Genomic epidemiology and evolution of rhinovirus in western Washington State, 2021-22.

BACKGROUND: Human rhinoviruses (RV) primarily cause the common cold, but infection outcomes vary from subclinical to severe cases, including asthma exacerbations and fatal pneumonia in immunocompromised individuals. To date, therapeutic strategies have been hindered by the high diversity of serotypes. Global surveillance efforts have traditionally focused on sequencing VP1 or VP2/VP4 genetic regions, leaving gaps in our understanding of RV genomic diversity. METHODS: We sequenced 1,078 RV genomes from nasal swabs of symptomatic and asymptomatic individuals to explore viral evolution during two epidemiologically distinct periods in Washington State: when the COVID-19 pandemic affected the circulation of other seasonal respiratory viruses except for RV (February - July 2021), and when the seasonal viruses reemerged with the severe RSV and influenza outbreak (November-December 2022). We constructed maximum likelihood and BEAST-phylodynamic trees to characterize intra-genotype evolution. RESULTS: We detected 99 of 168 known genotypes and observed inter-genotypic recombination and genotype cluster swapping from 2021 to 2022. We found a significant association between the presence of symptoms and viral load, but not with RV species or genotype. Phylodynamic trees, polyprotein selection pressure, and Shannon entropy revealed co-circulation of divergent clades within genotypes with high amino acid constraints throughout polyprotein. DISCUSSION: Our study underscores the dynamic nature of RV genomic epidemiology within a localized geographic region, as more than 20% of existing genotypes within each RV species co-circulated each studied month. Our findings also emphasize the importance of investigating correlations between rhinovirus genotypes and serotypes to understand long-term immunity and cross-protection.

Human Herpesvirus-6 Reactivation and Disease Are Infrequent in Chimeric Antigen Receptor T-cell Therapy Recipients.

Human herpesvirus-6B (HHV-6B) reactivation and disease are increasingly reported after CAR-T-cell therapy (CARTx). HHV-6 reactivation in the CAR-T-cell product was recently reported, raising questions about product and patient management. Due to overlapping manifestations with immune effector cell-associated neurotoxicity syndrome, diagnosing HHV-6B encephalitis is challenging. We provide two lines of evidence assessing the incidence and outcomes of HHV-6B after CARTx. First, in a prospective study with weekly HHV-6B testing for up to 12 weeks post-infusion, HHV-6B reactivation occurred in eight of 89 participants; three had chromosomally integrated HHV-6 and were excluded, resulting in a cumulative incidence of HHV-6B reactivation of 6% (95% confidence interval (CI), 2.2-12.5%). HHV-6B detection was low level (median peak, 435 copies/mL; IQR, 164-979) and did not require therapy. Second, we retrospectively analyzed HHV-6B detection in blood and/or cerebrospinal fluid (CSF) within 12 weeks post-infusion in CARTx recipients. Of 626 patients, 24 had symptom-driven plasma testing with detection in one. Among 34 patients with CSF HHV-6 testing, one patient had possible HHV-6 encephalitis for a cumulative incidence of 0.17% (95% CI, 0.02-0.94%), although symptoms improved without treatment. Our data demonstrate that HHV-6B reactivation and disease are infrequent after CARTx. Routine HHV-6 monitoring is not warranted.

Hybrid Immunity to SARS-CoV-2 During Pregnancy Provides More Durable Infant Antibody Responses Compared to Natural Infection Alone.

BACKGROUND: Hybrid immunity (infection plus vaccination) may increase maternally derived SARS-CoV-2 antibody responses and durability versus infection alone. METHODS: Prospective cohort of pregnant participants with prior SARS-CoV-2 infection (anti-nucleocapsid IgG, RT-PCR, or antigen positive) and their infants had blood collected in pregnancy, at delivery/birth, and postpartum tested for anti-spike (anti-S) IgG and neutralizing antibodies (neutAb). RESULTS: Among 107 participants at enrollment, 40% were unvaccinated and 60% were vaccinated (received ≥1 dose); 102 had previous SARS-CoV-2 infection in pregnancy (median, 19 weeks' gestation); 5 were diagnosed just prior to pregnancy (median, 8 weeks). At delivery, fewer unvaccinated participants (87% anti-S IgG+, 86% neutAb) and their infants (86% anti-S IgG+, 75% neutAb) had anti-S IgG+ or neutAb compared to vaccinated participants and their infants (100%, P ≤ .01 for all). By 3-6 months postpartum, 50% of infants of unvaccinated participants were anti-S IgG+ and 14% had neutAb, versus 100% among infants of vaccinated participants (all P < .01), with lower median antibody responses (anti-S IgG log10 1.95 vs 3.84 AU/mL, P < .01; neutAb log10 1:1.34 vs 1:3.20, P = .11). CONCLUSIONS: In pregnant people with prior SARS-CoV-2, vaccination before delivery provided more durable maternally derived antibody responses than infection alone in infants through 6 months.

Evaluation and clinical validation of monkeypox (mpox) virus real-time PCR assays.

BACKGROUND: In spring of 2022, an outbreak of monkeypox (mpox) spread worldwide. Here, we describe performance characteristics of monkeypox virus (MPXV)-specific and pan-orthopoxvirus qPCR assays for clinical use. METHODS: We validated probe-based qPCR assays targeting MPXV-specific loci F3L and G2R (genes MPXVgp052/OPG065 and MPXVgp002 and gp190/OPG002, respectively) and a pan-orthopoxvirus assay targeting the E9L locus (MPXVgp057/OPG071). Clinical samples and synthetic controls were extracted using the Roche MP96 or Promega Maxwell 48 instrument. qPCR was performed on the AB7500 thermocycler. Synthetic control DNA and high concentration clinical samples were quantified by droplet PCR. Cross-reactivity was evaluated for camelpox and cowpox genomic DNA, vaccinia culture supernatant, and HSV- and VZV-positive clinical specimens. We also tested the performance of the F3L assay using dry swabs, Aptima vaginal and rectal swabs, nasopharyngeal, rectal, and oral swabs, cerebrospinal fluid, plasma, serum, whole blood, breastmilk, urine, saliva, and semen. RESULTS: The MPXV-F3L assay is reproducible at a limit of detection (LoD) of 65.6 copies/mL of viral DNA in viral transport medium/universal transport medium (VTM/UTM), or 3.3 copies/PCR reaction. No cross-reactivity with herpesviruses or other poxviruses was observed. MPXV-F3L detects MPXV DNA in alternative specimen types, with an LoD ranging between 260-1000 copies/mL, or 5.7-10 copies/PCR reaction. In clinical swab VTM specimens, MPXV-F3L and MPXV-G2R assays outperformed OPXV-E9L by an average of 2.4 and 2.8 Cts, respectively. MPXV-G2R outperformed MPXV-F3L by 0.4 Cts, consistent with presence of two copies of G2R present in labile inverted terminal repeats (ITRs) of MPXV genome. CONCLUSIONS: MPXV is readily detected by qPCR using three clinically validated assays.

Repeated introductions and intensive community transmission fueled a mumps virus outbreak in Washington State.

In 2016/2017, Washington State experienced a mumps outbreak despite high childhood vaccination rates, with cases more frequently detected among school-aged children and members of the Marshallese community. We sequenced 166 mumps virus genomes collected in Washington and other US states, and traced mumps introductions and transmission within Washington. We uncover that mumps was introduced into Washington approximately 13 times, primarily from Arkansas, sparking multiple co-circulating transmission chains. Although age and vaccination status may have impacted transmission, our data set could not quantify their precise effects. Instead, the outbreak in Washington was overwhelmingly sustained by transmission within the Marshallese community. Our findings underscore the utility of genomic data to clarify epidemiologic factors driving transmission and pinpoint contact networks as critical for mumps transmission. These results imply that contact structures and historic disparities may leave populations at increased risk for respiratory virus disease even when a vaccine is effective and widely used.

COVID-19 Response Efforts of Washington State Public Health Laboratory: Lessons Learned.

Laboratory diagnostics play an essential role in pandemic preparedness. In January 2020, the first US case of COVID-19 was confirmed in Washington State. At the same time, the Washington State Public Health Laboratory (WA PHL) was in the process of building upon and initiating innovative preparedness activities to strengthen laboratory testing capabilities, operations, and logistics. The response efforts of WA PHL, in conjunction with the Centers for Disease Control and Prevention, to the COVID-19 outbreak in Washington are described herein-from the initial detection of severe acute respiratory syndrome coronavirus 2 through the subsequent 2 months.Factors that contributed to an effective laboratory response are described, including preparing early to establish testing capacity, instituting dynamic workforce solutions, advancing information management systems, refining laboratory operations, and leveraging laboratory partnerships. We also report on the challenges faced, successful steps taken, and lessons learned by WA PHL to respond to COVID-19.The actions taken by WA PHL to mount an effective public health response may be useful for US laboratories as they continue to respond to the COVID-19 pandemic and may help inform current and future laboratory pandemic preparedness activities.

Cost of Responding to the 2017 University of Washington Mumps Outbreak: A Prospective Analysis.

OBJECTIVES: To estimate costs of labor and materials by the University of Washington (UW) and state and local public health departments (PHDs) to respond to the February to June 2017 UW mumps outbreak, where 42 cases were identified among students (primarily sorority and fraternity members), staff, and associated community members. DESIGN: We applied standard cost analysis methodology using a combined public health and university perspective to examine the cost of responding to the outbreak. SETTING: UW's Seattle campus encompasses 703 acres with approximately 32 000 undergraduate students. Nearly 15% of the undergraduate population are members of fraternities or sororities. Housing for the fraternities and sororities is adjacent to the UW campus and consists of 50 houses. PARTICIPANTS: During the outbreak, customized costing tools based on relevant staff or faculty positions and activities were provided to the UW and Public Health-Seattle & King County, populated by each person participating in the outbreak response, and then collected and analyzed. Laboratory hours and material costs were collected from the Washington Department of Health and the Minnesota Department of Health. MAIN OUTCOME MEASURE: Labor and material costs provided by the UW and PHDs during the outbreak were collected and categorized by payer and activity. RESULTS: Total costs to the UW and PHDs in responding to the outbreak were $282 762 ($6692 per case). Of these, the UW spent $160 064, while PHDs spent $122 098. Labor accounted for 77% of total outbreak costs, and UW response planning and coordination accounted for the largest amount of labor costs ($75 493) overall. CONCLUSIONS: Given the current university and public health department budget constraints, the response to the outbreak amounted to a significant use of resources. Labor was the largest driver of costs for the outbreak response; UW labor costs-related to campus response planning and coordination-dominated the total economic burden from public health and university perspectives.

Laboratory Review of Foodborne Disease Investigations in Washington State 2007-2017.

The Washington State Department of Health Public Health Laboratories (WAPHL) has tested 11,501 samples between 2007 and 2017 for a foodborne disease using a combination of identification, serotyping, and subtyping tools. During this period there were 8037 total clinical and environmental samples tested by pulsed-field gel electrophoresis (PFGE), including 512 foodborne disease clusters and 2176 PFGE patterns of Salmonella enterica subsp. enterica. There were 2446 Shiga toxin-producing Escherichia coli samples tested by PFGE, which included 158 foodborne disease clusters and 1174 PFGE patterns. There were 332 samples of Listeria monocytogenes tested by PFGE, including 35 foodborne disease clusters and 104 PFGE patterns. Sources linked to outbreaks included raw chicken, unpasteurized dairy products, various produce types, and undercooked beef among others. As next-generation sequencing (NGS) replaces PFGE, the impact of this transition is expected to be significant given the enhanced cluster detection power NGS brings. The measures presented here will be a reference baseline in future years.

Cooperating H3N2 Influenza Virus Variants Are Not Detectable in Primary Clinical Samples.

The high mutation rates of RNA viruses lead to rapid genetic diversification, which can enable cooperative interactions between variants in a viral population. We previously described two distinct variants of H3N2 influenza virus that cooperate in cell culture. These variants differ by a single mutation, D151G, in the neuraminidase protein. The D151G mutation reaches a stable frequency of about 50% when virus is passaged in cell culture. However, it is unclear whether selection for the cooperative benefits of D151G is a cell culture phenomenon or whether the mutation is also sometimes present at appreciable frequency in virus populations sampled directly from infected humans. Prior work has not detected D151G in unpassaged clinical samples, but those studies have used methods like Sanger sequencing and pyrosequencing, which are relatively insensitive to low-frequency variation. We identified nine samples of human H3N2 influenza virus collected between 2013 and 2015 in which Sanger sequencing had detected a high frequency of the D151G mutation following one to three passages in cell culture. We deep sequenced the unpassaged clinical samples to identify low-frequency viral variants. The frequency of D151G did not exceed the frequency of library preparation and sequencing errors in any of the sequenced samples. We conclude that passage in cell culture is primarily responsible for the frequent observations of D151G in recent H3N2 influenza virus strains. IMPORTANCE Viruses mutate rapidly, and recent studies of RNA viruses have shown that related viral variants can sometimes cooperate to improve each other's growth. We previously described two variants of H3N2 influenza virus that cooperate in cell culture. The mutation responsible for cooperation is often observed when human samples of influenza virus are grown in the lab before sequencing, but it is unclear whether the mutation also exists in human infections or is exclusively the result of lab passage. We identified nine human isolates of influenza virus that had developed the cooperating mutation after being grown in the lab and performed highly sensitive deep sequencing of the unpassaged clinical samples to determine whether the mutation existed in the original human infections. We found no evidence of the cooperating mutation in the unpassaged samples, suggesting that the cooperation arises primarily under laboratory conditions.

Conservation and Recombination in the Genome Sequence of Haemophilus influenzae Type f WAPHL1.

We report here the second draft genome sequence of a bloodstream isolate of Haemophilus influenzae serotype f. Three discrete 3.1- to 7.8-kb sites contained 80% of the variability in the genome, consistent with recombination in known virulence factors.

Draft Genome Sequences of Listeria monocytogenes Strains from Listeriosis Outbreaks Linked to Soft Cheese in Washington State.

Listeria monocytogenes has caused listeriosis outbreaks linked to soft cheese. Here, we report the draft genome sequences of seven L. monocytogenes isolates from two possibly related outbreaks caused by soft cheese products in Washington State.

Whole genome sequencing analyses of Listeria monocytogenes that persisted in a milkshake machine for a year and caused illnesses in Washington State.

BACKGROUND: In 2015, in addition to a United States multistate outbreak linked to contaminated ice cream, another outbreak linked to ice cream was reported in the Pacific Northwest of the United States. It was a hospital-acquired outbreak linked to milkshakes, made from contaminated ice cream mixes and milkshake maker, served to patients. Here we performed multiple analyses on isolates associated with this outbreak: pulsed-field gel electrophoresis (PFGE), whole genome single nucleotide polymorphism (SNP) analysis, species-specific core genome multilocus sequence typing (cgMLST), lineage-specific cgMLST and whole genome-specific MLST (wgsMLST)/outbreak-specific cgMLST. We also analyzed the prophages and virulence genes. RESULTS: The outbreak isolates belonged to sequence type 1038, clonal complex 101, genetic lineage II. There were no pre-mature stop codons in inlA. Isolates contained Listeria Pathogenicity Island 1 and multiple internalins. PFGE and multiple whole genome sequencing (WGS) analyses all clustered together food, environmental and clinical isolates when compared to outgroup from the same clonal complex, which supported the finding that L. monocytogenes likely persisted in the soft serve ice cream/milkshake maker from November 2014 to November 2015 and caused 3 illnesses, and that the outbreak strain was transmitted between two ice cream production facilities. The whole genome SNP analysis, one of the two species-specific cgMLST, the lineage II-specific cgMLST and the wgsMLST/outbreak-specific cgMLST showed that L. monocytogenes cells persistent in the milkshake maker for a year formed a unique clade inside the outbreak cluster. This clustering was consistent with the cleaning practice after the outbreak was initially recognized in late 2014 and early 2015. Putative prophages were conserved among prophage-containing isolates. The loss of a putative prophage in two isolates resulted in the loss of the AscI restriction site in the prophage, which contributed to their AscI-PFGE banding pattern differences from other isolates. CONCLUSIONS: The high resolution of WGS analyses allowed the differentiation of epidemiologically unrelated isolates, as well as the elucidation of the microevolution and persistence of isolates within the scope of one outbreak. We applied a wgsMLST scheme which is essentially the outbreak-specific cgMLST. This scheme can be combined with lineage-specific cgMLST and species-specific cgMLST to maximize the resolution of WGS.

The Pseudomonas aeruginosa PAO1 Two-Component Regulator CarSR Regulates Calcium Homeostasis and Calcium-Induced Virulence Factor Production through Its Regulatory Targets CarO and CarP.

UNLABELLED: Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life-threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or artificial implants. During CF pulmonary infections, P. aeruginosa often encounters environments where the levels of calcium (Ca(2+)) are elevated. Previously, we showed that P. aeruginosa responds to externally added Ca(2+) through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca(2+) level, followed by its removal to the basal submicromolar level. However, the molecular mechanisms responsible for regulating Ca(2+)-induced virulence factor production and Ca(2+) homeostasis are not known. Here, we characterized the genome-wide transcriptional response of P. aeruginosa to elevated [Ca(2+)] in both planktonic cultures and biofilms. Among the genes induced by CaCl2 in strain PAO1 was an operon containing the two-component regulator PA2656-PA2657 (here called carS and carR), while the closely related two-component regulators phoPQ and pmrAB were repressed by CaCl2 addition. To identify the regulatory targets of CarSR, we constructed a deletion mutant of carR and performed transcriptome analysis of the mutant strain at low and high [Ca(2+)]. Among the genes regulated by CarSR in response to CaCl2 are the predicted periplasmic OB-fold protein, PA0320 (here called carO), and the inner membrane-anchored five-bladed ß-propeller protein, PA0327 (here called carP). Mutations in both carO and carP affected Ca(2+) homeostasis, reducing the ability of P. aeruginosa to export excess Ca(2+). In addition, a mutation in carP had a pleotropic effect in a Ca(2+)-dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca(2+) and responding through its regulatory targets that modulate Ca(2+) homeostasis, surface-associated motility, and the production of the virulence factor pyocyanin. IMPORTANCE: During infectious disease, Pseudomonas aeruginosa encounters environments with high calcium (Ca(2+)) concentrations, yet the cells maintain intracellular Ca(2+) at levels that are orders of magnitude less than that of the external environment. In addition, Ca(2+) signals P. aeruginosa to induce the production of several virulence factors. Compared to eukaryotes, little is known about how bacteria maintain Ca(2+) homeostasis or how Ca(2+) acts as a signal. In this study, we identified a two-component regulatory system in P. aeruginosa PAO1, termed CarRS, that is induced at elevated Ca(2+) levels. CarRS modulates Ca(2+) signaling and Ca(2+) homeostasis through its regulatory targets, CarO and CarP. The results demonstrate that P. aeruginosa uses a two-component regulatory system to sense external Ca(2+) and relays that information for Ca(2+)-dependent cellular processes.

Comparative analytical evaluation of the respiratory TaqMan Array Card with real-time PCR and commercial multi-pathogen assays.

In this study, a multicenter evaluation of the Life Technologies TaqMan(®) Array Card (TAC) with 21 custom viral and bacterial respiratory assays was performed on the Applied Biosystems ViiA™ 7 Real-Time PCR System. The goal of the study was to demonstrate the analytical performance of this platform when compared to identical individual pathogen specific laboratory developed tests (LDTs) designed at the Centers for Disease Control and Prevention (CDC), equivalent LDTs provided by state public health laboratories, or to three different commercial multi-respiratory panels. CDC and Association of Public Health Laboratories (APHL) LDTs had similar analytical sensitivities for viral pathogens, while several of the bacterial pathogen APHL LDTs demonstrated sensitivities one log higher than the corresponding CDC LDT. When compared to CDC LDTs, TAC assays were generally one to two logs less sensitive depending on the site performing the analysis. Finally, TAC assays were generally more sensitive than their counterparts in three different commercial multi-respiratory panels. TAC technology allows users to spot customized assays and design TAC layout, simplify assay setup, conserve specimen, dramatically reduce contamination potential, and as demonstrated in this study, analyze multiple samples in parallel with good reproducibility between instruments and operators.

Two Listeria monocytogenes Pseudo-outbreaks Caused by Contaminated Laboratory Culture Media.

Listeriosis is a serious foodborne infection that disproportionately affects elderly adults, pregnant women, newborns, and immunocompromised individuals. Diagnosis is made by culturing Listeria monocytogenes from sterile body fluids or from products of conception. This report describes the investigations of two listeriosis pseudo-outbreaks caused by contaminated laboratory media made from sheep blood.

Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification.

Improved access to effective tests for diagnosing tuberculosis (TB) has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA) is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC) DNA in <20 minutes at 39 °C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110) and 20 fg (IS1081)were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9) and 86.1% (95%CI: 78.1, 94.1) respectively (n = 71). Specificities were 100% and 88.6% (95% CI: 80.8, 96.1) respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2) and 70.8% (95%CI: 62.9, 78.7) were obtained (n = 90). Specificities were 95.4 (95% CI: 92.3,98.1) and 88% (95% CI: 83.6, 92.4) respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB assays could be of use for integration into a point-of-care test for use in resource constrained settings.

Heterogeneity in Pseudomonas aeruginosa biofilms includes expression of ribosome hibernation factors in the antibiotic-tolerant subpopulation and hypoxia-induced stress response in the metabolically active population.

Bacteria growing in biofilms are physiologically heterogeneous, due in part to their adaptation to local environmental conditions. Here, we characterized the local transcriptome responses of Pseudomonas aeruginosa growing in biofilms by using a microarray analysis of isolated biofilm subpopulations. The results demonstrated that cells at the top of the biofilms had high mRNA abundances for genes involved in general metabolic functions, while mRNA levels for these housekeeping genes were low in cells at the bottom of the biofilms. Selective green fluorescent protein (GFP) labeling showed that cells at the top of the biofilm were actively dividing. However, the dividing cells had high mRNA levels for genes regulated by the hypoxia-induced regulator Anr. Slow-growing cells deep in the biofilms had little expression of Anr-regulated genes and may have experienced long-term anoxia. Transcripts for ribosomal proteins were associated primarily with the metabolically active cell fraction, while ribosomal RNAs were abundant throughout the biofilms, indicating that ribosomes are stably maintained even in slowly growing cells. Consistent with these results was the identification of mRNAs for ribosome hibernation factors (the rmf and PA4463 genes) at the bottom of the biofilms. The dormant biofilm cells of a P. aeruginosa Δrmf strain had decreased membrane integrity, as shown by propidium iodide staining. Using selective GFP labeling and cell sorting, we show that the dividing cells are more susceptible to killing by tobramycin and ciprofloxacin. The results demonstrate that in thick P. aeruginosa biofilms, cells are physiologically distinct spatially, with cells deep in the biofilm in a viable but antibiotic-tolerant slow-growth state.

Rapid identification of mycobacteria and drug-resistant Mycobacterium tuberculosis by use of a single multiplex PCR and DNA sequencing.

Tuberculosis (TB) remains a significant global health problem for which rapid diagnosis is critical to both treatment and control. This report describes a multiplex PCR method, the Mycobacterial IDentification and Drug Resistance Screen (MID-DRS) assay, which allows identification of members of the Mycobacterium tuberculosis complex (MTBC) and the simultaneous amplification of targets for sequencing-based drug resistance screening of rifampin-resistant (rifampin(r)), isoniazid(r), and pyrazinamide(r) TB. Additionally, the same multiplex reaction amplifies a specific 16S rRNA gene target for rapid identification of M. avium complex (MAC) and a region of the heat shock protein 65 gene (hsp65) for further DNA sequencing-based confirmation or identification of other mycobacterial species. Comparison of preliminary results generated with MID-DRS versus culture-based methods for a total of 188 bacterial isolates demonstrated MID-DRS sensitivity and specificity as 100% and 96.8% for MTBC identification; 100% and 98.3% for MAC identification; 97.4% and 98.7% for rifampin(r) TB identification; 60.6% and 100% for isoniazid(r) TB identification; and 75.0% and 98.1% for pyrazinamide(r) TB identification. The performance of the MID-DRS was also tested on acid-fast-bacterium (AFB)-positive clinical specimens, resulting in sensitivity and specificity of 100% and 78.6% for detection of MTBC and 100% and 97.8% for detection of MAC. In conclusion, use of the MID-DRS reduces the time necessary for initial identification and drug resistance screening of TB specimens to as little as 2 days. Since all targets needed for completing the assay are included in a single PCR amplification step, assay costs, preparation time, and risks due to user errors are also reduced.

Heterogeneous rpoS and rhlR mRNA levels and 16S rRNA/rDNA (rRNA gene) ratios within Pseudomonas aeruginosa biofilms, sampled by laser capture microdissection.

The local environmental conditions in biofilms are dependent on the impinging aqueous solution, chemical diffusion, and the metabolic activities of cells within the biofilms. Chemical gradients established in biofilms lead to physiological heterogeneities in bacterial gene expression. Previously, we used laser capture microdissection (LCM) and quantitative reverse transcription (RT)-PCR to target defined biofilm subpopulations for gene expression studies. Here, we combined this approach with quantitative PCR of bacterial DNA to normalize the amount of gene expression per cell. By comparing the ratio of 16S rRNA to 16S rDNA (rRNA gene), we demonstrated that cells at the top of thick Pseudomonas aeruginosa biofilms have 16S rRNA/genome ratios similar to those of cells in a transition from the exponential phase to the stationary phase. Cells in the middle and bottom layers of these biofilms have ratios that are not significantly different from those of stationary-phase planktonic cultures. Since much of each biofilm appeared to be in a stationary-phase-like state, we analyzed the local amounts of the stationary-phase sigma factor rpoS gene and the quorum-sensing regulator rhlR gene per cell. Surprisingly, the amount of rpoS mRNA was largest at the top of the biofilms at the air-biofilm interface. Less than one rpoS mRNA transcript per cell was observed in the middle or base of the biofilms. The rhlR mRNA content was also greatest at the top of the biofilms, and there was little detectable rhlR expression at the middle or bottom of the biofilms. While the cell density was slightly greater at the bottom of the biofilms, expression of the quorum-sensing regulator occurred primarily at the top of the biofilms, where the cell metabolic activity was greatest, as indicated by local expression of the housekeeping gene acpP and by expression from a constitutive P(trc) promoter. The results indicate that in thick P. aeruginosa biofilms, cells in the 30 microm adjacent to the air-biofilm interface actively express genes associated with stationary phase, while cells in the interior portions do not express these genes and therefore are in a late-stationary-phase-like state and may be dormant.