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To examine the association between isoniazid preventive therapy (IPT) or nontuberculous mycobacteria (NTM) sputum culture positivity and tuberculosis (TB) transcriptional signatures in people with HIV.Cross-sectional study.We enrolled adults living with HIV who were IPT-naive or had completed IPT more than 6 months prior at HIV care clinics in western Kenya. We calculated TB signatures using gene expression data from qRT-PCR. We used multivariable linear regression to analyze the association between prior receipt of IPT or NTM sputum culture positivity with a transcriptional TB risk score, RISK6 (range 0-1). In secondary analyses, we explored the association between IPT or NTM positivity and four other TB transcriptional signatures.Among 381 participants, 99.7% were receiving antiretroviral therapy and 86.6% had received IPT (completed median of 1.1 years prior). RISK6 scores were lower (mean difference 0.10; 95% confidence interval (CI): 0.06-0.15; P 0.001) among participants who received IPT than those who did not. In a model that adjusted for age, sex, duration of ART, and plasma HIV RNA, the RISK6 score was 52.8% lower in those with a history of IPT ( P 0.001). No significant association between year of IPT receipt and RISK6 scores was detected. There was no association between NTM sputum culture positivity and RISK6 scores.In people with HIV, IPT was associated with significantly lower RISK6 scores compared with persons who did not receive IPT. These data support investigations of its performance as a TB preventive therapy response biomarker.

The National Institute of Allergy and Infectious Diseases organized a symposium in June 2022, to facilitate discussion of the environmental risks for nontuberculous mycobacteria exposure and disease. The expert researchers presented recent studies and identified numerous research gaps. This report summarizes the discussion and identifies six major areas of future research related to culture-based and culture independent laboratory methods, alternate culture media and culturing conditions, frameworks for standardized laboratory methods, improved environmental sampling strategies, validation of exposure measures, and availability of high -quality spatiotemporal data. Intramural Research Program at the National Institute of Allergy and Infectious Diseases, National Institutes of Health Published version This work was supported in part by the Intramural Research Program at the National Institute of Allergy and Infectious Diseases, National Institutes of Health. The authors thank the conference organizers, speakers, and attendees for their contributions to the symposium and manuscript.

This study assesses and compares the performance of different swab types and specimen collection sites for SARS-CoV-2 testing, to reference standard real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and viral culture.Symptomatic adults with COVID-19 who visited routine COVID-19 testing sites used spun polyester and FLOQSwabs to self-collect specimens from the anterior nares and tongue. We evaluated the self-collected specimen from anterior nares and tongue swabs for the nucleocapsid (N) or spike (S) antigen of SARS-CoV-2 by RT-PCR and then compared these results with results from RT-PCR and viral cultures from nurse-collected nasopharyngeal swabs.Diagnostic sensitivity was highest for RT-PCR testing conducted using specimens from the anterior nares collected on FLOQSwabs (84%; 95% CI 68-94%) and spun polyester swabs (82%; 95% CI 66-92%), compared to RT-PCR tests conducted using specimens from nasopharyngeal swabs. Relative to viral culture from nasopharyngeal swabs, diagnostic sensitivities were higher for RT-PCR and antigen testing of anterior nares swabs (91-100%) than that of tongue swabs (18-81%). Antigen testing of anterior nares swabs had higher sensitivities against viral culture (91%) than against nasopharyngeal RT-PCR (38-70%). All investigational tests had high specificity compared with nasopharyngeal RT-PCR. Spun polyester swabs are equally effective as FLOQSwabs for anterior nasal RT-PCR testing.We found that anterior nares specimens were more sensitive than tongue swab specimens or antigen testing for detecting SARS-CoV-2 by RT-PCR. Thus, self-collected anterior nares specimens may represent an alternative method for diagnostic SARS-CoV-2 testing in some settings.

Healthcare workers (HCW) who come into contact with tuberculosis (TB) patients are at elevated risk of TB infection and disease. The collection and handling of sputum samples for TB diagnosis poses exposure risks to HCW, particularly in settings where aerosol containment is limited. An alternative sample collection method, tongue swabbing, was designed to help mitigate this risk, and is under evaluation in multiple settings. This study assessed risk perceptions among South African HCW who used tongue swabbing in TB diagnostic research during the COVID-19 pandemic. We characterized their context-specific preferences as well as the facilitators and barriers of tongue swab use in clinical and community settings. Participants (n=18) were HCW with experience using experimental tongue swabbing methods at the South African Tuberculosis Vaccine Initiative (SATVI). We used key informant semi-structured interviews to assess attitudes toward two tongue swab strategies: Provider-collected swabbing (PS) and supervised self-swabbing (SSS). Responses from these interviews were analyzed by rapid qualitative analysis and thematic analysis methods. Facilitators included aversion to sputum (PS and SSS), perceived safety of the method (SSS), and educational resources to train patients (SSS). Barriers included cultural stigmas, as well as personal security and control of their work environment when collecting swabs in community settings. COVID-19 risk perception was a significant barrier to the PS method. Motivators for HCW use of tongue swabbing differed substantially by use case, and whether the HCW has the authority and agency to implement safety precautions in specific settings. These findings point to a need for contextually specific educational resources to enhance safety of and adherence to the SSS collection method.

The COVID-19 pandemic prompted widespread closures of primary and secondary schools. Routine testing of asymptomatic students and staff members, as part of a comprehensive mitigation program, can help schools open safely. “Pooling in a pod” is a public health surveillance strategy whereby testing cohorts (pods) are based on social relationships and physical proximity. Pooled testing provides a single laboratory test result for the entire pod, rather than a separate result for each person in the pod. During the 2020-2021 school year, an independent preschool–grade 12 school in Washington, DC, used pooling in a pod for weekly on-site point-of-care testing of all staff members and students. Staff members and older students self-collected anterior nares samples, and trained staff members collected samples from younger students. Overall, 12 885 samples were tested in 1737 pools for 863 students and 264 staff members from November 30, 2020, through April 30, 2021. The average pool size was 7.4 people. The average time from sample collection to pool test result was 40 minutes. The direct testing cost per person per week was $24.24, including swabs. During the study period, 4 surveillance test pools received positive test results for COVID-19. A post-launch survey found most parents (90.3%), students (93.4%), and staff members (98.8%) were willing to participate in pooled testing with confirmatory tests for pool members who received a positive test result. The proportion of students in remote learning decreased by 62.2% for students in grades 6-12 ( P < .001) and by 92.4% for students in preschool to grade 5 after program initiation ( P < .001). Pooling in a pod is a feasible, cost-effective surveillance strategy that may facilitate safe, sustainable, in-person schooling during a pandemic.

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.

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). We evaluated urine LAM testing conducted in parallel with tests for Mycobacterium tuberculosis DNA in oral swabs. In a cohort of 131 South Africans (92% with HIV), combined urine LAM and oral swab testing was significantly more sensitive than either sample tested alone (57% vs. 35% and 39%, respectively), and 97% specific, compared to reference sputum testing (TB culture and Xpert Ultra). Complementary non-sputum sample testing increased sensitivity of TB diagnosis, without sacrificing specificity.

To facilitate treatment and limit transmission of tuberculosis (TB), new methods are needed to enable rapid and affordable diagnosis of the disease in high-burden low-resource settings. We have developed a prototype integrated nucleic acid testing device to detect Mycobacterium tuberculosis (M.tb) in sputum. The device consists of a disposable cartridge and compact, inexpensive instrument that automates pathogen lysis, nucleic acid extraction, isothermal DNA amplification and lateral flow detection. A liquefied and disinfected sputum sample is manually injected into the cartridge, and all other steps are automated, with a result provided in

Rationale: Nontuberculous mycobacteria (NTM), including Mycobacterium avium complex (MAC), are emerging pathogens that can opportunistically cause debilitating pulmonary disease in susceptible huma...

Patient-Collected Swabs for SARS-CoV-2 Testing Tongue, nasal, and mid-turbinate swabs to detect SARS-CoV-2 were collected by 530 outpatients before a health care worker collected nasopharyngeal swa...

Efforts to control transmissible infectious diseases rely on the ability to screen large populations, ideally in community settings. These efforts can be limited by the requirement for invasive or logistically difficult collection of patient samples, such as blood, urine, stool, sputum, and nasopharyngeal swabs. Oral sampling is an appealing, noninvasive alternative that could greatly facilitate high-throughput sampling in community settings. Oral sampling has been described for the detection of dozens of human pathogens, including pathogens whose primary sites of infection are outside of the oral cavity, such as the respiratory pathogens Mycobacterium tuberculosis and SARS-CoV-2. Oral sampling can demonstrate active infections as well as resolving or previous infections, the latter through the detection of antibodies. Its potential applications are diverse, including improved diagnosis in special populations (e.g., children), population surveillance, and infectious disease screening. In this minireview, we address the use of oral samples for the detection of diseases that primarily manifest outside the oral cavity. Focusing on well-supported examples, we describe applications for such methods and highlight their potential advantages and limitations in medicine, public health, and research.

The COVID-19 pandemic has prompted widespread primary and secondary school closures. Routine testing of asymptomatic students and staff, as part of a comprehensive program, can help schools open safely. “Pooling-in-a-pod” is a public health surveillance strategy whereby testing cohorts are composed based on social relationships and physical proximity. Pooling-in-a-pod allowed for weekly on-site point-of-care testing of all staff and students at an independent preschool to grade 12 school in Washington, D.C. Staff and older students self-collected anterior nares samples, and trained staff collected samples from younger students. Overall, 6,746 samples were tested for 815 students and 276 staff between November 30, 2020, and March 3, 2021. The average pool size was 7.3 people. Sample collection to pool result time averaged 40 minutes. The direct testing cost per person per week was $24.77, including swabs. One surveillance test pool was positive. During the study period, daily new cases in Washington, D.C., ranged from 24 – 46 per 100,000 population. A post-launch survey found most parents (90.3%), students (93.4%), and staff (98.8%) were willing to participate in pooled testing with confirmatory tests for positive pool members. The school reported a 32.6% decrease in virtual learning after initiation of the program. Pooling-in-a-pod is feasible, cost-effective, and an acceptable COVID-19 surveillance strategy for schools. School officials and policymakers can leverage this strategy to facilitate safe, sustainable, in-person schooling. SUMMARY 1) What is the current understanding of this subject? Routine COVID-19 testing as part of a comprehensive strategy to operate schools safely is currently not widely implemented. 2) What does this report add to the literature? “Pooling-in-a-pod,” is a public health surveillance strategy whereby cohorts are composed based on social relationships and physical proximity. 6,746 samples were tested in 969 pools (average pool size 7.3 people) in a Washington, D.C. school, thereby requiring fewer test kits and less expense. The program was widely acceptable. 3) What are the implications for public health practice? Pooling-in-a-pod allows for more accessible testing to facilitate safe in-person schooling and minimize the negative effects of distance learning.

Oral swab analysis (OSA) has been shown to detectMycobacterium 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.

Oral swabs are emerging as a non-invasive sample type for diagnosing infectious diseases including Ebola, tuberculosis (TB), and COVID-19. To assure proper sample collection, sample adequacy controls (SACs) are needed that detect substances indicative of samples collected within the oral cavity. This study evaluated two candidate SACs for this purpose. One detected representative oral microbiota (Streptococcus species DNA) and the other, human cells (human mitochondrial DNA, mtDNA). Quantitative PCR (qPCR) assays for the two target cell types were applied to buccal swabs (representing samples collected within the oral cavity) and hand swabs (representing improperly collected samples) obtained from 51 healthy U.S. volunteers. Quantification cycle (Cq) cutoffs that maximized Youden’s index were established for each assay. The streptococcal target at a Cq cutoff of ≤34.9 had 99.0% sensitivity and specificity for oral swab samples, whereas human mtDNA perfectly distinguished between hand and mouth swabs with a Cq cutoff of 31.3. The human mtDNA test was then applied to buccal, tongue, and gum swabs that had previously been collected from TB patients and controls in South Africa, along with “air swabs” collected as negative controls (total N = 292 swabs from 71 subjects). Of these swabs, 287/292 (98%) exhibited the expected Cq values. In a paired analysis the three oral sites yielded indistinguishable amounts of human mtDNA, however PurFlock™ swabs collected slightly more human mtDNA than did OmniSwabs™ (p = 0.012). The results indicate that quantification of human mtDNA cannot distinguish swabs collected from different sites within the mouth. However, it can reliably distinguish oral swabs from swabs that were not used orally., which makes it a useful SAC for oral swab-based diagnosis.

Nanoyeast single-chain variable fragments (nanoyeast-scFv) are a new class of low-cost and stable protein capture agents developed as alternatives to full length monoclonal antibodies for use in immunosensors. Physical characteristics that impart nanoyeast-scFv with these advantages have yet to be investigated. We investigate the structure, size and surface loading of nanoyeast-scFv to better understand its ability to specifically and sensitively capture proteins of interest while retaining activity in solution. Nanoyeast-scFv fragments were found to be globular in structure and heterogeneous in size, typically

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.

Non-tuberculous Mycobacterium species (NTMs) are considered opportunistic pathogens as they cause disease in animals as well as in susceptible humans. Infections can vary from asymptomatic nodules to chronic, debilitating lung and skin infections. Depending on species- and strain-specific characteristics, some NTM infections respond readily to antibiotic treatment, while others exhibit intrinsic and non-intrinsic resistance to multiple antibiotics. Intrinsic drug resistance by NTMs may have evolutionary roots in the soil.

The time and costs associated with monoclonal antibody production limit the potential for portable diagnostic devices to penetrate the market. Replacing the antibody with a low-cost alternate affinity reagent would reduce the costs of diagnostic development and use, and lead to new portable diagnostic devices towards many diseases. Herein, we present low-cost affinity reagents, nano-yeast–scFv, on commercially available, inexpensive, and portable screen-printed electrodes for the label-free electrochemical detection of Entamoeba histolytica cyst antigens. The biosensor was able to detect antigen at concentrations down to 10 pg mL −1 in buffer with an inter-assay reproducibility of (% RSD, n =3) 4.1%. The applicability of two differently engineered nano-yeast–scFv to each specifically detect their cognant E. histolytica cyst antigens was demonstrated in a biological matrix derived from human stool. Because of the simple, inexpensive, and sensitive nature of this methodology, it may offer a low-cost alternative to immunosensors based on antibody-target recognition.

Immunoassays analyzing interactions between antigens and antibodies can be affected by capillary action together with binding affinity. This paper studies contact-angle changes of bacterial suspensions on antibody immobilized surfaces. The capillary action and the dried pattern of the bacterial suspensions are analyzed and correlated with specific- and nonspecific bindings between bacteria and antibodies.

Rapid progress in disease biomarker discovery has increased the need for robust detection technologies. In the past several years, the designs of many immunoaffinity reagents have focused on lowering costs and improving specificity while also promoting stability. Antibody fragments (scFvs) have long been displayed on the surface of yeast and phage libraries for selection; however, the stable production of such fragments presents challenges that hamper their widespread use in diagnostics. Membrane and cell wall proteins similarly suffer from stability problems when solubilized from their native environment. Recently, cell envelope compositions that maintain membrane proteins in native or native-like lipid environment to improve their stability have been developed. This cell envelope composition approach has now been adapted toward stabilizing antibody fragments by retaining their native cell wall environment. A new class of immunoaffinity reagents has been developed that maintains antibody fragment attachment to yeast cell wall. Herein, we review recent strategies that incorporate cell wall fragments with functional scFvs, which are designed for easy production while maintaining specificity and stability when in use with simple detection platforms. These cell wall based antibody fragments are globular in structure, and heterogeneous in size, with fragments ranging from tens to hundreds of nanometers in size. These fragments appear to retain activity once immobilized onto biosensor surfaces for the specific and sensitive detection of pathogen antigens. They can be quickly and economically generated from a yeast display library and stored lyophilized, at room temperature, for up to a year with little effect on stability. This new format of scFvs provides stability, in a simple and low-cost manner toward the use of scFvs in biosensor applications. The production and “panning” of such antibody cell wall composites are also extremely facile, enabling the rapid adoption of stable and inexpensive affinity reagents for emerging infectious threats.

Porphyromonas gingivalis and Filifactor alocis are fastidious anaerobic bacteria strongly associated with chronic forms of periodontitis. Our understanding of the growth activities of these microorganisms in situ is very limited. Previous studies have shown that copy numbers of ribosomal-RNA precursor (pre-rRNA) of specific pathogen species relative to genomic-DNA (gDNA) of the same species (P:G ratios) are greater in actively growing bacterial cells than in resting cells. The method, so-called steady-state pre-rRNA-analysis, represents a novel culture-independent approach to study bacteria. This study employed this technique to examine the in situ growth activities of oral bacteria in periodontitis before and after non-surgical periodontal therapy. Sub-gingival paper-point samples were taken at initial and re-evaluation appointments. Pre-rRNA and gDNA levels of P. gingivalis and F. alocis were quantified and compared using reverse-transcriptase qPCR. The results indicate significantly reduced growth activity of P. gingivalis, but not F. alocis, after therapy. The P:G ratios of P. gingivalis and F. alocis were compared and a low-strength, but statistically significant inter-species correlation was detected. Our study demonstrates that steady-state pre-rRNA-analysis can be a valuable culture-independent approach to studying opportunistic bacteria in periodontitis.

Entamoeba histolytica causes intestinal disease in endemic settings throughout the world. Diagnosis of E. histolytica infection would be improved by the identification of biomarkers that are expressed by cysts of E. histolytica, but not by cysts of closely related commensal species of Entamoeba. Herein, we describe two novel monoclonal antibodies (1A4 and 1D3) produced against a spacer region of the E. histolytica Jacob2 lectin, an outer cyst wall protein. These reagents demonstrated no cross-reaction to E. dispar recombinant antigen and low picomolar molecular detection limits when paired in ELISA sandwich assays. In an immunofluorescence microscopy assay, the α-Jacob2 murine antibodies labeled cysts of three xenically cultured E. histolytica isolates but did not label cysts of three E. bangladeshi isolates. Monoclonal antibody 1A4 did not cross-react with xenic cultures of three E. dispar isolates, demonstrating specificity to E. histolytica, while monoclonal antibody 1D3 cross-reacted with two out of three E. dispar isolates. Both antibodies labeled cysts in formalin-fixed slides, a potential logistical advantage in some settings. The monoclonal antibody 1A4 was also used in an immunofluorescence microscopy assay with formalin-fixed stool specimens. Seven out of ten ELISA-positive stool specimens exhibited 1A4-labeled cyst-like objects, compared to one out of seven ELISA-negative specimens. These results demonstrate that antibodies generated against the flexible spacer of E. histolytica Jacob2 lectin recognize and bind to Jacob2 protein in whole cysts and are capable of differentiating Entamoeba species in fixed specimens. Thus, Jacob2 is a promising biomarker for use in diagnosing E. histolytica infection., Author Summary Entamoeba histolytica is a prevalent human parasite requiring sensitive and specific detection. Assays available for Entamoeba detection utilize antibodies to detect parasite protein in stool, and they distinguish E. histolytica from nonpathogenic commensal amoeba. However, these tests have exhibited suboptimal sensitivity in some studies. This may have occurred because the tests do not detect the cyst stage of the parasite, which is more prevalent and stable in stool. Moreover, they cannot be used on formalin-fixed material, which creates logistical problems in some settings. Here, we have generated antibodies against a region of an E. histolytica cyst surface protein that has a unique sequence relative to nonpathogenic amoeba. One of these antibodies bound to E. histolytica cysts in fixed, xenic cultures without cross-reacting to nonpathogenic E. dispar and E. bangladeshi, while another antibody bound to both E. histolytica and E. dispar. The antibody that selectively bound to E. histolytica cysts also labeled cyst-like objects in fixed stool specimens from E. histolytica-positive patients. As they detected fixed organisms, these antibodies could ultimately make specimen collection and processing for Entamoeba diagnosis a simpler process. Additionally, this study indicates that the antibodies’ cyst protein target may be a useful biomarker for E. histolytica detection.

Nucleic acid amplification testing (NAAT) enables rapid and sensitive diagnosis of tuberculosis (TB), which facilitates treatment and mitigates transmission. Nucleic acid extraction from sputum constitutes the greatest technical challenge in TB NAAT for near-patient settings. This report presents preliminary data for a semi-automated sample processing method, wherein sputum is disinfected and liquefied, followed by PureLyse® mechanical lysis and solid-phase nucleic acid extraction in a miniaturized, battery-operated bead blender. Sputum liquefaction and disinfection enabled a >104 fold reduction in viable load of cultured Mycobacterium tuberculosis (M.tb) spiked into human sputum, which mitigates biohazard concerns. Sample preparation via the PureLyse® method and a clinically validated manual method enabled positive PCR-based detection for sputum spiked with 104 and 105 colony forming units (cfu)/mL M.tb. At 103 cfu/mL sputum, four of six and two of six samples amplified using the comparator and PureLyse® method, respectively. For clinical specimens from TB cases and controls, the two methods provided 100% concordant results for samples with "Equation missing"1 mL input volume (N = 41). The semi-automated PureLyse® method therefore performed similarly to a validated manual comparator method, but is faster, minimally instrumented and can be integrated into TB molecular diagnostic platforms designed for near-patient low-resource settings.

Mycobacterium avium complex lung disease is an increasingly common and chronically debilitating problem. Several host traits have been suggested or confirmed as risk factors. Potential environmental and behavioral risk factors have also been proposed. Few have been evaluated in comparative studies.To determine if aerosol-generating activities in the home and garden, features of the home water supply, or several pulmonary and immune-compromising conditions are associated with Mycobacterium avium complex lung disease.Cases were recruited from academic medical centers and by informal referrals from nonuniversity practices in Washington and Oregon. Control subjects were recruited by random-digit dialing and matched to cases by age, sex, and partial telephone number. Associations were measured as odds ratios (OR) estimated using conditional logistic regression.Known and potential risk factors were measured by in-home interview. Fifty-two matched pairs were studied. Six of 12 examined host traits were associated with disease, including history of chronic obstructive pulmonary disease (OR, 10; 95% confidence interval [CI], 1.2-80), pneumonia hospitalization (OR, 3.4; 95% CI, 1.1-11), and steroid use (OR, 8; 95% CI, 1.6-41). In contrast, 11 of the 14 aerosol-generating activities and all five features of home water supply studied bore little or no association with disease.Aerosol-generating activities seem not to be risk factors for Mycobacterium avium complex lung disease in HIV-negative adults, but prior lung disease and immune-suppressing drugs seem to be associated with susceptibility.

Simple, high-yield concentration of DNA is important for high-throughput genetic analysis and disease diagnosis. Glass-based microfilters are popular but the process requires centrifugation steps with cumbersome chemical processes. As an alternative, a concentration method using an electric field has been explored previously, but with limited efficiency. In this paper, electric field-induced concentration and capture of DNA are studied by using high-aspect-ratio microtips coated with a gold layer. The microtips are immersed longitudinally into a solution of 100 μL containing λ-phage DNA. After DNA concentration using an electric field, the microtips are withdrawn from the solution. Under AC- and biased AC fields, DNA is concentrated by electrophoresis (EP), dielectrophoresis (DEP), and electroosmotic flow (EOF). To reduce capillary effects in the withdrawal process, the microtips are coated with positively charged poly-l-lysine (PLL). The pattern of captured DNA is analyzed by fluorescence microscopy. DEP attracts DNA molecules at the edges of microtips, where the highest gradient of electric field exists. EP attracts DNA onto the surface of microtips following the vectors of an electric field. EOF generates vortexes that deliver DNA onto microtips. Using this method, 85% of DNA is captured on the PLL-coated microtips after three sequential captures. The concentration mechanism can potentially facilitate rapid and simple preparation of DNA for downstream analysis.

New high-performance detection technologies and more robust protein capture agents can be combined to both rapidly and specifically capture and detect protein biomarkers associated with disease in complex biological samples. Here we demonstrate the use of recently developed recombinant affinity reagents, namely nanoyeast-scFv, in combination with alternating current electrohydrodynamic (ac-EHD)-induced shear forces, to enhance capture performance during protein biomarker analysis. The use of ac-EHD significantly improves fluid transport across the capture domain, resulting in enhanced sensor-target interaction and simultaneous displacement of nonspecific molecules from the electrode surface. We demonstrate this simple proof-of-concept approach for the capture and detection of Entamoeba histolytica antigens from disinfected stool, within a span of 5 min using an ac-EHD microfluidic device. Under an ac-EHD field, antigens were captured on a nanoyeast-scFv immobilized device and subsequently detected using a quantum dot conjugated antibody. This immunosensor specifically detected antigen in disinfected stool with low background noise at concentrations down to 58.8 fM with an interassay reproducibility (%RSD of n = 3) < 17.2%, and in buffer down to 5.88 fM with an interassay reproducibility (% RSD, n = 3) of 8.4%. Furthermore, antigen detection using this immunosensor was 10 times more sensitive than previously obtained with the same nanoyeast-scFv reagents in a microfluidic device employing surface-enhanced Raman scattering (SERS) detection in buffer and at least 200 times more sensitive than methods using screen printed gold electrodes in disinfected stool. We predict this rapid and sensitive approach using these stable affinity reagents may offer a new methodology to detect protein disease biomarkers from biological matrices.

A non-immune library of human single chain fragment variable (scFv) antibodies displayed on Sacchar- omyces cerevisiae was screened for binding to the Clostridium botulinum neurotoxin serotype A binding domain (BoNT/A (Hc)) with the goal of identifying scFv to novel epitopes. To do this, an antibody-mediated labeling strategy was used in which antigen-binding yeast clones were selected after label- ing with previously characterized monoclonal antibodies (MAbs) specific to the Hc. Twenty unique scFv clones were isolated that bound Hc. Of these, 3 also bound to full-length BoNT/A toxin complex with affinities ranging from 5 to 48 nM. Epitope binning showed that the three unique clones recognized at least two epitopes distinct from one another as well as from the detection MAbs. After production in E. coli, scFv were coupled to magnetic particles and tested for their ability to capture BoNT/A holotoxin using an Endopep-MS assay. In this assay, toxin captured by scFv coated magnetic particles was detected by incubation of the complex with a peptide containing a BoNT/A-specific cleavage sequence. Mass spectrometry was used to detect the ratio of intact peptide to cleavage products as evidence for toxin capture. When tested individually, each of the scFv showed a weak positive Endopep-MS result. However, when the particles were coated with all three scFv simultaneously, they exhib- ited significantly higher Endopep-MS activity, consistent with synergistic binding. These results demonstrate novel approaches toward the isolation and characterization of scFv antibodies specific to unlabeled antigens. They also provide evidence that distinct scFv antibodies can work synergisti- cally to increase the efficiency of antigen capture onto a solid support. Biotechnol. Bioeng. 2011;108: 2456-2467.

BACKGROUND: Bacterial contamination or platelet (PLT) metabolism can change the pH of stored PLT concentrates (PCs). Measurement of pH for quality control is currently done on a limited basis. An easy noninvasive method was developed to obtain sequential pH measurements over time, without risking contamination and/or consuming PCs.

Mycobacterium avium is an environmental organism and opportunistic pathogen with inherent resistance to drugs, environmental stresses, and the host immune response. To adapt to these disparate conditions, M. avium must control its transcriptional response to environmental cues. M. avium forms biofilms in various environmental settings, including drinking water pipes and potable water reservoirs. In this study, we investigated the role of the universal signaling molecule autoinducer-2 (AI-2) in biofilm formation by M. avium . The addition of the compound to planktonic M. avium cultures resulted in increased biofilm formation. Microarray and reverse transcriptase PCR studies revealed an upregulation of the oxidative stress response upon addition of AI-2. This suggests that the response to AI-2 might be related to oxidative stress, rather than quorum sensing. Consistent with this model, addition of hydrogen peroxide, a known stimulus of the oxidative stress response, to M. avium cultures resulted in elevated biofilm formation. These results suggest that AI-2 does not act as a quorum-sensing signal in M. avium . Instead, biofilm formation is triggered by environmental stresses of biotic and abiotic origins and AI-2 may exert effects on that level.

Genotypic analysis of Mycobacterium tuberculosis isolates is increasingly applied in direct support of tuberculosis outbreak control activities. This is facilitated by PCR-based strain typing methods that enable the genotypic characterization of samples containing small numbers of M. tuberculosis cells. By using DNA extracted directly from primary diagnostic cultures, PCR-based methods were applied to a tuberculosis outbreak investigation and to surveillance in King County, Washington. In the outbreak investigation, five epidemiologically linked M. tuberculosis isolates had a unique pattern at mycobacterial interspersed repeating unit (MIRU) loci 10 and 23 when the pattern was compared to the patterns in a local MIRU locus database. In order to quickly identify new cases involving this strain (termed SBRI10), targeted genotyping at these two loci was performed with cultures from epidemiologically associated tuberculosis cases. Isolates with the characteristic genotypes at loci 10 and 23 were further analyzed by use of a 12-locus MIRU panel and by repetitive-unit-sequence-based PCR (rep-PCR). Between May 2004 and January 2005, 82 cases were screened, of which 14 were identified for further analysis and 13 were confirmed to be infected with SBRI10. Between September 2005 and August 2006, surveillance universal genotyping was performed by using the 12-locus MIRU panel with DNA from primary diagnostic enrichment cultures. A total of 161 samples were submitted for analysis, and 156 were successfully typed. Fifty-one cases formed 18 presumptive clusters by MIRU locus typing. Of these, 30 cases were confirmed to be members of 11 clusters by rep-PCR. Presumptive genotypic data were available rapidly, sometimes within 2 weeks of diagnosis. In this fashion, PCR-based genotyping provided data that can be used to prioritize disease control activities.

Readily accessible affinity reagents are critical to the validation of biomarkers and to the development of new diagnostic tests. As alternatives to monoclonal antibodies, yeast-bound single chain fragment variable antibody (yeast-scFv) can be rapidly selected from yeast display libraries. An important characteristic for any diagnostic reagent is its stability or ability to store it. A lyophilization procedure that has extended the shelf life of yeast-scFv by a factor of ≥10-fold relative to previous reports is reported. Real time stability for three yeast-scFv clones to three distinct Entamoeba histolytica potential diagnostic antigen targets for one year at room temperature as well as at 37°C and 45°C. Retention of full binding activity and specificity of the yeast-scFv clones for their cognate antigens is shown by flow cytometry. Lyophilization can easily be carried out in batches and in single-use vials.

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-Guerin, 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.

Diagnosis of pulmonary tuberculosis (TB) usually includes laboratory analysis of sputum, a viscous material derived from deep in the airways of patients with active disease. As a diagnostic sample matrix, sputum can be difficult to collect and analyze by microbiological and molecular techniques. An alternative, less invasive sample matrix could greatly simplify TB diagnosis. We hypothesized that Mycobacterium tuberculosis cells or DNA accumulate on the oral epithelia of pulmonary TB patients and can be collected and detected by using oral (buccal) swabs. To test this hypothesis, 3 swabs each were collected from 20 subjects with active pulmonary TB and from 20 healthy controls. Samples were tested by using a polymerase chain reaction (PCR) specific to the M. tuberculosis IS6110 insertion element. Eighteen out of 20 confirmed case subjects (90%) yielded at least 2 positive swabs. Healthy control samples were 100% negative. This case-control study supports past reports of M. tuberculosis DNA detection in oral swabs. Oral swab samples are non-invasive, non-viscous and easy to collect with or without active TB symptoms. These characteristics may enable simpler and more active TB case finding strategies.

A immunofluorescence microtip sensor was developed for specific detection of Mycobacterium cells in sputum samples by the combination of electric field, streaming flow, and immuno-affinity binding. The detection limit was 200 CFU/mL in human sputum, which was comparable to PCR but without requiring bacteriological culture, centrifugation, or nucleic acid amplification. In spite of the complex nature of physical, chemical, and biological mechanisms, the simple operation of "dipping and withdrawal" of tips will allow for screening by minimally trained personnel within 30 min. In addition, the minimal power requirement (5 W) combined with low assay cost is ideal for point-of-care (POC) screening in resource-limited settings.

A immunofluorescence microtip sensor was developed for specific detection of Mycobacterium cells in sputum samples by the combination of electric field, streaming flow, and immuno-affinity binding. The detection limit was 200 CFU/mL in human sputum, which was comparable to PCR but without requiring bacteriological culture, centrifugation, or nucleic acid amplification. In spite of the complex nature of physical, chemical, and biological mechanisms, the simple operation of "dipping and withdrawal" of tips will allow for screening by minimally trained personnel within 30 min. In addition, the minimal power requirement (5 W) combined with low assay cost is ideal for point-of-care (POC) screening in resource-limited settings.

The N and W-Beijing families of Mycobacterium tuberculosis are phylogenetically closely related. The ability of the W-Beijing family to rapidly cause widespread disease is well described; however, few outbreaks involving the N family have been reported outside the New York City, N.Y., area. During 2002 to 2003, Seattle, Wash., experienced a rapidly expanding tuberculosis outbreak involving 38 persons in a 23-month period. The outbreak strain, SBRI9, exhibited the genotypic properties of the N family. Its IS 6110 restriction fragment length polymorphism pattern was identical or nearly identical to those of two N family strains that were responsible for clusters of tuberculosis cases, including a large nosocomial outbreak, in New York City and New Jersey from 1989 to 1990. It was also identical to strains involved in late 1990s tuberculosis cases in Michigan, Maryland, and Arkansas. Further monitoring of the N family may show that it shares with the W-Beijing family the propensity to spread rapidly, suggesting that this characteristic evolved prior to the divergence of the two genetic lineages.

Nucleic acid-based analytical methods, ranging from species-targeted PCRs to metagenomics, have greatly expanded our understanding of microbiological diversity in natural samples. However, these methods provide only limited information on the activities and physiological states of microorganisms in samples. Even the most fundamental physiological state, viability, cannot be assessed cross-sectionally by standard DNA-targeted methods such as PCR. New PCR-based strategies, collectively called molecular viability analyses, have been developed that differentiate nucleic acids associated with viable cells from those associated with inactivated cells. In order to maximize the utility of these methods and to correctly interpret results, it is necessary to consider the physiological diversity of life and death in the microbial world. This article reviews molecular viability analysis in that context and discusses future opportunities for these strategies in genetic, metagenomic, and single-cell microbiology.