Comparison of Quantitative Techniques including Xpert MTB/RIF to Evaluate Mycobacterial Burden

Introduction: Accurate quantification of mycobacterial load is important for the evaluation of patient infectiousness, disease severity and monitoring treatment response in human and in-vitro laboratory models of disease. We hypothesized that newer techniques would perform as well as solid media culture to quantify mycobacterial burden in laboratory specimens. Methods: We compared the turn-around-time, detection-threshold, dynamic range, reproducibility, relative discriminative ability, of 4 mycobacterial load determination techniques: automated liquid culture (BACTEC-MGIT-960), [3H]-uracil incorporation assays, luciferase-reporter construct bioluminescence, and quantitative PCR(Xpert -MTB/RIF) using serial dilutions of Mycobacterium bovis and Mycobacterium tuberculosis H37RV. Mycobacterial colony-forming-units(CFU) using 7H10-Middlebrook solid media served as the reference standard. Results: All 4 assays correlated well with the reference standard, however, bioluminescence and uracil assays had a detection threshold ≥1×103 organisms. By contrast, BACTEC-MGIT-960 liquid culture, although only providing results in days, was user-friendly, had the lowest detection threshold (<10 organisms), the greatest discriminative ability (1 vs. 10 organisms; p = 0.02), and the best reproducibility (coefficient of variance of 2% vs. 38% compared to uracil incorporation; p = 0.02). Xpert-MTB/RIF correlated well with mycobacterial load, had a rapid turn-around-time (<2 hours), was user friendly, but had a detection limit of ~100 organisms. Conclusions: Choosing a technique to quantify mycobacterial burden for laboratory or clinical research depends on availability of resources and the question being addressed. Automated liquid culture has good discriminative ability and low detection threshold but results are only obtained in days. Xpert MTB/RIF provides rapid quantification of mycobacterial burden, but has a poorer discrimination and detection threshold

Constant stress layer characteristics in simulated stratified air flows: Implications for aeolian transport

Varying thermal atmospheric stability conditions and their effects on shearing flows has long been a subject of interest for researchers working in atmospheric science. The development of new instrument technologies now offers an opportunity to study flows with high spatial and temporal resolutions in wind tunnel atmospheric boundary layers. In the presented study, we use a laser Doppler anemometer within the Trent Environmental Wind Tunnel Laboratory to investigate the influence of thermal stratification on the constant stress layer. Analyses of the thermal stratification represented by the gradient Richardson number and the apparent von Kármán parameter, shear velocity, and the slope of the streamwise velocity profiles reveal strong linear relationships. An exponential relationship between thermal stability and the apparent roughness length is also revealed. Profiles of the streamwise and vertical velocity and turbulence intensity, as well as the dimensionless Reynolds stress, are influenced by the gradient Richardson number. These findings have implications for producing accurate models of sediment entrainment and transport by wind in non-neutral conditions

Clinical, biochemical, and genetic spectrum of MADD in a South African cohort: an ICGNMD study

\ua9 2024, The Author(s).Background: Multiple acyl-CoA dehydrogenase deficiency (MADD) is an autosomal recessive disorder resulting from pathogenic variants in three distinct genes, with most of the variants occurring in the electron transfer flavoprotein-ubiquinone oxidoreductase gene (ETFDH). Recent evidence of potential founder variants for MADD in the South African (SA) population, initiated this extensive investigation. As part of the International Centre for Genomic Medicine in Neuromuscular Diseases study, we recruited a cohort of patients diagnosed with MADD from academic medical centres across SA over a three-year period. The aim was to extensively profile the clinical, biochemical, and genomic characteristics of MADD in this understudied population. Methods: Clinical evaluations and whole exome sequencing were conducted on each patient. Metabolic profiling was performed before and after treatment, where possible. The recessive inheritance and phase of the variants were established via segregation analyses using Sanger sequencing. Lastly, the haplotype and allele frequencies were determined for the two main variants in the four largest SA populations. Results: Twelve unrelated families (ten of White SA and two of mixed ethnicity) with clinically heterogeneous presentations in 14 affected individuals were observed, and five pathogenic ETFDH variants were identified. Based on disease severity and treatment response, three distinct groups emerged. The most severe and fatal presentations were associated with the homozygous c.[1067G &gt; A];c.[1067G &gt; A] and compound heterozygous c.[976G &gt; C];c.[1067G &gt; A] genotypes, causing MADD types I and I/II, respectively. These, along with three less severe compound heterozygous genotypes (c.[1067G &gt; A];c.[1448C &gt; T], c.[740G &gt; T];c.[1448C &gt; T], and c.[287dupA*];c.[1448C &gt; T]), resulting in MADD types II/III, presented before the age of five years, depending on the time and maintenance of intervention. By contrast, the homozygous c.[1448C &gt; T];c.[1448C &gt; T] genotype, which causes MADD type III, presented later in life. Except for the type I, I/II and II cases, urinary metabolic markers for MADD improved/normalised following treatment with riboflavin and L-carnitine. Furthermore, genetic analyses of the most frequent variants (c.[1067G &gt; A] and c.[1448C &gt; T]) revealed a shared haplotype in the region of ETFDH, with SA population-specific allele frequencies of &lt; 0.00067–0.00084%. Conclusions: This study reveals the first extensive genotype–phenotype profile of a MADD patient cohort from the diverse and understudied SA population. The pathogenic variants and associated variable phenotypes were characterised, which will enable early screening, genetic counselling, and patient-specific treatment of MADD in this population

Correlation of Mycobacterium Tuberculosis Specific and Non-Specific Quantitative Th1 T-Cell Responses with Bacillary Load in a High Burden Setting

Measures of bacillary load in patients with tuberculosis (TB) may be useful for predicting and monitoring response to treatment. The relationship between quantitative T-cell responses and mycobacterial load remains unclear. We hypothesised that, in a HIV-prevalent high burden setting, the magnitude of mycobacterial antigen-specific and non-specific T-cell IFN-γ responses would correlate with (a) bacterial load and (b) culture conversion in patients undergoing treatment.We compared baseline (n = 147), 2 (n = 35) and 6 month (n = 13) purified-protein-derivative (PPD) and RD1-specific (TSPOT.TB and QFT-GIT) blood RD1-specific (TSPOT.TB; QFT-GIT) responses with associates of sputum bacillary load in patients with culture-confirmed TB in Cape Town, South Africa.IFN-γ responses were not associated with liquid culture time-to-positivity, smear-grade, Xpert MTB/RIF-generated cycle threshold values or the presence of cavities on the chest radiograph in patients with culture-confirmed TB and irrespective of HIV-status. 2-month IGRA conversion rates (positive-to-negative) were negligible [<11% for TSPOT.TB (3/28) and QFT-GIT (1/29)] and lower compared to culture [60% (21/35); p<0.01].In a high burden HIV-prevalent setting T-cell IFN-γ responses to M. tuberculosis-specific and non-specific antigens do not correlate with bacillary load, including Xpert MTB/RIF-generated C(T) values, and are therefore poorly suited for monitoring treatment and prognostication

Clinical, biochemical, and genetic spectrum of MADD in a South African cohort : an ICGNMD study

AVAILABILITY OF DATA AND MATERIALS : Previous data and samples were made available by the Centre for Human Metabolomics (NWU), SU, and UCT. New samples were collected with the help of paediatric and adult neurologists via Steve Biko Academic Hospital, Tygerberg Hospital, and Red Cross War Memorial Children’s Hospital. The datasets generated and/or analysed during the current study are not publicly available due to the data sharing policy of the ICGNMD study, but are available from the corresponding author on reasonable request.ADDITIONAL FILE 1 : Additional Clinical Information.ADDITIONAL FILE 2 : Additional Metabolic Information.ADDITIONAL FILE 3 : Additional Structural Information.BACKGROUND : Multiple acyl-CoA dehydrogenase deficiency (MADD) is an autosomal recessive disorder resulting from pathogenic variants in three distinct genes, with most of the variants occurring in the electron transfer flavoprotein-ubiquinone oxidoreductase gene (ETFDH). Recent evidence of potential founder variants for MADD in the South African (SA) population, initiated this extensive investigation. As part of the International Centre for Genomic Medicine in Neuromuscular Diseases study, we recruited a cohort of patients diagnosed with MADD from academic medical centres across SA over a three-year period. The aim was to extensively profile the clinical, biochemical, and genomic characteristics of MADD in this understudied population. METHODS : Clinical evaluations and whole exome sequencing were conducted on each patient. Metabolic profiling was performed before and after treatment, where possible. The recessive inheritance and phase of the variants were established via segregation analyses using Sanger sequencing. Lastly, the haplotype and allele frequencies were determined for the two main variants in the four largest SA populations. RESULTS : Twelve unrelated families (ten of White SA and two of mixed ethnicity) with clinically heterogeneous presentations in 14 affected individuals were observed, and five pathogenic ETFDH variants were identified. Based on disease severity and treatment response, three distinct groups emerged. The most severe and fatal presentations were associated with the homozygous c.[1067G > A];c.[1067G > A] and compound heterozygous c.[976G > C];c.[1067G > A] genotypes, causing MADD types I and I/II, respectively. These, along with three less severe compound heterozygous genotypes (c.[1067G > A];c.[1448C > T], c.[740G > T];c.[1448C > T], and c.[287dupA*];c.[1448C > T]), resulting in MADD types II/III, presented before the age of five years, depending on the time and maintenance of intervention. By contrast, the homozygous c.[1448C > T];c.[1448C > T] genotype, which causes MADD type III, presented later in life. Except for the type I, I/II and II cases, urinary metabolic markers for MADD improved/normalised following treatment with riboflavin and L-carnitine. Furthermore, genetic analyses of the most frequent variants (c.[1067G > A] and c.[1448C > T]) revealed a shared haplotype in the region of ETFDH, with SA population-specific allele frequencies of < 0.00067–0.00084%. CONCLUSIONS : This study reveals the first extensive genotype–phenotype profile of a MADD patient cohort from the diverse and understudied SA population. The pathogenic variants and associated variable phenotypes were characterised, which will enable early screening, genetic counselling, and patient-specific treatment of MADD in this population.Open access funding provided by North-West University. A Medical Research Council (MRC) strategic award; the National Research Foundation (NRF) of South Africa; the South African Medical Research Council (SAMRC); the Wellcome Centre for Mitochondrial Research; the Mitochondrial Disease Patient Cohort (UK); the Medical Research Council International Centre for Genomic Medicine in Neuromuscular Disease; the Lily Foundation; the UK NIHR Biomedical Research Centre for Ageing and Age-related Disease award to the Newcastle upon Tyne Foundation Hospitals NHS Trust; the UK NHS Highly Specialised Service for Rare Mitochondrial Disorders of Adults and Children; the MRC; Mito Foundation, and the Pathological Society (UK).https://ojrd.biomedcentral.comhj2024Paediatrics and Child HealthNon

Benefits of jasmonate-dependent defenses against vertebrate herbivores in nature

Endogenous jasmonates are important regulators of plant defenses. If and how they enable plants to maintain their reproductive output when facing community-level herbivory under natural conditions, however, remains unknown. We demonstrate that jasmonate-deficient Nicotiana attenuata plants suffer more damage by arthropod and vertebrate herbivores than jasmonate-producing plants in nature. However, only damage by vertebrate herbivores translates into a significant reduction in flower production. Vertebrate stem peeling has the strongest negative impact on plant flower production. Stems are defended by jasmonate-dependent nicotine, and the native cottontail rabbit Sylvilagus nuttallii avoids jasmonate-producing N. attenuata shoots because of their high levels of nicotine. Thus, endogenous jasmonates enable plants to resist different types of herbivores in nature, and jasmonate-dependent defenses are important for plants to maintain their reproductive potential when facing vertebrate herbivory. Ecological and evolutionary models on plant defense signaling should aim at integrating arthropod and vertebrate herbivory at the community level