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* From the Departments of Pathology (Drs. Mastorides and Sandin, and Mr. Kranik) and Internal Medicine (Drs. Oehler, Greene, and Sinnott), University of South Florida College of Medicine, H. Lee Moffitt Cancer Center and Research Institute, Tampa General Hospital, Tampa, FL.
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Abstract |
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Design: Prospective cohort analysis.
Setting: Samples of circulating air were collected over a 12-month period from within the rooms of 10 hospitalized patients who were under respiratory isolation to rule out MTb infection. A small laboratory pump was used to draw ambient air at a rate of 2 L/min over a 6-h period through a 0.2-µm polycarbonate membrane filter placed near the patient's bed. Analysis of the membrane filters was conducted using PCR. Sputum cultures for MTb were performed simultaneously, and the results of smears stained for acid-fast bacilli (AFB) were noted.
Measurements and results: MTb complex was successfully detected by PCR in six of seven patients in whom sputum MTb cultures were subsequently positive, and in zero of three with subsequently negative sputum cultures. Sampling in one patient with a positive culture, in whom PCR results were negative, was only carried out for 2 h due to pump malfunction. One of the six PCR-positive patients was AFB-smear negative at the time of air sampling.
Conclusions: Our preliminary findings indicate that the technique of Micropore membrane air sampling with PCR analysis has important applications in the epidemiology and diagnosis of MTb.
Key Words: airborne detection • membrane air sampling • Mycobacterium tuberculosis • polymerase chain reaction
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MTb bacilli are carried on airborne droplet nuclei produced by aerosolization that can occur from coughing, talking, or even singing.4 ,5 ,6 The most clinically significant respiratory droplets measure between 0.5 and 2.0 µm. Larger droplet nuclei settle rapidly. The smaller droplet nuclei are rapidly dispersed and can remain suspended for an extended length of time, though once settling on surfaces, they do not reaerosolize and are no longer considered infectious.7 Transmissibility of MTb infection appears to be dependent on several factors. Among these is the quantity of MTb in the sputum, the radiologic extent of disease especially cavitary lesions, cough frequency and other personal habits, the closeness and duration of exposure, host susceptibility factors, and the presence or absence of specific lesions such as laryngeal tuberculosis.8 Effective chemotherapy markedly reduces the release of infectious aerosolized particles.9
Droplet nuclei are small enough to bypass mucociliary defenses and settle out in terminal alveoli. Here, they multiply and infect adjacent lymph nodes and are eventually disseminated to the bloodstream. In most immunocompetent patients, the development of cell-mediated immunity after several weeks aborts the infection and prevents development of active disease. Individuals with compromised immune systems (eg, HIV-positive patients) may develop acute progression to active disease within weeks. The overall risk of reactivation of latent infection peaks at 2 years after exposure and is 5 to 10% over a lifetime,10 though in HIV-infected patients, this figure is substantially higher.11
Airborne droplet nuclei may contain as little as a single tubercle bacillus, yet because of their prolonged period of suspension, it may be reasonably assumed that they can be filtered from the air onto a porous medium, where, using an extremely sensitive technique such as polymerase chain reaction (PCR), even a single organism may be readily detected. Membrane filters (Micropore; 3M Health Care; Minneapolis, MN) are a relatively new technology involving the manufacture of durable polycarbonate membranes with extremely small (0.01 to 20 µm) pores. These membranes have a variety of industrial uses ranging from water filtration to air quality analysis. Filtration and collection of microorganisms from the air are possible due to the extremely small size of the pores. Air filtration sampling has been commonly employed to investigate industrial exposures, but its potential medical uses have only recently been explored. In this study, we wish to introduce a novel, noninvasive application of this technique to detect the environmental presence of aerosolized MTb. This rapid technique combines air filtration and PCR. The technique has been used by us previously to successfully document the presence of aerosolized MTb in our first patient,12 and to detect airborne Cytomegalovirus.13 Others have used similar setups to document aerosolized Varicella-zoster virus14 and Pneumocystis carinii.15 To our knowledge, this is the first large study reporting on the rapid noninvasive detection of airborne MTb in multiple patients through the combined use of air filtration and PCR.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Air Sampling
The air samples were taken from the negative pressure isolation
rooms of patients selected for the study. The air was filtered through
a 37-mm-diameter, 0.2-µm pore polycarbonate track etch
membrane (Poretics Corp, Livermore, CA). The membrane filter holder was
a 37-mm disposable styrene acrylonitrile two-piece assembly that holds
the membrane filter. The filters have been loaded into the membrane
holders with reinforcement backing in a class 100 clean room at the
manufacturer to ensure sterility. The filter holder was directly
connected to a battery-operated pump (Escort Sampling; Hazco Services
Inc; Dayton, OH). The pump and filter apparatus were placed within
1 m of the patient's bed on an adjacent nightstand. Pump and
filter were calibrated with a soap bubble apparatus (Gilibrator)
to filter air at the rate of 2.0 L/min. Room air was filtered through
the membrane for a 6-h period. After sampling, the membrane filter was
recovered by removal from the membrane holder using sterile technique
under an aseptic hood and stored at -70°C.
PCR Assay-Amplification Step
Filter membranes were minced under sterile conditions and the
fragments placed in sterile tubes containing 1 mL of Buffer AE (Qiagen
Inc; Chatsworth, CA) and approximately 60 mg of 0.3-µm
diameter glass beads (Fisher Scientific; Pittsburgh, PA). Tubes were
placed in a microprocessor-controlled ultrasonic water bath (Lab-Line
model 9331; Lab-Line Instruments Inc; Melrose Park, IL) and sonicated
at 35 Khz for 30 min.
A one-tube nested PCR amplification scheme was carried out on 10-µl aliquots of the sonicated product according to the method of Wilson et al,16 ,17 with some modifications following procedure optimization in our laboratory.18 The target for the PCR was the insertion sequence IS 6110 that is present in members of the MTb complex.19 ,20 All reactions were run with positive and negative controls; positive controls consisted of dilutions of culture-proven MTb clinical isolates and negative controls consisted of all PCR reagents without MTb DNA. The concentration of dsDNA within these MTb culture lysates was measured with an RNA/DNA spectrophotometric calculator (Gene Quant II; Pharmacia Biotech; Piscataway, NJ). The concentration obtained was then divided by 4.0 femtograms, the average amount of genomic material within a single organism. This calculated value represents the number of organisms of MTb (genomic equivalents) within the culture lysate. Outer oligonucleotide primers Tb294 (5-GGACAACGCCGAATTGCG-AAGGGC-3')16 ,17 and Tb850 (5'-TAGGCGTCGGTGACAAAGGCCACG-3')16 ,17 were used at a concentration of 200 nM (DNA Synthesis Laboratory; University of Florida; Gainesville, FL), while inner oligonucleotide primer Tb670 (5'-AGTTTGGTCATCAGCC-3')16 ,17 was used at a concentration of 10 µM (DNA Synthesis Laboratory; University of Florida; Gainesville, FL). The second inner oligonucleotide primer Tb505 (5'-ACGACCACATCAACC-3<29)>16 ,17 was used at a concentration of 20 µM (Oligos Etc Inc; Newton, CT), following multiple experiments during the optimization stage that showed it to be a more labile oligonucleotide than the others. PCR amplification was performed in a thermal cycler (Biometra TRIO-Thermoblock; Biometra Inc; Gottingen, Germany) programmed to amplify DNA in two stages. The first stage comprised 30 cycles of denaturation (93°C x 1 min), primer annealing (65°C x 2.5 min), and extension (72°C x 8 min). A 580-bp product was generated by the outer primers. The second stage consisted of 20 cycles of denaturation (93°C x 1 min), primer annealing (50°C x 2.5 min), and extension (72°C x 8 min), where product from the first 30 cycles became target for the inner primers. At the annealing temperature of 50°C, inner primers preferentially bind to their complementary target sequence and create a 181-bp product. To prevent contamination, preparation of reaction mixtures and detection of products were each performed in separate rooms. In addition, well-recognized approaches to decrease the risk of amplicon carryover such as aerosol-resistent pipette tips, frequent glove changes, master mixes, and single-tube reagent aliquots were used throughout the procedure.
PCR Assay Detection Step
A probe (5'-CGCAAAGTGTGGCTAACCCTGAACCGTGA-3')
complementary to a 30-base sequence located within the inner, 181-bp,
amplified product was designed in our laboratory. The probe was
end-labeled with [(-32P] adenosine triphosphate (DuPont
New England Nuclear; Boston, MA) as previously
described.12
,13
Hybridization mixtures were prepared by
combining the labeled probe with formamide (Clontech; Palo-Alto, CA),
sodium chloride, sodium phosphate, EDTA (SSPE) buffer (Fisher
Scientific; Pittsburgh, PA), and Gel Loading Dye II (Ambion Inc;
Austin, TX). Equal volumes of PCR amplicon and hybridization mixture
were mixed and hybridized by heating to 95°C for 10 min followed by
incubation at 37°C for 1 h. Hybridized samples were
electrophoresed at 200 V for 45 min on 10% polyacrylamide mini gels
(Bio-Rad Laboratories; Hercules, CA). Gels were sealed in plastic wrap,
placed in autoradiography cassettes (Fisher Scientific; Pittsburgh,
PA), overlaid with x-ray film (Kodak XAR; Eastman Kodak Company;
Rochester, NY), and cassettes were stored at -70°C for 24 h to
generate autoradiograms. The film was then processed in an automatic
developer. A 100-bp reference ladder (GibcoBRL; Gaithersburg, MD) was
run with patient samples on polyacrylamide gels.21
This
reference ladder displays band lengths in 100-bp increments ranging
from 100 to 1,500 bp.
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Results |
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Three of the study patients had negative sputum cultures. All also had negative MAS/PCR runs. Patient H was a 30-year-old HIV-positive individual with a CD4 count of <50 admitted to the hospital with fever, chills, and weakness. He was thought to have disseminated Mycobacterium avium complex, although this was not cultured in blood or sputum during his hospital admission. Patient I was a 27-year-old HIV-positive woman with a CD4 count <50 who presented with a left lung cavitary lesion, fever, and a productive cough. No culture-proven source of her infection was identified during her hospitalization and she responded to antibiotic therapy. Patient J was a 36-year-old HIV-positive man admitted to the hospital for mental status changes. No pulmonary source of infection was identified in this patient.
The results of smears for AFB were also noted in Table 1 and are of interest. AFB smear results were positive for patients A, C, D, and E, and negative for patients H, I, and J. Patient B had a history of positive smears and cultures for tuberculosis, and given his clinical presentation at the time of present hospital admission, he was placed on a regimen of treatment presumptively. However, of interest, patient F had been smear positive on August 23, 1996, but was smear negative on October 6, 1996, which was the date of air sampling. Patient G, in whom equipment problems forced us to abbreviate the sampling time to 120 min, only had rare AFBs seen on smear on the day before and the day after the air sampling.
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Prior to the start of the study, it was not known whether Micropore air filtration at the modest rate of 2 L/min could recover MTb organisms successfully. It was recognized that in a negative pressure isolation room with an average of 6 to 10 air exchanges per hour, HEPA filters within the air handling system were expected to remove 99.9% of all airborne contaminants within 69 min.7 It would be expected that nearly all droplet nuclei would be removed by ventilation and filtration systems. It is possible that the close proximity of the filtration pump to the patient's bed facilitated in the filtration of infectious droplet nuclei prior to its recirculation. In a previous MTb outbreak22 on a commercial aircraft cabin with HEPA filtration of recirculated air, purified protein derivative skin test conversions occurred in passengers who were in physical proximity to the index case, whereas passengers in the forward part of the aircraft were not exposed.
Smear-positive patients possess AFB concentration within their sputum of about 106 to 107 AFB per milliliter.9 A rapid decline in mycobacterial counts is expected with the institution of therapy. Most sputum-positive patients were sampled within 3 days of onset of a four-drug regimen. Patients who were sampled >3 days after therapy initiation who had positive membranes tended to be nonprimary presenters, ie, they were previously diagnosed and had either failed to respond to therapy or had been noncompliant.
Most membrane positive patients in the study (ie, five of six) had documented positive sputum smears on or after the date in which they were subjected to MAS/PCR. Two patients had documented positive sputum smears at 1 and 5 days prior to the sampling session, respectively. The significance of sputum smear/culture positivity in relationship to MAS/PCR result is difficult to assess, as patients did not provide sputum specimens daily but only periodically, but it is reasonable to presume that smear/culture positivity at or near the sampling date would increase assay sensitivity. Of interest is the fact that one of the patients, patient F, had been sputum smear positive several months prior to the present hospital admission, but was smear negative on the day of air sampling. This presumes that the lower level of AFB present in his sputum was still at levels detectable by a very sensitive amplification technique. Also of interest is the fact that patient E's empty isolation room was sampled approximately 3 weeks after the previous sampling session and 7 days following hospital discharge. PCR results on the membrane were negative, suggesting that no "background" level of circulating MTb existed in this frequently used isolation room.
Careful handling and storage of membrane filters was essential for MAS/PCR accuracy. Six additional patients sampled after the initial 10 were removed from the study because their membranes were not handled according to the exact stipulated protocol. Plastic containers with membranes inside were frozen prior to the DNA lysis procedure rather than being processed immediately following the filtration process. It is speculated that freezing of the membranes within their plastic holders, followed by thawing and refreezing of the membranes prior to removal from the membrane holders (a modification of original protocol to facilitate their processing), may render the MTb complex DNA on the membrane surface less available for amplification. Results on these membranes were negative. This second set of patients had also been sampled for various intervals ranging from 4 to 11 h and it was believed that this variability could also obscure our ability to reach conclusions once too many uncontrolled variables were included. In addition, therapy had already been underway at the sampling time of these patients.
Potentially, Micropore MAS/PCR could have numerous epidemiologic applications. The background levels of circulating MTb within hospital wards, microbiology laboratories, tuberculosis clinics, and other similar institutions (eg, correctional facilities) is not well characterized. In such settings, the use of a technique for detecting the presence of circulating MTb complex DNA could be used to document environmental contamination in nonisolated areas.
Hospital respiratory isolation rooms are designed to be effective barrier spaces for the containment of highly infectious respiratory pathogens. Air handling mechanisms and HEPA filtration units are designed to minimize levels of circulating microorganisms. We have shown, however, that significant detectable quantities of AFB are still present within the air of these rooms. Little is known, however, concerning how long AFB organisms may circulate within the isolation room prior to being filtered by ventilation or air handling mechanisms. We will soon be examining these issues and several others through additional studies. In certain noninstitutional settings, there has been great public concern involving the potential infectious risks associated with a group of individuals sharing a confined airspace with poor air circulation. Public transportation has received particular scrutiny. Most notably, Kenyon et al22 presented a well-researched epidemiologic investigation documenting the transmission of MTb aboard a transoceanic commercial airline flight. Transmission was documented by purified protein derivative conversion and appeared to have occurred to passengers in close proximity to the index case, rather than via recirculated air. Routine surveillance of commercial airline flights using the membrane sampling technique could improve our understanding of the relative incidence of exposure incidents within the commercial airline system.
Several limitations to the use of MAS/PCR exist. A potential limitation is the lack of a means for assessing the viability of airborne AFB. Because of the limited size of the membranes, the investigators were unable to culture portions of the membrane filters to determine whether any viable MTb organisms remained. Given the environmental exposure and desiccating effects of 6 h on the air filter, it is unlikely that any living organisms could have remained. Certain instruments, such as the Anderson air sampler (Graseby; Smyrna, GA), are commonly used for collecting and culturing airborne microorganisms and could potentially be used concomitantly for this purpose. A quantitative assay of the concentration of airborne AFB as detected by MAS/PCR using a curve of known controls, not performed at the time, could permit specific quantification of mycobacteria and could have many potential applications, such as helping us gauge the level of potentially infectious mycobacteria in room air. Despite some of the limitations presented in this study, we believe that MAS/PCR could be a clinically useful tool in the noninvasive diagnosis of infectious tuberculosis in patients who cannot physically or willingly provide sputum for analysis. Patients could very easily be sampled either within their isolation rooms or in controlled ventilation booths to allow for optimal collection of exhaled organisms. However, the greatest application of this technology is in the area of hospital epidemiology and infection control, and in the documentation of the transmissibility of MTB within noninstitutional settings where groups of people are involved. More must be known, however, regarding the most ideal sampling interval and filtration rates for clinical and other uses, and future studies will address these issues.
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Acknowledgements |
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Footnotes |
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Abbreviations: AFB = acid-fast bacilli; MAS/PCR = membrane air sampling with polymerase chain reaction analysis; MTb = Mycobacterium tuberculosis; PCR = polymerase chain reaction
Received for publication January 26, 1998. Accepted for publication July 29, 1998.
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