Non-actionable Results, Accuracy, and Effect of First- and Second-line Line Probe Assays for Diagnosing Drug-Resistant Tuberculosis, Including on Smear-Negative Specimens, in a High-Volume Laboratory.

BACKGROUND: Rapid tuberculosis (TB) drug susceptibility testing (DST) is crucial. Genotype MTBDRsl is a widely deployed World Health Organization (WHO)-endorsed assay. Programmatic performance data, including non-actionable results from smear-negative sputum, are scarce. METHODS: Sputa from Xpert MTB/RIF individuals (n = 951) were routinely-tested using Genotype MTBDRplus and MTBDRsl (both version 2). Phenotypic DST was the second-line drug reference standard. Discrepant results underwent Sanger sequencing. FINDINGS: 89% (849 of 951) of individuals were culture-positive (56%, 476 of 849 smear-negative). MTBDRplus had at least 1 nonactionable result (control and/or TB-detection bands absent or invalid, precluding resistance reporting) in 19% (92 of 476) of smear-negatives; for MTBDRsl, 40% (171 of 427) were nonactionable (28%, 120 of 427 false-negative TB; 17%, 51 of 427 indeterminate). In smear-negatives, MTBDRsl sensitivity for fluoroquinolones was 84% (95% confidence interval, 67%-93), 81% (54%-95%) for second-line injectable drugs, and 57% (28%-82%) for both. Specificities were 93% (89%-98%), 88% (81%-93%), and 97% (91%-99%), respectively. Twenty-three percent (172 of 746) of Xpert rifampicin-resistant specimens were MTBDRplus isoniazid-susceptible. Days-to-second-line-susceptibility reporting with the programmatic advent of MTBDRsl improved (6 [5-7] vs 37 [35-46]; P < .001). CONCLUSIONS: MTBDRsl did not generate a result in 4 of 10 smear-negatives, resulting in substantial missed resistance. However, if MTBDRsl generates an actionable result, that is accurate in ruling-in resistance. Isoniazid DST remains crucial. This study provides real-world, direct, second-line susceptibility testing performance data on non-actionable results (that, if unaccounted for, cause an overestimation of test utility), accuracy, and care cascade impact.

Xpert MTB/XDR for detection of pulmonary tuberculosis and resistance to isoniazid, fluoroquinolones, ethionamide, and amikacin.

BACKGROUND: The World Health Organization (WHO) End TB Strategy stresses universal access to drug susceptibility testing (DST). DST determines whether Mycobacterium tuberculosis bacteria are susceptible or resistant to drugs. Xpert MTB/XDR is a rapid nucleic acid amplification test for detection of tuberculosis and drug resistance in one test suitable for use in peripheral and intermediate level laboratories. In specimens where tuberculosis is detected by Xpert MTB/XDR, Xpert MTB/XDR can also detect resistance to isoniazid, fluoroquinolones, ethionamide, and amikacin. OBJECTIVES: To assess the diagnostic accuracy of Xpert MTB/XDR for pulmonary tuberculosis in people with presumptive pulmonary tuberculosis (having signs and symptoms suggestive of tuberculosis, including cough, fever, weight loss, night sweats). To assess the diagnostic accuracy of Xpert MTB/XDR for resistance to isoniazid, fluoroquinolones, ethionamide, and amikacin in people with tuberculosis detected by Xpert MTB/XDR, irrespective of rifampicin resistance (whether or not rifampicin resistance status was known) and with known rifampicin resistance. SEARCH METHODS: We searched multiple databases to 23 September 2021. We limited searches to 2015 onwards as Xpert MTB/XDR was launched in 2020. SELECTION CRITERIA: Diagnostic accuracy studies using sputum in adults with presumptive or confirmed pulmonary tuberculosis. Reference standards were culture (pulmonary tuberculosis detection); phenotypic DST (pDST), genotypic DST (gDST),composite (pDST and gDST) (drug resistance detection). DATA COLLECTION AND ANALYSIS: Two review authors independently reviewed reports for eligibility and extracted data using a standardized form. For multicentre studies, we anticipated variability in the type and frequency of mutations associated with resistance to a given drug at the different centres and considered each centre as an independent study cohort for quality assessment and analysis. We assessed methodological quality with QUADAS-2, judging risk of bias separately for each target condition and reference standard. For pulmonary tuberculosis detection, owing to heterogeneity in participant characteristics and observed specificity estimates, we reported a range of sensitivity and specificity estimates and did not perform a meta-analysis. For drug resistance detection, we performed meta-analyses by reference standard using bivariate random-effects models. Using GRADE, we assessed certainty of evidence of Xpert MTB/XDR accuracy for detection of resistance to isoniazid and fluoroquinolones in people irrespective of rifampicin resistance and to ethionamide and amikacin in people with known rifampicin resistance, reflecting real-world situations. We used pDST, except for ethionamide resistance where we considered gDST a better reference standard. MAIN RESULTS: We included two multicentre studies from high multidrug-resistant/rifampicin-resistant tuberculosis burden countries, reporting on six independent study cohorts, involving 1228 participants for pulmonary tuberculosis detection and 1141 participants for drug resistance detection. The proportion of participants with rifampicin resistance in the two studies was 47.9% and 80.9%. For tuberculosis detection, we judged high risk of bias for patient selection owing to selective recruitment. For ethionamide resistance detection, we judged high risk of bias for the reference standard, both pDST and gDST, though we considered gDST a better reference standard. Pulmonary tuberculosis detection - Xpert MTB/XDR sensitivity range, 98.3% (96.1 to 99.5) to 98.9% (96.2 to 99.9) and specificity range, 22.5% (14.3 to 32.6) to 100.0% (86.3 to 100.0); median prevalence of pulmonary tuberculosis 91.3%, (interquartile range, 89.3% to 91.8%), (2 studies; 1 study reported on 2 cohorts, 1228 participants; very low-certainty evidence, sensitivity and specificity). Drug resistance detection People irrespective of rifampicin resistance - Isoniazid resistance: Xpert MTB/XDR summary sensitivity and specificity (95% confidence interval (CI)) were 94.2% (87.5 to 97.4) and 98.5% (92.6 to 99.7) against pDST, (6 cohorts, 1083 participants, moderate-certainty evidence, sensitivity and specificity). - Fluoroquinolone resistance: Xpert MTB/XDR summary sensitivity and specificity were 93.2% (88.1 to 96.2) and 98.0% (90.8 to 99.6) against pDST, (6 cohorts, 1021 participants; high-certainty evidence, sensitivity; moderate-certainty evidence, specificity). People with known rifampicin resistance - Ethionamide resistance: Xpert MTB/XDR summary sensitivity and specificity were 98.0% (74.2 to 99.9) and 99.7% (83.5 to 100.0) against gDST, (4 cohorts, 434 participants; very low-certainty evidence, sensitivity and specificity). - Amikacin resistance: Xpert MTB/XDR summary sensitivity and specificity were 86.1% (75.0 to 92.7) and 98.9% (93.0 to 99.8) against pDST, (4 cohorts, 490 participants; low-certainty evidence, sensitivity; high-certainty evidence, specificity). Of 1000 people with pulmonary tuberculosis, detected as tuberculosis by Xpert MTB/XDR: - where 50 have isoniazid resistance, 61 would have an Xpert MTB/XDR result indicating isoniazid resistance: of these, 14/61 (23%) would not have isoniazid resistance (FP); 939 (of 1000 people) would have a result indicating the absence of isoniazid resistance: of these, 3/939 (0%) would have isoniazid resistance (FN). - where 50 have fluoroquinolone resistance, 66 would have an Xpert MTB/XDR result indicating fluoroquinolone resistance: of these, 19/66 (29%) would not have fluoroquinolone resistance (FP); 934 would have a result indicating the absence of fluoroquinolone resistance: of these, 3/934 (0%) would have fluoroquinolone resistance (FN). - where 300 have ethionamide resistance, 296 would have an Xpert MTB/XDR result indicating ethionamide resistance: of these, 2/296 (1%) would not have ethionamide resistance (FP); 704 would have a result indicating the absence of ethionamide resistance: of these, 6/704 (1%) would have ethionamide resistance (FN). - where 135 have amikacin resistance, 126 would have an Xpert MTB/XDR result indicating amikacin resistance: of these, 10/126 (8%) would not have amikacin resistance (FP); 874 would have a result indicating the absence of amikacin resistance: of these, 19/874 (2%) would have amikacin resistance (FN). AUTHORS' CONCLUSIONS: Review findings suggest that, in people determined by Xpert MTB/XDR to be tuberculosis-positive, Xpert MTB/XDR provides accurate results for detection of isoniazid and fluoroquinolone resistance and can assist with selection of an optimised treatment regimen. Given that Xpert MTB/XDR targets a limited number of resistance variants in specific genes, the test may perform differently in different settings. Findings in this review should be interpreted with caution. Sensitivity for detection of ethionamide resistance was based only on Xpert MTB/XDR detection of mutations in the inhA promoter region, a known limitation. High risk of bias limits our confidence in Xpert MTB/XDR accuracy for pulmonary tuberculosis. Xpert MTB/XDR's impact will depend on its ability to detect tuberculosis (required for DST), prevalence of resistance to a given drug, health care infrastructure, and access to other tests.

Frequent Suboptimal Thermocycler Ramp Rate Usage Negatively Impacts GenoType MTBDRsl VER 2.0 Performance for Second-Line Drug-Resistant Tuberculosis Diagnosis.

Strengthening second-line drug-resistant tuberculosis (TB) detection is a priority. GenoType MTBDRplus VER 2.0 performance is reduced with non-recommended ramp rate usage (temperature change speed between PCR cycles); however, ramp rate's effect on GenoType MTBDRsl VER 2.0 (MTBDRsl) performance, is unknown. Fifty-two Xpert MTB/RIF Ultra-positive rifampicin-resistant smear-negative sputa and a Mycobacterium tuberculosis dilution series were tested at a manufacturer-recommended (2.2°C/second) or suboptimal (4.0°C/second) ramp rate. M. tuberculosis-complex-DNA positivity, indeterminates, fluoroquinolone- and second-line injectable-resistance accuracy, banding differences, and, separately, inter-reader variability were assessed. Five (39%) of 13 re-surveyed laboratories did not use the manufacturer-recommended ramp rate. On sputum, 2.2°C/second improved indeterminates versus 4.0°C/second (0 of 52 versus 7 of 51; P = 0.006), incorrect drug-class diagnostic calls (0 of 104 versus 6 of 102; P = 0.013), and incorrect banding calls (0 of 1300 versus 54 of 1275; P < 0.001). Similarly, 2.2°C/second improved valid results [(52 of 52 versus 41 of 51; +21% (P = 0.001)] and banding call inter-reader variability [34 of 1300 (3%) versus 52 of 1300 (4%); P = 0.030]. At the suboptimal ramp rate, false-resistance and false-susceptible calls resulted from wild-type band absence rather than mutant band appearance, resulting in misclassification of moxifloxacin resistance level from high-to-low. Suboptimal ramp rate contributes to poor MTBDRsl performance. Laboratories must ensure that the manufacturer-recommended ramp rate is used.

Melting the eis: Nondetection of Kanamycin Resistance Markers by Routine Diagnostic Tests and Identification of New eis Promoter Variants.

Eis promoter mutations can confer reduced Mycobacterium tuberculosis kanamycin susceptibility. GenoType MTBDRsl, a widely used assay evaluating this region, wrongly classified 17/410 isolates as eis promoter wild type. Six out of seventeen isolates harbored mutations known to confer kanamycin resistance, and the remainder harbored either novel eis promoter mutations (7/11) or disputed mutations (4/11). GenoType MTBDRsl can miss established and new variants that cause reduced susceptibility. These data highlight the importance of reflex phenotypic kanamycin testing.

Mycobacterial genomic DNA from used Xpert MTB/RIF cartridges can be utilised for accurate second-line genotypic drug susceptibility testing and spoligotyping.

Xpert MTB/RIF (Xpert) is a widely-used test for tuberculosis (TB) and rifampicin-resistance. Second-line drug susceptibility testing (DST), which is recommended by policymakers, typically requires additional specimen collection that delays effective treatment initiation. We examined whether cartridge extract (CE) from used Xpert TB-positive cartridges was, without downstream DNA extraction or purification, suitable for both genotypic DST (MTBDRplus, MTBDRsl), which may permit patients to rapidly receive a XDR-TB diagnosis from a single specimen, and spoligotyping, which could facilitate routine genotyping. To determine the limit-of-detection and diagnostic accuracy, CEs from dilution series of drug-susceptible and -resistant bacilli were tested (MTBDRplus, MTBDRsl). Xpert TB-positive patient sputa CEs (n = 85) were tested (56 Xpert-rifampicin-susceptible, MTBDRplus and MTBDRsl; 29 Xpert-rifampicin-resistant, MTBDRsl). Spoligotyping was done on CEs from dilution series and patient sputa (n = 10). MTBDRplus had high non-valid result rates. MTBDRsl on CEs from dilutions ≥103CFU/ml (CT ≤ 24, >"low" Xpert semiquantitation category) was accurate, had low indeterminate rates and, on CE from sputa, highly concordant with MTBDRsl isolate results. CE spoligotyping results from dilutions ≥103CFU/ml and sputa were correct. MTBDRsl and spoligotyping on CE are thus highly feasible. These findings reduce the need for additional specimen collection and culture, for which capacity is limited in high-burden countries, and have implications for diagnostic laboratories and TB molecular epidemiology.

How and why to take a Martius labial interposition flap in female urology.

The Martius modified labial fat pad flap at nearly 90 years old is still very much part of the modern vaginal surgeon's armamentarium. Here we describe this straightforward and adaptable technique, the avoidance of pitfalls, its advantages and uses in vaginal surgery, and outcomes both short and long term. Specifically patient reported outcomes related to scar perception and sexual function are explored.

Sacral neuromodulation for refractory overactive bladder after prior intravesical onabotulinumtoxinA treatment.

AIMS: Sacral neuromodulation (SNM) is a well-established treatment modality for refractory overactive bladder (OAB). There is a paucity of evidence examining the use of SNM in patients who have received prior intravesical onabotulinumtoxinA (BTXA) treatment. We aim to review those patients who underwent SNM for refractory OAB following treatment with BTXA. METHODS: A retrospective review was conducted to identify patients who had undergone prior intradetrusor BTXA for refractory OAB, then subsequent first-stage SNM. Patient demographics, number/dosage of BTXA, voiding diaries, and patient global impression of improvement (PGI-I) scores were recorded. Successful first-stage SNM was defined as subjective patient improvement of greater than 50%. Patient satisfaction and device use at last follow-up was noted. RESULTS: Eighty-three patients were identified having undergone SNM for OAB, of which 36 had prior BTXA treatment and were included in the series. 23/36 (63.9%) of patients had successful first-stage SNM, and underwent insertion of implantable pulse generator, compared to 33/47 (70.2%) in those who had never been treated with BTXA (P = 0.5). Mean PGI-I score was 2.6 (range 1-4). With a mean follow up of 29.1 months (range 12-53), 17/23 (73.9%) were satisfied, and using the device at last follow-up. CONCLUSION: SNM is a suitable treatment option in those patients who have had prior BTXA treatment for refractory OAB, even in those for whom BTXA proved ineffective. Success rates were within the published range, and comparable to our own results, for SNM in OAB patients without prior BTXA treatment.

Conjoint Urological Society of Australia and New Zealand (USANZ) and Urogynaecological Society of Australasia (UGSA) Guidelines on the management of adult non-neurogenic overactive bladder.

Due to the myriad of treatment options available and the potential increase in the number of patients afflicted with overactive bladder (OAB) who will require treatment, the Female Urology Special Advisory Group (FUSAG) of the Urological Society of Australia and New Zealand (USANZ), in conjunction with the Urogynaecological Society of Australasia (UGSA), see the need to move forward and set up management guidelines for physicians who may encounter or have a special interest in the treatment of this condition. These guidelines, by utilising and recommending evidence-based data, will hopefully assist in the diagnosis, clinical assessment, and optimisation of treatment efficacy. They are divided into three sections: Diagnosis and Clinical Assessment, Conservative Management, and Surgical Management. These guidelines will also bring Australia and New Zealand in line with other regions of the world where guidelines have been established, such as the American Urological Association, European Association of Urology, International Consultation on Incontinence, and the National Institute for Health and Care Excellence guidelines of the UK.

Design, biometric simulation and optimization of a nano-enabled scaffold device for enhanced delivery of dopamine to the brain.

This study focused on the design, biometric simulation and optimization of an intracranial nano-enabled scaffold device (NESD) for the site-specific delivery of dopamine (DA) as a strategy to minimize the peripheral side-effects of conventional forms of Parkinson's disease therapy. The NESD was modulated through biometric simulation and computational prototyping to produce a binary crosslinked alginate scaffold embedding stable DA-loaded cellulose acetate phthalate (CAP) nanoparticles optimized in accordance with Box-Behnken statistical designs. The physicomechanical properties of the NESD were characterized and in vitro and in vivo release studies performed. Prototyping predicted a 3D NESD model with enhanced internal micro-architecture. SEM and TEM revealed spherical, uniform and non-aggregated DA-loaded nanoparticles with the presence of CAP (FTIR bands at 1070, 1242 and 2926 cm(-1)). An optimum nanoparticle size of 197 nm (PdI=0.03), a zeta potential of -34.00 mV and a DEE of 63% was obtained. The secondary crosslinker BaCl(2) imparted crystallinity resulting in significant thermal shifts between native CAP (T(g)=160-170 degrees C; T(m)=192 degrees C) and CAP nanoparticles (T(g)=260 degrees C; T(m)=268 degrees C). DA release displayed an initial lag phase of 24 h and peaked after 3 days, maintaining favorable CSF (10 microg/mL) versus systemic concentrations (1-2 microg/mL) over 30 days and above the inherent baseline concentration of DA (1 microg/mL) following implantation in the parenchyma of the frontal lobe of the Sprague-Dawley rat model. The strategy of coupling polymeric scaffold science and nanotechnology enhanced the site-specific delivery of DA from the NESD.

Patenting of nanopharmaceuticals in drug delivery: no small issue.

Nanotechnology is a rapidly evolving interdisciplinary field based on the manipulation of matter on a submicron scale, encompassing matter between 1 and 100 nanometers (nm). The currently registered nanotechnology patents comprise 35 countries being involved in the global distribution of these patents. Close to 3000 patents were issued in the USA since 1996 with the term 'nano' in the patents, with a considerable number having application in nanomedicine. The large majority of therapeutic patents are focused on drug delivery systems, highlighting an important application globally. Nanopharmaceutical patents are centered mainly on non-communicable diseases, with cancer receiving the greatest focus, followed by hepatitis. Drug delivery systems employing nanotechnology have the ability to allow superior drug absorption, controlled drug release and reduced side-effects, enhancing the effectiveness of existing drug delivery systems. Nanoparticle-based drug delivery systems may be among the first types of products to generate serious nanotechnology patent disputes as the multi-billion dollar pharmaceutical industry begins to adopt them. This review article aimed to locate patented nanopharmaceuticals in drug delivery online, employing pertinent key terms while searching the patent databases. Awarded and pending patents in the past 20 years pertaining to nanopharmaceutical or nano-enabled systems such as micelles, nanoemulsions, nanogels, liposomes, nanofibres, dendrimer technology and polymer therapeutics are presented in the review article, providing an overview of the diversity of the patent applications.