Standardisation is necessary in urogenital and extragenital bacterial load determination by quantitative PCR: a review of literature and retrospective study.

OBJECTIVES

Pathogen load has been linked to disease severity in patients infected with HIV, resulting in international standards to adequately and reproducibly quantify load. (CT) load has been inconsistently linked to disease severity since extensive differences exist in quantification methods (14 methods in 28 articles). Differences include normalisation for human cell load due to CT's intracellular nature, despite the inability to distinguish inflammatory cells from epithelial cells with molecular techniques. We compared the human cell load in CT-positive men and women at the genital and anal site to a CT-negative control group to estimate the impact of inflammatory cells in these samples.

METHODS

188 women (tested at genital and anal site) and 519 men (207 tested at the anal site and 312 tested at the urogenital site) were included from our STI-clinic in the Netherlands. Specimens were self-collected vaginal swabs, anal swabs and urine samples. Quantitative-PCR targeting the HLA-gene quantified human cell load. Mann-Whitney-U-test was used for statistical analyses.

RESULTS

The genital cell load had a similar range and median (6.5 log10) between CT-negative and CT-positive women . The urogenital cell load was significantly higher than the anal cell load (median 3.6 log10). The anal cell load was significantly higher in men with- than without anal CT infection (median 4.5 versus 3.9 respectively). The anal cell load is significantly higher in CT-positive men than in women. Both Neisseria gonorrhoeae-co-infections and reported anal intercourse significantly increased the human cell load in anal samples.

CONCLUSION

Standardisation in CT load studies is necessary as current studies show 14 different quantification methods in 28 studies . In this study we demonstrate the inappropriateness of normalising the CT load for the human cell load using molecular techniques, as the presence of inflammatory cells cannot be excluded.