Introducing bedaquiline: experiences from the Challenge TB Project.

BACKGROUND: Drug-resistant tuberculosis (DR-TB) remains a global public health crisis. In 2013, the World Health Organization recommended the introduction of bedaquiline (BDQ) for eligible DR-TB patients.METHODS: We conducted a retrospective review and analyses of project reports from 2016 to mid-2019 on the processes, activities implemented, available results on enrolment and interim treatment outcomes, across the 23 Challenge TB (CTB) supported countries.RESULTS: Initial introduction of BDQ-containing regimens in the 23 CTB-supported countries took on average 2 years, with subsequent nation-wide scale-up achieved in Ethiopia and Kyrgyzstan within a short time period. Successful implementation required critical interventions including advocacy, revision of policies and guidelines, capacity building of health care workers, and strengthening of laboratory networks. The number of countries providing BDQ increased from 9 to 23; 9398 patients were enrolled on bedaquiline containing regimens; 71% were culture-negative after 6 months of treatment; and the number of countries reporting serious adverse events increased (from 5 to 18). Major challenges included limited in-country coordination with drug regulatory agencies, unrealistic quantification and drug ordering, weak laboratory networks and reporting systems for drug safety.CONCLUSION: BDQ introduction required a systematic and programmatic approach. The initial time investment helped achieve initial introduction and scale-up of coverage, ownership and sustainability by National TB Programmes.

Hydrogen sulphide production by Saccharomyces cerevisiae UCD 522 in a synthetic grape juice medium deficient of thiamin (vitamin B1 ) and/or pyridoxine (vitamin B6 ).

The impacts of thiamin and pyridoxine along with YAN on alcoholic fermentation and hydrogen sulphide production by Saccharomyces cerevisiae were studied. Using a synthetic grape juice medium, three fermentation trials were conducted; (i) 2 × 3 factorial design with thiamin (0, 0·2, or 0·5 mg l-1 ) and YAN (60 or 250 mg l-1 ) as variables, (ii) 2 × 3 factorial design with pyridoxine (0, 0·25, or 0·5 mg l-1 ) and YAN (60 or 250 mg l-1 ) as variables, and (iii) 3 × 3 factorial design with thiamin (0, 0·2 or 0·5 mg l-1 ) and pyridoxine (0, 0·25 or 0·5 mg l-1 ) as variables in media containing 60 mg l-1 YAN. Although the progress of fermentations was affected by thiamin or pyridoxine, YAN had a larger impact than either vitamin. H2 S production was significantly lower with increasing amounts of thiamin in those fermentations under low YAN (60 mg l-1 ) while even lower amounts (<30 µg l-1 ) were produced under high YAN (250 mg l-1 ) with or without the vitamin. The highest amounts of H2 S were synthesized in those fermentations without any pyridoxine (>110 µg l-1 ), with the lowest production in media with pyridoxine and high YAN (<20 µg l-1 ). SIGNIFICANCE AND IMPACT OF THE STUDY: Concentrations of thiamin, pyridoxine and yeast assimilable nitrogen (YAN) influenced the synthesis of hydrogen sulphide (H2 S) by Saccharomyces cerevisiae in a synthetic grape juice medium. With a few exceptions, an increase in the concentration of either vitamin or YAN resulted in less H2 S released. This is the first report to demonstrate that both thiamin and pyridoxine along with YAN affected H2 S production, emphasizing the need to assess yeast nutrients to lower risks of off-odours during fermentation.

Interactive effects between total SO2 , ethanol and storage temperature against Brettanomyces bruxellensis.

Although Brettanomyces bruxellensis continues to be a problem during red winemaking due to formation of off-odours and flavours, few interactions between intrinsic and extrinsic conditions that would limit spoilage have been identified. Using a commercially prepared Merlot wine, a 3 × 2 × 2 complete factorial design was implemented with total SO2 (0, 60 or 100 mg l-1 ), ethanol (13% or 14·5% v v-1 ) and storage temperature (15° or 18°C) as variables. Populations of two strains of B. bruxellensis isolated from Washington wines (I1a and F3) were monitored for 100 days before concentrations of 4-ethylphenol, 4-ethylguaiacol and volatile acidity were measured. In wines with 13% v v-1 ethanol and stored at 15°C, addition of 100 mg l-1 total SO2 resulted in much longer lag phases (>40 days) compared with wines without sulphites. At 14·5% v v-1 ethanol, culturability did not recover from wines with 100 mg l-1 total SO2 regardless of the storage temperature (15° or 18°C). A few significant interactions were noted between these parameters which also affected synthesis of metabolites. Thus, SO2 , ethanol concentration and storage temperature should be together used as means to reduce infections by B. bruxellensis. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential for utilizing SO2 along with the ethanol and storage temperature was studied to inhibit the spoilage yeast, Brettanomyces bruxellensis, during cellar ageing of red wines. This report is the first to identify the existence of interactions between these parameters that affect growth and/or metabolism of the yeast (i.e., synthesis of 4-ethylphenol, 4-ethylguaiacol and volatile acidity). Based on current and past findings, recommendations are presented related to the use of potential antimicrobial synergies between SO2 , ethanol concentration and storage temperatures.

Advances in the control of wine spoilage by Zygosaccharomyces and Dekkera/Brettanomyces.

Understanding the characteristics of yeast spoilage, as well as the available control technologies, is vital to producing consistent, high-quality wine. Zygosaccharomyces bailii contamination may result in refermentation and CO2 production in sweet wines or grape juice concentrate, whereas Brettanomyces bruxellensis spoilage often contributes off-odors and flavors to red wines. Early detection of these yeasts by selective/differential media or genetic methods is important to minimize potential spoilage. More established methods of microbial control include sulfur dioxide, dimethyl dicarbonate, and filtration. Current research is focused on the use of chitosan, pulsed electric fields, low electric current, and ultrasonics as means to protect wine quality.

Comparison of media formulations used to selectively cultivate Dekkera/Brettanomyces.

AIMS: The objectives of this research were to (i) optimize the concentration of cycloheximide for use in WL media used in the wine industry and (ii) evaluate Dekkera/Brettanomyces differential medium (DBDM) as a means to detect Dekkera. METHODS AND RESULTS: Dekkera bruxellensis and other yeasts were transferred into WL broths containing 0, 10, 50 or 100 mg l(-1) of cycloheximide. While several grew in 10 mg l(-1) , only Hanseniaspora uvarum, Pichia guillermondii, Schizosaccharomyces pombe and D. bruxellensis tolerated ≥50 mg l(-1) of the antibiotic. On solidified WL media after 8-days incubation, colony sizes of two strains of D. bruxellensis (B1b and ATCC 52905) decreased with increased concentrations of cycloheximide, while others (F3 and P2) were unaffected. Although D. bruxellensis B1b did not grow well on another selective medium, DBDM, colony development was improved by the addition of sterilized red wine. CONCLUSIONS: Of the concentrations tested, 50 mg l(-1) cycloheximide inhibited many grape/wine yeasts yet generally yielded countable colonies of Dekkera (1-2.5 mm diameter). Several strains of Dekkera did not grow well on DBDM, probably due to the lack of an unidentified nutrient(s). SIGNIFICANCE AND IMPACT OF THE STUDY: Better media formulations will improve the detection of Dekkera, thereby increasing microbiological control during winemaking.

The influence of nitrogen and biotin interactions on the performance of Saccharomyces in alcoholic fermentations.

AIM: To study the impact of assimilable nitrogen, biotin and their interaction on growth, fermentation rate and volatile formation by Saccharomyces. METHODS AND RESULTS: Fermentations of synthetic grape juice media were conducted in a factorial design with yeast assimilable nitrogen (YAN) (60 or 250 mg l(-1)) and biotin (0, 1 or 10 microg l(-1)) as variables. All media contained 240 g l(-1) glucose + fructose (1 : 1) and were fermented using biotin-depleted Saccharomyces cerevisiae strains EC1118 or UCD 522. Both strains exhibited weak growth and sluggish fermentation rates without biotin. Increased nitrogen concentration resulted in higher maximum fermentation rates, while adjusting biotin from 1 to 10 microg l(-1) had no effect. Nitrogen x biotin interactions influenced fermentation time, production of higher alcohols and hydrogen sulfide (H(2)S). Maximum H(2)S production occurred in the medium containing 60 mg l(-1) YAN and 1 microg l(-1) biotin. CONCLUSIONS: Nitrogen x biotin interactions affect fermentation time and volatile production by Saccharomyces depending on strain. Biotin concentrations sufficient to complete fermentation may affect the organoleptic impact of wine. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the necessity to consider nutrient interactions when diagnosing problem fermentations.

Nonstarter lactic acid bacteria and aging temperature affect calcium lactate crystallization in cheddar cheese.

The occurrence of unappetizing calcium lactate crystals in Cheddar cheese is a challenge and expense to manufacturers, and this research was designed to understand their origin. It was hypothesized that nonstarter lactic acid bacteria (NSLAB) affect calcium lactate crystallization (CLC) by producing D(-)-lactate. This study was designed to understand the effect of NSLAB growth and aging temperature on CLC. Cheeses were made from milk inoculated with Lactococcus lactis starter culture, with or without Lactobacillus curvatus or L. helveticus WSU19 adjunct cultures. Cheeses were aged at 4 or 13 degrees C for 28 d, then half of the cheeses from 4 and 13 degrees C were transferred to 13 and 4 degrees C, respectively, for the remainder of aging. The form of lactate in cheeses without adjunct culture or with L. helveticus WSU19 was predominantly L(+)-lactate (> 95%, wt/wt), and crystals were not observed within 70 d. While initial lactate in cheeses containingL. curvatus was only L(+)-lactate, the concentration of D(-)-lactate increased during aging. After 28 d, a racemic mixture of D/L-lactate was measured in cheeses containing L. curvatus; at the same time, CLC was observed. The earliest and most extensive CLC occurred on cheeses aged at 13 degrees C for 28 d then transferred to 4 degrees C. These results showed that production of D(-)-lactate by NSLAB, and aging temperature affect CLC in maturing Cheddar cheese.

Fermentative activity and production of volatile compounds by Saccharomyces grown in synthetic grape juice media deficient in assimilable nitrogen and/or pantothenic acid.

AIMS: To understand the impact of assimilable nitrogen and pantothenic acid on fermentation rate and synthesis of volatile compounds by Saccharomyces under fermentative conditions. METHODS AND RESULTS: A 2 x 3 factorial experimental design was employed with the concentrations of yeast assimilable nitrogen (YAN) (60 and 250 mg l(-1)) and pantothenic acid (10, 50 and 250 microg l(-1)) as variables. In media containing 250 microg l(-1) pantothenic acid, H2S production by two different species of Saccharomyces decreased when YAN was increased from 60 to 250 mg l(-1). Conversely, H2S production was significantly higher when the concentration of assimilable nitrogen was increased if pantothenic acid was deficient (10 or 50 microg l(-1)). Yeast synthesis of other volatile compounds were impacted by both assimilable nitrogen and pantothenic acid. CONCLUSIONS: While growth and fermentative rate of Saccharomyces was more influenced by nitrogen than by pantothenic acid, complicated interactions exist between these nutrients that affect the synthesis of volatile compounds including H2S. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has important implications for the winemaking industry where a better understanding of the nutritional requirements of Saccharomyces is necessary to reduce fermentation problems and to improve final product quality.

Lactobacillus nagelii sp. nov., an organism isolated from a partially fermented wine.

A Gram-positive rod was isolated from a commercial grape wine undergoing a sluggish/stuck alcoholic fermentation. The organism produced DL-lactic acid from glucose without gas formation, produced dextran from sucrose, hydrolysed aesculin and fermented galactose, D-glucose, D-fructose, D-mannose, L-sorbose, rhamnose, mannitol, sorbitol, methyl alpha-D-glucoside, N-acetylglucosamine, amygdalin, salicin, cellobiose, maltose, sucrose, trehalose and beta-gentiobiose. 16S rRNA gene sequence analysis revealed that the isolate was phylogenetically a member of the genus Lactobacillus and formed a distinct subline within the Lactobacillus casei cluster of species. On the basis of phenotypic and phylogenetic evidence, Lactobacillus nagelii sp. nov. ATCC 700692T is proposed as a new species.

Lactobacillus kunkeei sp. nov.: a spoilage organism associated with grape juice fermentations.

A Gram-positive rod was isolated from a commercial grape wine undergoing a sluggish/stuck alcoholic fermentation. The organism produced L-lactic acid from glucose, possessed weak catalase activity, and fermented relatively few carbohydrates, i.e. glucose, fructose, sucrose, raffinose (weakly) and mannitol. The 16S rRNA gene sequence analysis revealed that the isolate was phylogenetically a member of the genus Lactobacillus and formed a distinct subline within the Lact. casei cluster of species. Based on phenotypic and phylogenetic evidence, a new species is proposed, Lact. kunkeei. The type strain of Lact. kunkeei is ATCC 700308.

Evaluation of Four Pea ( Pisum sativum ) Cultivars in PE-2 Medium for the MPN Enumeration of Anaerobic Spore-Forming Organisms.

Untreated 'Alaska' seed peas have traditionally been used to prepare PE-2 medium, a medium used to recover anaerobic bacteria. Three cultivars of seed peas, 'Columbian', 'Yellow', and 'Scotch' were compared to the cultivar 'Alaska' peas in PE-2 for recovery of Clostridium butyricum ATCC 860, Clostridium sporogenes ATCC 7955/NCA 3679, and Thermoanerobacterium thermosaccharolyticum ATCC 25773 determined by using the most probable number (MPN) technique. Organisms were grown in cooked meat medium (CMM) and enumerated by the three-tube MPN method in PE-2 media incubated at 37°C for 48 h. Recovery of C. butyricum and T. thermosaccharolyticum grown in homogenates of commercial cream-style corn and vegetable beef soup were also evaluated. Similar recovery of organisms from stock cultures or from foodstuffs were observed in PE-2 prepared with three of the pea cultivars ('Alaska', 'Columbian', and 'Yellow'). While the 'Scotch' cultivar PE-2 medium yielded recoveries comparable to PE-2 prepared with the other pea cultivars, turbid tubes were occasionally observed from which viable microorganisms could not be recovered. This cultivar is therefore not recommended due to the probability of yielding false positives in the MPN technique.

Thresholds for short-latency auditory-evoked potentials to tones in notched noise in normal-hearing and hearing-impaired subjects.

The thresholds for the short-latency auditory evoked potentials (SLAEPs) to short-duration tones presented in notched-noise masking were evaluated in 20 normal-hearing and 20 hearing-impaired subjects. The differences (dB) between these thresholds (dB nHL) and the pure-tone behavioral thresholds (dB HL) across all 40 subjects were 11.6, 6.1, 6.3 and 0.8 dB for 500, 1,000, 2,000 and 4,000 Hz, respectively. These differences were significantly smaller for the hearing-impaired subjects than for the normal-hearing subjects. Ninety-eight percent of the SLAEP threshold estimations were within 30 dB of the subjects' pure-tone behavioral thresholds and 91% were within 20 dB.

Inducing malolactic fermentation in wines.

Malolactic fermentation (MLF) in wine can be accomplished by relying on the natural microflora or by inducing through inoculation of a specific strain(s) of malolactic bacteria, primarily strains of Leuconostoc oenos. Problems with inducing MLF include intrinsic factors of the grape must such as pH, presence of sulfur dioxide, and ethanol in addition to antagonism of malolactic bacteria by wine yeast. Current methods and new technology to improve the predictability of MLF are discussed.

Total Sulfur Dioxide Residuals in Fresh Mushrooms Washed in Sulfite Solutions 1.

Freshly-harvested, cultivated mushrooms ( Agaricus bisporus ) were washed in 1000 ppm solutions of sodium sulfite and stored in ventilated, polyvinylchloride film-overwrapped packages at 5 and 15°C. Total sulfur dioxide of the mushrooms was measured by the modified Monier-Williams procedure numerous times during a 48-h storage period. Initial SO2 averaged 48.1 ppm and subsequently declined at a logarithmic rate. Total SO2 fell below detectable levels (<10 ppm) at about 11.8 and 15.6 h at 15 and 5°C, respectively.

Brainstem electric-response audiometry in infants of a neonatal intensive care unit.

Brainstem electric-response audiometry was used to assess the auditory function of 600 infants from a neonatal intensive care unit. Two groups of children were tested, one group as inpatients prior to discharge with a mean age at test of 39.4 weeks and one group as outpatients with a mean age at test of 55.4 weeks. Our results indicate that testing infants when they are older will reduce the incidence of failure on the initial evaluation and will reduce the identification of transient hearing losses which resolve spontaneously. Our results also indicate that a threshold of 30 dB nHL or less is probably normal and that some infants with a threshold of 40 dB nHL at first test require otological or audiological management.

Neonatal auditory brain stem responses from ipsilateral and contralateral recording montages.

Ipsilateral and contralateral auditory brain stem responses were recorded from both ears of 10 neonates between 37 and 43 weeks from conception with normal auditory brain stem function. The most prominent component of the contralateral response was a forehead positive deflection occurring 0.3 millisecond before the ipsilateral wave III followed by two forehead negative waves, the latter present at the same latency as the ipsilateral wave V. The amplitude of the contralateral response was small: at 30 dB nHL only 7 of the 20 ears showed clear responses from this montage. The morphology of the neonatal contralateral response is thus considerably different than the ipsilateral, unlike the adult. A neonate could be mistakenly diagnosed as hearing impaired or neurologically involved if the interpretation is based on the contralateral recording.

Auditory brainstem responses to clicks in neonates.

Normative data are required when interpreting brainstem electric response audiometry results. An abnormal auditory brainstem response (ABR) cannot be identified without knowledge of the normal ABR and the variability associated with technical and physiological factors. Normative data obtained from babies in a neonatal intensive care unit in response to click stimulation are presented in this paper.

Auditory brainstem response audiometry in neonatal hydrocephalus.

Sixteen hydrocephalic NICU babies and 16 age-matched, non-hydrocephalic NICU babies were tested with BERA. The latency and amplitude of waves I and V were compared between groups. Both waves were longer in latency and smaller in amplitude in the hydrocephalic group. The I-V latency interval was within normal limits, but the V/I amplitude ratio was significantly smaller. The reduced amplitude of wave V was the most noticeable ABR abnormality. A total of 11 hydrocephalic babies showed responses that were considered abnormal in amplitude, with all but one having an elevated ABR threshold in at least one ear. These threshold elevations may reflect a neurological condition rather than a peripheral hearing loss.