Pneumocystis jiroveci Internal Transcribed Spacer Types in Patients Colonized by the Fungus and in Patients with Pneumocystosis from the Same French Geographic Region

Abstract

Pneumocystis jiroveci (human-derived Pneumocystis) infections can display a broad spectrum of clinical presentations, of which pulmonary colonization with the fungus may represent an important part, occurring frequently in patients with various underlying diseases and presenting alternative diagnoses of acute pneumocystosis (Pneumocystis carinii pneumonia [PCP]). There are few data concerning the P. jiroveci genotypes involved in pulmonary colonization, whereas several genotypes responsible for PCP in immunocompromised patients have been described. In this study, P. jiroveci genotypes have retrospectively been investigated and compared in 6 colonized patients and in 11 patients with PCP who were in the same hospital. Seventeen archival bronchoalveolar lavage samples were genotyped at internal-transcribed spacer 1 (ITS1) and ITS2 of the nuclear rRNA operon. Fourteen different genotypes were identified, of which 1 was found only in colonized patients, 10 were found only in patients with PCP, and 3 were found in both patient populations. Mixed infections were diagnosed in 2 of the 6 colonized patients and in 6 of the 11 patients with PCP. The results show that similar genotypes can be responsible for PCP as well as pulmonary colonization. There is a high diversity of genotypes in colonized patients and in patients with PCP. Mixed infections may occur in these two patient populations. These shared features of P. jiroveci ITS genotypes in colonized patients and patients with PCP suggest that human populations infected by P. jiroveci, whatever the clinical manifestation, may play a role as a common reservoir for the fungus.

Over the past decade, the use of PCR has led to the detection of low numbers of Pneumocystis jiroveci (human-derived Pneumocystis) (9, 36) organisms in bronchoalveolar lavage (BAL) specimens from patients, showing an alternative diagnosis of acute Pneumocystis carinii pneumonia (PCP) (24, 30, 40). These low levels of P. jiroveci organisms were considered to reflect pulmonary colonization. These cases of colonization that have been described have occurred mainly in immunocompromised patients (24, 30) and less frequently in immunocompetent patients; nevertheless, cases of colonization in immunocompromised patients presenting with underlying pulmonary diseases have been described (4, 34). Cases of pulmonary colonization with P. jiroveci are frequently missed since PCR assays are not usually used for the routine detection of P. jiroveci in pulmonary specimens. Moreover, the small numbers of P. jiroveci organisms carried by colonized patients render molecular typing of the organisms difficult. For these reasons, little is known concerning the types of P. jiroveci carried by colonized patients whereas several types of P. jiroveci isolates obtained from patients developing PCP have been described (5, 14-17, 19-23, 25, 27, 28, 32, 37-39). Two different molecular studies, one using single-strand conformation polymorphism analysis (10) and another using sequencing of internal-transcribed spacer 1 (ITS1) and ITS2 of the nuclear rRNA operon (29) on three and one specimens, respectively, have shown that the P. jiroveci types identified in colonized patients have been described in previous reports concerning patients with PCP. These results, suggesting that P. jiroveci types responsible for pulmonary colonization do not differ from those responsible for PCP, need further validation. Particularly, it is not known if the prevalence and diversity of the P. jiroveci types are comparable in colonized patients and patients with PCP who are living in the same geographic location. In this study, we have identified and compared the P. jiroveci ITS types present in archival BAL specimens from 6 patients colonized by the fungus and from 11 patients developing PCP who were monitored in the same hospital. P. jiroveci ITS types identified in both patient populations were compared with those previously described in reports concerning patients with acute PCP (16, 23, 27, 28, 37-39).

(This work was partially presented in a conference report [31]).

MATERIALS AND METHODS

Patients and P. jiroveci isolates.

The 17 patients from whom P. jiroveci isolates were obtained and typed were monitored in the University Hospital of Amiens, Amiens, France, between March 1996 and March 1999. Clinical and biological data for each patient are detailed in Table 1.

TABLE 1.

Characteristics of 6 patients colonized by P. jiroveci and 11 patients who developed PCP, for all of whom the fungus detected in BAL fluids was typed

Patient no.	Age	Sexa	Date of BAL (mo-day-yr)	Underlying condition(s)	T-cell countb		Clinical presentation of P. jiroveci infection (alternative diagnosis of PCP, duration of follow-up without PCP)
					CD4+/CD8+	CD4+ (106/liter)
A.6	64	F	03-06-96	Myeloma	0.97	185	Pulmonary colonization (bacterial pneumonia, 3 months)
A.9c	44	M	03-08-96	Panhypopituitarism	0.46	389	Pulmonary colonization (sarcoidosis, 12 months)
A.10	44	F	03-13-96	Renal transplantation	ND	ND	Pulmonary colonization (bacterial pneumonia, 12 months)
A.20	44	F	04-17-96	HIV infection	0.08	6	PCP
A.36	35	M	05-22-96	HIV infection	0.18	31	PCP
A.64	33	M	08-30-96	HIV infection	0.06	108	PCP
A.93	50	M	02-06-97	HIV infection	0.19	59	PCP
N.20d	85	F	05-30-97	Chronic obstructive pulmonary disease	1.8	968	Pulmonary colonization (exacerbation of chronic bronchitis, 6 months)
N.24	52	F	07-09-97	Systemic lupus erythematosus	0.60	569	Pulmonary colonization (pulmonary lymphoma, 32 months)
N.28	67	M	07-17-97	HIV infection	0.20	68	PCP
N.60	29	F	10-03-97	Systemic lupus erythematosus, long-term corticosteroid treatment	0.97	332	PCP
N.61	34	M	01-26-98	HIV infection	0.04	7	PCP
N.51	41	F	03-26-98	Chronic lymphoid leukemia	1.64	71	Pulmonary colonization (bacterial pneumonia, 16 months)
N.50	60	M	05-20-98	Pemphigus vulgaris, long-term cortico- steroid treatment	0.92	638	PCP
N.69	33	M	11-27-98	HIV infection	0.09	17	PCP
N.70	35	M	02-18-99	Hepatic granulomatosis, long-term corticosteroid treatment	ND	ND	PCP
N.71	40	F	03-03-99	HIV infection	0.09	22	PCP

^aF, female; M, male.

^bThe normal values were as follows: for CD4⁺/CD8⁺ T cells, >1; for CD4⁺ T cells, >600. ND, not done.

^cThe case of patient A.9 was previously reported in reference 29.

^dPatient N.20 was the only immunocompetent colonized patient.

Six P. jiroveci isolates were obtained from six human immunodeficiency virus (HIV)-negative patients who were colonized by the fungus. The patients were submitted to BAL to investigate pulmonary symptoms (abnormal chest X ray, cough) or fever. P. jiroveci was not detected in BAL specimens by microscopic examination, whereas it was detected by a PCR assay performed after rapid DNA extraction (GeneReleaser; BioVentures, Inc., Murfreesboro, Tenn.) (33) with specific primers of the gene encoding the mitochondrial large subunit rRNA (mt LSU rRNA) (40). Alternative diagnoses of PCP were available for five patients, while symptom etiology was not clearly determined for the sixth patient, who presented exacerbation of chronic bronchitis. Clinical improvement was observed in the six patients after P. jiroveci detection despite the absence of specific treatment for the fungus. For these reasons, the patients were considered to be merely colonized by P. jiroveci.

Eleven other P. jiroveci isolates were obtained from 11 patients diagnosed with PCP according to criteria described for HIV-infected patients by the Centers for Disease Control and Prevention (3). P. jiroveci was detected in BAL specimens by microscopic examination with methanol-Giemsa stain and an immunofluorescence assay (MonofluoKit Pneumocystis; Bio-Rad, Marnes La Coquette, France). The underlying conditions were HIV infection or long-term corticosteroid therapy. Specimens from both the colonized and PCP patients were stored at −80°C until typing.

P. jiroveci typing.

Typing of P. jiroveci isolates was performed by analysis of the ITS1 and ITS2 sequences. A DNA extraction procedure using a commercially available kit (QIAamp DNA MiniKit; Qiagen, Valencia, Calif.) was performed on all BAL specimens. To amplify the ITS1 and ITS2 sequences, a nested PCR assay was performed with two pairs of primers specific for P. jiroveci, N18SF (5′-GGT CTT CGG ACT GGC AGC-3′) with N26SRX (5′-TTA CTA AGG GAA TCC TTG TTA-3′) (first PCR round) and ITSF3 (5′-CTG CGG AAG GAT CAT TAG AAA-3′) with ITS2R3 (5′-GAT TTG AGA TTA AAA TTC TTG-3′) (second PCR round), described by Tsolaki et al. (37, 38). The two rounds were performed under the same reagent conditions. The primers were used (1 μM each) in a reaction mixture (50 μl) containing 3 mM MgCl₂, 10 mM Tris HCl, a 200 μM concentration of each deoxynucleoside triphosphate (dNTP set; Eurogentec, Seraing, Belgium), and 0.4 U of polymerase (Goldstar DNA polymerase; Eurogentec). The second PCR round was done with 3 μl of the first-round PCR products. The two rounds of PCR were done with denaturation at 94°C for 1.30 min, annealing at 55°C for 1.30 min, and extension at 72°C for 2 min for 40 cycles. The first- and second-round PCR products were electrophoresed on a 1.5% agarose gel containing ethidium bromide to visualize the expected bands (580- and 530-bp bands, respectively). To avoid contamination due to environmental amplicons, extraction, reagent preparation, and amplification procedures were performed in separate rooms with different sets of micropipettes and using barrier tips. Extraction and reagent preparations were performed in flow cabinets. Rooms required for amplified DNA manipulation were continuously submitted to an airflow with UV decontamination (SPRW 30 GR4; Paragerm, Inc., Paris, France). To monitor for possible contamination, negative controls (ultra pure distilled water) were included in extraction and the first- and second-round PCR procedures.

The second-round PCR products were purified by using Microcon PCR columns (Millipore, Bedford, Mass.) and cloned into the plasmid pGEMT (pGEMT Vector System II kit; Promega Corporation, Madison, Wis.). In order to verify if the transformation was related to the correct recombinant plasmids, each positive clone was subjected to a PCR assay using T7 (5′-GTA ATA CGA CTC ACT ATA G-3′) and SP6 (5′-ATT TAG GTG ACA CTA TAG AA-3′), universal primers specific for the T7 and SP6 promoters flanking the cloning region. Recombinant plasmids were sequenced from the two strands by using the same universal primers with the dideoxy chain termination method and an automatic sequencer (BigDye Terminator method, model 3700 sequencer; Applied Biosystems, Foster City, Calif.). Sequence alignment was performed with Clustal W software version 1.81. ITS1 and ITS2 alleles were identified by using the score previously described by Tsolaki et al. (37, 38). P. jiroveci ITS types were defined by a combination of the alleles of the two loci.

Nucleotide sequence accession number.

The nucleotide sequence of the new ITS2 allele has been deposited in GenBank under accession number AF498265.

RESULTS

Results of the P. jiroveci ITS type identification are shown in Table 2. By applying the score described for typing by Tsolaki et al. (37, 38), four different types were identified in six colonized patients. Type B₁a₃ was detected in three patients (50%). Types B₂a₁ and A₃a₃ were each found in two patients (33%). Type A₃a₃ results in a new combination of the ITS1 allele A₃ with the ITS2 allele a₃ described previously (37, 38). This new allele combination was considered to reflect a new type. The ITS1 allele, which we designated ‘A,' was close to allele A₂ or A₃; however, it differed by T residues at positions 2 and 16 of the ITS1 locus. Applying the score described for typing by Lee et al. (23), type ‘A’a₃ is the same as type Gg (GenBank accession numbers AF013812 [ITS1G] and AF013827 [ITS2g]). More than one type was detected in two patients, showing the same type association, A₃a₃/B₁a₃.

TABLE 2.

Identification of P. jiroveci ITS types in 6 patients colonized by the fungus and in 11 patients who developed PCP in the University Hospital, Amiens, France

Patient no.	Clone no.	Nucleotide(s) at indicated position(s) in locusa:										Type(s)	Cb or PCP
		ITS1				ITS2
		2	16	74-75	111-113	54-56	63	67-71	122	169-172	176
A.6	A6.4	T	A	AG	TTA	TAA	-	AATAT	-	----	G	B2a1	C
A.9	A9.1	T	T	--	TTA	TAA	-	AATAA	-	----	A	‘A’a3c	C
	A9.2	T	T	--	TTA	TAA	-	AATAA	-	----	A	‘A’a3
	A9.3	T	T	--	TTA	TAA	-	AATAA	-	----	A	‘A’a3
A.10	A10.1	T	T	AG	TTA	TAA	-	AATAA	-	----	A	B1a3	C
	A10.3	T	T	AG	TTA	TAA	-	AATAA	-	----	A	B1a3
	A10.5	T	T	AG	TTA	TAA	-	AATAA	-	----	A	B1a3
A.20	A20.1	T	T	--	TTA	TAA	-	AATAA	-	----	A	‘A’3	PCP
	A20.2	T	T	AG	TTA	TAA	-	AATAA	-	----	A	B1a3
A.36	A36.2	T	A	AG	TTA	TAA	-	AATAT	-	----	G	B2a1	PCP
	A36.3	T	A	AG	TTA	TAA	-	AATAT	-	----	G	B2a1
	A36.7	T	A	AG	TTA	TAA	-	AATAT	-	----	G	B2a1
A.64	A64.1	C	T	--	TTA	TAA	-	AATAT	-	----	A	A2a4	PCP
	A64.2	T	A	--	TTA	TAA	-	AATAT	-	----	A	A3a4
	A64.3	C	T	--	TTA	---	A	-----	-	----	A	A2c1
A.93	A93.2	T	A	AG	TTA	TAA	-	AATAT	-	----	G	B2a1	PCP
	A93.3	T	A	AG	TTA	TAA	-	AATAT	-	----	G	B2a1
	A93.6	T	A	AG	TTA	TAA	-	AATAA	-	----	A	B2a3
	A93.8	T	A	AG	TTA	TAA	-	AATAT	-	----	G	B2a1
N.20	N20.2	T	A	AG	TTA	TAA	-	AATAT	-	----	G	B2a1	C
	N20.7	T	A	AG	TTA	TAA	-	AATAT	-	----	G	B2a1
N.24	N24.1	T	T	AG	TTA	TAA	-	AATAA	-	----	A	B1a3	C
	N24.2	T	T	AG	TTA	TAA	-	AATAA	-	----	A	B1a3
	N24.4	T	A	--	TTA	TAA	-	AATAA	-	----	A	A3a3
N.28	N28.1	T	T	AG	TTA	TAA	-	AATAT	C	--AT	G	B1b2	PCP
	N28.9	T	T	AG	TTA	TAA	-	AATAT	C	--AT	G	B1b2
	N28.10	T	T	AG	TTA	TAA	-	AATAT	C	--AT	G	B1b2
N.60	N60.3	T	T	AG	TTA	TAA	-	AATAA	-	----	A	B1a3	PCP
	N60.6	T	T	AG	TTA	TAA	-	AATAA	-	----	A	B1a3
	N60.9	T	T	AG	TTA	TAA	-	AATAA	-	----	A	B1a3
N.61	N61.6	C	T	--	TTA	---	A	-----	-	----	A	A2c1	PCP
	N61.7	C	T	--	TTA	---	A	-----	-	----	A	A2c1
	N61.10	C	T	--	TTA	---	A	-----	-	----	A	A2c1
N.51	N51.1	T	T	AG	TTA	TAA	-	AATAA	-	----	A	B1a3	C
	N51.6	T	T	AG	TTA	TAA	-	AATAA	-	----	A	B1a3
	N51.7	T	A	--	TTA	TAA	-	AATAA	-	----	A	A3a3
N.50	N50.2	T	A	AG	TTA	TAA	-	AATAT	-	----	G	B2a1	PCP
	N50.5	T	A	AG	TTA	TAA	-	AATAT	-	----	G	B2a1
N.69	N69.1	C	T	--	TTA	---	A	-----	-	----	A	A2c1	PCP
	N69.2	C	T	--	TTA	TAA	-	AATAT	-	----	A	A2a4
	N69.4	C	T	--	TTA	TAA	-	AATAT	-	--AT	A	A2‘b’d
N.70	N70.2	T	A	AG	TTA	TAA	-	AATAA	-	----	A	B2a3	PCP
	N70.3	T	T	AG	TTA	TAA	-	AATAT	-	--AT	G	B1b1
	N70.4	T	A	AG	TTA	TAA	-	AATAT	-	--AT	G	B2b1
N.71	N71.1	T	A	--	TTA	TAA	-	AATAA	-	----	G	A3a2	PCP
	N71.2	T	T	--	TTA	TAA	-	AATAA	-	----	A	‘A’a3
	N71.3	T	T	--	TTA	TAA	-	AATAA	-	----	A	‘A'a3
	N71.4	T	T	--	TTA	TAA	-	AATAA	-	----	G	‘A'a2
	N71.5	T	A	AG	TTA	TAA	-	AATAT	-	----	G	B2a1

^aScoring positions are described in references 37 and 38; complete sequence data are available upon request from the authors. Each dash represents one position for which the nucleotide is not specified.

^bC, colonized.

^cThe ITS1 allele designated ‘A’ differs from the A₂ and A₃ previously described (37, 38) by having T residues at positions 2 and 16.

^dThe ITS2 allele designated ‘b’ (GenBank accession number AF498265) differs from the b₁ previously described (38) by having an A residue at position 176.

Thirteen different types were found in 11 patients with PCP. Type B₂a₁ was found in four patients (36%). Type A₂c₁ was identified in three patients (27%). Types B₁a₃, B₂a₃, A₂a₄, and ‘A’a₃ were each detected in two different patients (18%). Types A₃a₄, B₁b₂, and A₂‘b' were each identified in only one patient. Types B₁b₁ and B₂b₁ were found in the same patient. Both types A₃a₂ and ‘A'a₂ were found in another patient. Types B₁a₃, B₂a₁, A₂c₁, B₁b₂, B₁b₁, and B₂a₃ have been previously reported in patients with PCP (16, 27, 28, 37-39). Type B₂b₁, which was not previously observed by Tsolaki et al. (37, 38), has previously been described by Lee et al. and designated Nb (23). Types A₂a₄, A₃a₄, and A₃a₂ result from new combinations of ITS1 and ITS2 alleles described previously (37, 38). These new allele combinations were considered to be new types. Type ‘A'a₂ results in a combination of the identical ITS1 allele ‘A' described above with the ITS2 allele a₂ described previously (37). The ITS2 allele that we designated ‘b' differed from the b₁ allele previously reported (37) by an A residue at position 176 of the ITS2 locus. Allele combinations ‘A'a₂ and A₂‘b' were also considered to be new types. More than one type was found in six patients with PCP, showing six different type associations.

DISCUSSION

In this study, specimens from 6 colonized patients and 11 patients who had PCP were typed. The hypothesis that low levels of P. jiroveci initially detected only by PCR in the six colonized patients can be related to sample contamination by the fibroscopes previously used for other patients who had PCP was ruled out. Indeed, a PCP case was diagnosed 16 weeks before the first colonized patient enrolled in the study (data not shown). The other PCP cases were diagnosed at least 8 weeks before each case of pulmonary colonization with P. jiroveci (Table 1). Positive results of P. jiroveci detection in the six colonized patients were obtained by using two PCR assays performed with specific primers from two different loci (at the mt LSU rRNA and the ITS regions) after two different extraction procedures (with the GeneReleaser and QIAamp DNA MiniKit). As different rooms, flow cabinets, and air UV decontamination procedures as well as several negative controls were used, contamination of specimens within PCR processing was excluded. These data are consistent with a genuine presence of P. jiroveci cells in the respiratory tracts of the patients.

Five colonized patients had an impairment of immunity. Thus, the presence of P. jiroveci was consistent with their immune status (24, 30), the fungus behaving opportunistically. In the sixth patient, who was immunocompetent and had chronic bronchitis, the presence of P. jiroveci may have been due to lung damage, as previously suggested by Armbruster et al. (4).

For the typing, the method based on ITS sequence analysis was chosen since these loci are more informative than the mt LSU rRNA (37). By using this method, previous reports have shown that the most frequent P. jiroveci types in patients with PCP from different regions of Europe or the United States (23) were identical (B₂a₁ and B₁a₃ [corresponding to Ne and Eg as described by Lee et al. {23}]). This identity of major types among nonepidemiologically linked isolates pleads in favor of ITS sequence analysis as a valuable method for typing the P. jiroveci cells involved in PCP. Our results show that this method also makes it possible to type organisms involved in pulmonary colonization. Nevertheless, ITS genotyping is quite difficult for colonized-patient samples that contain small quantities of P. jiroveci. Even after amplification by ITS PCR, the amount of P. jiroveci DNA obtained from colonized-patient samples remained small. For this reason, a prior cloning of ITS PCR products was preferred to direct sequencing in order to enhance the amount of DNA to be sequenced. Furthermore, cloning facilitates the detection of mixed infections revealed by the identification of more than one type in specimens.

It has recently been established that genotyping by PCR on BAL specimens may represent only the dominant types present within lungs (15). Consistently, our observations may also concern only dominant types in both PCP and colonized patients. In the present work, a high diversity of P. jiroveci ITS genotypes was observed in colonized patients close to that previously reported in studies on patients with PCP (28, 38). Nonetheless, the diversity of types in colonized patients appears to be lower than that in our patients with PCP. The results are linked to the low rate of mixed infections in the first patient population in comparison with that in the second one. This difference may be partially due to the fact that the sequencing of only one clone in one colonized patient (A.6) did not allow the detection of a mixed infection. Nonetheless, our results establish that mixed infections may occur in patients with pulmonary colonization and that this form of P. jiroveci infection may not be necessarily clonal as previously suggested for PCP (2, 38).

Among the four types identified in the colonized patients, three types (B₁a₃, B₂a₁, and ‘A'a₃) were also detected in our patients with PCP. It is noteworthy that types B₁a₃ and B₂a₁, two of the most frequent types previously identified in reports on patients who had PCP (28, 37-39), are also common in the two patient populations examined in our study. Type A₃a₃ was found only in colonized patients. As this type has not previously been reported, the hypothesis that it can be involved only in pulmonary colonization cannot be ruled out. Likewise, the fact that type A₂c₁, found in patients with PCP (references 28 and 37 and this study), was absent in colonized patients may be consistent with the results of Miller and Wakefield (28), who suggested that this type was responsible for severe PCP. In addition to type A₂c₁, nine other less frequent types (A₂a₄, B₂a₃, A₃a₄, B₁b₂, A₂‘b', B₁b₁, B₂b₁, A₃a₂, and ‘A'a₂) were detected only in patients with PCP, showing a high diversity of types in this patient population. In fact, this diversity of P. jiroveci types, as well as the occurrence of mixed infections in the two patient populations, renders it quite difficult to clearly establish a correlation between genotypes and clinical profiles (colonization versus PCP). The difficulties have previously been addressed to explain the conflicting results of previous studies on clinical correlation with P. jiroveci types in patients with PCP (11-14, 28). Our results indicate above all that shared features of P. jiroveci ITS types (diversity, partial commonality of types, and occurrence of mixed infections) can be observed in both colonized individuals and patients with PCP, monitored in the same geographic region at the same time.

Host-to-host airborne transmission of the fungus has been demonstrated in rodent models (7, 18, 35), and observations suggest that interindividual transmission occurs in man (26, 27). Moreover, it is now presently assumed that Pneumocystis organisms infecting mammalian hosts are host specific (1, 8). Consistently, an animal reservoir for P. jiroveci has been excluded. Although an exosaprophytic form of P. jiroveci cannot be ruled out, these data suggest that humans constitute the main reservoir and infection sources of P. jiroveci. New detection tools like PCR assays have revealed that P. jiroveci infection can display a large spectrum of clinical presentations, of which acute PCP in immunocompromised patients may represent only a small part, while pulmonary colonization may be a major part (6). The results of our P. jiroveci ITS type identification in colonized patients with those of previous studies on patients with PCP suggest that human populations infected by P. jiroveci, whatever the clinical context, may play a role as a common human reservoir.