Abstract
By DNA sequence analysis we identified two new strain types and five novel sporadic variations among 25 isolates of Pneumocystis carinii f. sp. hominis obtained from 19 human immunodeficiency virus-positive patients. Of these, 13 were infected with a single strain and 6 were coinfected. Fifteen different combination types were identified among the 18 strains for which complete molecular typing was accomplished.
Pneumocystis carinii f. sp. hominis pneumonia remains the most frequent opportunistic infection among AIDS patients, despite its decline in incidence following the introduction of active antiretroviral combination therapy (18).
Most cases are supposedly due to the reactivation of latent infections, but recent studies indicate the possibility of de novo introduction from human or environmental reservoirs (6, 8). Because an efficient continuous in vitro culture system is not available (4), molecular techniques are essential for epidemiological studies. Sequence analysis of some genetic loci demonstrated a marked heterogeneity of P. carinii isolated from humans or other mammalian species (6, 10, 11, 14, 15, 16, 19, 21). Coinfection with different strains of P. carinii in the same host has also been demonstrated (1, 5, 11, 15, 20).
In this study, we analyzed 25 P. carinii isolates from bronchoalveolar lavage (BAL) fluids obtained from 19 human immunodeficiency virus (HIV)-positive patients of the Amedeo di Savoia Hospital, Turin, Italy. Specimens were collected consecutively from June 1994 to September 1997, and the presence of P. carinii was assessed by microscopy. BAL fluids were centrifuged, and DNA was extracted as described by Moonens et al. (17) and stored at −20°C. Portions of five highly variable P. carinii genes, i.e., the mitochondrial 26S rRNA gene (mt26S), the intron of the nuclear 26S rRNA gene (26S), the β-tubulin intron 6 region (β-tub), and the internal transcribed spacers (ITS-1 and ITS-2) of the rRNA genes, were amplified by PCR. To amplify ITS-1, 26S, mt26S, and β-tub, we used the primer pairs and cycle conditions described by Hauser et al. (5). For ITS-2 amplification, the ITS2U and ITS2L primers and cycle conditions described by Keely and Stringer were used (7). Amplification products were electrophoresed on a 1% agarose gel (Bio-Rad Laboratories), visualized by ethidium bromide staining under UV light, and band purified using Concert Rapid Gel Extraction System (Life Technologies) purification columns. Purified products were directly sequenced from both ends, without cloning, using the Big Dye Terminator DNA Sequencing Kit (Perkin-Elmer) with the oligonucleotide primers used for PCR amplification, and then purified with Centrisep Spin Columns (Princeton Separation; Perkin-Elmer). Products were run on an ABI PRISM 377 DNA sequencer and analyzed with Factura and Sequence Navigator software (all from Perkin-Elmer). Some samples showed double electrophoretic peaks at one or more of the nucleotide positions reported as variable, indicating that the PCR product contained two mixed sequences (coinfection). When the two coinfecting strains differed in a few nucleotide positions, we identified their respective sequence types, whereas when they differed in many positions, the types involved were not resolved (not determinable types).
To show that sequences were not influenced by PCR errors, the ITS-1 loci from all 19 samples were amplified twice. The sequences of the PCR products were identical in all duplicate samples.
β-tub.
All samples were positive for the expected 309-bp fragment of the β-tub locus. Sequence polymorphisms were all detected at positions previously reported to be variable (3, 5), except for a novel variant at nucleotide position 96, where a G-to-A change was detected (Table 1). On the basis of the variations, we divided the samples into three types: type 1, which is identical to the prototypic sequence reported by Edlind et al. (3); type 3, previously described by Hauser et al. (5); and type 4, a novel sporadic variation reported here for the first time, found in fewer than three specimens and therefore not considered a distinct type according to Lu et al. (14). Two coinfections were observed, as A and G were both found at position 96 in one sample (suggesting coinfection with types 3 and 4) and at position 282 in another (suggesting coinfection with types 1 and 3) (Table 1).
TABLE 1.
P. carinii f. sp. hominis β-tub sequence types
TypeH, typing according to Hauser et al. (5).
Novel sporadic variation.
New nucleotide variation.
26S.
Sequence analysis of all samples for the 26S intron (a 426-bp fragment) revealed four types differing at several nucleotide positions (Table 2). We noted new polymorphisms: an A-to-G change at position 34 in one sample and a 2-bp insertion (GG) at position 356 in another. Sequences identical to the 26S prototype reported by Liu and Leibowitz (13), corresponding to Hauser type 1, are designated type α in Table 2. Types β and δ are novel types, and γ is a sporadic variation. Greek letters have been adopted for classification to avoid confusion with terms (Arabic numerals and Roman letters) already in use for other loci.
TABLE 2.
P. carinii f. sp. hominis 26S sequence types
| Typea | Nucleotide at position (bp):
|
No. of observations | ||||||
|---|---|---|---|---|---|---|---|---|
| 3 | 34 | 78 | 212 | 296 | 305 | 356 | ||
| α | A | A | A | A | T | C | G | 15 |
| βb | A | Gc | A | A | —d | C | G | 4 |
| γe | A | A | A | A | — | CA | GGc | 1 |
| δb | A | A | A | A | — | CT | G | 5 |
Greek letters adopted by authors.
New sequence type.
New nucleotide variation.
—, missing base.
Novel sporadic variation.
mt26S.
In all samples we amplified mt26S (a 340-bp fragment). All sequences differed from the prototype sequence reported by Sinclair et al. (19) by a G-to-A change at position 288. On the basis of polymorphisms at nucleotides 85 and 248, we distinguished four sequence types (Table 3), all of them previously described (5, 9, 10, 11). In one specimen, two different nucleotides (C and T) were present at position 248, suggesting coinfection with types 2 and 3.
TABLE 3.
P. carinii f. sp. hominis mt26S sequence types
| TypeHa | Nucleotide at position (bp):
|
No. of observations | ||
|---|---|---|---|---|
| 85 | 248 | 288 | ||
| 2 | C | C | A | 9 |
| 3 | C | T | A | 5 |
| 7 | A | C | A | 2 |
| 8 | T | C | A | 9 |
TypeH, typing according to Hauser et al. (5).
ITS-1.
Sequence comparison revealed that ITS-1 sequences (a 204-bp fragment) can be classified into six types (Table 4). The analysis was based on polymorphisms at nine different positions, including the poly(T) tract at positions 54 to 62. It has been reported that the number of T's may vary when one is resequencing the same sample (21). However, since we did not encounter this problem in two sequencing attempts, we used the poly(T) tract in typing. We found one novel sporadic variation, referred to as B5, and five previously identified types (5, 12) (Table 4). None of the sequences were identical to the prototype sequence reported by Lu et al. (14); types A3, B, and B2 have been described previously by Hauser et al. (5) using the same terms, and by Lee et al., who defined them as A, E, and N, respectively (12). Types B1 and B3 have been described previously by Hauser et al. (5) and by Latouche et al. (9) using the same terms. We also observed four samples with mixed types. For two of these, listed in Table 6 as belonging to patients 159 and 48, we identified one of the two ITS-1 types involved, while for the other two patients, 45 and 158, only PCR cloning could permit the discrimination and identification of the strains.
TABLE 4.
P. carinii f. sp. hominis ITS-1 sequence types
| TypeHa | Nucleotide at position (bp):
|
No. of observations | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| 2 | 8–10 | 11 | 17 | 22 | 46–47 | 54–62 | 71–72 | 111–113 | ||
| A3 | C | 3 T's | A | T | T | TC | 10 T's | - -b | TTA | 2 |
| B | T | 2 T's | A | T | T | TC | 10 T's | AG | TTA | 3 |
| B1 | T | 2 T's | A | T | T | TC | 9 T's | AG | TTA | 4 |
| B2 | T | 2 T's | A | A | T | TC | 10 T's | AG | TTA | 4 |
| B5c | T | 2 T's | A | T | C | TC | 9 T's | AG | TTA | 2 |
| B3 | T | 2 T's | C | T | C | TATC | 9 T's | AG | TTA | 4 |
| Unresolved | 6 | |||||||||
TABLE 6.
Genetic profiles of P. carinii f. sp. hominis strains
| Patient no. | Infection type | ITS-1 typeHa | ITS-2 typeLb | 26S typec | mt26S typeH | β-tub typeH |
|---|---|---|---|---|---|---|
| 3 | Single | A3 | c1 | δ | 3 | 1 |
| 47, 154 | Single | B3 | a4 | β | 8 | 3 |
| 64 | Single | B3 | a4 | β | 3 | 1 |
| 63 | Single | B | a2 | δ | 2 | 3 |
| 4 | Single | B3 | a4 | β | 7 | 1 |
| 55 | Single | B | b2 | α | 3 | 1 |
| 80, 152, 163 | Single | B2 | a1 | α | 2 | 3 |
| 89 | Single | A3 | c1 | δ | 7 | 1 |
| 106 | Single | B1 | a2 | γ | 3 | 3 |
| 153 | Single | B2 | a5 | α | 8 | 3 |
| 48 | Coinfection | B1 | a2 | α | 8 | 1 |
| NDd | a2 | α | 8 | 1 | ||
| 45 | Coinfection | ND | ND | δ | 3 | 1 |
| ND | ND | δ | 2 | 1 | ||
| 91 | Coinfection | B5 | a4 | α | 2 | 1 |
| B5 | a4 | α | 2 | 3 | ||
| 159 | Coinfection | B | ND | α | 8 | 1 |
| ND | ND | α | 8 | 1 | ||
| 157 | Coinfection | B1 | a2 | α | 2 | 3 |
| B1 | a2 | α | 2 | 4 | ||
| 158 | Coinfection | ND | ND | α | 8 | 3 |
| ND | ND | α | 8 | 3 |
ITS-2.
The ITS-2 region (a 327-bp fragment) was amplified in all samples. On the basis of polymorphisms observed in eight previously reported positions, we found six ITS-2 sequence types (Table 5). Types a1, a2, a4, and c1 have been described by Latouche et al., and a1 has been designated the prototype sequence by Lu et al. (9, 14). Types a5 and b2 are novel sporadic variations. We also detected three coinfections, but we were unable to resolve any of the six ITS-2 types involved (Table 6).
TABLE 5.
P. carinii f. sp. hominis ITS-2 sequence types
The combined results obtained from the analysis of the five loci led to a highly accurate characterization of P. carinii strains (Table 6). Of the 25 strains, complete molecular typing was possible for 18, among which 15 different combination types were demonstrated. Thirteen out of 19 patients were infected with a single strain, whereas 6 were coinfected, as indicated by demonstration of mixed genotypes in at least one of the studied loci. The coinfection percentage (31%) in our series is in agreement with that observed by other authors (2, 5); however, we do not know if it represents an underestimate. Indeed, the sensitivity of any molecular typing method depends on the number of microorganisms present in the specimen, which at the moment cannot be easily assessed by conventional methods.
We found two new distinct types and five novel sporadic variations, on the basis of new polymorphic positions identified in our study and/or of new variations at nucleotide positions previously reported as variable.
From the data shown in Table 6 it can be inferred that the most significant association genes for strain typing is ITS-1 and mt26S, whereas the 26S locus consented the identification of new variants but was not useful for strain discrimination. However, it can be supposed that this locus could be significant in a larger series of cases or as part of a different set of indicator genes.
Our results demonstrate great genetic diversity among the isolates. Only two combination types (B3/a4/β/8/3 and B2/a1/α/2/3) were present in more than one patient. Comparison of our sequence data with those reported by other authors revealed the presence of combination types similar to those circulating in other parts of Italy and in other countries. To date, few authors have examined the mt26S locus; therefore, the only possible comparison in these cases has been with the ITS loci. Moreover, there is at present no general agreement on strain classification terms: the strain that we designate B2/a1/2 is identical to the strain designated B2/a1/3 by Latouche et al. (10). Likewise, our strain B/a2/2 is identical to Latouche strain B6/a2/3 (9). Strain B1/a2/8, present in one of the six coinfection samples, is identical to strain B1/a2/1 found by Latouche et al. (10). However, taking into account the ITS type, our B2/a1 strains correspond to the N/e strains described by Lee (12) and to the B2/a1 strains described by Margutti et al. (15) and by Tsolaki et al. (20), even if these authors did not consider the poly(T) tract in typing. The B1/a3 strain described by Margutti et al. and Tsolaki et al. corresponds to our B/a2 or B1/a2. Our B/a2 corresponds to the E/g strain described by Lee. Finally, the ITS combination A3/c1 corresponds to A/1 in the Lee classification.
We think it would be highly desirable to standardize the terms used to define sequence types and genetic loci in order to permit easier comparison of the circulating strains. In this paper we used the Hauser terms to define sequence types of β-tub, mt26S, and ITS-1; we used Latouche terms for ITS-2; and we introduced Greek letters for 26S to avoid confusion with Arabic numerals and Roman letters already in use.
Acknowledgments
This work was supported in part by the Italian Ministry of University and Scientific Research (ex-60% Funds) and Denegri Foundation, Turin, Italy (www.cdfound.to.it).
We thank Mario Zucca for helpful discussions and Enza Ferrero for careful review of the manuscript.
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