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Molecular surveillance of rifampin-resistant Mycobacterium tuberculosis can help to monitor the transmission of the disease. The Xpert MTB/RIF Ultra assay detects mutations in the rifampin resistance-determining region (RRDR) of the rpoB gene by the use of melting temperature (Tm ) information from 4 rpoB probes which can fall in one of the 9 different assay-specified Tm windows. The large amount of Tm data generated by the assay offers the possibility of an RRDR genotyping approach more accessible than whole-genome sequencing. In this study, we developed an automated algorithm to specifically identify a wide range of mutations in the rpoB RRDR by utilizing the pattern of the Tm of the 4 probes within the 9 windows generated by the Ultra assay. The algorithm builds a RRDR mutation-specific "Tm signature" reference library from a set of known mutations and then identifies the RRDR genotype of an unknown sample by measuring the Tm distances between the test sample and the reference Tm values. Validated using a set of clinical isolates, the algorithm correctly identified RRDR genotypes of 93% samples with a wide range of rpoB single and double mutations. Our analytical approach showed a great potential for fast RRDR mutation identification and may also be used as a stand-alone method for ruling out relapse or transmission between patients. The algorithm can be further modified and optimized for higher accuracy as more Ultra data become available.

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The Xpert MTB/RIF assay (Xpert) is a rapid test for tuberculosis (TB) and rifampin resistance (RIF-R) suitable for point-of-care testing. However, it has decreased sensitivity in smear-negative sputum, and false identification of RIF-R occasionally occurs. We developed the Xpert MTB/RIF Ultra assay (Ultra) to improve performance. Ultra and Xpert limits of detection (LOD), dynamic ranges, and RIF-R rpoB mutation detection were tested on Mycobacterium tuberculosis DNA or sputum samples spiked with known numbers of M. tuberculosis H37Rv or Mycobacterium bovis BCG CFU. Frozen and prospectively collected clinical samples from patients suspected of having TB, with and without culture-confirmed TB, were also tested. For M. tuberculosis H37Rv, the LOD was 15.6 CFU/ml of sputum for Ultra versus 112.6 CFU/ml of sputum for Xpert, and for M. bovis BCG, it was 143.4 CFU/ml of sputum for Ultra versus 344 CFU/ml of sputum for Xpert. Ultra resulted in no false-positive RIF-R specimens, while Xpert resulted in two false-positive RIF-R specimens. All RIF-R-associated M. tuberculosis rpoB mutations tested were identified by Ultra. Testing on clinical sputum samples, Ultra versus Xpert, resulted in an overall sensitivity of 87.5% (95% confidence interval [CI], 82.1, 91.7) versus 81.0% (95% CI, 74.9, 86.2) and a sensitivity on sputum smear-negative samples of 78.9% (95% CI, 70.0, 86.1) versus 66.1% (95% CI, 56.4, 74.9). Both tests had a specificity of 98.7% (95% CI, 93.0, 100), and both had comparable accuracies for detection of RIF-R in these samples. Ultra should significantly improve TB detection, especially in patients with paucibacillary disease, and may provide more-reliable RIF-R detection.IMPORTANCE The Xpert MTB/RIF assay (Xpert), the first point-of-care assay for tuberculosis (TB), was endorsed by the World Health Organization in December 2010. Since then, 23 million Xpert tests have been procured in 130 countries. Although Xpert showed high overall sensitivity and specificity with pulmonary samples, its sensitivity has been lower with smear-negative pulmonary samples and extrapulmonary samples. In addition, the prediction of rifampin resistance (RIF-R) in paucibacillary samples and for a few rpoB mutations has resulted in both false-positive and false-negative results. The present study is the first demonstration of the design features and operational characteristics of an improved Xpert Ultra assay. This study also shows that the Ultra format overcomes many of the known shortcomings of Xpert. The new assay should significantly improve TB detection, especially in patients with paucibacillary disease, and provide more-reliable detection of RIF-R.

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Extensively drug-resistant (XDR) tuberculosis (TB) cannot be easily or quickly diagnosed. We developed a rapid, automated assay for the detection of XDR-TB plus resistance to the drug isoniazid (INH) for point-of-care use. Using a simple filter-based cartridge with an integrated sample processing function, the assay identified a wide selection of wild-type and mutant sequences associated with XDR-TB directly from sputum. Four new large-Stokes-shift fluorophores were developed. When these four Stokes-shift fluorophores were combined with six conventional fluorophores, 10-color probe detection in a single PCR tube was enabled. A new three-phase, double-nested PCR approach allowed robust melting temperature analysis with enhanced limits of detection (LODs). Finally, newly designed sloppy molecular beacons identified many different mutations using a small number of probes. The assay correctly distinguished wild-type sequences from 32 commonly occurring mutant sequences tested in gyrA, gyrB, katG, and rrs genes and the promoters of inhA and eis genes responsible for resistance to INH, the fluoroquinolone (FQ) drugs, amikacin (AMK), and kanamycin (KAN). The LOD was 300 CFU of Mycobacterium tuberculosis in 1 ml sputum. The rate of detection of heteroresistance by the assay was equivalent to that by Sanger sequencing. In a blind study of 24 clinical sputum samples, resistance mutations were detected in all targets with 100% sensitivity, with the specificity being 93.7 to 100%. Compared to the results of phenotypic susceptibility testing, the sensitivity of the assay was 75% for FQs and 100% each for INH, AMK, and KAN and the specificity was 100% for INH and FQ and 94% for AMK and KAN. Our approach could enable testing for XDR-TB in point-of-care settings, potentially identifying highly drug-resistant TB more quickly and simply than currently available methods.

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BACKGROUND: Mifepristone became available in New Zealand in 2001, and was first used for second trimester terminations at the Level J Unit, Wellington Hospital. The protocol is based on that published by Ashok et al. in Aberdeen. AIMS: To describe the use of mifepristone prior to misoprostol induction of labour for mid-trimester termination and to compare outcomes with the published data. METHODS: A retrospective audit of prospectively collected notes for 272 women presenting for mid-trimester termination of pregnancy in a hospital termination clinic. Data collection included age, ethnicity, previous pregnancies, gestational age, induction-to-abortion interval, analgesia, and complications. Data were entered into an Access database and imported into Excel and Epi Info for the computation of descriptive statistics. RESULTS: Data on completed abortion were available for 271 women (one chose not to continue the abortion following mifepristone). The median time to abortion was 6 h, and mean number of doses of misoprostol was three. The proportion of women who aborted within 24 h was 95.9%. Immediate surgical evacuation of retained placenta was required in 22 women (8.1%). Heavy bleeding occurred in 22 women (8.1%), and seven required a transfusion (2.6%). The proportion of women who required parenteral narcotics was 78.2%. CONCLUSIONS: Outcomes for women in the present review were comparable with those for previous publications using the same regimen, with the exception of a higher transfusion rate. Our experience supports the finding that the use of mifepristone as pretreatment to misoprostol results in a shorter induction-to-delivery interval than the use of misoprostol alone as has been reported by other groups.

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BACKGROUND: In New Zealand, mifepristone became available in 2001, but because of uncertainty about the law, the first 67 cases were carried out under a very strict protocol. Once the prostaglandin had been administered it was necessary that the woman remain in the unit until the products of conception (POC) had been passed and, if this had not occurred within 8 h, she underwent suction curettage. AIMS: To demonstrate that an early medical termination of pregnancy (EMTOP) service could be offered as a safe option for women, despite the constraints of the law. METHODS: An audit of patient notes was carried out on the first 67 patients undergoing an EMTOP at the Level J Unit (LJU), Wellington Hospital. Data collected included age, ethnicity, parity, previous abortions, gestational age, length of time between the administration of mifepristone and misoprostol, length of time after administration of misoprostol to the completion of abortion, whether a fetal sac was seen, analgesia required, extent of heavy bleeding and any adverse effects. Patient characteristics were compared with those of the 3052 women who underwent surgical termination during the same time period. Data were analysed using EpiInfo 2000 (Centers for Disease Control and Prevention, Atlanta, GA) and Chi square tests for significance. RESULTS: Successful completion of EMTOP occurred in 63 of 67 cases (94%). Only four cases (6%) required completion by suction curettage and this was performed for legal and financial reasons, rather than for medical reasons. Clinical events requiring management, mainly bleeding problems, occurred in 11 patients (16%). CONCLUSIONS: EMTOP with mifepristone and misoprostol was successfully introduced and the experience provides useful data for others contemplating a similar service.

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