Restriction Fragment Length Polymorphism Typing Demonstrates Substantial Diversity among Pneumocystis jirovecii Isolates
Restriction Fragment Length Polymorphism Typing Demonstrates Substantial Diversity among Pneumocystis jirovecii Isolates
Chiara Ripamonti 2
Abigail Orenstein 2
Geetha Kutty 2
Laurence Huang 0
Regina Schuhegger 4
Andreas Sing 4
Giovanna Fantoni 2
Chiara Atzori 3
Carol Vinton 2
Charles Huber 1
Patricia S. Conville 1
Joseph A. Kovacs () 2
0 Department of Medicine, San Francisco General Hospital, University of California San Francisco , San Francisco
1 Department of Laboratory Medicine, National Institutes of Health Clinical Center, National Institutes of Health , Bethesda, Maryland
2 Critical Care Medicine Department
3 Department of Infectious Diseases, Luigi Sacco Hospital , Milan , Italy
4 Bavarian Health and Food Safety Authority (Bavarian LGL) , Oberschleissheim , Germany
Better understanding of the epidemiology and transmission patterns of human Pneumocystis should lead to improved strategies for preventing Pneumocystis pneumonia (PCP). We have developed a typing method for Pneumocystis jirovecii that is based on restriction fragment length polymorphism (RFLP) analysis after polymerase chain reaction amplification of an ∼1300 base-pair region of the msg gene family, which comprises an estimated 50-100 genes/genome. The RFLP pattern was reproducible in samples containing 11000 msg copies/ reaction and was stable over time, based on analysis of serial samples from the same patient. In our initial analysis of 48 samples, we found that samples obtained from different individuals showed distinct banding patterns; only samples obtained from the same patient showed an identical RFLP pattern. Despite this substantial diversity, samples tended to cluster on the basis of country of origin. In an evaluation of samples obtained from an outbreak of PCP in kidney transplant recipients in Germany, RFLP analysis demonstrated identical patterns in samples that were from 12 patients previously linked to this outbreak, as well as from 2 additional patients. Our results highlight the presence of a remarkable diversity in human Pneumocystis strains. RFLP may be very useful for studying clusters of PCP in immunosuppressed patients, to determine whether there is a common source of infection.
Pneumocystis jirovecii is an opportunistic fungus that
causes pneumonia in immunocompromised patients,
especially those with HIV infection, in whom it remains
a major cause of morbidity and mortality [
Understanding the epidemiology of human Pneumocystis
infection can be important in minimizing the clinical
impact of this pathogen.
Recently, a number of molecular biologic methods
have been developed to study the epidemiology,
transmission patterns, and potential emergence of
antimicrobial resistance of this organism [
]. At least 14
unique genetic loci have been evaluated using a
variety of typing methods, including DNA sequence,
single strand conformation polymorphism, and
restriction fragment length polymorphism (RFLP). In general,
genes that evolve very slowly, such as the mitochondrial
small subunit ribosomal RNA (rRNA) and 5.8S rRNA
genes, have been used in evolutionary studies or to
examine genetic diversity. Genes that show greater
diversity, such as the internal transcribed spacer region
of the nuclear rRNA genes, have been used to evaluate
case clustering and to look for evidence of direct
person-to-person transmission. Genes that are targets of
therapeutic drugs, such as the dihydropteroate synthetase and
dihydrofolate reductase genes, have been used to study the
development of drug resistance.
The major surface glycoprotein is a Pneumocystis surface
protein encoded by a multicopy msg gene family with an estimated
50–100 copies of related but unique msg genes per genome; only
a single msg variant, which is present at a unique (single copy)
expression site, the upstream conserved sequence, is expressed
in a given organism [
]. We previously showed that a region
in the intron of the upstream conserved sequence of P. jirovecii
contains variable numbers of 10 base-pair (bp) tandem repeats
that can be easily quantitated and used in epidemiologic studies
]. More recently, we have demonstrated by RFLP analysis that
the repertoire of msg genes (50–100 genes per genome) shows
substantial variation among P. jirovecii isolates but not among
Pneumocystis carinii (infecting rats) or Pneumocystis murina
(infecting mice) isolates [
]. This suggests that RFLP analysis
of msg genes could be used as an epidemiologic tool to
investigate human Pneumocystis infection. The current study was
undertaken to determine whether msg repertoire variability
could be used as a typing method to distinguish among
different human isolates of Pneumocystis.
MATERIALS AND METHODS
Patient specimens. A total of 48 samples with isolates of P.
jirovecii, which included autopsy lung, bronchoalveolar lavage
(BAL), sputum, and oral wash samples, were obtained from 40
patients from San Francisco, California (n p 8); other parts of
the United States (n p 8); the Netherlands (n p 3) [
Milan (n p 21) [
]. An additional 26 amplifiable samples were
obtained from 22 patients from Germany [
]. Guidelines of
the US Department of Health and Human Services were
followed in the conduct of these studies.
DNA amplification of msg variable region. Genomic DNA
was extracted using the QIAamp DNA mini Kit (Qiagen),
according to the manufacturer’s instructions. Nested (initial
studies) or semi-nested polymerase chain reaction (PCR) was used
to amplify ∼1.3 kb of the msg variable region with use of the
following primers: for the first round, GK 126, 5
-GTGGCGCGGGCGGT-3 (corresponding to 2138–2151 bp of P. jirovecii msg
upstream conserved sequence; GB#AF367050; for initial
studies) or GK 472, 5 -TGGATCAAAAGMGAGAYTTYCCRACAG-3
(corresponding to 1576–1602 bp of P. jirovecii msg HuMSG14;
GB#AF033209; for later studies) and GK 452, 5
-AATGCACTTTCMATTGATGCT-3 (complementary to 479–499 bp of P.
jirovecii msg; partial coding system; GB#AF372980); and for the
second round (all studies), GK 472 and GK 195, 5
-GTGTGTGTCGATGTCTGTG-3 (complementary to 2875–2893 bp of P.
jirovecii msg HuMSG14). PCR was performed using HotStart
Taq (Qiagen). The conditions were 15 min at 95 C, followed
by 35 cycles of 30 s at 94 C, 30 s at 60 C, and 4 min (for the
first round) or 2 min (for the second round) at 72 C, with a
final extension of 10 min at 72 C. msg Copy number was
quantitated by a previously described real-time quantitative PCR
Restriction enzyme treatment. PCR products were purified
using QuickStep 2 PCR Purification Kit (Edge BioSystems),
according to the manufacturer’s instructions. The purified products
were digested with Dra1, HindIII, or Xba1 restriction enzymes
for 5–6 h at 37 C. The digested products were analyzed on a
1.2% TBE agarose gel and were visualized by SYBR green
staining (Molecular Probes). After transfer to a Nytran membrane
(Whatman), the blot was hybridized with a
digoxigenin-labeled DNA probe (PCR DIG Probe Synthesis Kit; Roche). The
PCR probe (spanning ∼1.3 Kb) was an equal mixture of 4
products obtained by PCR amplification (as described above) of lung
samples from 4 P. jirovecii–infected individuals. The hybridization
signal was detected by chemiluminescence with use of alkaline
phosphatase–conjugated anti-digoxigenin antibody and CDPstar
(Roche). The results were recorded with a Luminescent Imager
(Kodak Image Station 440CF; PerkinElmer).
Analysis of gels and blots The gels and blots were analyzed
using BioNumerics software (version 4.01; Applied Maths). The
pattern of banding among different gels and/or blots was
normalized using internal standards that were included in each run:
Lambda/HindIII molecular weight markers for gels and a clinical
sample (sample number 385) for blots. Molecular weights were
assigned to the bands of the standards, and sample bands were
identified manually. The Dice coefficient was used to analyze the
similarity of the patterns of bands with a position tolerance of
]. The unweighted pair group method with average
linkages was used by the BioNumerics software for cluster
analysis. DNA samples with banding patterns with 100% similarity
(Dice coefficient, 1) were considered to be identical.
RFLP assay development and reproducibility. We previously
demonstrated substantial variation in RFLP patterns among a
small number of P. jirovecii isolates by using analysis of the
entire msg sequence (∼3200 nucleotides) from autopsy lung
]. Because of difficulties in amplifying the entire
sequence when using samples with lower organism loads (eg,
induced sputum), we developed a semi-nested PCR that
amplified an ∼1300 nucleotide region of msg with use of primers
from conserved regions of msg (based on alignment of available
P. jirovecii msg clones).
In preliminary studies, RFLP analysis of this shorter msg
fragment also showed substantial variation among isolates. To
investigate the reproducibility of this assay, we ran replicate
PCRs using Pneumocystis DNA extracted from lung tissue
and oral wash samples. The PCR products were digested with
HindIII or Dra1 restriction enzymes and were analyzed by
agarose gel electrophoresis or Southern blotting. We found that
the pattern was reproducible with use of lung tissue samples
but not consistently with use of oral wash samples (data not
shown), suggesting that the reproducibility of the RFLP pattern
is dependent on organism load. By serial dilution of a single
sample, we found that this assay lost reproducibility at !1000
msg copies per PCR (as determined by quantitative PCR [
data not shown). On the basis of these data, only samples with
11000 msg copies per PCR were considered to be reliably
reproducible, and samples with !1000 msg copies were used with
RFLP pattern stability over time. To investigate the
stability of the RFLP pattern in samples obtained from 1 individual
over time, we examined paired samples collected over varying
periods. Samples collected at close time points (eg, !3 months
apart) likely represent the same episode of PCP and likely would
exhibit the greatest stability. We analyzed 16 samples from 8
individuals (10 BAL samples, 2 sputum samples, and 4 oral
wash samples). The collection time between samples ranged
from 1 day to 111 days. All BAL sample pairs (from patients
1 and 2) had an identical RFLP pattern (Figure 1), and the
pair of sputum samples (from patient 3) had an identical RFLP
pattern (Figure 1); however, there were differences in banding
intensity among the paired isolates, which may represent
variation in the proportions of coinfecting P. jirovecii types. The
oral wash sample pairs (from patients 4 and 5) (Figure 1) had
highly similar patterns; however, for patient 4, the patterns were
not identical, possibly because of low msg copy numbers. These
data demonstrate that the RFLP pattern is stable over at least
a period of days to weeks and that recurrent episodes of PCP
(in patient 2) (Figure 1) can result from relapse rather than
from reinfection with a new strain.
RFLP analysis of multiple isolates. We then performed
RFLP studies to compare a larger number (n p 48) of
Pneumocystis isolates that we had collected over time. Because RFLP
analysis with Dra1 alone appeared sufficient to distinguish
among isolates, these studies used only Dra1 in the initial
analysis. Because samples needed to be run on different gels, a
known sample (sample number 385) that would hybridize to
the probe during Southern blotting, as well as commercial
molecular weight standards, were included in each gel to allow
comparison among different runs.
On the basis of visual examination, gels and blots showed
an identical RFLP banding pattern only in samples collected
from the same individual and a distinct RFLP pattern in
samples obtained from different individuals. To allow comparison
of samples run on different gels, we used BioNumerics software,
with standardization using molecular weight markers (for gels)
and sample 385 (for blots). Figure 2 shows a dendrogram
created by BioNumerics software from different gels, after
normalization using the internal standards. For occasional samples,
1618 • JID 2009:200 (15 November) • Ripamonti et al
there were differences in the precise clustering between gels and
blots, which may be related to differences following
hybridization with the probe specific for P. jirovecii (data not shown).
The dendrogram reveals that, in general, only samples
obtained from the same patient showed 100% similarity. However,
this analysis identified 4 Italian samples that formed 2 pairs,
with each pair showing 100% similarity (samples 2999 and 3960
and samples 2428 and 7780). All the Italian samples were
collected in the same city (Milan) during 1994–1999; however,
because of unlinking, we cannot further analyze the patient
data to determine whether these samples were collected from
the same patient or at close time points. To explore this
further, we digested 1 pair of samples (2999 and 3960) with
another enzyme, Xba1. Xba1 was used rather than HindIII as the
second enzyme in our later studies, because it generated greater
variability in the RFLP pattern than did HindIII. These samples
clearly showed different RFLP patterns (data not shown),
demonstrating that they were not identical. Unfortunately, there
was inadequate material to run the other 2 samples.
The Pneumocystis isolates that we analyzed were collected in
the United States, Italy, and the Netherlands. Of interest, the
samples collected in the same country clustered more closely
to each other than to samples from other countries (Figure 2).
In particular, the samples from Italy and the United States,
which accounted for the majority of isolates, were not randomly
Application of RFLP to investigate an outbreak of PCP.
Because of the substantial variability among Pneumocystis
isolates from different patients and the stability of RFLP patterns
within individuals over time, RFLP analysis appeared to provide
a method for easily demonstrating whether isolates from a
potential outbreak of PCP were identical. A recent study of an
outbreak of PCP among renal transplant recipients in Germany
provided molecular evidence, primarily by single nucleotide
polymorphism analysis, that all patients were infected with a
single Pneumocystis strain [
]. Pneumocystis DNA from 22
German patients was provided to us for RFLP analysis: 13
isolates were from patients linked to the outbreak, and 9 were
unrelated to the outbreak. In 12 isolates previously identified
as a single strain of Pneumocystis by single nucleotide
polymorphism analysis, RFLP analysis showed an identical banding
pattern (Figure 3A), although patient 8 (!1000 msg copies/
assay) had an additional band not seen in the other 11 isolates.
RFLP analysis with a second enzyme, Xba1, confirmed that the
banding pattern was identical in all 12 isolates (data not shown).
One additional isolate from the outbreak had a different
banding pattern, but the sample contained !200 msg copies/assay.
Of the 9 isolates from patients who were not previously linked
to the outbreak, 6 showed a unique pattern, 2 showed a pattern
identical to the outbreak pattern (Figure 3B), and 1 was similar
but had !1000 msg copies/assay; RFLP analysis with Xba1 again
confirmed these results (data not shown). Clinical history
obtained after these results confirmed that all 3 of the latter
patients were renal transplant recipients who had been seen at
the same clinic as the other outbreak patients and who
underwent bronchoscopy during the period of the outbreak.
When the samples from Germany were included in the
dendrogram, the non–outbreak-related samples did not cluster
with the outbreak strain but tended to cluster with samples
from Italy (data not shown).
We developed a reproducible and easy-to-perform method to
type human Pneumocystis strains with use of RFLP analysis. By
using this method, we demonstrated a remarkable diversity
among human Pneumocystis strains: no 2 isolates from different
patients showed an identical RFLP pattern, other than those
from a cohort of German patients previously linked to a
nosocomial outbreak of PCP. In contrast, samples from the same
patient (that were obtained within 111 days of each other)
showed an identical pattern. Thus, this method may be very
useful for studying transmission patterns and potential
outbreaks of PCP among immunosuppressed patients. Moreover,
our data strongly support the previously published
conclusions that the renal transplant recipients from Germany were
infected with the same Pneumocystis strain, and we identified
at least 2 additional renal transplant recipients who were
likely to have been part of the same outbreak.
A strength of RFLP analysis is that, rather than examining
a single or very limited number of nucleotide polymorphisms,
as is the case with many available typing methods, it interrogates
the entire msg repertoire of the Pneumocystis genome, which
is estimated to include 50–100 genes, with ∼40% of each msg
of ∼3200 nucleotides being evaluated in the RFLP analysis.
Because there are multiple msg copies per genome and there
is a high level of sequence conservation in short stretches across
msg genes, it is likely that recombination in Pneumocystis can
lead to rearrangements and establishment of unique msg
repertoires, as we and others have previously shown [
conservation in RFLP pattern among isolates from the same
patient, as well as the conserved pattern among a cohort of
patients linked epidemiologically, suggest that recombination
does not occur within a period of days to weeks. Previously,
we revealed that the RFLP pattern in mouse and rat
Pneumocystis isolates obtained from inbred animals at 2 locations
over a period of years were identical or highly similar. If we
assume that human Pneumocystis strains are biologically
similar, it appears likely that repertoire evolution is not rapid and
that the observed diversity is related to recombination that
has evolved over many, perhaps thousands or millions of years.
One potential disadvantage of RFLP analysis is that it is
unable to distinguish the contributions of individual isolates
to the banding pattern in patients infected with 11 isolate. If
only one of multiple strains is transmitted to a new host,
however, the RFLP bands should be easily distinguishable as a
subset of those in the first host. In addition, the reproducibility
of RFLP analysis was lost in samples with !1000 msg copies.
This likely represents a sampling bias in a specimen with a
low organism load that resulted in uneven distribution of msg
variants in different aliquots.
On the basis of the dendrogram analysis, isolates from
samples obtained from patients from the same geographic area at
approximately the same time did not show 100% similarity
(other than 1 pair of Italian samples and those from the renal
transplant recipients), suggesting that interhuman
transmission among these patients did not occur to any significant
extent. These data are in agreement with the results of a previous
] in which the authors showed, using PCR–single
strand conformation polymorphism typing, that transmission
of Pneumocystis from patients with active PCP to susceptible
persons is rare. However, outbreaks with the same strain can
occur, as shown among the renal transplant recipients. It is
noteworthy that samples collected in the same country
clustered more closely with each other than with samples from
other countries. This clustering may represent local strain
variation. It is intriguing to speculate that host immune pressures
at the population level (eg, human leukocyte antigen
mediated) are driving the diversity of the msg repertoire, as has been
reported for HIV [
In summary, this study revealed a broad diversity in
Pneumocystis strains, provided a method for rapidly typing strains,
and provided confirmatory evidence that an outbreak of PCP
was caused by a single strain of Pneumocystis. Larger studies
using this approach are needed to better define the
epidemiology of PCP and to determine whether any predominant
strains, as defined by RFLP analysis, can be identified.
We thank Dr. Pieter J. A. Beckers for providing samples of Pneumocystis
jirovecii and allowing us to include them in this study.
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