This study demonstrates the excellent diagnostic accuracy of the Xpert MTB/RIF test in patients with tuberculous lymphadenitis. The test sensitivity and specificity were 96.7% (95% confidence interval [CI], 86.6 to 100%) and 88.9% (95% CI, 69.6 to 100%), respectively, and it correctly identified 6/6 (100%) of the cytology smear-negative/culture-positive cases and 1 of 2 (50%) rifampin-resistant cases.Tuberculous lymphadenitis is the most common extrapulmonary manifestation of tuberculosis (TB) (4,8), and the majority of cases have no active lung involvement. Fineneedle aspiration biopsy (FNAB) offers a feasible and safe option for specimen collection (11,15). The use of cytology together with the confirmation of acid fastness by ZiehlNeelsen (ZN) staining and Papanicolaou stain-induced fluorescence microscopy as well as mycobacterial detection by culture offers excellent yields (13, 14) but remains limited by the absence of species confirmation, slow turnaround times, and/or lack of drug resistance guidance. Conventional microbiological culture and drug susceptibility testing are not always available and in rare instances may take 6 weeks or longer (10).The World Health Organization (WHO)-endorsed Xpert MTB/RIF combines sample processing and real-time PCR in a fully automated platform and detects Mycobacterium tuberculosis complex and rifampin resistance in less than 2 h (2, 3, 9). Xpert MTB/RIF has been used successfully on various extrapulmonary specimens, including urine and stool (6), but has not been rigorously evaluated with the use of tissue specimens, such as FNAB specimens.To determine the diagnostic utility of the Xpert MTB/RIF, FNAB specimens were collected from 50 consenting patients by aspirating two passes of a 23-or 25-gauge needle attached to a 10-ml syringe. Two smears were prepared from each aspirate, one fixed with commercial cytology fixative for Papanicolaou staining and evaluation by fluorescence microscopy and the other air dried for Giemsa and subsequent ZN staining.Smears were evaluated for adequacy and for a morphological diagnosis, and cases were excluded from the analysis if either one or both passes had inadequate cellular material on smears. Both ZN and Papanicolaou stain-induced fluorescence microscopy evaluations were performed for direct detection of mycobacteria on all specimens. Residual material from one of the aspirates was rinsed in a mycobacterial growth indicator tube (MGIT 960; Becton Dickinson) by aspirating a small volume of fluid into the syringe and expressing it back into the MGIT 960 tube, followed by incubation in a MGIT 960 instrument for mycobacterial culture. Positive cultures were identified as belonging to the Mycobacterium tuberculosis complex, and genotypic drug susceptibility testing was done using the genotype MTBDRplus assay (Hain Lifescience, Germany) (1).The residual material from the remaining aspirate was rinsed as described above into 0.7 ml sterile phosphate-buffered saline (PBS) in a 10-ml headspace glass vial sealed with a TFE/Sil Septa and alumin...

IS6110 restriction fragment length polymorphism (RFLP) genotyping is the most widely used genotyping method to study the epidemiology of Mycobacterium tuberculosis. However, due to the complexity of the IS6110 RFLP genotyping technique, and the interpretation of RFLP data, mycobacterial interspersed repetitive-unitvariable-number tandem-repeat (MIRU-VNTR) genotyping has been proposed as the new genotyping standard. This study aimed to determine the discriminatory power of different MIRU-VNTR locus combinations relative to IS6110 RFLP genotyping, using a collection of Beijing genotype M. tuberculosis strains with a well-established phylogenetic history. Clustering, diversity index, clustering concordance, concordance among unique genotypes, and divergent and convergent evolution were calculated for seven combinations of 27 different MIRU-VNTR loci and compared to IS6110 RFLP results. Our results confirmed previous findings that MIRU-VNTR genotyping can be used to estimate the extent of recent or ongoing transmission. However, molecular epidemiological linking of cases varied significantly depending on the genotyping method used. We conclude that IS6110 RFLP and MIRU-VNTR loci evolve independently and at different rates, which leads to discordance between transmission chains predicted by the respective genotyping methods. Concordance between the two genotyping methods could be improved by the inclusion of genetic distance (GD) into the clustering formulae for some of the MIRU-VNTR loci combinations. In summary, our findings differ from previous reports, which may be explained by the fact that in settings of low tuberculosis incidence, the genetic distance between epidemiologically unrelated isolates was sufficient to define a strain using either marker, whereas in settings of high incidence, continuous evolution and persistence of strains revealed the weaknesses inherent to these markers.Over the past 2 decades, molecular genotyping methods have enhanced our understanding of the epidemiology of tuberculosis (TB) in numerous geographical settings. These methods have enabled geo-temporal tracking of Mycobacterium tuberculosis strains with the view to identifying source cases responsible for TB outbreaks (3), tracking of recent and ongoing disease transmission (31), distinguishing between reinfection and relapse (28), evaluating the effectiveness of direct observed therapy short-course-based TB control programs (5, 16), and identifying global genetic lineages (7). Ideally, molecular genotyping tools should be inexpensive, highly discriminative, deliver rapid results, be straightforward to perform, and produce easily interpretable results that allow for accurate interlaboratory comparisons (universally comparable databases).Three genotyping methods are currently widely used in molecular epidemiological studies of TB: IS6110 restriction fragment length polymorphism (RFLP) genotyping (27), spoligotyping (14), and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping (21,22). Cu...

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BackgroundStaphylococcus aureus (S. aureus) has established itself over the years as a major cause of morbidity and mortality both within the community and in healthcare settings. Methicillin resistant S. aureus (MRSA) in particular has been a major cause of nosocomial infections resulting in significant increase in healthcare costs. In Africa, the MRSA prevalence has been shown to vary across different countries. In order to better understand the epidemiology of MRSA in a setting, it is important to define its population structure using molecular tools as different clones have been found to predominate in certain geographical locations.MethodsWe carried out PFGE, MLST, SCCmec and spa typing of selected S. aureus isolates from a private and public referral hospital in Nairobi, Kenya.ResultsA total of 93 S. aureus isolates were grouped into 19 PFGE clonal complexes (A–S) and 12 singletons. From these, 55 (32 MRSA and 23 MSSA) representative isolates from each PFGE clonal complex and all singletons were spa typed. There were 18 different MRSA spa types and 22 MSSA spa types. The predominant MRSA spa type was t037 comprising 40.6 % (13/32) of all MRSA. In contrast, the MSSA were quite heterogeneous, only 2 out of 23 MSSA shared the same spa type. Two new MRSA spa types (t13149 and t13150) and 3 new MSSA spa types (t13182, t13193 and t13194) were identified. The predominant clonal complex was CC 5 which included multi-locus sequence types 1, 8 and 241.ConclusionIn contrast to previous studies published from Kenya, there’s marked genetic diversity amongst clinical MRSA isolates in Nairobi including the presence of well-known epidemic MRSA clones. Given that these clones are resident within our referral hospitals, adherence to strict infection control measures needs to be ensured to reduce morbidity and mortality associated with hospital acquired MRSA infections.

Recent developments in the field of TB diagnostics, including the introduction of the Xpert MTB/RIF assay in field testing, raise the hope for faster and more accurate identification of active TB patients. However, there are still many issues that need to be addressed as no point-of-care tests are yet available. Furthermore, no tests are available which are universally applicable to all patients. Improvements in the microbiological and molecular-based approaches are promising and the diagnostic pipeline is encouraging. Host markers associated with active disease may hold promise, especially in situations where sputum diagnostics are problematic, including in children, HIV-infected individuals and in the case of extrapulmonary TB.

Although commercial nucleic-acid amplification tests performed on CSF revealed incrementally improved diagnostic accuracy, providing rapid microbiological confirmation, they cannot serve as a rule-out test.

The emergence and transmission of multidrug-resistant tuberculosis (MDR-TB) and extensively drugresistant tuberculosis (XDR-TB) have raised concern about diagnostic delay associated with culture-based drug susceptibility testing methods. The association between rifampin resistance and MDR-TB or XDR-TB makes it an important genetic marker for genotypic drug susceptibility testing. In this article, we describe the analysis of the physical properties of the rifampin resistance-determining region (RRDR) in the rpoB gene by high-resolution thermal melt analysis as a method for detecting rifampin resistance in Mycobacterium tuberculosis complex. The RRDR from the M. tuberculosis complex was amplified by PCR from DNA templates extracted from sputum cultures of M. tuberculosis or the laboratory strain (H37Rv) in the presence of a fluorescent DNA binding dye. Subsequent mixing of the amplification products from the respective sputum cultures and the laboratory strain and thermocycling allowed the formation of DNA duplexes. The thermal denaturation properties of these DNA duplexes were determined by measuring the derivative of the intensity of fluorescence at different temperatures. Analysis of DNA extracted from 153 sputum cultures showed a sensitivity of 98% and a specificity of 100% for the detection of rifampin resistance compared to the "gold standard" culture-based phenotyping method. No statistical difference was detected in the performance of the method when applied to crude DNA from 134 boiled cultures. This method, named "FAST-Rif" ("fluorometric assay for susceptibility testing of rifampin"), allowed the rapid, reliable, and easy detection of genotypic rifampin resistance as a marker for MDR-TB and XDR-TB.

2 Declaration I, Preneshni Rochelle Naicker, hereby declare that the work contained in this assignment is my original work and that I have not previously submitted it, in its entirety or in part, at any university for a degree. The ability to form biofilms contributes significantly to the virulence of Staphylococcus aureus. Virulence factors may be associated with certain S. aureus lineages. However, the contribution of the genetic background of S. aureus to biofilm formation is poorly understood. This study investigated the association between the genetic background and the biofilm forming ability of clinical invasive S. aureus isolates. Secondary objectives included investigating any correlation with biofilm formation and methicillin resistance or the source of bacteraemia. Methods:The study was conducted at a 1300-bed tertiary hospital in Cape Town, South Africa. S. aureus isolates obtained from blood cultures between January 2010 and January 2012 were included. Genotypic characterization was performed by PFGE, spa typing, SCCmec typing and MLST. Thirty genotypically unique strains were assessed for phenotypic biofilm formation with the microtitre plate assay. All isolates were tested in triplicate and an average optical density, measured at a wavelength of 490nm, was determined. The biofilm forming ability of isolates with A 490 > 0.17 was considered 'weak positive' and A 490 > 0.34 'strong positive'. Isolates with A 490 ≤ 0.17 were considered non-adherent. ANOVA with Bonferroni-adjusted post-hoc tests and t-tests were used for statistical analysis of the association between biofilm forming ability and strain characteristics.Results: Fifty seven percent of isolates were capable of forming biofilms. Weak biofilm formation occurred in 40% (n=12) and strong biofilm formation in 17% (n=5) of isolates. Thirteen (43%) of the isolates were classified as non-adherent. All 5 isolates capable of strong biofilm formation belong to one spa clonal complex (spa-CC 064). Strains from spa-CC 064 were capable of higher biofilm formation than other spa clonal complexes (p=0.00002). These 5 strains belonged to MLST CC5 and CC8. Biofilm formation did not correlate with methicillin resistance and was not related to the source of bacteraemia. Conclusion:Biofilm formation correlates with the spa clonal lineage in our population of invasive S. aureus strains. High biofilm formation was associated with spa-CC 064. MLST CC5 and CC8 strains are capable of strong biofilm formation.