Mark P. Buttner

Enhanced Detection of Surface-Associated Bacteria in Indoor Environments by Quantitative PCR

ABSTRACT Methods for detecting microorganisms on surfaces are needed to locate biocontamination sources and to relate surface and airborne concentrations. Research was conducted in an experimental room to evaluate surface sampling methods and quantitative PCR (QPCR) for enhanced detection of a target biocontaminant present on flooring materials. QPCR and culture analyses were used to quantitateBacillus subtilis (Bacillus globigii) endospores on vinyl tile, commercial carpet, and new and soiled residential carpet with samples obtained by four surface sampling methods: a swab kit, a sponge swipe, a cotton swab, and a bulk method. The initial data showed that greater overall sensitivity was obtained with the QPCR than with culture analysis; however, the QPCR results for bulk samples from residential carpet were negative. The swab kit and the sponge swipe methods were then tested with two levels of background biological contamination consisting of Penicillium chrysogenum spores. The B. subtilis values obtained by the QPCR method were greater than those obtained by culture analysis. The differences between the QPCR and culture data were significant for the samples obtained with the swab kit for all flooring materials except soiled residential carpet and with the sponge swipe for commercial carpet. The QPCR data showed that there were no significant differences between the swab kit and sponge swipe sampling methods for any of the flooring materials. Inhibition of QPCR due solely to biological contamination of flooring materials was not evident. However, some degree of inhibition was observed with the soiled residential carpet, which may have been caused by the presence of abiotic contaminants, alone or in combination with biological contaminants. The results of this research demonstrate the ability of QPCR to enhance detection and enumeration of biocontaminants on surface materials and provide information concerning the comparability of currently available surface sampling methods.

Determination of the efficacy of two building decontamination strategies by surface sampling with culture and quantitative PCR analysis

ABSTRACT The efficacy of currently available decontamination strategies for the treatment of indoor furnishings contaminated with bioterrorism agents is poorly understood. Efficacy testing of decontamination products in a controlled environment is needed to ensure that effective methods are used to decontaminate domestic and workplace settings. An experimental room supplied with materials used in office furnishings (i.e., wood laminate, painted metal, and vinyl tile) was used with controlled dry aerosol releases of endospores of Bacillus atrophaeus (“Bacillus subtilis subsp. niger,” also referred to as BG), a Bacillus anthracis surrogate. Studies were performed using two test products, a foam decontaminant and chlorine dioxide gas. Surface samples were collected pre- and posttreatment with three sampling methods and analyzed by culture and quantitative PCR (QPCR). Additional aerosol releases with environmental background present on the surface materials were also conducted to determine if there was any interference with decontamination or sample analysis. Culture results indicated that 105 to 106 CFU per sample were present on surfaces before decontamination. After decontamination with the foam, no culturable B. atrophaeus spores were detected. After decontamination with chlorine dioxide gas, no culturable B. atrophaeus was detected in 24 of 27 samples (89%). However, QPCR analysis showed that B. atrophaeus DNA was still present after decontamination with both methods. Environmental background material had no apparent effect on decontamination, but inhibition of the QPCR assay was observed. These results demonstrate the effectiveness of two decontamination methods and illustrate the utility of surface sampling and QPCR analysis for the evaluation of decontamination strategies.

Evaluation of the Biological Sampling Kit (BiSKit) for Large-Area Surface Sampling

ABSTRACT Current surface sampling methods for microbial contaminants are designed to sample small areas and utilize culture analysis. The total number of microbes recovered is low because a small area is sampled, making detection of a potential pathogen more difficult. Furthermore, sampling of small areas requires a greater number of samples to be collected, which delays the reporting of results, taxes laboratory resources and staffing, and increases analysis costs. A new biological surface sampling method, the Biological Sampling Kit (BiSKit), designed to sample large areas and to be compatible with testing with a variety of technologies, including PCR and immunoassay, was evaluated and compared to other surface sampling strategies. In experimental room trials, wood laminate and metal surfaces were contaminated by aerosolization of Bacillus atrophaeus spores, a simulant for Bacillus anthracis, into the room, followed by settling of the spores onto the test surfaces. The surfaces were sampled with the BiSKit, a cotton-based swab, and a foam-based swab. Samples were analyzed by culturing, quantitative PCR, and immunological assays. The results showed that the large surface area (1 m2) sampled with the BiSKit resulted in concentrations of B. atrophaeus in samples that were up to 10-fold higher than the concentrations obtained with the other methods tested. A comparison of wet and dry sampling with the BiSKit indicated that dry sampling was more efficient (efficiency, 18.4%) than wet sampling (efficiency, 11.3%). The sensitivities of detection of B. atrophaeus on metal surfaces were 42 ± 5.8 CFU/m2 for wet sampling and 100.5 ± 10.2 CFU/m2 for dry sampling. These results demonstrate that the use of a sampling device capable of sampling larger areas results in higher sensitivity than that obtained with currently available methods and has the advantage of sampling larger areas, thus requiring collection of fewer samples per site.

Measurement of Airborne Fungal Spore Dispersal from Three Types of Flooring Materials

Research was conducted in an experimental roomto measure the effect of human activity onairborne dispersal of settled fungal sporesfrom carpet and vinyl tile flooring. A seriesof experiments were conducted in whichcommercial loop pile carpet, residential cutpile carpet, or vinyl tile installed in theexperimental room were contaminated with Penicillium chrysogenum spores. The flooringmaterials were contaminated to two differentlevels (106 and 107 colony formingunits per square meter [c.f.u./m2] offlooring surface). Airborne culturable andtotal P. chrysogenum concentrations weremeasured using Andersen single-stage impactorsamplers and Burkard personal slide impactorsamplers, respectively. Bioaerosolconcentrations were measured at floor level, 1meter, and the adult breathing zone (1.5 meter)heights before and after human activityconsisting of walking in a prescribed patternfor 1 minute in the room. Airborne P.chrysogenum concentrations were greater withthe higher surface loading for all threeflooring materials. For all flooring materialsthere was no significant difference betweensampler locations, although the data from the1-meter location were the highest, followed bythe floor level and the breathing zonelocations, respectively. The data from theseexperiments indicate that while a very smallfraction of culturable P. chrysogenumspores present on flooring materials wereaerosolized by walking, relatively highairborne concentrations of spores maybere-entrained from contaminated materials. Theairborne P. chrysogenum concentrationswere significantly higher after walking on cutpile carpet than with the other two flooringmaterials at both contamination levels, withthe differences in concentration often ≥ 2orders of magnitude. No differences weremeasured in airborne culturable P.chrysogenum between vinyl flooring and looppile carpet at both contamination levels. Total spore data from the experiments with the107 c.f.u./m2 contamination levelindicated that walking on loop pile carpetproduced higher airborne spore concentrationsthan similarly contaminated vinyl tile althoughno significant difference was observed at the106 c.f.u./m2 level.

Evaluation of two surface sampling methods for detection of Erwinia herbicola on a variety of materials by culture and quantitative PCR

ABSTRACT This research was designed to evaluate surface sampling protocols for use with culture and quantitative PCR (QPCR) amplification assay for detection of the gram-negative bacterial biothreat simulant Erwinia herbicola on a variety of surface materials. Surfaces selected for evaluation were wood laminate, glass and computer monitor screens, metal file cabinets, plastic arena seats, nylon seat cushions, finished concrete flooring, and vinyl tile flooring. Laboratory and test chamber studies were performed to evaluate two sampling methods, a sponge and a macrofoam swab, for detection of E. herbicola on surface materials. In laboratory trials, seven materials were inoculated with a known concentration of E. herbicola cells and samples were collected from the surfaces of the materials to determine sampling efficiencies. Culture analysis was ineffective for assessing E. herbicola collection efficiency because very few culturable cells were obtained from surface samples. QPCR demonstrated that E. herbicola DNA was present in high concentrations on all of the surface samples, and sampling efficiencies ranged from 0.7 to 52.2%, depending on the sampling method and the surface material. The swab was generally more efficient than the sponge for collection of E. herbicola from surfaces. Test chamber trials were also performed in which E. herbicola was aerosolized into the chamber and allowed to settle onto test materials. Surface sampling results supported those obtained in laboratory trials. The results of this study demonstrate the capabilities of QPCR to enhance the detection and enumeration of biocontaminants on surface materials and provide information on the comparability of sampling methods.

Dispersal of Fungal Spores from Three Types of Air Handling System Duct Material

Exposure to airborne microorganisms in indoor environments may result in infectious disease or elicit an allergic or irritant response. Air handling system components contaminated by fungi have been implicated in the dispersal of spores into the indoor environment, thereby serving as a route of exposure to occupants. This study was conducted to provide quantitative data on the dispersal of spores from fungal colonies growing on three types of duct material. Galvanized metal, rigid fibrous glass ductboard, and fiberglass duct liner were soiled and contaminated with a known concentration of Penicillium chrysogenum spores. The duct materials were incubated in humidity chambers to provide a matrix of growing, sporulating fungal colonies at a contamination level of 109 colony forming units (CFU) per duct section, consistent for all materials. For each experiment a contaminated duct section was inserted into the air handling system of an experimental room, and the air handling system was operated for three 5-minute cycles with an air flow of 4.2 m3 min−1. The duct air velocity was approximately 2.8 m sec−1. The airborne concentration of culturable P. chrysogenum spores (CFU m−3), total P. chrysogenum spores (spores m−3), and total P. chrysogenum-sized particles (particles m−3) were measured in the room using Andersen single-stage impactor samplers, Burkard slide impactor samplers, and an aerodynamic particle sizer, respectively. The highest airborne concentrations (104 CFU m−3; 105 spores m−3; 104 particles m−3) were measured during the first operating cycle of the air handling system for all duct materials with decreasing airborne concentrations measured during the second and third cycles. There was no significant difference in spore dispersal from the three contaminated duct materials. These data demonstrate the potential exposure for building occupants to high concentrations of spores dispersed from fungal colonies on air handling system duct materials during normal operation of the system.

Development and evaluation of a real-time quantitative PCR assay for Aspergillus flavus

Aspergillus flavus is a ubiquitous mold and the most common mold contaminating foodstuffs. Many strains of A. flavus produce aflatoxins. In addition it is an allergen and an opportunistic pathogen of animals and plants. A. flavus often is underestimated in traditional culture analyses due to the expertise required and the cost associated with speciating members of the genus Aspergillus. The goal of this study was to develop and validate a primer and probe set for the rapid detection and quantitation of A. flavus in pure culture using real-time quantitative polymerase chain reaction (QPCR) amplification. Unique DNA regions were located in the genome of the target organism by sequence comparison with the GenBank database, and several candidate oligonucleotides were identified from the scientific literature for potential use with the TaqMan® QPCR technology. Three primer and probe sets were designed and validated for specificity and sensitivity in laboratory experiments. Initial screening to test for sensitivity was performed with seven A. flavus isolates and selected nontarget fungi. Specificity testing was conducted with the selected primer and probe set, which amplified all nine A. flavus isolates tested, including an aflatoxin producing strain. The primers did not amplify DNA extracted from 39 other fungal species (comprising 16 genera), including 18 other Aspergillus species and six Penicillium species. No amplification of human or bacterial DNA was observed; however cross-reactivity was observed with Aspergillus oryzae. PCR analysis of DNA dilutions and the use of an internal positive control demonstrated that 67% of the fungal DNA samples assayed contained PCR inhibitors. The assay validated for the target organism is capable of producing PCR results in less than 1 h after DNA extraction. The results of this research demonstrate the capabilities of QPCR for the enhanced detection and enumeration of fungi of significance to human health.

Interventions to Reduce Loss to Follow-up During All Stages of the HIV Care Continuum in Sub-Saharan Africa: A Systematic Review

The continuum of care for successful HIV treatment includes HIV testing, linkage, engagement in care, and retention on antiretroviral therapy (ART). Loss to follow-up (LTFU) is a significant disruption to this pathway and a common outcome in sub-Saharan Africa. This review of literature identified interventions that have reduced LTFU in the HIV care continuum. A search was conducted utilizing terms that combined the disease state, stages of the HIV care continuum, interventions, and LTFU in sub-Saharan Africa and articles published between January 2010 and July 2015. Thirteen articles were included in the final review. Use of point of care CD4 testing and community-supported programs improved linkage, engagement, and retention in care. There are few interventions directed at LTFU and none that span across the entire continuum of HIV care. Further research could focus on devising programs that include a series of interventions that will be effective through the entire continuum.ResumenLa continuidad de la atención para el éxito del tratamiento del VIH incluye la prueba del VIH, la vinculación y el compromiso en el cuidado y mantenimiento de la terapia antirretroviral (TAR). Las pérdidas durante el seguimiento (LTFU) es una alteración significativa de esta vía y un resultado común en el África subsahariana. Esta revisión de la literatura identificó intervenciones que han reducido LTFU en el continuo de la atención del VIH. Se realizó una búsqueda utilizando términos que combinaban el estado de la enfermedad, las etapas del continuo de la atención del VIH, las intervenciones y LTFU en el África subsahariana y los artículos publicados entre enero de 2010 y julio de 2015. Trece artículos fueron incluidos en la revisión final. El uso del punto de atención pruebas de CD4 y los programas apoyados por la comunidad mejorar la articulación, compromiso, y la retención de los pacientes. Hay pocas intervenciones dirigidas a LTFU y ninguno que se extienden a lo largo de todo el continuo de la atención del VIH. La investigación adicional podría centrarse en la elaboración de programas que incluyen una serie de intervenciones que serán efectivas a través de toda la cadena.

Comparison of Clinical & Laboratory Standards Institute standards in antimicrobial susceptibility among the carbapenemase producing Enterobacteriaceae

Aim: Carbapenems are antibiotics reserved for treatment of severe infections. Carbapenem antimicrobial susceptibility testing profiles were determined in a population of Klebsiella pneumoniae, and their resistance assessed based on previous and current Clinical and Laboratory Standards Institute criteria. Materials & methods: Isolates were examined using an automated antimicrobial susceptibility testing method, and real time polymerase chain reaction to detect the resistance (blaKPC) gene. Results: The prevalence of blaKPC gene was 45/54 (83.3%). Five isolates that were susceptible under the previous criteria changed to nonsusceptible with the current standards. The overall difference in susceptibility between the standards was 8%. Conclusion: This study shows that the current Clinical and Laboratory Standards Institute criteria may not offer additional benefits in the fight against carbapenem-resistant Enterobacteriaceae.

Interventions that increase the intention to seek voluntary HIV testing in young people: a review

ABSTRACT Young people 15–24 years old represent 39% of new HIV infections globally. However, they are the least likely age demographic to seek HIV testing and the most likely to be unaware of their HIV status. The purpose of this systematic literature review was to identify interventions that increase either rates of HIV testing or intentions to seek HIV testing in young people 10–24 years old. In total, 1601 manuscripts were systematically examined and five manuscripts were included in the final review. Two common themes identified in the interventions were education and test delivery methods. Educational programs were found to be effective when delivered in classroom or entertainment-based formats. Health providers offering testing and home testing increased the rate of testing. Additional research is needed on programs aimed at young people not enrolled in schools, interventions that measure testing rates, and educating healthcare providers about offering HIV tests to young people.