Typing of Pneumocystis carinii f. sp. hominis by Single-Strand ...

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To better investigate Pneumocystis carinii f. sp. hominis epidemiology, we have developed ... Multitarget typing of P. carinii hominis by PCR-SSCP should allow.
JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1997, p. 3086–3091 0095-1137/97/$04.0010 Copyright © 1997, American Society for Microbiology

Vol. 35, No. 12

Typing of Pneumocystis carinii f. sp. hominis by Single-Strand Conformation Polymorphism of Four Genomic Regions PHILIPPE M. HAUSER,1* PATRICK FRANCIOLI,1 JACQUES BILLE,1 AMALIO TELENTI,2 1 AND DOMINIQUE S. BLANC Centre Hospitalier Universitaire Vaudois, 1011 Lausanne,1 and Institut fu ¨r Medizinische Mikrobiologie, Universita ¨t Bern, 3010 Bern,2 Switzerland Received 2 June 1997/Returned for modification 11 August 1997/Accepted 22 September 1997

To better investigate Pneumocystis carinii f. sp. hominis epidemiology, we have developed a molecular typing method. Because of the limited genetic variability of the P. carinii hominis genome, a multitarget approach was used. Four variable regions of the genome were amplified by PCR, polymorphism in each region was assessed by the single-strand conformation polymorphism (SSCP) technique, and the results for the four regions of each patient were combined. Bronchoalveolar lavage specimens collected from 11 patients were examined. Four patients were probably infected by a single strain, since their specimens yielded simple SSCP patterns (two bands corresponding to one allele). The combinations of these patterns were unique, suggesting that the strains which infected these patients were different. For the other seven patients, complex patterns were found (three or four bands corresponding to two alleles). The presence of more than one allele of a region in a patient is likely to be due to coinfection. Polymorphism was also assessed by sequencing, which revealed variations at nucleotide positions previously reported to vary. About half of the observed alleles had already been reported by laboratories in different countries. Multitarget typing of P. carinii hominis by PCR-SSCP should allow investigation of strain diversity and thus be useful for future epidemiological studies. Immunocompromised patients, such as human immunodeficiency virus (HIV)-infected individuals and transplant recipients, are at high risk for the development of P. carinii f. sp. hominis pneumonia (PcP) (8). The lack of a long-term in vitro culture system has hampered progress in understanding the epidemiology of this pathogen (27). For example, the relative contributions of reactivation of a latent previously acquired P. carinii hominis infection and of de novo infection are still unknown (24), thus precluding appropriate preventive measures. To further study P. carinii hominis epidemiology, typing methods are being developed. They generally rely on the amplification by PCR and sequencing of variable regions of the genome (14–17, 20, 26, 28) or the use of allele-specific PCRs and hybridizations (12, 21). However, sequencing is both fastidious and expensive, while the allele-specific approach is limited to the analysis of only two variable regions. In the present study, we assessed a typing method consisting of the amplification of four variable regions of the genome of P. carinii hominis from bronchoalveolar lavage (BAL) specimens of patients with PcP followed by the detection of polymorphisms by the single-strand conformation polymorphism (SSCP) technique. (Preliminary results of this work were presented in a conference report [10].)

min at 70°C to inactivate HIV and then at 55°C for 60 min in the presence of 0.5 mg of proteinase K/ml. BAL specimens were frozen in liquid nitrogen and thawed three times. Proteinase K was inactivated by incubation at 98°C for 15 min, and processed BAL specimens were stored at 220°C. To avoid crosscontamination, BAL specimens were introduced to and opened separately in a laminar air flow hood at the beginning of the procedure. In each experiment, controls were included to monitor cross-contamination. PCR conditions. One to three microliters of a processed BAL specimen was added to a 25-ml PCR mixture containing 0.2 mM each deoxynucleoside triphosphate, Mg21-free buffer (InVitrogen, Leek, Holland), 10 pmol of each primer, and 0.625 U of Taq DNA polymerase (Boehringer, Mannheim, Germany). Hot starting was performed with HotWax Mg21 beads (InVitrogen). The primers (Mycrosynth, Balgach, Switzerland), annealing temperature, magnesium concentration, and pH used for each PCR are given in Table 1. The 39 primer used for amplification of the intron of the nuclear 26S rRNA gene (hereafter referred to as 26S) has an 11-bp overlap with the previously described primer 4358 (19). The 59 primer used to amplify internal transcribed spacer 1 of the nuclear rRNA gene operon (hereafter called ITS1) was described previously (26). The primers used for the amplification of the variable region of the mitochondrial 26S rRNA gene (hereafter referred to as mt26S) were previously described primers (22, 29) shortened by 2 bp at the 39 end. No sequences homologous to the primers were found in the nucleotide database with the Basic Local Alignment Search Tool software (1). Forty cycles consisting of 30 s at 94°C, 1 min at the annealing temperature, and 1 min at 72°C were carried out. The reaction began with a 3-min denaturation at 94°C and ended with a 5-min extension at 72°C. Negative controls were included for each experiment. PCRs were set up and analyzed in separate rooms. For reamplification, SSCP bands scraped from the gel were resuspended in sterile H2O for 4 h and amplified with the Expand high-fidelity DNA polymerase (Boehringer) at room temperature. SSCP. Approximately 20 ng of a PCR product in loading buffer (gel buffer kit containing 2 mM EDTA and 0.002% xylene cyanol; Pharmacia, Uppsala, Sweden) was denatured at 95°C for 5 min and run in a nondenaturing 10% polyacrylamide gel (Pharmacia 48S Cleangel; bisacrylamide content, 5 2%), containing 5% glycerol, fitted on a Pharmacia Multiphor electrophoresis device. Temperatures, Pharmacia buffer kits, and times of migration used are given in Table 1. The current was 115 V for 45 min and 600 V for the rest of the run. Gels were stained by using a silver staining kit (Pharmacia). Cloning and sequencing of the PCR products. PCR products obtained with the Expand high-fidelity DNA polymerase were cloned into plasmid pCR-Script and introduced into Escherichia coli XL1-Blue MRF9 in accordance with the instructions accompanying an Amp SK(1) Cloning Kit (Stratagene, La Jolla, Calif.). About 1 ng of plasmid obtained according to the procedure of Del Sal et al. (6) was used for each PCR as described above. Universal M13 primers flanking the insert were used to generate PCR products which were sequenced bidirectionally by using an automated laser fluorescent sequencer and a T7 DNA polymerase kit (Pharmacia). Patient 2 provided the following alleles: 26S no. 1, mt26S no. 12

MATERIALS AND METHODS Specimens and their processing for PCR. Twelve BAL specimens were collected in 1994 or 1995 from 11 HIV-positive patients during their first PcP episode and were diagnosed as positive for P. carinii by Gomori staining. Native BAL specimen aliquots, stored frozen at 270°C, were thawed, incubated for 5

* Corresponding author. Mailing address: Centre Hospitalier Universitaire Vaudois, Division Autonome de Me´decine Pre ´ventive Hospitalie`re, IMU 218, Av. du Bugnon 44, 1011 Lausanne, Switzerland. Phone: 41 21 314 02 68. Fax: 41 21 340 40 60. E-mail: Philippe.Hauser @chuv.hospvd.ch. 3086

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TABLE 1. Conditions for PCR amplification and SSCP analysis of variable regions of the P. carinii hominis genomea PCR Variable region

ITS1 26S mt26S b-tub a

Primers (59, 39)

59-CTGCGGAAGGATCATTAGAAA-39, 59-CGCGAGAGCCAAGAGATC-39 59-GAAGAAATTCAACCAAGC-39, 59-ATTTGGCTACCTTAAGAG-39 59-GATGGCTGTTTCCAAGCC-39, 59-GTGTACGTTGCAAAGTAC-39 59-TCATTAGGTGGTGGAACGGG-39, 59-ATCACCATATCCTGGATCCG-39

SSCP 21

pH

Mg concn (mM)

Annealing temp (°C)

Product size (bp)

Pharmacia buffer kit

Temp (°C)

Migration time (min)

8.5

3.5

56

204

Delect

20

150

8.5

2.5

54

426

Delect

4

270

8.5

2.5

52

340

Disc

4

270

9.5

1.5

62

309

Delect

4

270

Constant parameters are given in Materials and Methods.

and 13, and b-tub no. 1. Patient 3 provided the alleles ITS1 A3 and B1 and b-tubulin intron 6 region (hereafter called b-tub) no. 2. Patient 6 provided 26S no. 2 and 3. Patient 8 provided mt26S no. 11.

RESULTS Amplification of variable regions of the P. carinii hominis genome from BAL specimens. We amplified by PCR several putative variable regions of the P. carinii hominis genome from 12 BAL specimens collected from a total of 11 PcP patients. We found four regions that showed variation in SSCP analysis: ITS1, 26S, mt26S, and b-tub. The sizes of all PCR products were in agreement with published sequences. SSCP analysis of the variable regions from 11 patients. Figure 1 shows the results of one representative SSCP experiment for each of the four regions amplified from the 12 BAL specimens collected from the 11 patients (patient 3 had two BAL specimens collected 16 days apart). A number of samples generated two SSCP bands for each region (for example, the ITS1 sample of patient 9). Each band of these “simple” patterns corresponds to one of the two single strands of the PCR product. That there are differences among these patterns indicates that there exist different nucleotide sequences within the regions investigated. Henceforth, each of these sequences will be called an allele of the region. For each region, some other samples generated SSCP patterns with three or four SSCP bands (for example, the ITS1 sample of patient 1). These “complex” patterns could correspond to (i) various conformations of the two single strands of a single allele or (ii) the presence of two alleles. To investigate these possibilities, SSCP bands were reamplified by PCR and analyzed by SSCP. If there was only one allele in the sample (hypothesis i), this allele should be regenerable from each of the SSCP bands, thus producing the same complex SSCP pattern. This was not the case for any of the complex SSCP patterns shown in Fig. 1, strongly suggesting the presence of two alleles (hypothesis ii). Each of the four reamplified bands of complex patterns with four bands generated a simple pattern (see, for example, the ITS1 sample of patient 2 in Fig. 2). In the case of complex patterns with three bands (Fig. 1), two bands generated simple patterns but one band could regenerate the complex pattern, most certainly because this band contained one single strand of each allele which had migrated to the same place (see, for example, the b-tub sample of patient 7 in Fig. 2). Thus, complex patterns are likely to be due to the superimposition of two simple patterns. Furthermore, a mixture of two b-tub samples which showed simple patterns when run separately generated a complex pattern corresponding to the superimposition of the two simple patterns (Fig. 2). The presence of two alleles was confirmed by the fact that inde-

pendent plasmid clones obtained by cloning the PCR product generated only simple patterns. There were small variations or faint bands in the SSCP patterns (for example, the small variation in ITS1 patterns 1 of samples of patients 5 and 6, the faint bands at the center of the pattern of the mt26S sample of patient 3, and the faint bands above the SSCP bands of the ITS1 sample of patient 4 [Fig. 1]). These small variations and faint bands were not observed in each SSCP analysis of the same PCR product. Analysis of independent plasmid clones obtained by cloning the PCR product revealed that the faint bands corresponded to rare conformations adopted by one of the single strands in some, but not all, SSCP analyses.

FIG. 1. SSCP analysis of variable regions of P. carinii hominis genomes of 12 BAL specimens collected from 11 unrelated patients. The number attributed to each pattern is shown at the bottom of each gel. Complex SSCP patterns with three or four bands, which signify the presence of two alleles of the region, are indicated by the two numbers of the simple patterns which constitute them (see the text). Footnotes: a, this BAL specimen was collected 16 days after the first BAL specimen was obtained from this patient, during the same PcP episode; b, this sample was run in a separate gel that included controls which identified the pattern; c, this sample was run in another region of the same gel.

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FIG. 2. SSCP analysis of reamplified SSCP bands showing the presence of two alleles in complex patterns. SSCP bands were cut out of the gel, reamplified by PCR, and analyzed by SSCP. b-tub samples from patient 7 were run in a different gel than those of patients 2 and 3. For the last b-tub lane, samples from patients 2 and 3 were mixed before SSCP analysis.

Cloning and sequencing of alleles of the variable regions. The results reported above suggested that among our samples there were two or three different simple SSCP patterns for each of the four regions. These patterns are represented schematically in Fig. 3. The PCR products of patients were cloned, and independent plasmid clones were analyzed. Sequencing of both strands of a single clone generating each of the SSCP patterns shown in Fig. 3 revealed 1- to 4-bp polymorphisms when compared to the reference allele (Tables 2 to 5) (all alleles of the regions reported to date are included). Polymorphisms observed in the ITS1, mt26S, and b-tub regions were all at positions previously reported to vary among human isolates, except that at positions 54 to 57 of mt26S (the same polymorphism at the latter position was reported, but this apparently went unnoticed [16]). About half of the observed alleles were previously reported.

DISCUSSION Among the 12 BAL specimens from the 11 Swiss patients investigated in the present study, only a few polymorphic positions were observed in the four regions analyzed. Moreover, most of these positions and many of the observed alleles have already been reported from other parts of the world, indicating that there exists only a limited range of variation of the P. carinii hominis genome. This indicates the need for a multipletarget approach to generate sufficient information to define isolates as unique. In other words, a discriminant typing method should involve the analysis of several variable regions for each strain. Moreover, given the low degree of genetic divergence between the alleles—only 2 to 4% in the most variable regions of the genome (this work and reference 23)— typing would require very sensitive methods for the detection

TABLE 2. Polymorphisms in P. carinii hominis ITS1 region reported to date Alleleb

Nucleotide(s) at position(s) (bp)a:

No. of observations

Countryd

TTA ● ●

5 1 3 2

USA F UK I

20 15 26 15

● GAGG GAGG

● ● ●

1 6 7 6 5 2 1

CH USA UK USA F I CH

This work (2) 20 26 20 15 15 This work (1)

103T

GAGG



8 1 1

UK USA F

26 20 15

TATC ●

● 103T

GAGG GAGG

● ●



103T

GAGG

—g

1 2 1 2

F UK F UK

15 26 15 26

8–10

11

17

22

Af A1 A2

C T ●

33T 23T 23T

A ● ●

T ● ●

T ● ●

TC ● ●

93T ● ●

GG ● ●

A3 B B1

● T T

● 23T 23T

● ● ●

● ● ●

● ● ●

● ● ●

103T 103T ●

B2

T

23T



A





B3 B4

T ●

23T 23T

C ●

● ●

C ●

C

T

23T







a

46–47

54–62c

2

71–72

111–113

Reference (SSCP pattern no.e)

Dots indicate homology to the reference allele (A) described in the first line. Designation previously used. B4 was called B3 by Tsolaki et al. (26) and C1 by Latouche et al. (15). A3 is a new allele observed in this work. Polymorphisms at this position were not taken into account for the alleles reported by Tsolaki et al. (26) since they claimed that PCR-induced errors can occur at this position. d Abbreviations: CH, Switzerland; F, France; I, Italy; UK, United Kingdom; USA, United States of America. e According to Fig. 1. f Reference allele (GenBank accession no. U07220). g —, base pairs are absent. b c

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TABLE 3. Polymorphisms in P. carinii hominis 26S region reported to date Alleleb

1e 2 3

Nucleotide position (bp)a 3

78

212

296

305

A

A

A

T

C

● G

G ●

f

● G

— —f

No. of observations

Countryc

Reference (SSCP pattern no.d)

5 1 1 1

USA CH CH CH

19 This work (1) This work (2) This work (3)

CT CA

a

Dots indicate homology to the reference allele described in the first line. Arbitrary numbers. Abbreviations: CH, Switzerland, USA, United States of America. d According to Fig. 1. e Reference allele (Genbank accession no. L13615). f —, base pairs are absent. b c

of polymorphisms. Typing by sequencing, which is the most discriminative method available and which has been widely used for P. carinii hominis (14–17, 20, 26, 28), is both fastidious and expensive, thus precluding analysis of many samples as well as of several variable regions for each strain. The second widely used typing approach is allele-specific PCR and hybridization of oligonucleotides derived from ITS1 and ITS2 (ITS2 is the second spacer of the rRNA genes) (2, 4, 12, 18, 21). This approach is limited to the analysis of only two variable regions. Moreover, differentiation of new ITS alleles would require the development of new PCRs or oligonucleotides. In the present study, we used PCR-SSCP, a simple technique which has been shown to be able to detect single-nucleotide polymorphisms (11). PCR-SSCP generated simple patterns with two bands and complex ones with three or four bands. In simple patterns, each band corresponds to a single strand of

the PCR product, allowing easy interpretation. Our results suggest that 4 of 11 patients were infected by a single strain, since they each showed a simple SSCP pattern for all four regions. When the patterns obtained for each region were combined, the strains that infected these patients appeared to be different because the combination of patterns for each patient was unique. Complex patterns were more difficult to interpret. Our study demonstrated that they represent a superimposition of two simple patterns generated by two alleles of the region. The presence of two alleles in a single sample could be due to (i) coinfection by two strains, with each allele belonging to one of the strains; (ii) heterozygosity in diploid organisms; or (iii) the presence of two or more copies of the same region per genome, with variation between the copies existing. The possibility of coinfection is likely since coinfections have been demonstrated in rats (5) and ferrets (3). The

TABLE 4. Polymorphisms in P. carinii hominis mt26S region reported to date Alleleb

Nucleotide position(s) (bp)a 54–57

85

248

288

1e

43A

C

C

G

2







A

3





T

A

4 5 6 7

● ● ● ●

● A A A

T ● T ●

● ● ● A

8



T



A

9 10 11

● ● 33A

T —f ●

● T ●

● A A

12

33A

A



A

13 14

33A 33A

A T

T ●

A A

a

No. of observations

Country c

Reference (SSCP pattern no.d)

9 5 5 6 5 2 1 1 2 7 2 2 2 3 2 1 2 1 15 1 7 1 1 6

USA UK USA F I USA USA F USA USA USA F I F I USA USA USA F CH F CH CH F

14 22 17 15 15 17 17 15 14 14 14 15 15 15 15 17 14 17 15 This work (3) 15 This work (1) This work (2) 15

Dots indicate homology to the reference allele described in the first line. Arbitrary numbers. Abbreviations: CH, Switzerland; F, France; I, Italy; UK, United Kingdom; USA, United States of America. d According to Fig. 1. e Reference allele (GenBank accession no. M58605). f —, base pairs are absent. b c

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FIG. 3. Schematic representation of simple SSCP patterns with two bands identified for the four variable regions used to type P. carinii hominis. Patterns are numbered according to the system shown in Fig. 1.

possibility of heterozygosity in diploid organisms has been investigated in P. carinii carinii, the special form infecting rats, via DNA content measurements by fluorescence microscopy. Preliminary results suggest that trophozoites, the predominant form during infection, are haploid (30). However, the possibility that the ploidy of P. carinii hominis differs from that of P. carinii carinii cannot be excluded. Finally, the possibility that there are several different copies of genes per genome has been investigated only in P. carinii carinii. In this organism, the presence of only one copy of the b-tubulin gene (7) and not more than two copies of the nuclear rRNA operon (9) has been demonstrated. As far as mt26S is concerned, the presence of two alleles in a single strain could also result from fusion of trophozoites during sexual reproduction. Thus, none of the three possibilities—coinfection, diploidy, and multicopy genes—which may account for the presence of two alleles in a single sample can be firmly excluded, although the possibility of coinfection is most likely. Moreover, the three possibilities are not mutually exclusive. Products of 7 of the 11 patients yielded a complex SSCP pattern for at least one of the four regions. Taking into account the two simple patterns that constitute each complex pattern, there are theoretically two to eight combinations of four simple patterns of the four regions for each of these patients. Since coinfection could occur, each of these combinations might correspond to a different coinfecting strain. To validate a typing system, many criteria must be taken into account (25). Because of the small number of specimens analyzed, only reproducibility, stability of markers, and specificity of our typing system could be evaluated. The results presented in Fig. 1 were consistently reproduced a minimum of three times for each BAL specimen. Two BAL specimens collected TABLE 5. Polymorphisms in P. carinii hominis b-tub region reported to date

b

Allele

Nucleotide position (bp)a 24

282

1

A

G

2 3

T ●

A A

e

a

No. of observations

Country c

Reference (SSCP pattern no.d)

1 1 1 1

USA CH USA CH

7 This work (1) 7 This work (2)

Dot indicates homology to the reference allele described in the first line. Arbitrary numbers. Abbreviations: CH, Switzerland; USA, United States of America. d According to Fig. 1. e Reference allele. b c

from patient 3 during the same PcP episode generated the same patterns for the four regions (Fig. 1), suggesting that the four regions analyzed are stable over a period of at least 16 days. This stability is also strongly suggested by the fact that the same polymorphic positions and alleles of the variable regions analyzed in the present work were observed by several laboratories from various countries (Tables 2 to 5). The absence of a high mutation rate is also supported by a study which demonstrated the stability of four genetic loci of P. carinii carinii over a 2-year period (13). In the present work, only the primers used to amplify ITS1 are specific to the P. carinii hominis genome; the other primers could use P. carinii carinii DNA as a template (results not shown). However, our results strongly suggest that none of the 11 patients was infected by P. carinii carinii. Indeed, in agreement with their 15 to 20% divergence (23), variable regions of P. carinii carinii and P. carinii hominis generated different SSCP patterns (results not shown). In conclusion, the described multitarget typing method for P. carinii hominis, involving the use of the PCR-SSCP technique, should allow investigation of strain diversity and thus be a useful tool for gaining a better understanding of the epidemiology of this pathogen. However, the possible occurrence of coinfections might complicate epidemiological studies. ACKNOWLEDGMENTS This work was supported by grant 94-7213 from the Swiss National Program on AIDS Research. We thank A. E. Wakefield for the gift of P. carinii carinii DNA and C. Bernasconi for technical assistance. REFERENCES 1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403–410. 2. Atzori, C., F. Agostoni, A. Cargnel, R. Perenboom, and P. Beckers. 1996. ITSs typing of P. carinii samples from Italy, The Netherlands and Tanzania. J. Eukaryot. Microbiol. 43:43S. 3. Banerji, S., E. B. Lugli, R. F. Miller, and A. E. Wakefield. 1995. Analysis of genetic diversity at the arom locus in isolates of Pneumocystis carinii. J. Eukaryot. Microbiol. 42:675–679. 4. Bartlett, M. S., J. J. Lu, C. H. Lee, P. J. Durant, S. F. Queener, and J. W. Smith. 1996. Types of Pneumocystis carinii detected in air samples. J. Eukaryot. Microbiol. 43:44S. 5. Cushion, M. T., J. Zhang, M. Kaselis, D. Giuntoli, S. L. Stringer, and J. R. Stringer. 1993. Evidence for two genetic variants of Pneumocystis carinii coinfecting laboratory rats. J. Clin. Microbiol. 31:1217–1223. 6. Del Sal, G., G. Manfioletti, and C. Schneider. 1988. A one-tube plasmid DNA mini-preparation suitable for sequencing. Nucleic Acids Res. 16:9878. 7. Edlind, T. D., M. S. Bartlett, G. A. Weinberg, G. N. Prah, and J. W. Smith. 1992. The b-tubulin gene from rat and human isolates of Pneumocystis carinii. Mol. Microbiol. 6:3365–3373. 8. Egger, M., J. von Overbeck, B. Janin, P. Francioli, and The Swiss HIV Cohort Study. 1996. Evolution du spectre des infections opportunistes de 1988 `a 1994: expe´rience de l’e ´tude suisse de cohorte HIV (SHCS). Med. Hyg. 54:306–310. 9. Guintoli, D., S. L. Stringer, and J. R. Stringer. 1994. Extraordinarily low number of ribosomal RNA genes in P. carinii. J. Eukaryot. Microbiol. 41:88S. 10. Hauser, P. M., D. S. Blanc, J. Bille, A. M. Telenti, and P. Francioli. 1996. Development of a molecular typing method for Pneumocystis carinii sp. f. hominis. J. Eukaryot. Microbiol. 43:34S. 11. Hayashi, K., and D. W. Yandell. 1993. How sensitive is PCR-SSCP? Hum. Mutat. 2:338–346. 12. Jiang, B., J.-J. Lu, B. Li, X. Tang, M. S. Bartlett, J. W. Smith, and C.-H. Lee. 1996. Development of type-specific PCR for typing Pneumocystis carinii f. sp. hominis based on nucleotide sequence variations of internal transcribed spacer regions of rRNA genes. J. Clin. Microbiol. 34:3245–3248. 13. Keely, S. P., M. T. Cushion, and J. R. Stringer. 1996. Stability of four genetic loci in Pneumocystis carinii sp. f. carinii. J. Eukaryot. Microbiol. 43:49S. 14. Keely, S. P., J. R. Stringer, R. P. Baughman, M. J. Linke, P. D. Walzer, and A. G. Smulian. 1995. Genetic variation among Pneumocystis carinii hominis isolates in recurrent pneumocystosis. J. Infect. Dis. 172:595–598. 15. Latouche, S., E. Ortona, E. Mazars, P. Margutti, E. Tamburrini, A. Siracusano, K. Guyot, M. Nigou, and P. Roux. 1997. Biodiversity of Pneumocystis carinii hominis: typing with different DNA regions. J. Clin. Microbiol. 35:383–387. 16. Latouche, S., P. Roux, J. L. Poirot, I. Lavrard, B. Hermelin, and V. Bertrand.

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