antibodies to Taenia solium cysticerci. Although the appearance of bands at any of these six sites is considered to be a positive result, a growing body of evidence suggests that the presence of a single 50-kDa band in this assay may not indicate infection. An audit of 984 samples tested over a 3-year period showed that only two (15.4%) of 13 samples with a single 50-kDa band were associated with a diagnosis of cysticercosis. Possible reasons for this include technical problems, cross-reactivity with other parasites or other diseases, or the presence of a non-specific band. The results suggest that the finding of a single 50-kDa band should be interpreted with caution

Comparison of PCR and antigen detection methods for diagnosis of Entamoeba histolytica infection

Aims: To assess different laboratory methods for the identification of Entamoeba histolytica in clinical samples. Methods: Antigen detection enzyme linked immunosorbent assay, polymerase chain reaction solution hybridisation enzyme linked immunoassay (PCR-SHELA), and a commercial Lightcycler PCR were compared using 101 stool and pus samples. Results: Fifteen of the 101 samples were positive for E histolytica by one or more method. There were discrepancies between the results in five of these 15 samples when the assays were compared. Conclusions: All three methods performed adequately, so that the choice of assay will depend on each individual laboratory’s budget and projected turnaround time

Molecular epidemiology of amebiasis.

Entamoeba histolytica, the causative agent of human amebiasis, remains a significant cause of morbidity and mortality in developing countries and is responsible for up to 100,000 deaths worldwide each year. Entamoeba dispar, morphologically indistinguishable from E. histolytica, is more common in humans in many parts of the world. Similarly Entamoeba moshkovskii, which was long considered to be a free-living ameba, is also morphologically identical to E. histolytica and E. dispar, and is highly prevalent in some E. histolytica endemic countries. However, the only species to cause disease in humans is E. histolytica. Most old epidemiological data on E. histolytica are unusable as the techniques employed do not differentiate between the above three Entamoeba species. Molecular tools are now available not only to diagnose these species accurately but also to study intra-species genetic diversity. Recent studies suggest that only a minority of all E. histolytica infections progress to the development of clinical symptoms in the host and there exist population level differences between the E. histolytica strains isolated from the asymptomatic and symptomatic individuals. Nevertheless the underlying factors responsible for variable clinical outcome of infection by E. histolytica remain largely unknown. We anticipate that the recently completed E. histolytica genome sequence and new molecular techniques will rapidly advance our understanding of the epidemiology and pathogenicity of amebiasis