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.

Anesthesia-related and perioperative mortality: An audit of 8493 cases at a tertiary pediatric teaching hospital in South Africa.

AIM: This study aimed to quantify the incidence of anesthesia-related and perioperative mortality at a large tertiary pediatric hospital in South Africa. METHODS: This study included all children aged <18 years who died prior to discharge from hospital and within 30 days of their last anesthetic at the Red Cross War Memorial Children's Hospital between January 1, 2015 to December 31, 2015. A panel of three senior anesthetists reviewed each death to reach a consensus as to whether: (i) anesthesia caused the death; (ii) anesthesia may have contributed to or influenced the timing of death; or (iii) anesthesia was entirely unrelated to the death. RESULTS: There were 47 deaths within 30 days of anesthesia prior to discharge from hospital during this 12-month period. The in-hospital mortality within 24 h of administration of anesthesia was 16.5 per 10 000 cases (95% confidence intervals [CI]=7.8-25.1) and within 30 days of administration of anesthesia was 55.3 per 10 000 cases (95% CI=39.5-71.2). Age under 1 year (OR 4.5; 95% CI=2.5-8.0, P=.012) and cardiac surgery and interventional cardiology procedures (OR 2.5; 95% CI=1.2-5.2, P<.01) were both independent predictors of increased risk of perioperative mortality. CONCLUSION: The overall 24-h and 30-day anesthesia-related and in-hospital perioperative mortality rates in our study are comparable with other similar studies from tertiary pediatric centers.

Comparison of two diagnostic intervention packages for community-based active case finding for tuberculosis: an open-label randomized controlled trial.

Two in every five patients with active tuberculosis (TB) remain undiagnosed or unreported. Therefore community-based, active case-finding strategies require urgent implementation. However, whether point-of-care (POC), portable battery-operated, molecular diagnostic tools deployed at a community level, compared with conventionally used POC smear microscopy, can shorten time-to-treatment initiation, thus potentially curtailing transmission, remains unclear. To clarify this issue, we performed an open-label, randomized controlled trial in periurban informal settlements of Cape Town, South Africa, where we TB symptom screened 5,274 individuals using a community-based scalable mobile clinic. Some 584 individuals with HIV infection or symptoms of TB underwent targeted diagnostic screening and were randomized (1:1) to same-day smear microscopy (n = 296) or on-site DNA-based molecular diagnosis (n = 288; GeneXpert). The primary aim was to compare time to TB treatment initiation between the arms. Secondary aims included feasibility and detection of probably infectious people. Of participants who underwent targeted screening, 9.9% (58 of 584) had culture-confirmed TB. Time-to-treatment initiation occurred significantly earlier in the Xpert versus the smear-microscopy arm (8 versus 41 d, P = 0.002). However, overall, Xpert detected only 52% of individuals with culture-positive TB. Notably, Xpert detected almost all of the probably infectious patients compared with smear microscopy (94.1% versus 23.5%, P = <0.001). Xpert was associated with a shorter median time to treatment of probably infectious patients (7 versus 24 d, P = 0.02) and a greater proportion of infectious patients were on treatment at 60 d compared with the probably noninfectious patients (76.5% versus 38.2%, P < 0.01). Overall, a greater proportion of POC Xpert-positive participants were on treatment at 60 d compared with all culture-positive participants (100% versus 46.5%, P < 0.01). These findings challenge the traditional paradigm of a passive case-finding, public health strategy and argues for the implementation of portable DNA-based diagnosis with linkage to care as a community-oriented, transmission-interruption strategy. The study was registered with the South African National Clinical Trials Registry (application ID 4367; DOH-27-0317-5367) and ClinicalTrials.gov (NCT03168945).

Bacterial and host determinants of cough aerosol culture positivity in patients with drug-resistant versus drug-susceptible tuberculosis.

A burgeoning epidemic of drug-resistant tuberculosis (TB) threatens to derail global control efforts. Although the mechanisms remain poorly clarified, drug-resistant strains are widely believed to be less infectious than drug-susceptible strains. Consequently, we hypothesized that lower proportions of patients with drug-resistant TB would have culturable Mycobacterium tuberculosis from respirable, cough-generated aerosols compared to patients with drug-susceptible TB, and that multiple factors, including mycobacterial genomic variation, would predict culturable cough aerosol production. We enumerated the colony forming units in aerosols (≤10 µm) from 452 patients with TB (227 with drug resistance), compared clinical characteristics, and performed mycobacterial whole-genome sequencing, dormancy phenotyping and drug-susceptibility analyses on M. tuberculosis from sputum. After considering treatment duration, we found that almost half of the patients with drug-resistant TB were cough aerosol culture-positive. Surprisingly, neither mycobacterial genomic variants, lineage, nor dormancy status predicted cough aerosol culture positivity. However, mycobacterial sputum bacillary load and clinical characteristics, including a lower symptom score and stronger cough, were strongly predictive, thereby supporting targeted transmission-limiting interventions. Effective treatment largely abrogated cough aerosol culture positivity; however, this was not always rapid. These data question current paradigms, inform public health strategies and suggest the need to redirect TB transmission-associated research efforts toward host-pathogen interactions.

Recovery of Mycobacterium tuberculosis from positive mycobacterium growth indicator tubes stored at room temperature for up to 6 years in low-income and High-Tuberculosis-Burden Country.

Background: Biobanking of Mycobacterium tuberculosis (Mtb) sputum samples for future research activities recommends the use of -70°C or -80°C freezers. Infrastructure for biobanking is not readily available in the majority of low-income countries. This study aimed to assess the recovery rate of Mtb isolates stored at room temperature for more than 6 years in Zimbabwe. Methods: Census samples of all confirmed rifampicin-resistant/multidrug-resistant tuberculosis isolates that were stored in mycobacterial growth indicator tubes (MGITs) at room temperature from 2011 to 2016 were identified and retrieved. The samples were subcultured on MGIT and 7H10 solid media for the extraction of genomic deoxyribonucleic acid using the phenol/chloroform method followed by precipitation with isopropanol. Results: A total of 248/400 (62%) isolates were successfully recovered. Recovery rates increased with declining time since the last culture, with 51% for samples stored for 6 years which increased to 77% for those stored for 1 year. The isolates that grew but were contaminated during the first subculture at the National Microbiology Reference Laboratory in Harare could not be recovered through decontamination because of limited resources. Decontamination was only possible during the second culture at the University of Stellenbosch. Conclusion: Storage of Mtb isolates at room temperature is a viable option in low-income countries where currently recommended biobanking procedures may not be available. This low-cost biobanking will facilitate research activities years later as new questions arise. Standard infection prevention and control when handling Mtb samples stored under room temperature for long periods is strongly recommended as these bacteria remain viable longer than previously reported.

Cytokinins in the xylem sap of the dioecious fynbos shrub, Leucadendron rubrum Burm. f.: seasonal fluctuations and their possible interaction with morphological characteristics as expressed in the two sexes.

Brandies of male and female plants of the dioecious Leucadendron rubrum Burm. f. were collected on the site of the Weikamp weather-station in the Jonkershoek State Forest, Stellenbosch, from July 1990 to September 1991. Xylem sap was extracted from the branches by vacuum extraction. HPLC was employed to purify the xylem sap and to separate the cytokinins prior to quantitation by ELISA. The cytokinin levels in the purified plant samples were determined using rabbit antiserum-based ELISAs for the determination of the levels of the cytokinins DHZR and IPA, and a trans-ZR specific monoclonal antibody-based ELISA for the determination of the levels of the cytokinin ZR. It was observed that the levels of the cytokinins DHZR and ZR did not differ significantly between male and female plants but did correlate with the seasonal growth patterns. The levels of the cytokinin IPA, in male and female plants of L. rubrum, not only correlated specifically with the different seasonal growth patterns observed but differed significantly between male and female plants. Peaks in the levels of all three cytokinins. i.e. ZR, DHZR and IPA, occurred during the study period. These peaks corresponded with the phenological development of L. rubrum, specifically with vegetative bud break and inflorescence differentiation and appeared to be synchronized with the growth and development. An attempt was also made to correlate cytokinin levels with the extreme differences in morphological characters exhibited by male and female plants, and possibly to explain to what extent these phytohormones influence sex expression in L. rubrum.

Safe Mycobacterium tuberculosis DNA extraction method that does not compromise integrity.

Concern has been raised about the efficacy of the heat killing of mycobacteria during the isolation of DNA. We demonstrate a method that allows for the efficient killing of Mycobacterium tuberculosis without compromising DNA integrity for subsequent molecular investigation. This method reduces the risk of infection to the research scientist.

Reduction of the rate of false-positive cultures of Mycobacterium tuberculosis in a laboratory with a high culture positivity rate.

Our laboratory, engaged in a prospective study of adult pulmonary tuberculosis, processed on average 1186 sputum samples per year for the detection of Mycobacterium tuberculosis (M. tuberculosis). Approximately 55% of all sputum samples were culture-positive. The study protocol required that all patients had their M. tuberculosis isolates DNA fingerprinted at diagnosis, and at subsequent time points if the patients either failed treatment or presented again with tuberculosis. Over a 22-month period, there were 14 apparent treatment failures from 109 patients who had completed 6 months of therapy. Only two of these were true treatment failures, while the other 12 had DNA fingerprints that were different from those obtained at diagnosis. It was concluded that these 12 cultures represented episodes of laboratory cross-contamination. Retrospective DNA fingerprinting of patient isolates was done so that each patient had at least two independent isolates fingerprinted. This survey revealed that 7.3% of DNA fingerprints were discordant. False-positive cultures with discordant DNA fingerprints generally arose late in chemotherapy and the isolates were usually co-processed with other strongly smear-positive sputum samples. Simple modifications of laboratory procedures were made, and over a following 10.5-month period the false-positive rate was reduced to 2.1%. These modifications did not increase the workload or the cost of processing samples and can thus be used successfully by any laboratory, and particularly by those in resource-poor settings.