xii. pcr for detecting pneumocystis carinii in clinical or environmental samples

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XII. PCR for detecting Pneumocystis carinii in clinical or environmental samples Bettina Lundgren a , Ann E. Wake¢eld b; * a Department of Clinical Microbiology, Statens Serum Institut, Copenhagen, Denmark b Molecular Infectious Diseases Group, Department of Paediatrics, Institute of Molecular Medicine, Oxford, UK Abstract Since Pneumocystis carinii cannot be cultured in vitro, the introduction of polymerase chain reaction (PCR) has been an enormous advantage for research purposes. It is now possible to detect P. carinii in specimens containing low numbers of organisms where conventional detection methods using microscopic examination of histochemical stains has been insufficient. PCR has been used to detect P. carinii in bronchoalveolar lavage, induced sputum, spontaneous expectorates, oropharyngeal gargles, nasopharyngeal aspirates, serum, blood and in environmental samples. The use of PCR will enable the study of the epidemiology of P. carinii infection by detecting the organism in environmental samples, permitting molecular typing and thereby the study of the transmission of the organism. Furthermore PCR will facilitate studies on the response to therapy, studies monitoring for the emergence of drug resistant strains of P. carinii, and in the diagnosis of P. carinii pneumonia in non- invasive specimens, in patients unable to undergo more invasive diagnostic procedures. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Pneumocystis carinii ; DNA ampli¢cation ; Polymerase chain reaction ; Clinical sample ; Environmental sample Development of DNA ampli¢cation using the po- lymerase chain reaction (PCR) has initiated a large number of reports on the use of DNA based molec- ular methodology in the detection of Pneumocystis carinii. The potential advantages of using PCR in detecting P. carinii in clinical and environmental samples are the possibility of detecting low numbers of organisms in specimens where detection by con- ventional histochemical staining methods would be insu/cient. The clinical application of a PCR assay was ¢rst described by Wake¢eld et al. [1], amplifying a portion of the gene encoding the P. carinii mito- chondrial large subunit ribosomal RNA (mt LSU rRNA). Human-derived P. carinii (P. carinii sp. f. hominis) DNA has been detected by PCR in bronchoalveolar lavage (BAL), induced sputum (IS), spontaneous ex- pectorates, oropharyngeal gargles (OG), nasophar- yngeal aspirates, serum, blood and in environmental samples [2^16]. In preparing the sample for PCR, several di¡erent DNA extraction methods have been used, ranging from simple boiling to more la- borious extraction methods using phenol/chloroform and puri¢cation kits [1,13,15,17^22]. It is important to be aware that specimens may contain inhibitors of the PCR assay and therefore generate false negative results. In one study, inhibition was present in 36% of 102 IS specimens, 5% of 83 BAL specimens and in 0928-8244 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII:S0928-8244(98)00061-3 * Corresponding author. Tel.: +44 (1865) 222344; Fax: +44 (1865) 222626; E-mail: [email protected] FEMS Immunology and Medical Microbiology 22 (1998) 97^101

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XII. PCR for detecting Pneumocystis carinii in clinical orenvironmental samples

Bettina Lundgren a, Ann E. Wake¢eld b;*a Department of Clinical Microbiology, Statens Serum Institut, Copenhagen, Denmark

b Molecular Infectious Diseases Group, Department of Paediatrics, Institute of Molecular Medicine, Oxford, UK

Abstract

Since Pneumocystis carinii cannot be cultured in vitro, the introduction of polymerase chain reaction (PCR) has been anenormous advantage for research purposes. It is now possible to detect P. carinii in specimens containing low numbers oforganisms where conventional detection methods using microscopic examination of histochemical stains has been insufficient.PCR has been used to detect P. carinii in bronchoalveolar lavage, induced sputum, spontaneous expectorates, oropharyngealgargles, nasopharyngeal aspirates, serum, blood and in environmental samples. The use of PCR will enable the study of theepidemiology of P. carinii infection by detecting the organism in environmental samples, permitting molecular typing andthereby the study of the transmission of the organism. Furthermore PCR will facilitate studies on the response to therapy,studies monitoring for the emergence of drug resistant strains of P. carinii, and in the diagnosis of P. carinii pneumonia in non-invasive specimens, in patients unable to undergo more invasive diagnostic procedures. z 1998 Federation of EuropeanMicrobiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Pneumocystis carinii ; DNA ampli¢cation; Polymerase chain reaction; Clinical sample; Environmental sample

Development of DNA ampli¢cation using the po-lymerase chain reaction (PCR) has initiated a largenumber of reports on the use of DNA based molec-ular methodology in the detection of Pneumocystiscarinii. The potential advantages of using PCR indetecting P. carinii in clinical and environmentalsamples are the possibility of detecting low numbersof organisms in specimens where detection by con-ventional histochemical staining methods would beinsu¤cient. The clinical application of a PCR assaywas ¢rst described by Wake¢eld et al. [1], amplifyinga portion of the gene encoding the P. carinii mito-

chondrial large subunit ribosomal RNA (mt LSUrRNA).

Human-derived P. carinii (P. carinii sp. f. hominis)DNA has been detected by PCR in bronchoalveolarlavage (BAL), induced sputum (IS), spontaneous ex-pectorates, oropharyngeal gargles (OG), nasophar-yngeal aspirates, serum, blood and in environmentalsamples [2^16]. In preparing the sample for PCR,several di¡erent DNA extraction methods havebeen used, ranging from simple boiling to more la-borious extraction methods using phenol/chloroformand puri¢cation kits [1,13,15,17^22]. It is importantto be aware that specimens may contain inhibitors ofthe PCR assay and therefore generate false negativeresults. In one study, inhibition was present in 36%of 102 IS specimens, 5% of 83 BAL specimens and in

0928-8244 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 9 2 8 - 8 2 4 4 ( 9 8 ) 0 0 0 6 1 - 3

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* Corresponding author. Tel. : +44 (1865) 222344;Fax: +44 (1865) 222626;E-mail: [email protected]

FEMS Immunology and Medical Microbiology 22 (1998) 97^101

50% of 6 spontaneous expectorates, but disappearedby diluting the specimen [19]. Recently a P. cariniiPCR containing an internal process control has beendesigned, to directly detect inhibition [13].

A number of genes from the P. carinii genome hasbeen cloned and sequenced, and some have beenused as gene targets for the PCR assay (Table 1).Unfortunately, not all these primers are speci¢c forP. carinii, the 18S rRNA primers are capable of am-plifying Saccharomyces cerevisiae and Candida albi-cans DNA, and the thymidylate synthase primersampli¢ed C. albicans and C. neoformans [23]. Whenused for the detection of P. carinii sp. f. hominis, therat-derived P. carinii (P. carinii sp. f. carinii) dihy-drofolate reductase (DHFR) gene-speci¢c primersampli¢ed a non-speci¢c region of DNA with a se-quence unrelated to the P. carinii sp. f. hominisDHFR gene [24].

Both single round as well as nested PCR methodshave been evaluated for the detection of P. carinii inclinical samples. Two studies have compared di¡er-ent PCR methods. Lu and colleagues [23] comparedsix di¡erent PCR methods and found that twonested PCR methods, amplifying the internal tran-scribed spacer (ITS) regions of the nuclear rRNAoperon and a portion of the 18S rRNA gene of P.carinii were more sensitive than single round PCR.In both studies single round PCR using the mt LSUrRNA primers were the most sensitive [23,25]. How-ever, for routine clinical diagnostic use it is impor-

tant where possible to avoid nested PCR, to mini-mize excess handling and by this an increased risk oflaboratory contamination which is known to be aproblem using nested PCR.

Several studies have evaluated the use of PCR onBAL specimens and have found that PCR was assensitive as conventional stains but not as speci¢c,due to detection of P. carinii DNA in specimensobtained from patients without clinical PCP, sug-gesting the presence of colonization or subclinicalinfection [17,20,26^29], or early detection of develop-ing disease [30]. Routine diagnostic staining methodsof BAL specimens have a high sensitivity and specif-icity and therefore only in rare cases it would benecessary to apply the sensitive PCR method onthese specimens [20,31].

The sensitivity of conventional staining of inducedsputum (IS) is variable (45^78%) [18,26,32]. Sincethere are low numbers of P. carinii organisms in ISsamples, PCR has been shown to increase the sensi-tivity [4,7,17,18,21,26]. A clinical and cost analysisstudy comparing routine stains of BAL and IS toPCR on staining negative IS found that routineBAL was more cost e¡ective when the prevalenceof PCP was low [18]. However, another group foundthat PCR on IS is less expensive than immuno£uo-rescence [33].

Oropharyngeal gargles (OG) are easy to perform,and can be carried out on severely ill patients unableto sustain more invasive diagnostic procedures. PCR

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Table 1Gene targets for detection of P. carinii with PCR ampli¢cation

Gene targets Nuclearencoded

Mitochondrialencoded

P. cariniisp. f. carinii

P. cariniisp. f. hominis

Reference

Single copy genesDihydrofolate reductase (DHFR) + + 3 [11,24]Dihydropteroate synthase (DHPS) + + + [47]Thymidylate synthase (TS) + + + [23,26]arom + + + [48]Internal transcribed spacer (ITS) + + + [23,39,44]5S rRNA + + + [25,49]18S rRNA + + + [23]

Multicopy genesMitochondrial large subunit rRNA(mt LSU rRNA)

+ + + [1,4,14,38]

Mitochondrial small subunit rRNA(mt SSU rRNA)

+ + + [48,50]

Major surface antigen (MSG) + + + [51^53]

B. Lundgren, A.E. Wake¢eld / FEMS Immunology and Medical Microbiology 22 (1998) 97^10198

on OG was ¢rst reported by Wake¢eld et al. [14] in astudy detecting P. carinii DNA in 78% of 18 HIVpositive patients with PCP. Using a nested PCR onOG from 10 HIV positive patients, the sensitivitywas 90% [16], and among 26 patients with haemato-logical malignancies P. carinii DNA was detected inOG in all 8 patients with veri¢ed PCP using a simpleextraction method and a `touch down' PCR [13]. Inorder to properly evaluate the diagnostic sensitivityand speci¢city of OG in diagnosing PCP a largernumber of samples needs to be evaluated.

PCR on serum or blood samples has shown con-£icting results with sensitivities ranging from 0 to100% (Table 2) [7^12,34]. Thus the use of bloodproducts as a non-invasive specimen in the diagnosisof PCP remains to be established, although in rarecases of disseminated P. carinii infection PCR onblood samples may consistently show P. cariniiDNA [7,34].

PCR has also been found to be a sensitive tool forthe detection of P. carinii DNA in environmentalsamples in which the number of P. carinii organismsis very low. P. carinii DNA has been found in airsamples collected in animal facilities housing P. ca-rinii infected rats [35^37]. P. carinii sp. f. hominisDNA has been detected in air samples from hospitalrooms of patients with PCP, using nested PCR at theITS regions [36,39]. In the majority of cases the ITSsequence types of the P. carinii DNA in the air sam-ples from the patient room matched those from thepatient BAL samples. Air samples have also beencollected in the countryside and both P. carinii sp.f. carinii and P. carinii sp. f. hominis DNA have beenfound, using nested PCR at the mt LSU rRNA[35,38].

Because it is not possible to culture P. carinii invitro, the PCR technique has been an advantage tothe research. It is now possible to detect P. cariniiDNA in specimens where it has not previously beenidenti¢ed. The diagnosis of PCP, using PCR, onnon-invasive specimens from patients unable toundergo more invasive diagnostic procedures is beingdeveloped [6,13,14]. PCR methodology will also en-able further epidemiological studies, including theinvestigation of the transmission of P. carinii infec-tion, through the detection and genotyping of P.carinii organisms in air samples [35^48]. It will alsofacilitate studies on response to therapy as well asthe emergence of drug-resistant strains of P. carinii[48].

Acknowledgments

The study was supported in part by the AIDSFoundation, the Royal Society and the EuropeanConcerted Action Biomed 1 #PL 941118.

References

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Table 2PCR on serum/blood as a non-invasive specimen for diagnosis of PCP

Primers Specimen Patients with PCP Observed sensitivity (%) Reference

DHFR Serum 14 86 [11]18S rRNA Blood 14 21 [7]mt LSU rRNA Serum/blood 14 0 [9]ITS Serum 27 100 [12]mt LSU rRNA Serum 2 0 [34]mt LSU rRNA Serum 15 0 [8]

PBMC 15 7PMNC 15 33

PBMC, peripheral blood mononuclear cells ; PMNC, polymorphonuclear cells.

B. Lundgren, A.E. Wake¢eld / FEMS Immunology and Medical Microbiology 22 (1998) 97^101 99

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