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utilizing codon bias. Gene 129:167–174. 9. Giuntoli, D., S. L. Stringer, and J. R. Stringer. 1994. Extraordinarily low number of ribosomal RNA genes in P. carinii.
JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1995, p. 2785–2788 0095-1137/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 33, No. 10

Comparison of Six Different PCR Methods for Detection of Pneumocystis carinii JANG-JIH LU,1 CHI-HSIANG CHEN,1 MARILYN S. BARTLETT,2 JAMES W. SMITH,2 2 AND CHAO-HUNG LEE * Department of Pathology, Tri-Service General Hospital and National Defense Medical Center, Taipei, Taiwan, Republic of China,1 and Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana 462022 Received 8 May 1995/Returned for modification 13 June 1995/Accepted 18 July 1995

We have recently developed a nested PCR method which amplifies internal transcribed spacers (ITS) of the ribosomal RNA genes of Pneumocystis carinii. To determine whether this PCR method can be used to diagnose P. carinii infections, we examined 30 bronchoalveolar lavage (BAL) specimens that were shown microscopically to contain P. carinii organisms by the P. carinii ITS PCR (Pc-ITS-PCR) and five other PCR methods that have been described for detecting P. carinii in clinical specimens. The targets of these PCR methods are portions of 18S rRNA, mitochondrial (mt) rRNA, 5S rRNA, thymidylate synthase (TS), and dihydrofolate reductase (DHFR). We also examined five different fungi, including Saccharomyces cerevisiae, Candida albicans, Histoplasma capsulatum, Cryptococcus neoformans, and Aspergillus fumigatus to determine the specificity of these six PCR methods for P. carinii. All 30 BAL specimens were positive by both the Pc-ITS-PCR and the 18S rRNA gene PCR, whereas only 26 (87%), 18 (60%), 10 (33%), and 7 (23%) of 30 BAL specimens were positive by mt rRNA gene PCR, TS gene PCR, 5S rRNA gene PCR, and DHFR gene PCR, respectively. Although the 18S rRNA gene PCR had the same sensitivity as the Pc-ITS-PCR, it nonspecifically amplified S. cerevisiae and C. albicans. The TS gene PCR also produced false-positive PCR results with C. albicans and C. neoformans. None of the other PCR methods (Pc-ITS-PCR, mt rRNA gene, 5S rRNA gene, and DHFR gene PCR) amplified the control fungal DNA. Considering both sensitivity and specificity, we conclude that Pc-ITS-PCR is the most effective of the six PCR methods evaluated in this study for the detection of P. carinii in BAL specimens. Pc-ITS-PCR with that of PCR methods targeting genes of 18S rRNA, mt rRNA, 5S rRNA, TS, and DHFR. Specimens used for this study included 50 BAL specimens and five different fungi: Saccharomyces cerevisiae, Candida albicans, Histoplasma capsulatum, Cryptococcus neoformans, and Aspergillus fumigatus. The mycelial forms of H. capsulatum and A. fumigatus were used, whereas the yeast forms of S. cerevisiae, C. albicans, and C. neoformans were used. The BAL specimens were designated with numbers. Numbers 1 to 30 were shown microscopically to contain P. carinii organisms, and the remaining 20 were shown to contain cytomegalovirus (no. 31), adenovirus (no. 32), Legionella pneumophila (no. 33), no organisms (no. 34 to 40), or H. capsulatum (no. 40 to 50). All of these specimens were obtained from the Clinical Microbiology Laboratory, Department of Pathology and Laboratory Medicine, Indiana University Medical Center, from January to December 1994. The fungal specimens were pure cultures. BAL specimens sent to the clinical laboratory for detection of pathogens were centrifuged at 1,000 3 g for 5 min to pellet cells and organisms. Drops of sediment (50 to 100 ml) were transferred to sterile 1- by 3-in. (2.54- by 7.62-cm) glass slides (eight per specimen), spread to form thin films, fixed in methanol, and stained with four stains, including Giemsa, Diff-Quik, methenamine silver nitrate, and modified acid-fast. Slides were examined by personnel experienced in identifying microorganisms in smears and tissues. The organism load in each specimen was indicated as few (an average of 1 clump of P. carinii organisms in 30 fields at 31,000), moderate (an average of 1 clump of organisms in 10 to 20 fields at 31,000), or heavy (an average of 5 to 10 clumps of organisms in 1 field at 31,000). Portions of specimens were cultured to detect bacteria, fungi, viruses, and mycoplasma, and remaining portions were sent for PCR evaluation.

The diagnosis of Pneumocystis carinii pneumonia is usually achieved by examining stained smears of bronchoalveolar lavage (BAL) for the presence of the organism. The cloning and sequencing of P. carinii genes offer an alternative method for the diagnosis of P. carinii pneumonia. Many P. carinii genes have been completely or partially cloned and sequenced (2–8, 12, 14, 17, 20). Although most of these genes were isolated from P. carinii organisms that are commonly found in rats, the nucleotide sequences of these genes show significant homology with those of P. carinii organisms that are commonly found in humans. Therefore, some of the PCR methods developed on the basis of rat P. carinii gene sequences are able to amplify specific regions of the genome of human P. carinii. These PCR methods include those targeted to genes encoding 18S rRNA (11), mitochondrial (mt) rRNA (18), 5S rRNA (10), thymidylate synthase (TS) (15), and dihydrofolate reductase (DHFR) (16). Recently, we have developed a nested PCR method which amplifies internal transcribed spacers (ITS) of the rRNA genes of P. carinii. This method, designated Pc-ITS-PCR, was initially developed for typing P. carinii organisms that infect humans (13). In the P. carinii typing study, we found that all BAL specimens from patients with documented P. carinii pneumonia were Pc-ITS-PCR positive and that all BAL specimens from patients with opportunistic infections other than P. carinii were negative by the same method (13). Therefore, Pc-ITSPCR showed promise as a method for the diagnosis of P. carinii infection. In this study, we compared the efficacy of the * Corresponding author. Mailing address: Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, 1120 South Dr., FH 419, Indianapolis, IN 46202. Phone: (317) 2742596. Fax: (317) 278-0643. 2785

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J. CLIN. MICROBIOL. TABLE 1. Conditions and probes for various PCR methods

Target (product size [bp])

ITS (nested) First PCR (693) Second PCR (550)

18S rRNA (nested) First PCR (378)

Second PCR (312) mt rRNA (346) TS (403) 5S rRNA (120)

DHFR (273)

Universal fungal 18S rRNA gene (597)

Primers

PCR condition

1724F: 59-AAGTTGATCAAATTTGGTC-39 ITS2R: 59-CTCGGACGAGGATCCTCGCC-39 ITS1F: 59-CGTAGGTGAACCTGCGGAAGGA TC-39 ITS2R1: 59-GTTCAGCGGGTGATCCTGCCTG-39

(948C, 1 min; 478C, 1 min; 728C, 2 min) 3 35 (948C, 1 min; 588C, 1 min; 728C, 2 min) 3 35

340a: 59-CCAGATTAGCTTTTGCTGATCGCG GG-39 708: 59-ACTTTCCAGTAATAGGCTTATCG-39 JK1: 59-TGTTGGCATGAAGCCAATGGAA-39 JK2: 59-CAATAACCCATCACCAGTCCGAAG-39 pAZ102-E: 59-GATGGCTGTTTCCAAGCCCA-39 pAZ102-H: 59-GTGTACGTTGCAAAGTACTC-39 PC1: 59-ATTTATGGGTTTCAATGG-39 PC2: 59-TGCAATATTAAAGGGAAC-39 Sense primer: 59-AGTTACGGCCATACCTCA GA-39 Antisense primer: 59-AAAGCTACAGCACGTC GTAT-39 Sense primer: 59-CTGCAAAATCCTTGGATC AT-39 Antisense primer: 59-CTTTAGTACCAACCCAA GAT-39 NS3: 59-GCAAGTCTGGTGCCAGCAGCC-39 NS4: 59-CTTCCGTCAATTCCTTTAAG-39

(948C, 1 min; 508C, 1 min; 728C, 2 min) 3 36

Approximately 1 ml of each BAL specimen was used for PCR. The BAL was centrifuged to pellet cells and organisms. The pellet was resuspended in 400 ml of proteinase K buffer containing 500 mg of proteinase K (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) per ml. After an overnight incubation at 378C, the DNA was isolated by phenol-chloroform extraction, after which ethanol precipitation was performed as described previously (13). To obtain DNA samples from fungal cultures for PCR, the organisms were scraped from agar slants, suspended in 400 ml of a lysis buffer (100 mM Tris [pH 7.5], 0.5% sodium dodecyl sulfate [SDS], 30 mM EDTA), and boiled in a water bath for 15 min. Proteinase K was then added to the cell lysate to a final concentration of 500 mg/ml. The mixtures were incubated at 378C overnight and then extracted with a mixture of phenol-chloroform (24:1). DNA in the aqueous layer was precipitated with ethanol as described previously (13). One hundred nanograms of DNA isolated from BAL specimens and 10 ng of DNA isolated from fungal cultures were used as templates in PCRs. Each sample was examined by six different PCR methods, including the Pc-ITS-PCR, the 18S rRNA gene PCR, the mt rRNA gene PCR, the 5S rRNA gene PCR, the TS gene PCR, and the DHFR gene PCR. The PcITS-PCR and the 18S rRNA gene PCR were nested PCRs and were performed as described by Lu et al. (13) and Lipschik et al. (11), respectively. The mt rRNA gene PCR was performed as described by Wakefield et al. (18). The 5S rRNA gene PCR was done according to the method of Kitada et al. (10). The TS gene PCR and the DHFR gene PCR were performed as described by Olsson et al. (15) and Schluger et al. (16), respectively. The universal fungal 18S rRNA gene PCR was performed as described by White et al. (19). Sequences of primers and PCR conditions used are summarized in Table 1. The PCR products were electrophoresed on 6% polyacrylamide gels to detect amplified products and to determine the size of the products. The expected sizes of PCR products were

Probe

Cloned DNA fragment

(948C, 1 min; 508C, 1 min; 728C, 1 min) 3 36 (948C, 1.5 min; 508C, 1.5 min; 728C, 2 min) 3 40 (948C, 30 s; 508C, 25 s; 728C, 1 min) 3 40 (948C, 1.5 min; 508C, 2.5 min; 728C, 2 min) 3 40

372a: 59-AAGGAAAATGAACTTGCTG GCTCT-39 pAZ102-L2: 59-ATAAGGTAGATAGTC GAAAG-39 59-CTTTCAGCATGGAATCCTGCAGG CATA-39 Cloned DNA fragment

(948C, 2 min; 508C, 1 min; 728C, 1 min) 3 30

59-GATAGAATTATGGCTACAATA-39

(948C, 2 min; 508C, 1 min; 728C, 1 min) 3 30

500 bp by Pc-ITS-PCR, 312 bp by 18S rRNA gene PCR, 346 bp by mt rRNA gene PCR, 120 bp by 5S rRNA gene PCR, 403 bp by TS gene PCR, and 273 bp by DHFR gene PCR. All PCR products, except those of the universal fungal PCR, were also probed with specific probes on dot blots. Probes used for each PCR product are listed in Table 1. Oligonucleotide probes were end labelled with [g-32P]ATP (Du Pont, NEN Research Products, Wilmington, Del.) with the T4 kinase, and the cloned DNA fragments were labelled with [a-32P]dCTP (Du Pont, NEN Research Products) by primer extension with random primers. Ten microliters of the PCR products was mixed with 200 ml of 0.4 N NaOH to denature the DNA. The samples were applied onto a Nytran membrane (Schleicher & Schuell, Keene, N.H.) with a dot blot apparatus. The membranes were prehybridized in a solution containing 63 SSC (13 SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 53 Denhardt’s solution, 0.5% SDS, 0.05% sodium pyrophosphate, and 100 mg of denatured calf thymus DNA per ml at 378C overnight and then hybridized in the same solution containing specific probes at 378C overnight. The membranes that were probed with oligonucleotide probes were washed with 23 SSC–0.1% SDS at temperatures 58C below the melting temperature of each probe. Those membranes (the products of the Pc-ITS-PCR and the 5S rRNA gene PCR) that were probed with cloned PCR products were washed with 0.13 SSC–0.1% SDS at 658C. All membranes were washed until the background counts became undetectable with a handheld Geiger counter. The BAL specimens (no. 31 to 40) that were shown by microscopy to contain no P. carinii organisms were negative by all six PCRs (Table 2). Among the six PCR methods used, the Pc-ITS-PCR and the 18S rRNA gene PCR were nested PCRs. Both nested PCR methods gave positive results on all 30 (100%) P. carinii-positive BAL specimens after the second reaction (Table 2). The first reaction of the Pc-ITS-PCR and the 18S rRNA gene PCR gave positive results on 16 (53%) and 15 (50%) of 30 specimens, respectively. The mt rRNA gene

VOL. 33, 1995

NOTES

TABLE 2. Results of six different PCR methods for the detection of P. carinii from 50 BAL specimens Pc-ITS-PCR

18S rRNA gene

Reaction of BAL specimen by PCR methodb

Speci- P. carinii mt 5S men loada First Second First Second TS DHFR rRNA rRNA PCR PCR PCR PCR gene gene gene gene

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31–50 a b

11 1 111 11 11 1 111 1 111 1 111 1 111 111 1 111 1 111 1 111 111 111 1 111 11 11 1 1 111 111 NA

2 2 1 1 1 2 1 2 1 2 1 2 1 1 2 1 2 1 2 1 2 1 2 1 2 1 2 2 1 1 2

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2

1 2 1 2 2 2 1 2 1 2 1 2 1 1 2 1 2 1 2 1 2 1 1 1 2 2 2 2 1 1 2

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2

1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 2 1 2 1 1 1 1 1 1 1 1 2 1 1 2

2 2 1 2 2 2 2 2 2 2 1 2 2 1 2 1 2 1 2 1 1 1 2 1 2 1 2 2 2 2 2

2 2 1 2 2 1 1 2 1 1 2 2 1 1 2 1 2 1 2 1 1 1 2 1 1 1 2 2 1 1 2

2 2 2 2 1 2 1 2 2 2 2 2 2 2 2 2 1 2 1 2 1 2 1 2 2 2 2 2 1 2

1, few; 11, moderate; 111, heavy; NA, not applicable. 2, negative; 1, positive.

PCR produced positive results on 26 specimens (87%); 5S rRNA gene PCR produced positive results on 10 specimens (33%); TS gene PCR produced positive results on 18 specimens (60%); and DHFR produced positive results on 7 specimens (23%) (Table 2). Since all six PCR methods generated negative results with the 20 P. carinii-negative BAL specimens, the PCR methods were tested with cultures of S. cerevisiae, C. albicans, H. capsulatum, C. neoformans, and A. fumigatus to determine their specificity. To ensure that the DNAs isolated from these fungi contained no PCR inhibitors, PCRs were performed on these fungal DNAs with primers NS3 and NS4 (19), which are capable of amplifying a portion (597 bp) of the 18S rRNA gene

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from all fungi, and a 597-bp fragment was produced from all the fungal DNAs tested. The six different P. carinii PCR methods were then performed on these specimens. The P. carinii 18S rRNA gene PCR produced positive results with S. cerevisiae and C. albicans, and the TS gene PCR produced positive results with C. albicans and C. neoformans. The Pc-ITS-PCR, mt rRNA gene PCR, 5S rRNA gene PCR, and DHFR gene PCR gave negative results with all five fungi examined (Table 3). The results described above indicate that the Pc-ITS-PCR was more sensitive and specific than other PCR methods tested. Experiments were then performed to determine the sensitivity of the Pc-ITS-PCR. A 10-ml aliquot of a BAL specimen known to contain P. carinii was placed on a microscopic slide. The smear was stained with Giemsa and examined under a microscope. The number of P. carinii organisms present in the entire smear was determined to be approximately 750. Another 10-ml aliquot of the same BAL specimen was examined by the Pc-ITS-PCR. DNA was isolated from this 10-ml BAL specimen, and a fivefold serial dilution (51, 52, 53, 54, 55, and 56) was done on the DNA sample. Each dilution was subjected to Pc-ITS-PCR. The 51, 52, 53, and 54 dilutions gave rise to a positive Pc-ITS-PCR, whereas 55 and 56 dilutions produced a negative Pc-ITS-PCR result. These results indicate that the Pc-ITS-PCR has the sensitivity to detect approximately three P. carinii organisms per reaction. The six different PCR methods examined in this study produced different results on the same set of specimens in the detection of P. carinii. The reasons why these PCR methods showed different sensitivities are uncertain. There may be multiple copies of mt in a P. carinii organism; therefore, the targets for the mt rRNA gene PCR may be more abundant than those of other PCR methods. This may explain why the mt rRNA gene PCR is the most sensitive single-step PCR method. Unlike most eukaryotic organisms, P. carinii appears to have only one copy of the nuclear rRNA genes (9), which may account for the necessity to perform nested PCR on the 18S rRNA gene or the ITS regions to achieve satisfactory sensitivity. The TS, 5S rRNA, and DHFR genes were more difficult to amplify, especially the DHFR gene. These genes may be more difficult to denature, making them inadequate for the PCR. All six PCR methods were specific to P. carinii when used with patient specimens. Testing with DNA isolated from fungal cultures revealed that all but the 18S rRNA gene PCR and the TS gene PCR were specific to P. carinii. The 18S rRNA gene nonspecifically amplified S. cerevisiae and C. albicans, and the TS gene PCR nonspecifically amplified C. albicans and C. neoformans. We conclude from the results of this study that the Pc-ITS-PCR is the most specific and sensitive nested PCR method and that the mt rRNA gene PCR is the most specific and sensitive single-step PCR method for the detection of P. carinii in clinical specimens. We do not recommend using the DHFR gene PCR for the detection of P. carinii.

TABLE 3. Summary of results of six PCR assays for P. carinii PCR method

Pc-ITS-PCR 18S rRNA gene mt rRNA gene TS gene 5S rRNA gene DHFR gene

Specificity of PCR method to fungus

PCR type

No. of positive samples (n 5 30) (% of total)

S. cerevisiae

C. albicans

H. capsulatum

C. neoformans

A. fumigatus

Nested Nested Single Single Single Single

30 (100) 30 (100) 26 (87) 18 (60) 10 (33) 7 (23)

2 1 2 2 2 2

2 1 2 1 2 2

2 2 2 2 2 2

2 2 2 1 2 2

2 2 2 2 2 2

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J. CLIN. MICROBIOL.

Currently, diagnosis of P. carinii pneumonia is achieved by demonstrating the presence of organisms in clinical specimens by microscopy, which requires experienced microscopists. With PCR, diagnosis may be made without microscopy and any laboratory that is doing PCR for other diagnoses can perform this test. In addition, PCR can use specimens which are not stored properly and become unusable for microscopy. The recent finding that the presence of P. carinii DNA in sera correlated well with a diagnosis of P. carinii pneumonia after examining BAL specimens (1) may make Pc-ITS-PCR an important assay for the diagnosis of P. carinii pneumonia. Serum specimens are much easier and less costly to collect than BAL specimens. Pc-ITS-PCR on serum specimens will enable the diagnosis of P. carinii pneumonia on patients for whom invasive bronchoscopy procedures are difficult; however, the performance of serum Pc-ITS-PCR for diagnostic purposes remains to be established.

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