Diagnostic accuracy of tongue swab testing on two automated tuberculosis diagnostic platforms, Cepheid Xpert MTB/RIF Ultra and Molbio Truenat MTB Ultima.

Tongue dorsum swabbing is a potential alternative to sputum collection for tuberculosis (TB) testing. Previous studies showed that Cepheid Xpert MTB/RIF Ultra (Xpert Ultra) can detect Mycobacterium tuberculosis DNA on tongue swabs stored in buffer, with 72% sensitivity and 100% specificity relative to a sputum microbiological reference standard (sputum MRS). The present study evaluated a more convenient sample collection protocol (dry swab storage), combined with streamlined sample processing protocols, for evaluating two commercial TB diagnostic tests: Xpert Ultra and Molbio Truenat MTB Ultima (MTB Ultima). Copan FLOQSwabs were self-collected or collected by study workers from 321 participants in Western Cape, South Africa. All participants had symptoms suggestive of TB, and 245 of them had sputum MRS-confirmed TB (by sputum MGIT culture and/or Xpert Ultra). One tongue swab per participant was tested on Xpert Ultra, and another tongue swab was tested with MTB Ultima. Xpert Ultra was 75.5% sensitive and 100% specific relative to sputum MRS, similar to previous methods that used swabs stored in buffer. MTB Ultima was 71.6% sensitive and 96.9% specific relative to sputum MRS. When sample lysates that were false-negative or invalid by MTB Ultima were frozen, thawed, and re-tested, MTB Ultima sensitivity rose to 79.1%. Both tests were more sensitive with swabs from participants with higher sputum Xpert Ultra semi-quantitative results. Although additional development could improve diagnostic accuracy, these results further support tongue swabs as easy-to-collect samples for TB testing. IMPORTANCE: Tongue dorsum swabbing is a promising alternative to sputum collection for tuberculosis (TB) testing. Our results lend further support for tongue swabs as exceptionally easy-to-collect samples for high-throughput TB testing.

Complementary Nonsputum Diagnostic Testing for Tuberculosis in People with HIV Using Oral Swab PCR and Urine Lipoarabinomannan Detection.

Testing for mycobacterial lipoarabinomannan (LAM) in urine is a practical but insensitive alternative to sputum testing to diagnose tuberculosis (TB) in people with HIV (PWH). Here, we evaluated urine LAM testing alongside PCR-based tests for Mycobacterium tuberculosis (MTB) DNA in tongue swabs. We hypothesized that the two nonsputum samples would deliver complementary, not redundant, results. The study included 131 South African patients of whom 64 (48.1%) were confirmed to have TB by GeneXpert MTB/RIF Ultra (Xpert Ultra) or culture analysis of sputum. A total of 120 patients (91.6%) were coinfected with HIV and 130 yielded a valid urine LAM result (Alere DETERMINE LAM Ag). Tongue swab samples were tested by IS6110-targeted qPCR with a quantification cycle (Cq) cutoff of 32. Relative to reference sputum testing (TB culture and Xpert Ultra), combined urine LAM and oral swab testing (either sample positive) was significantly more sensitive than either nonsputum sample alone (57% sensitivity for combined testing versus 35% and 39% sensitivity for urine LAM and tongue swabs; P = 0.01 and 0.04, respectively). Specificity of combined testing (neither sample positive) was 97%. On average, tongue swab-positive participants had higher sputum signal strength than urine-LAM positive participants, as measured by sputum Xpert Ultra Cq value (P = 0.037). A subset of tongue swabs (N = 18) was also tested by using Xpert Ultra, which reproduced true positive and true negative IS6110 qPCR results and resolved the two false-positive tongue swabs. With further development, tongue swabs and urine may feasibly serve as complementary nonsputum samples for diagnosis of TB in PWH.

Noninvasive Detection of Tuberculosis by Oral Swab Analysis.

Diagnostic tests for tuberculosis (TB) usually require collection of sputum, a viscous material derived from human airways. Sputum can be difficult and hazardous to collect and challenging to process in the laboratory. Oral swabs have been proposed as alternative sample types that are noninvasive and easy to collect. This study evaluated the biological feasibility of oral swab analysis (OSA) for the diagnosis of TB. Swabs were tested from South African adult subjects, including sputum GeneXpert MTB/RIF (GeneXpert)-confirmed TB patients (n = 138), sputum GeneXpert-negative but culture-positive TB patients (n = 10), ill non-TB patients (n = 37), and QuantiFERON-negative controls (n = 34). Swabs were analyzed by using a manual, nonnested quantitative PCR (qPCR) targeting IS6110 Two swab brands and three sites within the oral cavity were compared. Tongue swabbing yielded significantly stronger signals than cheek or gum swabbing. A flocked swab performed better than a more expensive paper swab. In a two-phase study, tongue swabs (two per subject) exhibited a combined sensitivity of 92.8% relative to sputum GeneXpert. Relative to all laboratory-diagnosed TB, the diagnostic yields of sputum GeneXpert (1 sample per subject) and OSA (2 samples per subject) were identical at 49/59 (83.1%) each. The specificity of the OSA was 91.5%. An analysis of "air swabs" suggested that most false-positive results were due to contamination of manual PCRs. With the development of appropriate automated methods, oral swabs could facilitate TB diagnosis in clinical settings and patient populations that are limited by the physical or logistical challenges of sputum collection.

PBDEs Altered Gut Microbiome and Bile Acid Homeostasis in Male C57BL/6 Mice.

Polybrominated diphenyl ethers (PBDEs) are persistent environmental contaminants with well characterized toxicities in host organs. Gut microbiome is increasingly recognized as an important regulator of xenobiotic biotransformation; however, little is known about its interactions with PBDEs. Primary bile acids (BAs) are metabolized by the gut microbiome into more lipophilic secondary BAs that may be absorbed and interact with certain host receptors. The goal of this study was to test our hypothesis that PBDEs cause dysbiosis and aberrant regulation of BA homeostasis. Nine-week-old male C57BL/6 conventional (CV) and germ-free (GF) mice were orally gavaged with corn oil (10 mg/kg), BDE-47 (100 µmol/kg), or BDE-99 (100 µmol/kg) once daily for 4 days (n = 3-5/group). Gut microbiome was characterized using 16S rRNA sequencing of the large intestinal content in CV mice. Both BDE-47 and BDE-99 profoundly decreased the alpha diversity of gut microbiome and differentially regulated 45 bacterial species. Both PBDE congeners increased Akkermansia muciniphila and Erysipelotrichaceae Allobaculum spp., which have been reported to have anti-inflammatory and antiobesity functions. Targeted metabolomics of 56 BAs was conducted in serum, liver, and small and large intestinal content of CV and GF mice. BDE-99 increased many unconjugated BAs in multiple biocompartments in a gut microbiota-dependent manner. This correlated with an increase in microbial 7α-dehydroxylation enzymes for secondary BA synthesis and increased expression of host intestinal transporters for BA absorption. Targeted proteomics showed that PBDEs downregulated host BA-synthesizing enzymes and transporters in livers of CV but not GF mice. In conclusion, there is a novel interaction between PBDEs and the endogenous BA-signaling through modification of the "gut-liver axis".

Molecular Viability Testing of UV-Inactivated Bacteria.

PCR is effective in detecting bacterial DNA in samples, but it is unable to differentiate viable bacteria from inactivated cells or free DNA fragments. New PCR-based analytical strategies have been developed to address this limitation. Molecular viability testing (MVT) correlates bacterial viability with the ability to rapidly synthesize species-specific rRNA precursors (pre-rRNA) in response to brief nutritional stimulation. Previous studies demonstrated that MVT can assess bacterial inactivation by chlorine, serum, and low-temperature pasteurization. Here, we demonstrate that MVT can detect inactivation of Escherichia coli, Aeromonas hydrophila, and Enterococcus faecalis cells by UV irradiation. Some UV-inactivated E. coli cells transiently retained the ability to synthesize pre-rRNA postirradiation (generating false-positive MVT results), but this activity ceased within 1 h following UV exposure. Viable but transiently undetectable (by culture) E. coli cells were consistently detected by MVT. An alternative viability testing method, viability PCR (vPCR), correlates viability with cell envelope integrity. This method did not distinguish viable bacteria from UV-inactivated bacteria under some conditions, indicating that the inactivated cells retained intact cell envelopes. MVT holds promise as a means to rapidly assess microbial inactivation by UV treatment.IMPORTANCE UV irradiation is increasingly being used to disinfect water, food, and other materials for human use. Confirming the effectiveness of UV disinfection remains a challenging task. In particular, microbiological methods that rely on rapid detection of microbial DNA can yield misleading results, due to the detection of remnant DNA associated with dead microbial cells. This report describes a novel method that rapidly distinguishes living microbial cells from dead microbial cells after UV disinfection.

Dielectrophoretic characterization of antibiotic-treated Mycobacterium tuberculosis complex cells.

Multi-drug resistant tuberculosis (MDR-TB) has become a serious concern for proper treatment of patients. As a phenotypic method, dielectrophoresis can be useful but is yet to be attempted to evaluate Mycobacterium tuberculosis complex cells. This paper investigates the dielectrophoretic behavior of Mycobacterium bovis (Bacillus Calmette-Guérin, BCG) cells that are treated with heat or antibiotics rifampin (RIF) or isoniazid (INH). The experimental parameters are designed on the basis of our sensitivity analysis. The medium conductivity (σ(m)) and the frequency (f) for a crossover frequency (f(xo1)) test are decided to detect the change of σ(m)-f(xo1) in conjunction with the drug mechanism. Statistical modeling is conducted to estimate the distributions of viable and nonviable cells from the discrete measurement of f (xo1). Finally, the parameters of the electrophysiology of BCG cells, C(envelope) and σ(cyto), are extracted through a sampling algorithm. This is the first evaluation of the dielectrophoresis (DEP) approach as a means to assess the effects of antimicrobial drugs on M. tuberculosis complex cells.

Amperometric immunosensor for rapid detection of Mycobacterium tuberculosis.

Tuberculosis (TB) has been a major public health problem, which can be better controlled by using accurate and rapid diagnosis in low-resource settings. A simple, portable, and sensitive detection method is required for point-of-care (POC) settings. This paper studies an amperometric biosensor using a microtip immunoassay for a rapid and low cost detection of Mycobacterium Tuberculosis (MTB) in sputum. MTB in sputum is specifically captured on the functionalized microtip surface and detected by electric current. According to the numerical study, the current signal on microtip surface is linearly changed with increasing immersion depth. Using a reference microtip, the immersion depth is compensated for a sensing microtip. On the microtip surface, target bacteria are concentrated and organized by a coffee ring effect, which amplifies the electric current. To enhance the signal-to-noise ratio, both the sample processing- and rinsing steps are presented with use of deionized water as a medium for the amperometric measurement. When applied to cultured MTB cells spiked into human sputum, the detection limit was 100 CFU/mL, comparable to a more labor-intensive fluorescence detection method reported previously.

Detection of Mycobacterium tuberculosis from tongue swabs using sonication and sequence-specific hybridization capture.

Tongue swabs hold promise as a non-invasive sample for diagnosing tuberculosis (TB). However, their utility as replacements for sputum has been limited by their varied diagnostic performance in PCR assays compared to sputum. The use of silica-based DNA extraction methods may limit sensitivity due to incomplete lysis of Mycobacterium tuberculosis (MTB) cells and co-extraction of non-target nucleic acid, which may inhibit PCR. Specificity may also be compromised because these methods are labor-intensive and prone to cross-contamination. To address these limitations, we developed a sample preparation method that combines sonication for MTB lysis and a sequence-specific MTB DNA capture method using hybridization probes immobilized on magnetic beads. In spiked tongue swabs, our hybridization capture method demonstrated a 100-fold increase in MTB DNA yield over silica-based Qiagen DNA extraction and ethanol precipitation. In a study conducted on clinical samples from South Africa, our protocol had 74% (70/94) sensitivity and 98% (41/42) specificity for detecting active pulmonary TB with sputum Xpert MTB/RIF Ultra as the reference standard. While hybridization capture did not show improved sensitivity over Qiagen DNA extraction and ethanol precipitation, it demonstrated better specificity than previously reported methods and was easier to perform. With integration into point-of-care platforms, these strategies have the potential to help enable rapid non-sputum-based TB diagnosis across key underserved patient populations.

Molecular detection of pre-ribosomal RNAs of Mycobacterium bovis bacille Calmette-Guérin and Mycobacterium tuberculosis to enhance pre-clinical tuberculosis drug and vaccine development.

Efforts are underway globally to develop effective vaccines and drugs against M. tuberculosis (Mtb) to reduce the morbidity and mortality of tuberculosis. Improving detection of slow-growing mycobacteria could simplify and accelerate efficacy studies of vaccines and drugs in animal models and human clinical trials. Here, a real-time reverse transcription PCR (RT-PCR) assay was developed to detect pre-ribosomal RNA (pre-rRNA) of Mycobacterium bovis bacille Calmette-Guérin (BCG) and Mtb. This pre-rRNA biomarker is indicative of bacterial viability. In two different mouse models, the presence of pre-rRNA from BCG and Mtb in ex vivo tissues showed excellent agreement with slower culture-based colony-forming unit assays. The addition of a brief nutritional stimulation prior to molecular viability testing further differentiated viable but dormant mycobacteria from dead mycobacteria. This research has set the stage to evaluate pre-rRNA as a BCG and/or Mtb infection biomarker in future drug and vaccine clinical studies.

High-sensitivity detection of Mycobacterium tuberculosis DNA in tongue swab samples.

Tongue swab (TS) sampling combined with qPCR to detect Mycobacterium tuberculosis (MTB) DNA is a promising alternative to sputum testing for tuberculosis (TB) diagnosis. In prior studies, the sensitivity of tongue swabbing has usually been lower than sputum. In this study, we evaluated two strategies to improve sensitivity. In one, centrifugation was used to concentrate tongue dorsum bacteria from 2-mL suspensions eluted from high-capacity foam swab samples. The pellets were resuspended as 500-µL suspensions, and then mechanically lysed prior to dual-target qPCR to detect MTB insertion elements IS6110 and IS1081. Fractionation experiments demonstrated that most of the MTB DNA signal in clinical swab samples (99.22% ± 1.46%) was present in the sedimentable fraction. When applied to archived foam swabs collected from 124 South Africans with presumptive TB, this strategy exhibited 83% sensitivity (71/86) and 100% specificity (38/38) relative to sputum MRS (microbiological reference standard; sputum culture and/or Xpert® Ultra). The second strategy used sequence-specific magnetic capture (SSMaC) to concentrate DNA released from MTB cells. This protocol was evaluated on archived Copan FLOQSwabs® flocked swab samples collected from 128 South African participants with presumptive TB. Material eluted into 500 µL buffer was mechanically lysed. The suspensions were digested by proteinase K, hybridized to biotinylated dual-target oligonucleotide probes, and then concentrated ~20-fold using magnetic separation. Upon dual-target qPCR testing of concentrates, this strategy exhibited 90% sensitivity (83/92) and 97% specificity (35/36) relative to sputum MRS. These results point the way toward automatable, high-sensitivity methods for detecting MTB DNA in TS. Importance: Improved testing for tuberculosis (TB) is needed. Using a more accessible sample type than sputum may enable the detection of more cases, but it is critical that alternative samples be tested appropriately. Here, we describe two new, highly accurate methods for testing tongue swabs for TB DNA.

Shared Mycobacterium avium genotypes observed among unlinked clinical and environmental isolates.

Our understanding of the sources of Mycobacterium avium infection is partially based on genotypic matching of pathogen isolates from cases and environmental sources. These approaches assume that genotypic identity is rare in isolates from unlinked cases or sources. To test this assumption, a high-resolution PCR-based genotyping approach, large-sequence polymorphism (LSP)-mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR), was selected and used to analyze clinical and environmental isolates of M. avium from geographically diverse sources. Among 127 clinical isolates from seven locations in North America, South America, and Europe, 42 genotypes were observed. Among 12 of these genotypes, matches were seen in isolates from apparently unlinked patients in two or more geographic locations. Six of the 12 were also observed in environmental isolates. A subset of these isolates was further analyzed by alternative strain genotyping methods, pulsed-field gel electrophoresis and MIRU-VNTR, which confirmed the existence of geographically dispersed strain genotypes. These results suggest that caution should be exercised in interpreting high-resolution genotypic matches as evidence for an acquisition event.

Tongue swab testing on two automated tuberculosis diagnostic platforms, Cepheid Xpert® MTB/RIF Ultra and Molbio Truenat® MTB Ultima.

Tongue dorsum swabbing is a potential alternative to sputum collection for tuberculosis (TB) testing. Previous studies showed that Cepheid Xpert® MTB/RIF Ultra (Xpert Ultra) can detect Mycobacterium tuberculosis (MTB) DNA in tongue swabs stored in buffer, with 72% sensitivity and 100% specificity relative to a sputum microbiological reference standard (sputum MRS). The present study evaluated a more convenient sample collection protocol (dry swab storage), combined with streamlined sample processing protocols, for side-by-side analysis using two commercial TB diagnostic tests: Xpert Ultra and Molbio Truenat® MTB Ultima (MTB Ultima). Copan FLOQSwabs were self-collected, or collected by study workers, from 321 participants in Western Cape, South Africa. All participants had symptoms suggestive of TB, and 245 of them had sputum MRS-confirmed TB (by sputum culture and/or Xpert Ultra). One tongue swab per participant was tested on Xpert Ultra and another tongue swab was tested with MTB Ultima. Xpert Ultra was 75.4% sensitive and 100% specific, and MTB Ultima was 71.6% sensitive and 96.9% specific, relative to sputum MRS. When sample lysates that were false-negative by MTB Ultima were frozen, thawed, and re-tested, MTB Ultima sensitivity rose to 79.1%. Both tests were more sensitive with swabs from participants with higher sputum Xpert semi-quantitative results. The protocol for Xpert Ultra enabled fast and easy testing of dry-stored swabs with no loss of accuracy relative to previous methods. MTB Ultima testing of dry-stored swabs exhibited comparable performance to Xpert Ultra. These results further support tongue swabs as easy-to-collect samples for high-throughput TB testing.

Cryopreservation of Mycobacterium tuberculosis complex cells.

Successful long-term preservation of Mycobacterium tuberculosis cells is important for sample transport, research, biobanking, and the development of new drugs, vaccines, biomarkers, and diagnostics. In this report, Mycobacterium bovis bacillus Calmette-Guérin and M. tuberculosis H37Ra were used as models of M. tuberculosis complex strains to study cryopreservation of M. tuberculosis complex cells in diverse sample matrices at different cooling rates. Cells were cryopreserved in diverse sample matrices, namely, phosphate-buffered saline (PBS), Middlebrook 7H9 medium with or without added glycerol, and human sputum. The efficacy of cryopreservation was quantified by microbiological culture and microscopy with BacLight LIVE/DEAD staining. In all sample matrices examined, the microbiological culture results showed that the cooling rate was the most critical factor influencing cell viability. Slow cooling (a few degrees Celsius per minute) resulted in much higher M. tuberculosis complex recovery rates than rapid cooling (direct immersion in liquid nitrogen) (P < 0.05). Among the three defined cryopreservation media (PBS, 7H9, and 7H9 plus glycerol), there was no significant differential effect on viability (P = 0.06 to 0.87). Preincubation of thawed M. tuberculosis complex cells in 7H9 broth for 20 h before culture on solid Middlebrook 7H10 plates did not help the recovery of the cells from cryoinjury (P = 0.14 to 0.71). The BacLight LIVE/DEAD staining kit, based on Syto 9 and propidium iodide (PI), was also applied to assess cell envelope integrity after cryopreservation. Using the kit, similar percentages of "live" cells with intact envelopes were observed for samples cryopreserved under different conditions, which was inconsistent with the microbiological culture results. This implies that suboptimal cryopreservation might not cause severe damage to the cell wall and/or membrane but instead cause intracellular injury, which leads to the loss of cell viability.

Understanding the physiological functions of the host xenobiotic-sensing nuclear receptors PXR and CAR on the gut microbiome using genetically modified mice.

Pharmacological activation of the xenobiotic-sensing nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) is well-known to increase drug metabolism and reduce inflammation. Little is known regarding their physiological functions on the gut microbiome. In this study, we discovered bivalent hormetic functions of PXR/CAR modulating the richness of the gut microbiome using genetically engineered mice. The absence of PXR or CAR increased microbial richness, and absence of both receptors synergistically increased microbial richness. PXR and CAR deficiency increased the pro-inflammatory bacteria Helicobacteraceae and Helicobacter. Deficiency in both PXR and CAR increased the relative abundance of Lactobacillus, which has bile salt hydrolase activity, corresponding to decreased primary taurine-conjugated bile acids (BAs) in feces, which may lead to higher internal burden of taurine and unconjugated BAs, both of which are linked to inflammation, oxidative stress, and cytotoxicity. The basal effect of PXR/CAR on the gut microbiome was distinct from pharmacological and toxicological activation of these receptors. Common PXR/CAR-targeted bacteria were identified, the majority of which were suppressed by these receptors. hPXR-TG mice had a distinct microbial profile as compared to wild-type mice. This study is the first to unveil the basal functions of PXR and CAR on the gut microbiome.

Chronic exposure to ambient traffic-related air pollution (TRAP) alters gut microbial abundance and bile acid metabolism in a transgenic rat model of Alzheimer's disease.

Background: Traffic-related air pollution (TRAP) is linked to increased risk for age-related dementia, including Alzheimer's disease (AD). The gut microbiome is posited to influence AD risk, and an increase in microbial-derived secondary bile acids (BAs) is observed in AD patients. We recently reported that chronic exposure to ambient TRAP modified AD risk in a sex-dependent manner in the TgF344 AD (TG) rat. Objectives: In this study, we used samples from the same cohort to test our hypothesis that TRAP sex-dependently produces gut dysbiosis and increases secondary BAs to a larger extent in the TG rat relative to wildtype (WT) controls. Methods: Male and female TG and age-matched WT rats were exposed to either filtered air (FA) or TRAP from 28 days up to 15 months of age (n = 5-6). Tissue samples were collected after 9 or 14months of exposure. Results: At 10 months of age, TRAP tended to decrease the alpha diversity as well as the beneficial taxa Lactobacillus and Ruminococcus flavefaciens uniquely in male TG rats as determined by 16 S rDNA sequencing. A basal decrease in Firmicutes/Bacteroidetes (F/B) ratio was also noted in TG rats at 10 months. At 15 months of age, TRAP altered inflammation-related bacteria in the gut of female rats from both genotypes. BAs were more affected by chronic TRAP exposure in females, with a general trend of increase in host-produced unconjugated primary and microbiota-produced secondary BAs. Most of the mRNAs of the hepatic BA-processing genes were not altered by TRAP, except for a down-regulation of the BA-uptake transporter Ntcp in males. Conclusion: In conclusion, chronic TRAP exposure produced distinct gut dysbiosis and altered BA homeostasis in a sex and host genotype-specific manner.

Mechanical disruption of lysis-resistant bacterial cells by use of a miniature, low-power, disposable device.

Molecular detection of microorganisms requires microbial cell disruption to release nucleic acids. Sensitive detection of thick-walled microorganisms such as Bacillus spores and Mycobacterium cells typically necessitates mechanical disruption through bead beating or sonication, using benchtop instruments that require line power. Miniaturized, low-power, battery-operated devices are needed to facilitate mechanical pathogen disruption for nucleic acid testing at the point of care and in field settings. We assessed the lysis efficiency of a very small disposable bead blender called OmniLyse relative to the industry standard benchtop Biospec Mini-BeadBeater. The OmniLyse weighs approximately 3 g, at a size of approximately 1.1 cm(3) without the battery pack. Both instruments were used to mechanically lyse Bacillus subtilis spores and Mycobacterium bovis BCG cells. The relative lysis efficiency was assessed through real-time PCR. Cycle threshold (C(T)) values obtained at all microbial cell concentrations were similar between the two devices, indicating that the lysis efficiencies of the OmniLyse and the BioSpec Mini-BeadBeater were comparable. As an internal control, genomic DNA from a different organism was spiked at a constant concentration into each sample upstream of lysis. The C(T) values for PCR amplification of lysed samples using primers specific to this internal control were comparable between the two devices, indicating negligible PCR inhibition or other secondary effects. Overall, the OmniLyse device was found to effectively lyse tough-walled organisms in a very small, disposable, battery-operated format, which is expected to facilitate sensitive point-of-care nucleic acid testing.

Steady-State Pre-rRNA Analysis to Investigate the Functional Microbiome.

The gut microbiome is recognized as a critical regulator of human diseases. Constituents of the microbiota and their individual activities can affect a broad range of disease states related to autoimmunity, cancer, infection, metabolism, mental health, and toxicant exposure. A substantial number of microbiome species are not culturable, limiting their study in vitro. Sequencing methods have allowed quantification of the composition of the microbiome, but methods to characterize the physiological status of bacterial species remain limited. Ribosomal RNA precursors (pre-rRNAs) are species-specific intermediates in bacterial ribosomal synthesis, and their levels are highly responsive to environmental changes. Immediately before and during active growth, pre-rRNA levels are high, whereas in non-dividing cells, copy numbers are orders of magnitude lower. These dynamics are conserved in all bacterial species and occur exclusively in viable cells, allowing the specific characterization of living and functional bacteria in their native states. Pre-rRNA analysis has been shown to yield valuable real-time information on the physiology of individual bacterial species within complex samples, beyond what traditional qPCR and sequencing methods can offer. Herein, we describe a PCR-based protocol to interrogate and quantify the in situ growth status of bacterial species of interest within a complex microbiome. We also describe an in vitro protocol to characterize the pre-rRNA/growth relationship for a given bacterial species to provide greater context for values obtained from natural samples. Improved understanding of microbial physiological responses to exposures could reveal novel toxicological mechanisms, biomarkers, and potential treatments. © 2021 Wiley Periodicals LLC. Basic Protocol: Targeted steady-state pre-rRNA analysis Support Protocol: Characterization of pre-rRNA/growth relationship © 2021 by John Wiley & Sons, Inc.

Characterization of oral swab samples for diagnosis of pulmonary tuberculosis.

Oral swab analysis (OSA) has been shown to detect Mycobacterium tuberculosis (MTB) DNA in patients with pulmonary tuberculosis (TB). In previous analyses, qPCR testing of swab samples collected from tongue dorsa was up to 93% sensitive relative to sputum GeneXpert, when 2 swabs per patient were tested. The present study modified sample collection methods to increase sample biomass and characterized the viability of bacilli present in tongue swabs. A qPCR targeting conserved bacterial ribosomal rRNA gene (rDNA) sequences was used to quantify bacterial biomass in samples. There was no detectable reduction in total bacterial rDNA signal over the course of 10 rapidly repeated tongue samplings, indicating that swabs collect only a small portion of the biomass available for testing. Copan FLOQSwabs collected ~2-fold more biomass than Puritan PurFlock swabs, the best brand used previously (p = 0.006). FLOQSwabs were therefore evaluated in patients with possible TB in Uganda. A FLOQSwab was collected from each patient upon enrollment (Day 1) and, in a subset of sputum GeneXpert Ultra-positive patients, a second swab was collected on the following day (Day 2). Swabs were tested for MTB DNA by manual IS6110-targeted qPCR. Relative to sputum GeneXpert Ultra, single-swab sensitivity was 88% (44/50) on Day 1 and 94.4% (17/18) on Day 2. Specificity was 79.2% (42/53). Among an expanded sample of Ugandan patients, 62% (87/141) had colony-forming bacilli in their tongue dorsum swab samples. These findings will help guide further development of this promising TB screening method.

Molecular detection of viable bacterial pathogens in water by ratiometric pre-rRNA analysis.

Ratiometric pre-rRNA analysis (RPA) detects the replenishment of rRNA precursors that occurs rapidly upon nutritional stimulation of bacterial cells. In contrast to DNA detection by PCR, RPA distinguishes viable from inactivated bacteria. It exhibits promise as a molecular viability test for pathogens in water and other environmental samples.

Flow cytometry-based methods for assessing soluble scFv activities and detecting antigens in solution.

Novel methods are reported for evaluating and utilizing single chain fragment variable (scFv) antibodies derived from yeast-display libraries. Yeast-display was used to select scFv specific to invariant surface glycoproteins (ISG) of Trypanosoma brucei. A limiting step in the isolation of scFv from non-immune libraries is the conversion of highly active yeast-displayed scFv into soluble antibodies that can be used in standard immunoassays. Challenges include limited solubility or activity following secretion and purification of scFv. For this reason, few scFv derived from yeast-display platforms have moved into development and implementation as diagnostic reagents. To address this problem, assays were developed that employ both yeast-displayed and -secreted scFv as analytical reagents. The first is a competitive inhibition flow cytometry (CIFC) assay that detects secreted scFv by virtue of their ability to competitively inhibit the binding of biotinylated antigen to yeast-displayed scFv. The second is an epitope binning assay that uses secreted scFv to identify additional yeast-displayed scFv that bind non-overlapping or non-competing epitopes on an antigen. The epitope binning assay was used not only to identify sandwich assay pairs with yeast-displayed scFv, but also to identify active soluble scFv present in low concentration in a crude expression extract. Finally, a CIFC assay was developed that bypasses entirely the need for soluble scFv expression, by using yeast-displayed scFv to detect unlabeled antigen in samples. These methods will facilitate the continued development and practical implementation of scFv derived from yeast-display libraries.