PCR detection methods are useful in studies of organisms not amenable to culture. Inhibitors in environmental samples can interfere with such assays. We describe a magnetic bead DNA capture protocol that removes inhibitors from outdoor air samples, maintaining the sensitivity of a 16S Pneumocystis carinii mitochondrial rRNA gene-based PCR.Pneumocystis carinii pneumonia (PCP) remains the most common opportunistic infection among individuals diagnosed with AIDS in the United States (5). However, basic knowledge of the ecology and epidemiology of the causative agent, P. carinii, is still lacking. The once widely accepted theory of PCP reactivation in severely immunosuppressed persons is currently being questioned in favor of the view that most episodes of PCP result from a de novo acquisition of the organism through inhalation of contaminated air (8, 11-13, 16, 18, 19, 21-24).There is anecdotal evidence supporting the hypothesis that patients with fulminant PCP are the source of infectious P. carinii, shedding large numbers of organisms into their immediate environment as is the case with tuberculosis (2,3,7,9,10,17). Alternatively, some studies suggest the existence of environmental reservoirs (20, 25), a transmission model which more closely resembles that of aspergillosis. Determining the source(s) of infectious P. carinii organisms is key to understanding when and how potential exposure control measures could be instituted.PCR-based detection methods used in conjunction with environmental sampling are key tools in understanding the ecology and epidemiology of pathogens, such as P. carinii, that are difficult or impossible to culture. However, PCR results may be compromised by the presence of inhibitors in environmental samples. Such inhibitors have been reported in air, soil, and water samples (1,4,15). These include humic acid, clays, various organics, and large amounts of nontarget DNA in the collected sample, although additional types of inhibitors are likely to exist (4). Here we describe a methodology that allows for successful amplification of a target P. carinii DNA sequence spiked onto on a membrane filter containing inhibitory materials collected from a suburban outdoor environment.Air samples were collected from an urban indoor environment and from a suburban outdoor environment during the autumn in the Southeastern United States. Air samples were collected over a 24-h period at approximately 0.4 liters per min using polyvinylidene difluoride filters (pore size, 0.45 m) for use with PCR analysis and at approximately 0.25 liters per min using polycarbonate filters (pore size, 0.45 m) for use with microscopic analysis. Indoor air sampling for viable fungi was accomplished using an Andersen impactor containing malt extract agar plates which were incubated at 25°C for approximately 1 week.Fungal concentrations (in CFU per cubic meter) in the indoor environment were performed to assess the potential for microbial contamination. The rationale for this was that a high indoor fungal concentration would be ind...

Pneumocystis carinii pneumonia (PCP) remains the most common opportunistic infection among human immunodeficiency virus-infected persons. Despite this, little is known concerning the transmission dynamics of this infection. In the absence of a reliable method to isolate and culture P. carinii from environmental samples, it has not been possible to assess the importance of person-to-person transmission in the epidemiology of PCP. A molecular viability assay was developed for the human form of P. carinii (P. carinii f sp hominis) that is applicable to both clinical specimens and environmental samples. This assay will enable the evaluation of the spread and persistence of viable human P. carinii in the environment.

Despite recent declines in incidence, Pneumocystis carinii pneumonia (PCP) remains the most commonly occurring opportunistic illness among persons with AIDS in the United States. While P. carinii DNA has been detected in patient respiratory specimens and in air samples collected from various indoor environments housing PCP patients, the viability of these organisms is unknown. For this reason, we have developed and evaluated a molecular viability assay for P. carinii. This method is based upon the detection of P. carinii mRNA by a reverse transcription-PCR that employs specific primers from a member of the heat shock protein 70 family. Under optimal assay conditions, these primers were capable of detecting as few as 100 viable trophozoites as determined by ethidium bromide staining, while no signal was obtained from 106 trophozoites killed by heat, desiccation, or UV radiation. This assay was also capable of distinguishing P. carinii from other common fungi present in the air. Therefore, this molecular viability assay may be useful in conjunction with standard bioaerosol collection devices and procedures for the detection of viable P. carinii collected from various indoor environments. It may also be useful in confirming the presence of viable trophozoites in respiratory specimens collected by noninvasive techniques from putatively infected individuals.

A polymerase chain reaction assay was optimized to detect P. carinii cysts in composite dust samples. The optimal assay was capable of detecting as few as 10(3) P. carinii cysts in 50 mg of dust. Two dust collection devices were evaluated for efficiency and precision of collection of bulk dust and compatibility with the optimized PCR protocol for P. carinii DNA detection. A handheld vacuum cleaner equipped with a high-retention bag was found to be superior to a 37-mm filter cassette attached to an electrically powered vacuum pump in terms of dust collection efficiency (87% [n = 37] versus 81% [n = 35]), although the precision of the two devices as assessed by the standard deviation was similar (6.2% versus 6.3%). However, the vacuum cleaner method was not as compatible with the PCR-based detection assay as the filter cassette method. The filter cassette appears to be a better device for use in conjunction with PCR-based detection of P. carinii DNA in bulk dust samples from both smooth and carpeted surfaces.

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