Development and validation of a molecular assay and evaluation of the GeneXpert® MTB/RIF assay for the rapid detection of genital tuberculosis
| dc.contributor.advisor | Goedhals, D. | |
| dc.contributor.advisor | Hoosen, A. A. | |
| dc.contributor.author | Sokhela, Maxwell Cebolenkosi | |
| dc.date.accessioned | 2017-08-11T05:57:31Z | |
| dc.date.available | 2017-08-11T05:57:31Z | |
| dc.date.issued | 2016-02 | |
| dc.description.abstract | 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. | en_ZA |
| dc.description.abstract | Afrikaans: Tuberkulose (TB) is ‘n aanmeldbare siekte wat veroorsaak word deur die bakterium Mycobacterium tuberculosis (MTB). Volgens die Wêreld Gesondheidsorganisasie, was daar wêreldwyd 10.4 miljoen nuwe gevalle en 1.4 miljoen sterftes as gevolg van TB in 2015. TB is een van die hoofoorsake van sterftes in Suid-Afrika en gevolglik verantwoordelik vir ongeveerd 8.4% van sterftes in 2014. Die mees algemene beeld van TB betrek die longe en word gedefinieer as pulmonale TB (PTB), terwyl TB wat ander organe affekteer gedefinieer word as ekstrapulmonale TB (EPTB). EPTB verteenwoordig slegs 20% van TB gevalle in menslike immuungebrekvirus-negatiewe individue. Ongeveer 1.8% van alle TB gevalle affekteer die genito-urinêre area met ‘n voorkoms van genitale TB (GTB) in Suid-Afrika van 6.2-21.0%. Een van die mees algemene simptome van GTB in dames is onvrugbaarheid wat gewoonlik is as gevolg van die betrokkenheid van die fallopiusbuise en endometrium. Ongeveer 40-80% van dames met GTB sal onvrugbaar word. Die opsporing van mikro-organismes deur mikroskopie is die oudste tegniek vir ‘n laboratorium diagnose. Mikroskopie is vining en goedkoop, maar benodig ‘n groot hoeveelheid bakterieë wat gewoonlik nie teenwoordig is in pauci-basillêre EPTB monsters nie. Kweking van MTB word wyd beskou as die goue standard vir die diagnose van TB. Alhoewel die metode ‘n lang omkeertyd het, bly kweking van die organisme belangrik aangesien dit meer sensitief as mikroskopie is. Kweking van die organisme word vereis vir spesie-identifikasie, middel vatbaarheidstoetsing en genotipering wat van waarde kan wees vir epidemiologiese studies. Huidiglik is daar beperkte kennis met betrekking tot watter tegniek, anders as kweking, die beste is vir die opsporing van GTB in kliniese monsters. Daar is ‘n moontlikheid dat molekulêre tegnieke gebruik kan word vir ‘n vinniger, tog akkurate diagnose van EPTB. Die doel van hierdie projek was die ontwikkeling en validering van ‘n in-huis geneste polimerase kettingreaksie (PKR) toets, asook die validering van die GeneXpert® MTB/RIF (GeneXpert) toets vir die laboratorium diagnose van GTB. In total was daar 54 monsters ingedien vir GTB toetsing van dames wat huidiglik ondersoek word vir onvrugbaarheid by die Eenheid van Menslike Voortplanting, Universitas Akademiese Hospitaal, Bloemfontein. Hierdie monsters het 44 endometriale weefsel en 10 menstruele vloeistof monsters ingesluit. Alle monsters was getoets met die GeneXpert toets, die in-huis geneste PKR toets en met kweking. ‘n Geneste PKR toets was ontwerp wat die IS6110 area gevind in die MTB kompleks teiken. Die analitiese sensitiwiteit/limiet van opsporing (LVO) vir die GeneXpert toets was 250pg, terwyl die LVO vir die geneste PKR toets 62,5fg was. Beide toetse het uitstekende analitiese spesifisiteit om tussen TB Deoksiribose nukleïensuur (DNS) en ander bakteriële en nie-tuberkuleuse mikobakteriële DNS te onderskei. Die diagnostiese sensitiwiteit en spesifisiteit was bepaal met kweking as verwysingsmetode. Kweking kon GTB in 2 van die 54 monsters opspoor wat een menstruele vloeistof en een endometriale weefsel monster ingesluit het. Die voorkoms van GTB was dus 3.7%. Die GeneXpert toets het 1 van die 54 monsters as positief getoon met ‘n sensitiwiteit 50% en ‘n spesifisiteit van 100%. Die geneste PKR toets kon beide positiewe monsters opspoor met ‘n sensitiwiteit en spesifisiteit van 100%. Die GeneXpert toets het ‘n positiewe voorspellingswaarde (PVW) van 100% en ‘n negatiewe voorspellingswaarde (NVW) van 98.1%, terwyl die geneste PKR toets ‘n PVW en NVW van 100% gehad het. Genotipering in die vorm van spoligotipering en mikobakteriële verspreide herhalende eenheid – veranderlike aantal tandem herhalings (MIRU-VNTR) was bepaal. Die menstruele vloeistof isolaat was gekarakteriseer as ‘n Beijing stam en die endometiale weefsel isolaat as ‘n X3 stam. Die geneste PKR toets het hoër sensitiwiteit as die GeneXpert toets getoon en gevolglik ook ‘n beter LVO. Ten spyte hiervan, kan beide tegnieke in kombinasie met kweking gebruik word vir GTB opsporing. Toetsing van menstruele vloeistof monsters vir GTB met behulp van die GeneXpert toets sal gepas wees vir die opsporing van GTB in areas met beperkte hulpbronne. | af |
| dc.description.sponsorship | National Health Laboratory Service Research Trust Fund | en_ZA |
| dc.description.sponsorship | National Research Foundation (NRF) | en_ZA |
| dc.description.sponsorship | University of the Free State, School of Medicine | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/11660/6532 | |
| dc.language.iso | en | en_ZA |
| dc.publisher | University of the Free State | en_ZA |
| dc.rights.holder | University of the Free State | en_ZA |
| dc.subject | Extrapulmonary TB | en_ZA |
| dc.subject | Genital TB | en_ZA |
| dc.subject | Nested PCR | en_ZA |
| dc.subject | IS6110 | en_ZA |
| dc.subject | GeneXpert® MTB/RIF | en_ZA |
| dc.subject | MGIT culture | en_ZA |
| dc.subject | Predictive values | en_ZA |
| dc.subject | Cohen’s Kappa | en_ZA |
| dc.subject | Spoligotyping | en_ZA |
| dc.subject | Mycobacterial interspersed repetitive unit – variable number of tandem repeats | en_ZA |
| dc.subject | Tuberculosis | en_ZA |
| dc.subject | Communicable diseases | en_ZA |
| dc.subject | Mycobacterial diseases | en_ZA |
| dc.subject | Dissertation (M.Sc. (Medical Physics))--University of the Free State, 2016 | en_ZA |
| dc.title | Development and validation of a molecular assay and evaluation of the GeneXpert® MTB/RIF assay for the rapid detection of genital tuberculosis | en_ZA |
| dc.type | Dissertation | en_ZA |