Rotor gene platform

The mRNA quantification of IL1B, IL6, and TNFA was performed by qPCR using TaqMan® Gene Expression Assay (Life Technologies, Carlsbad, USA) and RotorGene® platform (QIAGEN, Hilden, Germany). The GAPDH gene was chosen as an endogenous reference using GeNorm (qbase+, Biogazelle). The PCR was performed in a multiplex format using Quantifast® Multiplex PCR assay (QIAGEN, Hilden, Germany). Thus, target genes and GAPDH were labeled with fluorescent dye FAM and VIC, respectively. Data were analyzed using Rotor-Gene® Q -Pure detection software (QIAGEN, Hilden, Germany). The results were obtained by relative quantification method (2 -ΔΔCt ) 18 .

Cervical specimens were collected as part of an evaluation study of cervical cancer screening strategies in Burkina Faso and South Africa, as previously described. [6] Inclusion criteria were WLHIV, aged 25-50 years, and resided in Ouagadougou or Johannesburg. This analysis focuses on WLHIV recruited in South Africa, as MG rates were found to be low in Burkina Faso(4/615). WLHIV were recruited from primary health care centres and surrounding communities in Johannesburg from December 2011 to October 2012. For all women at baseline and endline, a cervical swab was collected to detect Chlamydia trachomatis, Neisseria gonorrhoeae, MG, and Trichomonas vaginalis using a validated in-house realtime multiplex polymerase chain reaction(mPCR) assay. For the endline, not all samples could be tested for cervical PCR, and we only report MG testing for those found positive at baseline. Specimens that tested MG positive with the in-house mPCR assay were confirmed with the commercial Sacace MG real-time PCR assay(Sacace Biotechnologies, Como, Italy). All real-time PCR assays were performed using the Rotor-Gene platform(Qiagen, Hilden, Germany). A vaginal smear was Gram-stained for the diagnosis of bacterial vaginosis(Nugent's score of at least 7) and Candida spp.

For all women, a cervical swab was collected to detect Mycoplasma genitalium, Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis using a validated in-house realtime multiplex polymerase chain reaction (mPCR) assay. Specimens that tested MG positive with the in-house mPCR assay were confirmed with the commercial Sacace MG real-time PCR assay (Sacace Biotechnologies, Como, Italy). All real-time PCR assays were performed using the Rotor-Gene platform (Qiagen, Hilden, Germany). We collected an ecto/endocervical swab using
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Specimens were transported to the STI reference laboratory at the NICD in Johannesburg. DNA was extracted from endocervical swabs using two automated DNA extractors (X-tractor Gene and QIAxtractor platforms; Qiagen, Germany). Swab-extracted DNA was tested using a validated in-house real-time multiplex polymerase chain reaction assay on the RotorGene platform (Qiagen) to detect the presence of the STI pathogens N. gonorrhoeae, C. trachomatis, T. vaginalis and M. genitalium. Gram-stained smears of vaginal swab specimens were assessed microscopically for presence of BV (Nugent scoring) and Candida species. HIV seropositivity was determined using two rapid immunochromatographic assays, Unigold (Trinity Biotech, Ireland) and Alere Determine (Alere Medical Co. Ltd, Japan).