![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Previous Article | Next Article 
Journal of Clinical Microbiology, August 2001, p. 2995-2998, Vol. 39, No. 8
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.8.2995-2998.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Genetic Diversity of Pneumocystis
carinii Isolated from Human Immunodeficiency Virus-Positive
Patients in Turin, Italy
Gisella
Volpe,1
Luca
Sbaiz,1
Claudio
Avanzini,1
Pietro
Caramello,2 and
Dianella
Savoia1,*
Department of Clinical and Biological
Sciences, University of Turin,1 and
Clinic of Infectious Diseases, Amedeo di Savoia
Hospital,2 Turin, Italy
Received 1 February 2001/Returned for modification 1 April
2001/Accepted 10 May 2001
 |
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.
| |
TEXT |
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 
-tubulin intron 6 region (-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
-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.
-tub.
All samples were positive for the expected 309-bp
fragment of the -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).
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 
in Table 2.
Types and 
are novel types, and 
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.
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.
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.
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).
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//8/3 and
B2/a1//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 -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.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Clinical and Biological Sciences
University of Turin, San Luigi
Hospital, 10043 Orbassano (Turin), Italy. Phone: 39-011-6708127. Fax:
39-011-9038639. E-mail: dianella.savoia{at}unito.it.
| |
REFERENCES |
| 1.
|
Atzori, C.,
F. Agostoni,
A. Cargnel,
R. Perenboom, and P. Beckers.
1996.
ITS typing of Pneumocystis carinii samples from Italy, the Netherlands and Tanzania.
J. Eukaryot. Microbiol.
43:43S[Medline].
|
| 2.
|
Beard, C. B,
J. L. Carter,
S. P. Keely,
L. Huang,
N. J. Pieniazek,
I. N. Moura,
J. M. Roberts,
A. W. Hightower,
M. S. Bens,
A. R. Freeman,
S. Lee,
J. R. Stringer,
J. S. Duchin,
C. del Rio,
D. Rimland,
R. P. Baughman,
D. A. Levy,
V. J. Diets,
P. Simon, and T. R. Navin.
2000.
Genetic variation in Pneumocystis carinii isolates from different geographic regions: implications for transmission.
Emerg. Infect. Dis.
6:265-271[Medline].
|
| 3.
|
Edlind, T. D.,
M. S. Bartlett,
G. A. Weinberg,
G. N. Prah, and J. W. Smith.
1992.
The -tubulin gene from rat and human isolates of Pneumocystis carinii.
Mol. Microbiol.
6:3365-3373[CrossRef][Medline].
|
| 4.
|
European Concerted Action on Pneumocystis carinii.
1996.
In vitro systems in Pneumocystis research.
Parasitol. Today
12:245-249.
|
| 5.
|
Hauser, P. M.,
P. Francioli,
J. Bille,
A. Telenti, and D. S. Blanc.
1997.
Typing of Pneumocystis carinii f. sp. hominis by single-strand conformation polymorphism of four genomic regions.
J. Clin. Microbiol.
35:3086-3091[Abstract].
|
| 6.
|
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[Medline].
|
| 7.
|
Keely, S. P., and J. R. Stringer.
1997.
Sequences of Pneumocystis carinii f. sp. hominis strains associated with recurrent pneumonia vary at multiple loci.
J. Clin. Microbiol.
35:2745-2747[Abstract].
|
| 8.
|
Latouche, S.,
J. L. Poirot,
I. Lavrard,
M. Miltgen,
M. Nigou, and P. Roux.
1996.
Usefulness of molecular biology for Pneumocystis carinii hominis pneumonia epidemiology.
J. Eukaryot. Microbiol.
43:56S[Medline].
|
| 9.
|
Latouche, S.,
J. L. Poirot,
C. Bernard, and P. Roux.
1997.
Study of internal transcribed spacer and mitochondrial large-subunit genes of Pneumocystis carinii f. sp. hominis isolated by repeated bronchoalveolar lavage from human immunodeficiency virus-infected patients during one or several episodes of pneumonia.
J. Clin. Microbiol.
35:1687-1690[Abstract].
|
| 10.
|
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 different DNA regions.
J. Clin. Microbiol.
35:383-387[Abstract].
|
| 11.
|
Lee, C. H.,
J. J. Lu,
M. S. Bartlett,
M. M. Durlin,
T. H. Liu,
J. Wang,
B. Jiang, and J. W. Smith.
1993.
Nucleotide sequence variation in Pneumocystis carinii strains that infect humans.
J. Clin. Microbiol.
31:754-757[Abstract/Free Full Text].
|
| 12.
|
Lee, C. H.,
J. Helweg-Larsen,
X. Tang,
S. Jin,
B. Li,
M. S. Bartlett,
J. J. Lu,
B. Lundgren,
J. D. Lundgren,
M. Olsson,
S. B. Lucas,
P. Roux,
A. Cargnel,
C. Atzori,
O. Matos, and J. W. Smith.
1998.
Update on Pneumocystis carinii f. sp. hominis typing based on nucleotide sequence variations in internal transcribed spacer regions of rRNA genes.
J. Clin. Microbiol.
36:734-741[Abstract/Free Full Text].
|
| 13.
|
Liu, Y., and M. J. Leibowitz.
1993.
Variation and in vitro splicing of group introns in rRNA genes of Pneumocystis carinii.
Nucleic Acids Res.
21:2415-2421[Abstract/Free Full Text].
|
| 14.
|
Lu, J. J.,
M. S. Bartlett,
M. Shaw,
J. W. Smith,
M. Ortiz-Rivera,
M. J. Leibowitz, and C. H. Lee.
1994.
Typing of Pneumocystis carinii strains that infect humans based on nucleotide sequence variations of internal transcribed spacers of rRNA genes.
J. Clin. Microbiol.
32:2904-2912[Abstract/Free Full Text].
|
| 15.
|
Margutti, P.,
E. Visconti,
P. Mencarini,
M. Zolfo,
S. Marinaci,
E. Tamburrini,
A. Siracusano, and E. Ortona.
1998.
Typing with internal transcribed spacer regions of Pneumocystis carinii from AIDS patients with recurrent pneumonia.
Res. Microbiol.
149:595-599[Medline].
|
| 16.
|
Mazars, E., and E. Dei-Cas.
1998.
Epidemiological and taxonomic impact of Pneumocystis biodiversity.
FEMS Immunol. Med. Microbiol.
22:75-80[CrossRef][Medline].
|
| 17.
|
Moonens, F.,
C. Liesnard,
F. Brancart,
J. P. Van Vooren, and E. Serruys.
1995.
Rapid simple and nested polymerase chain reaction for the diagnosis of Pneumocystis carinii pneumonia.
Scand. J. Infect. Dis.
27:358-362[Medline].
|
| 18.
|
Palella, F. J., Jr.,
K. M. Delaney,
A. C. Moorman,
M. O. Loveless,
J. Fuhrer,
G. A. Satten,
D. J. Aschman,
S. D. Holmberg, and the HIV Outpatient Study Investigators.
1998.
Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection.
N. Engl. J. Med.
338:853-860[Abstract/Free Full Text].
|
| 19.
|
Sinclair, K.,
A. E. Wakefield,
S. Banerji, and J. M. Hopkin.
1991.
Pneumocystis carinii organisms derived from rat and human hosts are genetically distinct.
Mol. Biochem. Parasitol.
45:183-184[CrossRef][Medline].
|
| 20.
|
Tsolaki, A. G.,
R. F. Miller,
A. P. Underwood,
S. Banerji, and A. E. Wakefield.
1996.
Genetic diversity at the internal transcribed spacer regions of the rRNA operon among isolates of Pneumocystis carinii from AIDS patients with recurrent pneumonia.
J. Infect. Dis.
174:141-156[Medline].
|
| 21.
|
Wakefield, A. E.,
C. C. Fritscher,
A. S. Malin,
L. Gwanzura,
W. T. Hughes, and R. F. Miller.
1994.
Genetic diversity in human-derived Pneumocystis carinii isolates from four geographical locations shown by analysis of mitochondrial rRNA gene sequences.
J. Clin. Microbiol.
32:2959-2961[Abstract/Free Full Text].
|
Journal of Clinical Microbiology, August 2001, p. 2995-2998, Vol. 39, No. 8
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.8.2995-2998.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Top
Abstract
Text
