Computed Tomography of the Head Before Lumbar Puncture in Adults With Suspected Meningitis in High-HIV Prevalence Settings.

Background: The role of computed tomography (CT) before lumbar puncture (LP) is unclear, with limited evidence for a causal link between LP and cerebral herniation or for the ability of CT to identify individuals at risk of herniation. The risks of LP delay or deferral are potentially greater in high-HIV prevalence, resource-limited settings; we analyzed data from such a setting to determine the impact of CT on time to LP and treatment, as well as mortality. Methods: Adults with suspected central nervous system (CNS) infection were enrolled prospectively into the Botswana National Meningitis Survey between 2016 and 2019. Inpatient mortality and clinical data including time of treatment initiation and CT were captured from medical records. Associations between preceding CT and outcomes were assessed using logistic regression. Results: LPs were performed in 711 patients with suspected CNS infection; 27% had a CT before LP, and 73% were HIV positive. Time from admission to LP and time from admission to appropriate treatment were significantly longer in patients who had a CT before LP compared with those who did not (2.8 hours and 13.2 hours, respectively). There was some evidence for treatment delays being associated with increased mortality; however, there was no significant difference in mortality between those who had or did not have CT. Conclusions: Patients who had a CT had delays to diagnostic LP and initiation of appropriate treatment; although treatment delays were associated with increased mortality, our observational study could not demonstrate a causal association between delays in diagnosis and treatment introduced by CT and mortality.

The Impact of GeneXpert Cerebrospinal Fluid Testing on Tuberculous Meningitis Diagnosis in Routine Care in Botswana.

Background: Tuberculous meningitis (TBM) disproportionately impacts high-HIV prevalence, resource-limited settings where diagnosis is challenging. The GeneXpert platform has utility in TBM diagnosis, but uptake remains limited. In Botswana, before the introduction of GeneXpert, tuberculosis (TB) testing was only available through mycobacterial culture at the National TB Reference Laboratory. Data describing routine use of Xpert MTB/RIF for cerebrospinal fluid (CSF) testing in resource-limited settings are scarce. Methods: Electronic records for patients with CSF tested in government facilities in Botswana between 2016 and 2022 were obtained from a central online repository as part of ongoing national meningitis surveillance. Samples were excluded from 1 site where Xpert MTB/RIF is performed universally. The proportion receiving TB-specific investigation on CSF and the number positive for Mycobacterium tuberculosis following increased Xpert MTB/RIF capacity were determined. Results: The proportion of CSF samples receiving TB-specific investigation increased from 4.5% (58/1288) in 2016 to 29.0% (201/693) in 2022, primarily due to increased analysis with Xpert MTB/RIF from 0.9% (11/1288) to 23.2% (161/693). There was an overall decline in the annual number of CSF samples analyzed, but the proportion with microbiologically confirmed TBM increased from 0.4% to 1.2%. The proportion of samples tested for TB that were collected from health care facilities >100 km from the National TB Reference Laboratory increased with Xpert MTB/RIF rollout from 65.9% (87/132) to 78.0% (494/633). Conclusions: In Botswana, access to TB culture is challenging in remote populations; more accessible near-patient testing using Xpert MTB/RIF increased the number of patients receiving TB-specific testing on CSF and the number of confirmed TBM cases.

Ex vivo host transcriptomics during Cryptococcus neoformans, Cryptococcus gattii, and Candida albicans infection of PBMCs from South African volunteers.

Cryptococcus neoformans, Cryptococcus gattii and Candida albicans are opportunistic fungal pathogens associated with infections in immunocompromised hosts. Cryptococcal meningitis (CM) is the leading fungal cause of HIV-related deaths globally, with the majority occurring in Africa. The human immune response to C. albicans infection has been studied extensively in large genomics studies whereas cryptococcal infections, despite their severity, are comparatively understudied. Here we investigated the transcriptional response of immune cells after in vitro stimulation with in vitro C. neoformans, C. gattii and C. albicans infection of peripheral blood mononuclear cells (PBMCs) collected from healthy South African volunteers. We found a lower transcriptional response to cryptococcal stimuli compared to C. albicans and unique expression signatures from all three fungal stimuli. This work provides a starting point for further studies comparing the transcriptional signature of CM in immunocompromised patients, with the goal of identifying biomarkers of disease severity and possible novel treatment targets.

Using 16s rRNA sequencing to characterize the microbiome of tropical cutaneous ulcer disease: insights into the microbial landscape and implications for diagnosis and treatment.

Cutaneous ulcers are common in yaws-endemic areas. Although often attributed to 'Treponema pallidum subsp. pertenue' and Haemophilus ducreyi, quantitative PCR has highlighted a significant proportion of these ulcers are negative for both pathogens and are considered idiopathic. This is a retrospective analysis utilising existing 16S rRNA sequencing data from two independent yaws studies that took place in Ghana and the Solomon Islands. We characterized bacterial diversity in 38 samples to identify potential causative agents for idiopathic cutaneous ulcers. We identified a diverse bacterial profile, including Arcanobacterium haemolyticum, Campylobacter concisus, Corynebacterium diphtheriae, Staphylococcus spp. and Streptococcus pyogenes, consistent with findings from previous cutaneous ulcer microbiome studies. No single bacterial species was universally present across all samples. The most prevalent bacterium, Campylobacter ureolyticus, appeared in 42% of samples, suggesting a multifactorial aetiology for cutaneous ulcers in yaws-endemic areas. This study emphasizes the need for a nuanced understanding of potential causative agents. The findings prompt further exploration into the intricate microbial interactions contributing to idiopathic yaw-like ulcers, guiding future research toward comprehensive diagnostic and therapeutic strategies.

Gluten-Free Diet Induces Rapid Changes in Phenotype and Survival Properties of Gluten-Specific T Cells in Celiac Disease.

BACKGROUND & AIMS: The treatment of celiac disease (CeD) with gluten-free diet (GFD) normalizes gut inflammation and disease-specific antibodies. CeD patients have HLA-restricted, gluten-specific T cells persisting in the blood and gut even after decades of GFD, which are reactivated and disease driving upon gluten exposure. Our aim was to examine the transition of activated gluten-specific T cells into a pool of persisting memory T cells concurrent with normalization of clinically relevant biomarkers during the first year of treatment. METHODS: We followed 17 CeD patients during their initial GFD year, leading to disease remission. We assessed activation and frequency of gluten-specific CD4+ blood and gut T cells with HLA-DQ2.5:gluten tetramers and flow cytometry, disease-specific serology, histology, and symptom scores. We assessed gluten-specific blood T cells within the first 3 weeks of GFD in 6 patients and serology in an additional 9 patients. RESULTS: Gluten-specific CD4+ T cells peaked in blood at day 14 while up-regulating Bcl-2 and down-regulating Ki-67 and then decreased in frequency within 10 weeks of GFD. CD38, ICOS, HLA-DR, and Ki-67 decreased in gluten-specific cells within 3 days. PD-1, CD39, and OX40 expression persisted even after 12 months. IgA-transglutaminase 2 decreased significantly within 4 weeks. CONCLUSIONS: GFD induces rapid changes in the phenotype and number of gluten-specific CD4+ blood T cells, including a peak of nonproliferating, nonapoptotic cells at day 14. Subsequent alterations in T-cell phenotype associate with the quiescent but chronic nature of treated CeD. The rapid changes affecting gluten-specific T cells and disease-specific antibodies offer opportunities for clinical trials aiming at developing nondietary treatments for patients with newly diagnosed CeD.

The diagnosis of central nervous system infections in resource-limited settings and the use of novel and molecular diagnostic platforms to improve diagnosis.

INTRODUCTION: Central nervous system infections (CNSI) disproportionately affect individuals in low-resource settings where diagnosis is challenging; large proportions of patients never receive a confirmed microbiological diagnosis resulting in inadequate management and high mortality. The epidemiology of CNSI varies globally and conventional diagnostics deployed in resource-limited settings have significant limitations, with an urgent need for improved diagnostic strategies. AREAS COVERED: This review describes molecular platforms and other novel diagnostics used in the diagnosis of CNSI that are applicable to resource-limited settings. An extensive literature search of Medline and PubMed was performed. The emphasis is on investigations targeting infections of relevance to resource-limited settings either due to variation in regional CNSI epidemiology or due to increased prevalence in patients with immunosuppression. This includes commercially available multiplex PCR platforms, mycobacterial PCR platforms, and rapid diagnostics tests. To offer a framework for the optimal implementation in clinical settings, existing evidence highlighting the advantages and limitations of available platforms is reviewed. EXPERT OPINION: The implementation of molecular platforms and other novel diagnostics has the potential to transform CNSI diagnosis in resource-limited settings, with several examples of successful rollout of novel diagnostics such as Xpert MTB/RIF Ultra and cryptococcal antigen testing.

Evaluation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Molecular Typing of Acinetobacter baumannii in Comparison with Orthogonal Methods.

Colonization and subsequent health care-associated infection (HCAI) with Acinetobacter baumannii are a concern for vulnerable patient groups within the hospital setting. Outbreaks involving multidrug-resistant strains are associated with increased patient morbidity and mortality and poorer overall outcomes. Reliable molecular typing methods can help to trace transmission routes and manage outbreaks. In addition to methods deployed by reference laboratories, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) may assist by making initial in-house judgments on strain relatedness. However, limited studies on method reproducibility exist for this application. We applied MALDI-TOF MS typing to A. baumannii isolates associated with a nosocomial outbreak and evaluated different methods for data analysis. In addition, we compared MALDI-TOF MS with whole-genome sequencing (WGS) and Fourier transform infrared spectroscopy (FTIR) as orthogonal methods to further explore their resolution for bacterial strain typing. A related subgroup of isolates consistently clustered separately from the main outbreak group by all investigated methods. This finding, combined with epidemiological data from the outbreak, indicates that these methods identified a separate transmission event unrelated to the main outbreak. However, the MALDI-TOF MS upstream approach introduced measurement variability impacting method reproducibility and limiting its reliability as a standalone typing method. Availability of in-house typing methods with well-characterized sources of measurement uncertainty could assist with rapid and dependable confirmation (or denial) of suspected transmission events. This work highlights some of the steps to be improved before such tools can be fully integrated into routine diagnostic service workflows for strain typing. IMPORTANCE Managing the transmission of antimicrobial resistance necessitates reliable methods for tracking outbreaks. We compared the performance of MALDI-TOF MS with orthogonal approaches for strain typing, including WGS and FTIR, for Acinetobacter baumannii isolates correlated with a health care-associated infection (HCAI) event. Combined with epidemiological data, all methods investigated identified a group of isolates that were temporally and spatially linked to the outbreak, yet potentially attributed to a separate transmission event. This may have implications for guiding infection control strategies during an outbreak. However, the technical reproducibility of MALDI-TOF MS needs to be improved for it to be employed as a standalone typing method, as different stages of the experimental workflow introduced bias influencing interpretation of biomarker peak data. Availability of in-house methods for strain typing of bacteria could improve infection control practices following increased reports of outbreaks of antimicrobial-resistant organisms during the COVID-19 pandemic, related to sessional usage of personal protective equipment (PPE).

Human Herpesvirus-6 Detection in Cerebrospinal Fluid on the BioFire FilmArray Meningitis/Encephalitis Panel in a High Human Immunodeficiency Virus-Prevalence African Setting.

The prevalence and clinical relevance of human herpesvirus-6 (HHV-6) detection in cerebrospinal fluid (CSF) using multiplex polymerase chain reaction (PCR) testing in patients with suspected meningoencephalitis in high human immunodeficiency virus-prevalence African settings are not known. We describe the clinical and laboratory characteristics of 13 patients with HHV-6 CSF PCR positivity in Botswana.

Whole-genome sequencing of Chlamydia trachomatis isolates from persistently infected patients.

BACKGROUND: Current understanding of the causes of treatment failure in Chlamydia trachomatis is poor and antimicrobial susceptibility data are lacking. We used genome sequencing to seek evidence of antimicrobial resistance in isolates sourced from patients who were persistently infected. METHODS: Genomic DNA was extracted from C. trachomatis isolates cultured in McCoy cell monolayers. Sequencing libraries were prepared using the SureSelectXT Illumina paired-end protocol. Paired reads were mapped against a reference genome and single nucleotide variants (SNVs) were identified. RESULTS: Seven isolates from persistently infected patients and five isolates from successfully treated patients were sequenced. No previously reported SNVs associated with antimicrobial resistance were found. A unique SNV was identified in the gyrA gene of one treatment failure isolate but was located outside of the quinolone resistance determining region; this SNV has been previously reported in other members of the Chlamydiaceae family. CONCLUSION: No genomic evidence was found to explain the differences in clinical outcome for our two groups of patients. A mutation unrelated to antimicrobial susceptibility was found in an isolate from a persistently infected patient. The cause of these persistent infections with C. trachomatis remains unclear.

An inter-laboratory study to investigate the impact of the bioinformatics component on microbiome analysis using mock communities.

Despite the advent of whole genome metagenomics, targeted approaches (such as 16S rRNA gene amplicon sequencing) continue to be valuable for determining the microbial composition of samples. Amplicon microbiome sequencing can be performed on clinical samples from a normally sterile site to determine the aetiology of an infection (usually single pathogen identification) or samples from more complex niches such as human mucosa or environmental samples where multiple microorganisms need to be identified. The methodologies are frequently applied to determine both presence of micro-organisms and their quantity or relative abundance. There are a number of technical steps required to perform microbial community profiling, many of which may have appreciable precision and bias that impacts final results. In order for these methods to be applied with the greatest accuracy, comparative studies across different laboratories are warranted. In this study we explored the impact of the bioinformatic approaches taken in different laboratories on microbiome assessment using 16S rRNA gene amplicon sequencing results. Data were generated from two mock microbial community samples which were amplified using primer sets spanning five different variable regions of 16S rRNA genes. The PCR-sequencing analysis included three technical repeats of the process to determine the repeatability of their methods. Thirteen laboratories participated in the study, and each analysed the same FASTQ files using their choice of pipeline. This study captured the methods used and the resulting sequence annotation and relative abundance output from bioinformatic analyses. Results were compared to digital PCR assessment of the absolute abundance of each target representing each organism in the mock microbial community samples and also to analyses of shotgun metagenome sequence data. This ring trial demonstrates that the choice of bioinformatic analysis pipeline alone can result in different estimations of the composition of the microbiome when using 16S rRNA gene amplicon sequencing data. The study observed differences in terms of both presence and abundance of organisms and provides a resource for ensuring reproducible pipeline development and application. The observed differences were especially prevalent when using custom databases and applying high stringency operational taxonomic unit (OTU) cut-off limits. In order to apply sequencing approaches with greater accuracy, the impact of different analytical steps needs to be clearly delineated and solutions devised to harmonise microbiome analysis results.

Genome-Wide Association Study Identifies Novel Colony Stimulating Factor 1 Locus Conferring Susceptibility to Cryptococcosis in Human Immunodeficiency Virus-Infected South Africans.

BACKGROUND: Cryptococcus is the most common cause of meningitis in human immunodeficiency virus (HIV)-infected Africans. Despite universal exposure, only 5%-10% of patients with HIV/acquired immune deficiency syndrome and profound CD4+ T-cell depletion develop disseminated cryptococcosis: host genetic factors may play a role. Prior targeted immunogenetic studies in cryptococcosis have comprised few Africans. METHODS: We analyzed genome-wide single-nucleotide polymorphism (SNP) genotype data from 524 patients of African descent: 243 cases (advanced HIV with cryptococcal antigenemia and/or cryptococcal meningitis) and 281 controls (advanced HIV, no history of cryptococcosis, negative serum cryptococcal antigen). RESULTS: Six loci upstream of the colony-stimulating factor 1 (CSF1) gene, encoding macrophage colony-stimulating factor (M-CSF) were associated with susceptibility to cryptococcosis at P < 10-6 and remained significantly associated in a second South African cohort (83 cases; 128 controls). Meta-analysis of the genotyped CSF1 SNP rs1999713 showed an odds ratio for cryptococcosis susceptibility of 0.53 (95% confidence interval, 0.42-0.66; P = 5.96 × 10-8). Ex vivo functional validation and transcriptomic studies confirmed the importance of macrophage activation by M-CSF in host defence against Cryptococcus in HIV-infected patients and healthy, ethnically matched controls. CONCLUSIONS: This first genome-wide association study of susceptibility to cryptococcosis has identified novel and immunologically relevant susceptibility loci, which may help define novel strategies for prevention or immunotherapy of HIV-associated cryptococcal meningitis.

The role of IS in the covid-19 pandemic: A liquid-modern perspective.

History shows that pandemics can catalyse enormous change, fundamentally transforming the way people make sense of the world. Technologies can also be catalysts of change. While digital technologies are playing a vital role in tackling the covid-19 pandemic, the pandemic also presents a significant opportunity for digital technologies. Some experts believe the pandemic may permanently normalise the comprehensive societal use of digital technologies. This article casts a critical eye over the potential implications of this opportunity in the context of information systems (IS) research and development. We introduce and outline selected principles of Zygmunt Bauman's theory of liquid modernity. We then apply the liquid-modern principles to illustrative examples drawn from the covid-19 literature by focussing on three areas of established information systems interest: control, big data and information privacy. We show that traditional conceptualisations of scientific and societal order and control need to be reassessed; that big data alone cannot order clear and safe paths out of the current crisis and that information privacy regulations are irrelevant when undermined or circumvented by public and private actors. We conclude by making four recommendations for IS pandemic researchers and five practical recommendations in the context of the pandemic.

Discordant bioinformatic predictions of antimicrobial resistance from whole-genome sequencing data of bacterial isolates: an inter-laboratory study.

Antimicrobial resistance (AMR) poses a threat to public health. Clinical microbiology laboratories typically rely on culturing bacteria for antimicrobial-susceptibility testing (AST). As the implementation costs and technical barriers fall, whole-genome sequencing (WGS) has emerged as a 'one-stop' test for epidemiological and predictive AST results. Few published comparisons exist for the myriad analytical pipelines used for predicting AMR. To address this, we performed an inter-laboratory study providing sets of participating researchers with identical short-read WGS data from clinical isolates, allowing us to assess the reproducibility of the bioinformatic prediction of AMR between participants, and identify problem cases and factors that lead to discordant results. We produced ten WGS datasets of varying quality from cultured carbapenem-resistant organisms obtained from clinical samples sequenced on either an Illumina NextSeq or HiSeq instrument. Nine participating teams ('participants') were provided these sequence data without any other contextual information. Each participant used their choice of pipeline to determine the species, the presence of resistance-associated genes, and to predict susceptibility or resistance to amikacin, gentamicin, ciprofloxacin and cefotaxime. We found participants predicted different numbers of AMR-associated genes and different gene variants from the same clinical samples. The quality of the sequence data, choice of bioinformatic pipeline and interpretation of the results all contributed to discordance between participants. Although much of the inaccurate gene variant annotation did not affect genotypic resistance predictions, we observed low specificity when compared to phenotypic AST results, but this improved in samples with higher read depths. Had the results been used to predict AST and guide treatment, a different antibiotic would have been recommended for each isolate by at least one participant. These challenges, at the final analytical stage of using WGS to predict AMR, suggest the need for refinements when using this technology in clinical settings. Comprehensive public resistance sequence databases, full recommendations on sequence data quality and standardization in the comparisons between genotype and resistance phenotypes will all play a fundamental role in the successful implementation of AST prediction using WGS in clinical microbiology laboratories.

Cross-transmission Is Not the Source of New Mycobacterium abscessus Infections in a Multicenter Cohort of Cystic Fibrosis Patients.

BACKGROUND: Mycobacterium abscessus is an extensively drug-resistant pathogen that causes pulmonary disease, particularly in cystic fibrosis (CF) patients. Identifying direct patient-to-patient transmission of M. abscessus is critically important in directing an infection control policy for the management of risk in CF patients. A variety of clinical labs have used molecular epidemiology to investigate transmission. However, there is still conflicting evidence as to how M. abscessus is acquired and whether cross-transmission occurs. Recently, labs have applied whole-genome sequencing (WGS) to investigate this further and, in this study, we investigated whether WGS can reliably identify cross-transmission in M. abscessus. METHODS: We retrospectively sequenced the whole genomes of 145 M. abscessus isolates from 62 patients, seen at 4 hospitals in 2 countries over 16 years. RESULTS: We have shown that a comparison of a fixed number of core single nucleotide variants alone cannot be used to infer cross-transmission in M. abscessus but does provide enough information to replace multiple existing molecular assays. We detected 1 episode of possible direct patient-to-patient transmission in a sibling pair. We found that patients acquired unique M. abscessus strains even after spending considerable time on the same wards with other M. abscessus-positive patients. CONCLUSIONS: This novel analysis has demonstrated that the majority of patients in this study have not acquired M. abscessus through direct patient-to-patient transmission or a common reservoir. Tracking transmission using WGS will only realize its full potential with proper environmental screening, as well as patient sampling.

1,4-dihydroxy quininib attenuates growth of colorectal cancer cells and xenografts and regulates the TIE-2 signaling pathway in patient tumours.

Colorectal cancer (CRC) is the second leading cause of cancer associated deaths in developed countries. Cancer progression and metastatic spread is reliant on new blood vasculature, or angiogenesis. Tumour-related angiogenesis is regulated by pro- and anti-angiogenic factors secreted from malignant tissue in a stepwise process. Previously we structurally modified the small anti-angiogenic molecule quininib and discovered a more potent anti-angiogenic compound 1, 4 dihydroxy quininib (Q8), an antagonist of cysteinyl leukotriene receptor-1 with VEGF-independent bioactivity. Here, Q8, quininib (Q1) and five structural analogues were assayed for anti-tumorigenic effects in pre-clinical cancer models. Q8 reduced clone formation of the human colorectal cancer cell line HT29-Luc2. Gene silencing of CysLT1 in HT29-Luc2 cells significantly reduced expression of calpain-2. In human ex vivo colorectal cancer tumour explants, Q8 significantly decreased the secretion of both TIE-2 and VCAM-1 expression. In vivo Q8 was well tolerated up to 50 mg/kg by Balb/C mice and significantly more effective at reducing tumour volume in colorectal tumour xenografts compared to the parent drug quininib. In tumour xenografts, Q8 significantly reduced expression of the angiogenic marker calpain-2. In summary, we propose Q8 may act on the TIE-2-Angiopoietin signalling pathway to significantly inhibit the process of tumour angiogenesis in colorectal cancer.

Intraoperative and major postoperative complications and survival of dogs undergoing surgical management of epiglottic retroversion: 50 dogs (2003-2017).

OBJECTIVE: To report intraoperative and major postoperative complications in dogs treated surgically for epiglottic retroversion (ER), compare the incidence of major postoperative complications between procedures, and report survival of surgically treated dogs. STUDY DESIGN: Multi-institutional retrospective study. SAMPLE POPULATION: Fifty dogs treated with 78 procedures. METHODS: Medical records of dogs diagnosed and surgically treated for ER from 2003 to 2017 at 11 institutions were reviewed. Complications were divided into intraoperative and major postoperative complications. RESULTS: Intraoperative complications occurred during 2 of 78 (2.6%) procedures. Thirty-six major postoperative complications were documented in 22 dogs after 36 of 74 (48.7%) procedures. Postoperative complications occurred after 7 of 12 (58.3%) nonincisional epiglottopexy, 23 of 43 (53.5%) incisional epiglottopexy, 2 of 4 (50%) partial epiglottectomy, 2 of 12 (16.7%) subtotal epiglottectomy, and 2 of 3 (66.7%) other surgical procedures. Epiglottopexy failure was the most common major postoperative complication. The incidence of major postoperative complications did not differ between procedures (P = .1239), although, when combined, epiglottopexy procedures (30/55) had a higher incidence of complications than epiglottectomy procedures (4/16; P = .048). Thirty (60%) dogs were alive at a median of 928 days (range, 114-2805), 8 (16%) were lost to follow-up after 411 days (range, 43-1158), and 12 (24%) were dead/euthanized after 301.5 days (range, 3-1212). Median survival time was not reached after a median of 716 days. CONCLUSION: Although intraoperative complications were uncommon, major postoperative complications were common, especially after epiglottopexy procedures. CLINICAL SIGNIFICANCE: Although surgical treatment of ER is associated with a high rate of major postoperative complications, especially epiglottopexy procedures, long-term survival can be achieved.

Correction to: Whole genome sequencing Mycobacterium tuberculosis directly from sputum identifies more genetic diversity than sequencing from culture.

He authors reported that one of the authors' names was typeset incorrectly in the authorship list.

Whole genome sequencing Mycobacterium tuberculosis directly from sputum identifies more genetic diversity than sequencing from culture.

BACKGROUND: Repeated culture reduces within-sample Mycobacterium tuberculosis genetic diversity due to selection of clones suited to growth in culture and/or random loss of lineages, but it is not known to what extent omitting the culture step altogether alters genetic diversity. We compared M. tuberculosis whole genome sequences generated from 33 paired clinical samples using two methods. In one method DNA was extracted directly from sputum then enriched with custom-designed SureSelect (Agilent) oligonucleotide baits and in the other it was extracted from mycobacterial growth indicator tube (MGIT) culture. RESULTS: DNA directly sequenced from sputum showed significantly more within-sample diversity than that from MGIT culture (median 5.0 vs 4.5 heterozygous alleles per sample, p = 0.04). Resistance associated variants present as HAs occurred in four patients, and in two cases may provide a genotypic explanation for phenotypic resistance. CONCLUSIONS: Culture-free M. tuberculosis whole genome sequencing detects more within-sample diversity than a leading culture-based method and may allow detection of mycobacteria that are not actively replicating.

Children With Cystic Fibrosis Are Infected With Multiple Subpopulations of Mycobacterium abscessus With Different Antimicrobial Resistance Profiles.

BACKGROUND: Children with cystic fibrosis (CF) can develop life-threatening infections of Mycobacterium abscessus. These present a significant clinical challenge, particularly when the strains involved are resistant to antibiotics. Recent evidence of within-patient subclones of M. abscessus in adults with CF suggests the possibility that within-patient diversity may be relevant for the treatment of pediatric CF patients. METHODS: We performed whole-genome sequencing (WGS) on 32 isolates of M. abscessus that were taken from multiple body sites of 2 patients with CF who were undergoing treatment at Great Ormond Street Hospital, United Kingdom, in 2015. RESULTS: We found evidence of extensive diversity within patients over time. A clustering analysis of single nucleotide variants revealed that each patient harbored multiple subpopulations, which were differentially abundant between sputum, lung samples, chest wounds, and pleural fluid. The sputum isolates did not reflect the overall within-patient diversity and did not allow for the detection of subclones with mutations previously associated with macrolide resistance (rrl 2058/2059). Some variants were present at intermediate frequencies before the lung transplants. The time of the transplants coincided with extensive variation, suggesting that this event is particularly disruptive for the microbial community, but the transplants did not clear the M. abscessus infections and both patients died as a result of these infections. CONCLUSIONS: Isolates of M. abscessus from sputum do not always reflect the entire diversity present within the patient, which can include subclones with differing antimicrobial resistance profiles. An awareness of this phenotypic variability, with the sampling of multiple body sites in conjunction with WGS, may be necessary to ensure the best treatment for this vulnerable patient group.