Development and validation of a molecular assay and evaluation of the GeneXpert® MTB/RIF assay for the rapid detection of genital tuberculosis

[摘要] English: Tuberculosis (TB) is a communicable disease which is caused by the bacterium Mycobacterium tuberculosis (MTB). According to the World Health Organization, globally in 2015 there were 10.4 million new cases and 1.4 million deaths due to TB. TB is one of the leading causes of death in South Africa resulting in approximately 8.4% of deaths in 2015. The most common manifestation of TB involves the lungs, defined as pulmonary TB (PTB), while TB affecting other organs is defined as extrapulmonary TB (EPTB). EPTB accounts for only 20% of all TB cases in human immunodeficiency virus negative individuals. Approximately 1.8% of all TB cases have a genitourinary site, with the prevalence of genital TB (GTB) in South Africa reported to range from 6.2-21.0%. One of the leading symptoms of GTB in females is infertility, usually resulting from the involvement of the fallopian tubes and endometrium. Approximately 40-80% of women with GTB will become infertile.The detection of microorganisms through microscopy is the oldest technique for laboratory diagnosis. While microscopy is rapid and inexpensive, it requires a high bacterial load which is not present in paucibacillary EPTB samples. Culture of MTB is widely regarded as the gold standard for TB diagnosis. While culture has a long turnaround time, culture remains important since it is more sensitive than microscopy. In addition, growth is required for species identification, drug susceptibility testing and genotyping of cultured organisms may be useful for epidemiological studies. Little is known regarding which technique is best for the detection of GTB from clinical samples apart from culture. Molecular based techniques hold the promise of a more rapid and accurate diagnosis of EPTB.The aim of this project was the development and validation of an in-house nested PCR assay and the validation of the GeneXpert® MTB/RIF (GeneXpert) assay for the laboratory diagnosis of GTB. In total 54 samples were submitted for GTB screening from women being investigated for infertility at the Unit for Human Reproduction, Universitas Academic Hospital, Bloemfontein. This included 44 endometrial tissue samples and 10 menstrual fluid samples. All samples underwent testing with the GeneXpert, the in-house nested PCR and culture. The nested PCR was designed targeting the insertion sequence element 6110 (IS6110) found in members of the MTB complex. The analytical sensitivity/limit of detection (LOD) for the GeneXpert was determined to be 250pg while the LOD for the nested polymerase chain reaction (PCR) was 62.5fg. Both assays displayed excellent analytical specificity by discriminating TB deoxyribose nucleic acid (DNA) from other bacterial and nontuberculous mycobacterial DNA. The diagnostic sensitivity and specificity was determined using culture as the reference method. Culture was able to detect GTB in 2 of the 54 samples including one menstrual fluid and one endometrial tissue sample, thus indicating a GTB prevalence of 3.7%. The GeneXpert detected 1 of the 54 samples as positive indicating a sensitivity of 50% and a specificity of 100%. The nested PCR detected both positive samples resulting in a sensitivity and specificity of 100%. The GeneXpert obtained a positive predictive value (PPV) of 100% and a negative predictive value (NPV) of 98.1%, while the nested PCR obtained a PPV and NPV of 100%. The two GTB isolates underwent genotyping using spoligotyping and mycobacterial interspersed repetitive unit �?variable number of tandem repeats (MIRU-VNTR). The menstrual fluid isolate was characterised as a Beijing strain and the endometrial tissue isolate as an X3 strain.The nested PCR showed a greater sensitivity than the GeneXpert as a result of the better LOD. Despite this, both techniques could be implemented for GTB screening in combination with culture. Screening of menstrual fluid samples using the GeneXpert assay would be well suited for GTB screening in resource limited areas.

[发布机构] University of the Free State