Remco P. H. Peters

Improved Detection by Next-Generation Sequencing of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates

ABSTRACT The technical limitations of common tests used for detecting pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates pose challenges for comprehensive and accurate descriptions of drug resistance in patients with multidrug-resistant tuberculosis (MDR-TB). In this study, a 606-bp fragment (comprising the pncA coding region plus the promoter) was sequenced using Ion Torrent next-generation sequencing (NGS) to detect associated PZA resistance mutations in 88 recultured MDR-TB isolates from an archived series collected in 2001. These 88 isolates were previously Sanger sequenced, with 55 (61%) designated as carrying the wild-type pncA gene and 33 (37%) showing mutations. PZA susceptibility of the isolates was also determined using the Bactec 460 TB system and the Wayne test. In this study, isolates were recultured and susceptibility testing was performed in Bactec 960 MGIT. Concordance between NGS and MGIT results was 93% (n = 88), and concordance values between the Bactec 460, the Wayne test, or pncA gene Sanger sequencing and NGS results were 82% (n = 88), 83% (n = 88), and 89% (n = 88), respectively. NGS confirmed the majority of pncA mutations detected by Sanger sequencing but revealed several new and mixed-strain mutations that resolved discordancy in other phenotypic results. Importantly, in 53% (18/34) of these isolates, pncA mutations were located in the 151 to 360 region and warrant further exploration. In these isolates, with their known resistance to rifampin, NGS of pncA improved PZA resistance detection sensitivity to 97% and specificity to 94% using NGS as the gold standard and helped to resolve discordant results from conventional methodologies.

Evaluation of syndromic management guidelines for treatment of sexually transmitted infections in South African women.

To evaluate the performance of three different guidelines for the management of vaginal discharge syndrome (VDS) for women living in a rural setting in South Africa.

Laboratory evaluation of a specimen transport medium for downstream molecular processing of sputum samples to detect Mycobacterium tuberculosis

BACKGROUNDnModern molecular-based approaches for the detection of Mycobacterium tuberculosis in sputum samples promise quicker and more accurate detection of cases. However, processing sputum samples at central diagnostic facilities provides a diagnostic approach, but requires a safe and efficient system that is not affected by transport delays and ambient temperature to be feasible. We evaluated the technical properties of PrimeStore®-Molecular Transport Medium (PS-MTM) for its ability to inactivate mycobacteria, ensuring stability of DNA over time at ambient temperatures and to assess the compatibility of the transport medium with DNA extraction systems.nnnMETHODSnAssessment of the transport medium for application of sputum samples processed for the detection of M. tuberculosis included the inactivation of M. tuberculosis in spiked sputum samples, compatibility of the medium with three commercial nucleic extraction systems and stability of DNA in the medium at ambient temperature over 28 days. We further performed a clinical laboratory evaluation on 256 sputum specimens sent for tuberculosis investigation.nnnRESULTSnComplete inactivation of M. tuberculosis occurred within 30 min of exposure at a ratio of 1:3 for sputum to PS-MTM. Sputum specimen in PS-MTM showed very good compatibility with automated bead-based extraction systems, producing high DNA output (estimated lower limits of detection: ~170 CFU/ml). Furthermore, PS-MTM samples remained stable over 28 days at ambient temperature displaying no significant change over time in Ct-values (<5% on a mean starting value of 22.47). Of the 256 clinical sputum specimens, 10.2% were culture positive and 11.0% were positive by real-time PCR of PS-MTM samples.nnnCONCLUSIONSnCollecting and transporting sputum from TB suspects in PS-MTM offer safe transport at ambient temperature, DNA stability for extended periods without cooling and specimens directly suitable for molecular testing. This novel approach may support introduction and further scale-up of molecular diagnostics for TB in resource-limited settings.

High prevalence of asymptomatic sexually transmitted infections among human immunodeficiency virus-infected pregnant women in a low-income South African community:

There is a lack of evidence on the burden of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Trichomonas vaginalis (TV) among HIV-infected pregnant women in South Africa. We conducted a cross-sectional analysis of HIV-infected pregnant women in two healthcare facilities in a South African township to determine the prevalence of CT, NG and TV. HIV-infected pregnant women were recruited during the first antenatal care visit for their current pregnancy and requested to self-collect vulvovaginal swab specimens. Specimens were tested for CT, NG and TV using the Xpert® assay (Cepheid, Sunnyvale, CA). Of 247 tested for CT, NG and TV, 47.8% tested positive for at least one organism; CTu2009=u200936.8%, TVu2009=u200923.9%, NGu2009=u20096.9%. Forty three (17.4%) had multiple infections, of which 42 included CT as one of the infecting organisms. Of the 118 participants who tested positive for at least one sexually transmitted infection (STI), 23.7% reported STI-like symptoms. Among women who tested positive for CT, 29.7% reported symptoms while 47.1 and 27.1% of those who tested positive for NG and TV, respectively, reported symptoms. The high STI prevalence coupled with the low symptom prevalence among infected individuals justifies the use of diagnostic screening approaches rather than syndromic management of STIs in this setting.

Microbiological Characteristics of Chlamydia trachomatis and Neisseria gonorrhoeae Infections in South African Women

ABSTRACT We analyzed data of 263 women with at least one genital or anorectal sexually transmitted infection from a cross-sectional study conducted in rural South Africa. We provide new insights concerning the concurrence of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, and Trichomonas vaginalis infections as well as the characteristics of bacterial loads.

Uveitis is predominantly of infectious origin in a high HIV and TB prevalence setting in rural South Africa.

Aims To determine the burden of disease in a unique sample of patients with uveitis from a rural South African setting. Methods Data in this cross-sectional study were collected from patients presenting with uveitis (n=103) at the ophthalmology outpatient department of three hospitals in rural South Africa. Demographic and clinical data were collected, and laboratory analysis of aqueous humour, serological evaluation and routine diagnostics for tuberculosis (TB) were performed. Results Sixty-six (64%) participants were HIV infected. Uveitis was predominantly of infectious origin (72%) followed by idiopathic (16%) and autoimmune (12%). Infectious uveitis was attributed to herpes virus (51%), Mycobacterium tuberculosis (24%) and Treponema pallidum (7%) infection. HIV-infected individuals were more likely to have infectious aetiology of uveitis compared with HIV-uninfected individuals (83% vs 51%; p=0.001). Conclusions Microbial aetiology of uveitis is common in areas where HIV and TB are endemic. In these settings, a high index of suspicion for infectious origin of uveitis is warranted.

Evaluation of Presto(plus) assay and LightMix kit Trichomonas vaginalis assay for detection of Trichomonas vaginalis in dry vaginal swabs.

This is an evaluation study of the Presto(plus) Assay for T. vaginalis by comparing to the TIB MOLBIOL LightMix Kit Trichomonas vaginalis Assay using 615 dry collected vaginal and rectal swabs. Discordant samples were analyzed by the Qiagen® Microbial DNA qPCR for TV Assay. Both assays showed comparable performances (McNemar p>0.05).

Early- and late-stage ocular complications of herpes zoster ophthalmicus in rural South Africa.

To describe the spectrum of ocular complications of herpes zoster ophthalmicus (HZO) in rural South Africa.

Clinical and corneal microbial profile of infectious keratitis in a high HIV prevalence setting in rural South Africa

The purpose of this investigation was to determine the clinical and corneal microbial profile of infectious keratitis in a high human immunodeficiency virus (HIV) prevalence setting in rural South Africa. Data in this cross-sectional study were collected from patients presenting with symptoms of infectious keratitis (nu2009=u200946) at the ophthalmology outpatient department of three hospitals in rural South Africa. Corneal swabs were tested for herpes simplex virus type 1 (HSV-1) and 2 (HSV-2), varicella zoster virus (VZV) and adenovirus DNA by real-time polymerase chain reaction (PCR) and for bacteria and fungi by culture. Based on clinical history, disease characteristics and laboratory results, 29 (63xa0%) patients were diagnosed as viral keratitis, including 14 (48xa0%) viral keratitis cases complicated by bacterial superinfection, and 17 (37xa0%) as bacterial keratitis. VZV and HSV-1 DNA was detected in 11 (24xa0%) and 5 (11xa0%) corneal swabs, respectively. Among clinically defined viral keratitis cases, a negative viral swab was predominantly (93xa0%) observed in cases with subepithelial inflammation and was significantly associated with an increased duration of symptoms (pu2009=u20090.003). The majority of bacteria cultured were Gram-positive (24/35), including Staphylococcus epidermidis and S. aureus. Viral aetiology was significantly associated with a history of herpes zoster ophthalmicus (pu2009<u20090.001) and a trend was observed between viral aetiology and HIV infection (pu2009=u20090.06). Twenty-one (47xa0%) keratitis cases were complicated by anterior uveitis, of which 18 (86xa0%) were HIV-infected cases with viral keratitis. The data implicate a high prevalence of herpetic keratitis, in part complicated by bacterial superinfection and/or uveitis, in HIV-infected individuals presenting with infectious keratitis in rural South Africa.

Molecular detection of Mycobacterium tuberculosis from sputum transported in PrimeStore(®) from rural settings.

SETTINGnMopani District, South Africa.nnnOBJECTIVEnTo explore remote, molecular detection of Mycobacterium tuberculosis from sputum transported using PrimeStore(®) Molecular Transport Medium (PS-MTM) compared to settings where microscopy or Xpert(®) MTB/RIF is used as the baseline test.nnnDESIGNnTwo sputum specimens were collected from patients with cough of ⩾ 2 weeks at clinics in rural South Africa. Shortly after expectoration and before processing using Xpert, microscopy and liquid culture, a flocked swab was swirled in each of these specimens and placed in PS-MTM. Swabs were stored and transported to the United States at ambient temperature for real-time PrimeMix(®) polymerase chain reaction (PM-PCR).nnnRESULTSnOf 132 patients, 23 (17%) were positive on microscopy, 39 (30%) on Xpert and 44 (33%) by PS-MTM/PM-PCR. Concordance of PS-MTM/PM-PCR with positive microscopy and Xpert was respectively 96% and 85%. Of 107 microscopy-negative samples, 22 (21%) were positive using PS-MTM/PM-PCR, while 11/91 (12%) Xpert-negative samples were PS-MTM/PM-PCR-positive. PS-MTM/PM-PCR positivity was significantly higher than smear microscopy positivity (P < 0.001), but similar to Xpert (P = 0.33).nnnCONCLUSIONnPCR testing of specimens transported in PS-MTM would enhance TB diagnosis in settings where smear microscopy is the baseline diagnostic test, and could provide an alternative in settings where Xpert testing is not available.