Clinical Integration of a Highly Accurate Polymerase Chain Reaction Point-of-Care Test Can Inform Immediate Treatment Decisions for Chlamydia, Gonorrhea, and Trichomonas.

BACKGROUND: Accurate same-day sexually transmitted infection (STI) diagnostic testing is generally unavailable, leading to syndromic management with high rates of overtreatment and undertreatment. We analyzed the ease of integration of the Visby STI Panel into clinical practice, studied acceptance by patients and clinic personnel, and assessed the potential to inform accurate treatment decisions. METHODS: In a cross-sectional single-visit study of 55 women aged 18 to 56 years, women self-collected vaginal swab samples that were analyzed using the Visby STI Panel for Chlamydia trachomatis, Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV). Results were compared with standard-of-care clinic results from send-out laboratory polymerase chain reaction tests. Surveys assessed patient and device operator experiences with the Visby STI Panel and clinicians' perceived need for and acceptance of the device. Time parameters were measured to evaluate the impact on clinical workflow, and syndromic treatment decisions were compared with anticipated treatment based on the Visby STI Panel results. RESULTS: Patients strongly agreed that sample self-collection was easy, and operators reported the device easy to use. Clinicians valued the rapid return of results, and patients were comfortable waiting up to 30 minutes to receive them. In 13 of 15 cases, the Visby STI Panel correctly identified undertreated patients as infected and correctly identified all 33 incidences of overtreatment. CONCLUSIONS: Clinical adoption of the Visby STI Panel into primary care clinics and doctors' offices could reduce overtreatment and undertreatment of STIs. If integrated efficiently into the clinical workflow, the test would have minimal impact on staff time and visit duration for patients.

Identification of Widespread Antibiotic Exposure in Patients With Cholera Correlates With Clinically Relevant Microbiota Changes.

BACKGROUND: A first step to combating antimicrobial resistance in enteric pathogens is to establish an objective assessment of antibiotic exposure. Our goal was to develop and evaluate a liquid chromatography-ion trap mass spectrometry (LC/MS) method to determine antibiotic exposure in patients with cholera. METHODS: A priority list for targeted LC/MS was generated from medication-vendor surveys in Bangladesh. A study of patients with and those without cholera was conducted to collect and analyze paired urine and stool samples. RESULTS: Among 845 patients, 11% (90) were Vibrio cholerae positive; among these 90 patients, analysis of stool specimens revealed ≥1 antibiotic in 86% and ≥2 antibiotics in 52%. Among 44 patients with cholera and paired urine and stool specimens, ≥1 antibiotic was detected in 98% and ≥2 antibiotics were detected in 84%, despite 55% self-reporting medication use. Compared with LC/MS, a low-cost antimicrobial detection bioassay lacked a sufficient negative predictive value (10%; 95% confidence interval, 6%-16%). Detection of guideline-recommended antibiotics in stool specimens did (for azithromycin; P = .040) and did not (for ciprofloxacin) correlate with V. cholerae suppression. A nonrecommended antibiotic (metronidazole) was associated with decreases in anaerobes (ie, Prevotella organisms; P < .001). CONCLUSION: These findings suggest that there may be no true negative control group when attempting to account for antibiotic exposure in settings like those in this study.

The Mycobacterium tuberculosis regulatory network and hypoxia.

We have taken the first steps towards a complete reconstruction of the Mycobacterium tuberculosis regulatory network based on ChIP-Seq and combined this reconstruction with system-wide profiling of messenger RNAs, proteins, metabolites and lipids during hypoxia and re-aeration. Adaptations to hypoxia are thought to have a prominent role in M. tuberculosis pathogenesis. Using ChIP-Seq combined with expression data from the induction of the same factors, we have reconstructed a draft regulatory network based on 50 transcription factors. This network model revealed a direct interconnection between the hypoxic response, lipid catabolism, lipid anabolism and the production of cell wall lipids. As a validation of this model, in response to oxygen availability we observe substantial alterations in lipid content and changes in gene expression and metabolites in corresponding metabolic pathways. The regulatory network reveals transcription factors underlying these changes, allows us to computationally predict expression changes, and indicates that Rv0081 is a regulatory hub.

Does discovery of differentially culturable M tuberculosis really demand a new treatment paradigm? Longitudinal analysis of DNA clearance from sputum.

BACKGROUND: According to the traditional tuberculosis (TB) treatment paradigm, the initial doses of treatment rapidly kill most Mycobacterium tuberculosis (Mtb) bacilli in sputum, yet many more months of daily treatment are required to eliminate a small, residual subpopulation of drug-tolerant bacilli. This paradigm has recently been challenged following the discovery that up to 90% of Mtb bacilli in sputum are culturable only with growth-factor supplementation. These "differentially culturable" bacilli are hypothesized to be more drug-tolerant than routinely culturable bacilli. This hypothesis implies an alternative paradigm in which TB treatment does not rapidly reduce the total Mtb population but only the small, routinely culturable subpopulation. To evaluate these competing paradigms, we developed a culture-independent method for quantifying the viable fraction of Mtb bacilli in sputum during treatment. METHODS: We used GeneXpert MTB/RIF to quantify Mtb DNA in sputa collected longitudinally from Ugandan adults taking standard 4-drug treatment for drug-susceptible pulmonary TB. We modeled GeneXpert cycle thresholds over time using nonlinear mixed-effects regression. We adjusted these models for clearance of DNA from killed-but-not-yet-degraded bacilli, assuming clearance half-lives ranging from 0 to 1.25 days. We used a convolution integral to quantify DNA from viable bacilli only, and converted cycle thresholds to Mtb genomic equivalents. We replicated our results in a South African cohort. RESULTS: We enrolled 41 TB patients in Uganda. Assuming a DNA-clearance half-life of 0 days, genomic equivalents of viable sputum bacilli decreased by 0.22 log/day until 8.8 days, then by 0.07 log/day afterwards. Assuming a DNA-clearance half-life of 1.25 days, genomic equivalents of viable bacilli decreased by 0.36 log/day until 5.0 days, then by 0.06 log/day afterwards. By day 7, viable Mtb had decreased by 97.2-98.8%. We found similar results for 19 TB patients in South Africa. DISCUSSION: Using a culture-independent method, we found that TB treatment rapidly eliminates most viable Mtb in sputum. These findings are incompatible with the hypothesis that differentially culturable bacilli are drug-tolerant. CONCLUSIONS: A culture-independent method for measuring viable Mtb in sputum during treatment corroborates the traditional TB treatment paradigm in which a rapid bactericidal phase precedes slow, elimination of a small, residual bacillary subpopulation.

A Fully Integrated CMOS Fluorescence Biochip for DNA and RNA Testing.

Design and successful implementation of a fully-integrated CMOS fluorescence biochip for DNA/RNA testing in molecular diagnostics (MDx) is presented. The biochip includes a 32×32 array of continuous wave fluorescence detection biosensing elements. Each biosensing element is capable of having unique DNA probe sequences, wavelength-selective multi-dielectric emission filter (OD of 3.6), resistive heater for thermal cycling, and a high performance and programmable photodetector. The dimension of each biosensor is 100µm×100µm with a 50µm×50µm Nwell-Psub photodiode acting as the optical transducer, and a ΣΔ modulator based photocurrent sensor. The measured photodetector performance shows ~116 dB detection dynamic range (10fA - 10nA) over the 25°C - 100°C temperature range, while being ~1 dB away from the fundamental shot-noise limit. To empirically demonstrate the compatibility of this biochip with MDx applications, we have successfully utilized the array and its thermal cycling capability to adopt a 7-plex panel for detection of 6 human upper respiratory viruses.

Tuberculosis vaccine with high predicted population coverage and compatibility with modern diagnostics.

A central goal in vaccine research is the identification of relevant antigens. The Mycobacterium tuberculosis chromosome encodes 23 early secretory antigenic target (ESAT-6) family members that mostly are localized as gene pairs. In proximity to five of the gene pairs are ESX secretion systems involved in the secretion of the ESAT-6 family proteins. Here, we performed a detailed and systematic investigation of the vaccine potential of five possible Esx dimer substrates, one for each of the five ESX systems. On the basis of gene transcription during infection, immunogenicity, and protective capacity in a mouse aerosol challenge model, we identified the ESX dimer substrates EsxD-EsxC, ExsG-EsxH, and ExsW-EsxV as the most promising vaccine candidates and combined them in a fusion protein, H65. Vaccination with H65 gave protection at the level of bacillus Calmette-Guérin, and the fusion protein exhibited high predicted population coverage in high endemic regions. H65 thus constitutes a promising vaccine candidate devoid of antigen 85 and fully compatible with current ESAT-6 and culture filtrate protein 10-based diagnostics.

Adaptation of Mycobacterium tuberculosis to Impaired Host Immunity in HIV-Infected Patients.

BACKGROUND: It is unknown whether immunosuppression influences the physiologic state of Mycobacterium tuberculosis in vivo. We evaluated the impact of host immunity by comparing M. tuberculosis and human gene transcription in sputum between human immunodeficiency virus (HIV)-infected and uninfected patients with tuberculosis. METHODS: We collected sputum specimens before treatment from Gambians and Ugandans with pulmonary tuberculosis, revealed by positive results of acid-fast bacillus smears. We quantified expression of 2179 M. tuberculosis genes and 234 human immune genes via quantitative reverse transcription-polymerase chain reaction. We summarized genes from key functional categories with significantly increased or decreased expression. RESULTS: A total of 24 of 65 patients with tuberculosis were HIV infected. M. tuberculosis DosR regulon genes were less highly expressed among HIV-infected patients with tuberculosis than among HIV-uninfected patients with tuberculosis (Gambia, P < .0001; Uganda, P = .037). In profiling of human genes from the same sputa, HIV-infected patients had 3.4-fold lower expression of IFNG (P = .005), 4.9-fold higher expression of ARG1 (P = .0006), and 3.4-fold higher expression of IL10 (P = .0002) than in HIV-uninfected patients with tuberculosis. CONCLUSIONS: M. tuberculosis in HIV-infected patients had lower expression of the DosR regulon, a critical metabolic and immunomodulatory switch induced by NO, carbon monoxide, and hypoxia. Our human data suggest that decreased DosR expression may result from alternative pathway activation of macrophages, with consequent decreased NO expression and/or by poor granuloma formation with consequent decreased hypoxic stress.

Transcriptional Adaptation of Drug-tolerant Mycobacterium tuberculosis During Treatment of Human Tuberculosis.

BACKGROUND: Treatment initiation rapidly kills most drug-susceptible Mycobacterium tuberculosis, but a bacterial subpopulation tolerates prolonged drug exposure. We evaluated drug-tolerant bacilli in human sputum by comparing messenger RNA (mRNA) expression of drug-tolerant bacilli that survive the early bactericidal phase with treatment-naive bacilli. METHODS: M. tuberculosis gene expression was quantified via reverse-transcription polymerase chain reaction in serial sputa from 17 Ugandans treated for drug-susceptible pulmonary tuberculosis. RESULTS: Within 4 days, bacterial mRNA abundance declined >98%, indicating rapid killing. Thereafter, the rate of decline slowed >94%, indicating drug tolerance. After 14 days, 16S ribosomal RNA transcripts/genome declined 96%, indicating slow growth. Drug-tolerant bacilli displayed marked downregulation of genes associated with growth, metabolism, and lipid synthesis and upregulation in stress responses and key regulatory categories-including stress-associated sigma factors, transcription factors, and toxin-antitoxin genes. Drug efflux pumps were upregulated. The isoniazid stress signature was induced by initial drug exposure, then disappeared after 4 days. CONCLUSIONS: Transcriptional patterns suggest that drug-tolerant bacilli in sputum are in a slow-growing, metabolically and synthetically downregulated state. Absence of the isoniazid stress signature in drug-tolerant bacilli indicates that physiological state influences drug responsiveness in vivo. These results identify novel drug targets that should aid in development of novel shorter tuberculosis treatment regimens.

Comprehensive insights into transcriptional adaptation of intracellular mycobacteria by microbe-enriched dual RNA sequencing.

BACKGROUND: The human pathogen Mycobacterium tuberculosis has the capacity to escape eradication by professional phagocytes. During infection, M. tuberculosis resists the harsh environment of phagosomes and actively manipulates macrophages and dendritic cells to ensure prolonged intracellular survival. In contrast to other intracellular pathogens, it has remained difficult to capture the transcriptome of mycobacteria during infection due to an unfavorable host-to-pathogen ratio. RESULTS: We infected the human macrophage-like cell line THP-1 with the attenuated M. tuberculosis surrogate M. bovis Bacillus Calmette-Guérin (M. bovis BCG). Mycobacterial RNA was up to 1000-fold underrepresented in total RNA preparations of infected host cells. We employed microbial enrichment combined with specific ribosomal RNA depletion to simultaneously analyze the transcriptional responses of host and pathogen during infection by dual RNA sequencing. Our results confirm that mycobacterial pathways for cholesterol degradation and iron acquisition are upregulated during infection. In addition, genes involved in the methylcitrate cycle, aspartate metabolism and recycling of mycolic acids were induced. In response to M. bovis BCG infection, host cells upregulated de novo cholesterol biosynthesis presumably to compensate for the loss of this metabolite by bacterial catabolism. CONCLUSIONS: Dual RNA sequencing allows simultaneous capture of the global transcriptome of host and pathogen, during infection. However, mycobacteria remained problematic due to their relatively low number per host cell resulting in an unfavorable bacterium-to-host RNA ratio. Here, we use a strategy that combines enrichment for bacterial transcripts and dual RNA sequencing to provide the most comprehensive transcriptome of intracellular mycobacteria to date. The knowledge acquired into the pathogen and host pathways regulated during infection may contribute to a solid basis for the deployment of novel intervention strategies to tackle infection.

An unbiased genome-wide Mycobacterium tuberculosis gene expression approach to discover antigens targeted by human T cells expressed during pulmonary infection.

Mycobacterium tuberculosis is responsible for almost 2 million deaths annually. Mycobacterium bovis bacillus Calmette-Guérin, the only vaccine available against tuberculosis (TB), induces highly variable protection against TB, and better TB vaccines are urgently needed. A prerequisite for candidate vaccine Ags is that they are immunogenic and expressed by M. tuberculosis during infection of the primary target organ, that is, the lungs of susceptible individuals. In search of new TB vaccine candidate Ags, we have used a genome-wide, unbiased Ag discovery approach to investigate the in vivo expression of 2170 M. tuberculosis genes during M. tuberculosis infection in the lungs of mice. Four genetically related but distinct mouse strains were studied, representing a spectrum of TB susceptibility controlled by the supersusceptibility to TB 1 locus. We used stringent selection approaches to select in vivo-expressed M. tuberculosis (IVE-TB) genes and analyzed their expression patterns in distinct disease phenotypes such as necrosis and granuloma formation. To study the vaccine potential of these proteins, we analyzed their immunogenicity. Several M. tuberculosis proteins were recognized by immune cells from tuberculin skin test-positive, ESAT6/CFP10-responsive individuals, indicating that these Ags are presented during natural M. tuberculosis infection. Furthermore, TB patients also showed responses toward IVE-TB Ags, albeit lower than tuberculin skin test-positive, ESAT6/CFP10-responsive individuals. Finally, IVE-TB Ags induced strong IFN-γ(+)/TNF-α(+) CD8(+) and TNF-α(+)/IL-2(+) CD154(+)/CD4(+) T cell responses in PBMC from long-term latently M. tuberculosis-infected individuals. In conclusion, these IVE-TB Ags are expressed during pulmonary infection in vivo, are immunogenic, induce strong T cell responses in long-term latently M. tuberculosis-infected individuals, and may therefore represent attractive Ags for new TB vaccines.

A minimum variance method for genome-wide data-driven normalization of quantitative real-time polymerase chain reaction expression data.

Advances in multiplex qRT-PCR have enabled increasingly accurate and robust quantification of RNA, even at lower concentrations, facilitating RNA expression profiling in clinical and environmental samples. Here we describe a data-driven qRT-PCR normalization method, the minimum variance method, and evaluate it on clinically derived Mycobacterium tuberculosis samples with variable transcript detection percentages. For moderate to significant amounts of nondetection (∼50%), our minimum variance method consistently produces the lowest false discovery rates compared to commonly used data-driven normalization methods.

Chaperone activity of small heat shock proteins underlies therapeutic efficacy in experimental autoimmune encephalomyelitis.

To determine whether the therapeutic activity of αB crystallin, small heat shock protein B5 (HspB5), was shared with other human sHsps, a set of seven human family members, a mutant of HspB5 G120 known to exhibit reduced chaperone activity, and a mycobacterial sHsp were expressed and purified from bacteria. Each of the recombinant proteins was shown to be a functional chaperone, capable of inhibiting aggregation of denatured insulin with varying efficiency. When injected into mice at the peak of disease, they were all effective in reducing the paralysis in experimental autoimmune encephalomyelitis. Additional structure activity correlations between chaperone activity and therapeutic function were established when linear regions within HspB5 were examined. A single region, corresponding to residues 73-92 of HspB5, forms amyloid fibrils, exhibited chaperone activity, and was an effective therapeutic for encephalomyelitis. The linkage of the three activities was further established by demonstrating individual substitutions of critical hydrophobic amino acids in the peptide resulted in the loss of all of the functions.

Comparative analysis of Mycobacterium and related Actinomycetes yields insight into the evolution of Mycobacterium tuberculosis pathogenesis.

BACKGROUND: The sequence of the pathogen Mycobacterium tuberculosis (Mtb) strain H37Rv has been available for over a decade, but the biology of the pathogen remains poorly understood. Genome sequences from other Mtb strains and closely related bacteria present an opportunity to apply the power of comparative genomics to understand the evolution of Mtb pathogenesis. We conducted a comparative analysis using 31 genomes from the Tuberculosis Database (TBDB.org), including 8 strains of Mtb and M. bovis, 11 additional Mycobacteria, 4 Corynebacteria, 2 Streptomyces, Rhodococcus jostii RHA1, Nocardia farcinia, Acidothermus cellulolyticus, Rhodobacter sphaeroides, Propionibacterium acnes, and Bifidobacterium longum. RESULTS: Our results highlight the functional importance of lipid metabolism and its regulation, and reveal variation between the evolutionary profiles of genes implicated in saturated and unsaturated fatty acid metabolism. It also suggests that DNA repair and molybdopterin cofactors are important in pathogenic Mycobacteria. By analyzing sequence conservation and gene expression data, we identify nearly 400 conserved noncoding regions. These include 37 predicted promoter regulatory motifs, of which 14 correspond to previously validated motifs, as well as 50 potential noncoding RNAs, of which we experimentally confirm the expression of four. CONCLUSIONS: Our analysis of protein evolution highlights gene families that are associated with the adaptation of environmental Mycobacteria to obligate pathogenesis. These families include fatty acid metabolism, DNA repair, and molybdopterin biosynthesis. Our analysis reinforces recent findings suggesting that small noncoding RNAs are more common in Mycobacteria than previously expected. Our data provide a foundation for understanding the genome and biology of Mtb in a comparative context, and are available online and through TBDB.org.

A bistable switch and anatomical site control Vibrio cholerae virulence gene expression in the intestine.

A fundamental, but unanswered question in host-pathogen interactions is the timing, localization and population distribution of virulence gene expression during infection. Here, microarray and in situ single cell expression methods were used to study Vibrio cholerae growth and virulence gene expression during infection of the rabbit ligated ileal loop model of cholera. Genes encoding the toxin-coregulated pilus (TCP) and cholera toxin (CT) were powerfully expressed early in the infectious process in bacteria adjacent to epithelial surfaces. Increased growth was found to co-localize with virulence gene expression. Significant heterogeneity in the expression of tcpA, the repeating subunit of TCP, was observed late in the infectious process. The expression of tcpA, studied in single cells in a homogeneous medium, demonstrated unimodal induction of tcpA after addition of bicarbonate, a chemical inducer of virulence gene expression. Striking bifurcation of the population occurred during entry into stationary phase: one subpopulation continued to express tcpA, whereas the expression declined in the other subpopulation. ctxA, encoding the A subunit of CT, and toxT, encoding the proximal master regulator of virulence gene expression also exhibited the bifurcation phenotype. The bifurcation phenotype was found to be reversible, epigenetic and to persist after removal of bicarbonate, features consistent with bistable switches. The bistable switch requires the positive-feedback circuit controlling ToxT expression and formation of the CRP-cAMP complex during entry into stationary phase. Key features of this bistable switch also were demonstrated in vivo, where striking heterogeneity in tcpA expression was observed in luminal fluid in later stages of the infection. When this fluid was diluted into artificial seawater, bacterial aggregates continued to express tcpA for prolonged periods of time. The bistable control of virulence gene expression points to a mechanism that could generate a subpopulation of V. cholerae that continues to produce TCP and CT in the rice water stools of cholera patients.

Bacterial genetic signatures of human social phenomena among M. tuberculosis from an Aboriginal Canadian population.

Despite a widespread global distribution and highly variable disease phenotype, there is little DNA sequence diversity among isolates of Mycobacterium tuberculosis. In addition, many regional population genetic surveys have revealed a stereotypical structure in which a single clone, lineage, or clade makes up the majority of the population. It is often assumed that dominant clones are highly adapted, that is, the overall structure of M. tuberculosis populations is the result of positive selection. In order to test this assumption, we analyzed genetic data from extant populations of bacteria circulating in Aboriginal communities in Saskatchewan, Canada. Demographic parameters of the bacterial population were estimated from archival epidemiological data collected over approximately 130 years since the onset of epidemic tuberculosis in the host communities. Bacterial genetic data were tested against neutral theory expectations and the local evolutionary history of M. tuberculosis investigated by phylogenetic analysis. Our findings are not consistent with positive selection on the bacterial population. Instead, we uncovered founder effects persisting over decades and barriers to gene flow within the bacterial population. Simulation experiments suggested that a combination of these neutral influences could result in the stereotypical structure of M. tuberculosis populations. Some aspects of population structure were suggestive of background selection, and data were on the whole consistent with combined effects of population bottlenecks, subdivision, and background selection. Neutral phenomena, namely, bottlenecks and partitions within populations, are prominent influences on the evolution of M. tuberculosis and likely contribute to restricted genetic diversity observed within this species. Given these influences, a complex evolutionary model will be required to define the relative fitness of different M. tuberculosis lineages and, ultimately, to uncover the genetic basis for its success as a pathogen.

TB database: an integrated platform for tuberculosis research.

The effective control of tuberculosis (TB) has been thwarted by the need for prolonged, complex and potentially toxic drug regimens, by reliance on an inefficient vaccine and by the absence of biomarkers of clinical status. The promise of the genomics era for TB control is substantial, but has been hindered by the lack of a central repository that collects and integrates genomic and experimental data about this organism in a way that can be readily accessed and analyzed. The Tuberculosis Database (TBDB) is an integrated database providing access to TB genomic data and resources, relevant to the discovery and development of TB drugs, vaccines and biomarkers. The current release of TBDB houses genome sequence data and annotations for 28 different Mycobacterium tuberculosis strains and related bacteria. TBDB stores pre- and post-publication gene-expression data from M. tuberculosis and its close relatives. TBDB currently hosts data for nearly 1500 public tuberculosis microarrays and 260 arrays for Streptomyces. In addition, TBDB provides access to a suite of comparative genomics and microarray analysis software. By bringing together M. tuberculosis genome annotation and gene-expression data with a suite of analysis tools, TBDB (http://www.tbdb.org/) provides a unique discovery platform for TB research.

Severe Posaconazole-Induced Glucocorticoid Deficiency with Concurrent Pseudohyperaldosteronism: An Unfortunate Two-for-One Special.

A 56-year-old Hispanic man with a history of disseminated coccidioidomycosis was diagnosed with persistent glucocorticoid insufficiency and pseudohyperaldosteronism secondary to posaconazole toxicity. This case was notable for unexpected laboratory findings of both pseudohyperaldosteronism and severe glucocorticoid deficiency due to posaconazole's mechanism of action on the adrenal steroid synthesis pathway. Transitioning to fluconazole and starting hydrocortisone resolved the hypokalemia but not his glucocorticoid deficiency. This case highlights the importance of recognizing iatrogenic glucocorticoid deficiency with azole antifungal agents and potential long term sequalae.

Plasma as an alternative COVID-19 diagnostic specimen in a hospitalized patient negative for SARS-CoV-2 by nasopharyngeal swab.

We present the case of an inpatient with pneumonia and repeatedly negative nasopharyngeal SARS-CoV-2 testing. In such challenging cases, alternative diagnostic options include lower respiratory tract and plasma SARS-CoV-2 RNA testing, of which the latter may be particularly useful where bronchoscopy is deferred due to clinical factors or transmission risk.

High-throughput low-cost nl-qPCR for enteropathogen detection: A proof-of-concept among hospitalized patients in Bangladesh.

BACKGROUND: Diarrheal disease is a leading cause of morbidity and mortality globally, especially in low- and middle-income countries. High-throughput and low-cost approaches to identify etiologic agents are needed to guide public health mitigation. Nanoliter-qPCR (nl-qPCR) is an attractive alternative to more expensive methods yet is nascent in application and without a proof-of-concept among hospitalized patients. METHODS: A census-based study was conducted among diarrheal patients admitted at two government hospitals in rural Bangladesh during a diarrheal outbreak period. DNA was extracted from stool samples and assayed by nl-qPCR for common bacterial, protozoan, and helminth enteropathogens as the primary outcome. RESULTS: A total of 961 patients were enrolled; stool samples were collected from 827 patients. Enteropathogens were detected in 69% of patient samples; More than one enteropathogen was detected in 32%. Enteropathogens most commonly detected were enteroaggregative Escherichia coli (26.0%), Shiga toxin-producing E.coli (18.3%), enterotoxigenic E. coli (15.5% heat stable toxin positive, 2.2% heat labile toxin positive), Shigella spp. (14.8%), and Vibrio cholerae (9.0%). Geospatial analysis revealed that the median number of pathogens per patient and the proportion of cases presenting with severe dehydration were greatest amongst patients residing closest to the study hospitals." CONCLUSIONS: This study demonstrates a proof-of-concept for nl-qPCR as a high-throughput low-cost method for enteropathogen detection among hospitalized patients.

Mycobacterium tuberculosis precursor rRNA as a measure of treatment-shortening activity of drugs and regimens.

There is urgent need for new drug regimens that more rapidly cure tuberculosis (TB). Existing TB drugs and regimens vary in treatment-shortening activity, but the molecular basis of these differences is unclear, and no existing assay directly quantifies the ability of a drug or regimen to shorten treatment. Here, we show that drugs historically classified as sterilizing and non-sterilizing have distinct impacts on a fundamental aspect of Mycobacterium tuberculosis physiology: ribosomal RNA (rRNA) synthesis. In culture, in mice, and in human studies, measurement of precursor rRNA reveals that sterilizing drugs and highly effective drug regimens profoundly suppress M. tuberculosis rRNA synthesis, whereas non-sterilizing drugs and weaker regimens do not. The rRNA synthesis ratio provides a readout of drug effect that is orthogonal to traditional measures of bacterial burden. We propose that this metric of drug activity may accelerate the development of shorter TB regimens.