Medical Mycology, 2014, 52, 804–809
doi: 10.1093/mmy/myu059
Advance Access Publication Date: 6 October 2014
Original Article
Original Article
High prevalence of Pneumocystis jirovecii
colonization among HIV-positive patients in
southern Brazil
Robson M. Pereira1 , André L. Müller1 , Ricardo A. Zimerman1 ,
Denise B. Antunes1 , Vitor F. Zinn1 , Vicente Friaza2 , Carmen de la Horra2 ,
∗
Enrique J. Calderón2, and Gustavo Wissmann1
1
Pneumocystis Study Group, Infectious Diseases Unit, Hospital de Clı́nicas de Porto Alegre, Porto Alegre,
Federal University of Rio Grande do Sul, Brazil and and 2 Centro de Investigación Biomédica en Red de
Epidemiologı́a y Salud Pública and Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del
Rocı́o/Consejo Superior de Investigaciones Cientı́ficas/Universidad de Sevilla, Seville, Spain
*To whom correspondence should be addressed. Enrique J. Calderón, Instituto de Biomedicina de Sevilla, Hospital
Universitario Virgen del Rocı́o, Avda. Manuel Siurot s/n, 41013 Seville, Spain. Tel: +34 955 923096; Fax: +34 955 01 3292;
E-mail: [email protected]
Received 4 June 2014; Revised 14 August 2014; Accepted 22 August 2014
Abstract
A high prevalence of Pneumocystis jirovecii colonization was observed in patients positive for the human immunodeficiency virus (HIV) admitted to a tertiary hospital in southern Brazil between August 2012 and December 2012. Amplification of the mitochondrial
large subunit ribosomal RNA gene in oropharyngeal samples through nested polymerase
chain reaction identified P. jirovecii colonization in 26 of 58 (44.8%) HIV-positive patients
admitted for causes other than Pneumocystis pneumonia. Colonization was more frequent among patients with an absolute CD4 count ≤200 cells/µl. These findings suggest
that the HIV-infected population is a major reservoir and source of P. jirovecii infection and
that identification of such individuals may contribute to future strategies for improving
management of HIV-infected patients.
Key words: Pneumocystis jirovecii, colonization, HIV.
Introduction
Pneumocystis jirovecii is an atypical fungus that causes
Pneumocystis pneumonia (PcP), an opportunistic infection associated with high morbidity and mortality in patients infected with the human immunodeficiency virus
(HIV) [1].
The advent of trimethoprim-sulfamethoxazole (TMPSMX) prophylaxis and antiretroviral therapy (ART) has
804
resulted in a major reduction in the incidence of PcP among
HIV-positive patients. However, PcP remains frequently associated with acquired immunodeficiency syndrome (AIDS)
in patients who are unaware of their HIV status and/or lack
access to ART. Recent data indicate that PcP remains common in developing countries. For example, autopsy studies
of AIDS patients in Brazil in the post-ART era show a PcP
prevalence ranging from 17.3% to 27.0% [2].
C The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal
Mycology. All rights reserved. For permissions, please e-mail: [email protected]
Pereira et al.
Polymerase chain reaction (PCR) methods have been
used to demonstrate that humans can be colonized by
P. jirovecii. This techniques can detect this microorganism
in respiratory specimens such as bronchoalveolar lavage
(BAL), sputum, oropharyngeal washings, and nasopharyngeal swabs from patients with no clinical or radiographic
manifestations of PcP. Nested PCR amplification of the mitochondrial large subunit ribosomal RNA (mtLSUrRNA)
gene is the most sensitive technique for detecting P. jirovecii
colonization in respiratory samples [3].
The prevalence of P. jirovecii colonization has been reported in several populations, for example, 40.5% in patients with chronic obstructive pulmonary disease (COPD),
37.8% in patients with idiopathic interstitial pneumonias,
28.8% in individuals with cystic fibrosis, 15.5% in pregnant women, and 29% in patients with rheumatologic diseases treated with infliximab, an antitumor necrosis factor
agent [4–8].
To date, few studies have described P. jirovecii colonization in HIV-positive patients, and estimates of the
colonization rate vary considerably from 10% to 68.8%
[9]. A landmark study in which BAL samples from
50 patients were analyzed showed colonization in 16%,
but the prevalence increased to 40% when analysis was
confined to patients with a CD4 count <60 cells/µl [10].
Another study described colonization in 68% of 172 patients through the use of nested PCR in BAL or inducedsputum samples. These individuals had CD4 counts <50
cells/µl and TMP-SMX prophylaxis was absent, both of
which have been reported as risk factors for the presence
of P. jirovecii [11]. In a multicenter AIDS cohort study,
colonization was detected in autopsy specimens of 46%
of HIV-infected men dying of non-PcP, and only smoking was associated with an increased risk of Pneumocystis
colonization [12].
Regarding developing countries, researchers reported a
low prevalence (6%) of P. jirovecii colonization in 124 HIVpositive patients undergoing BAL in Uganda, sub-Saharan
Africa [13]. In another study conducted in Cameroon
among patients without pulmonary symptoms treated at
a regional hospital, the Pneumocystis colonization rate in
HIV-positive individuals was 42.9% but ranged from 20%
in patients with CD4 counts >500 cells/µl to 57.1% in
patients with CD4 counts <200 cells/µl [14].
Colonized individuals are possibly at risk of developing
PcP. Infections in immunosuppressed rats with Pneumocystis were shown to progress from colonization to pneumonia [15]. In a human study, Leigh et al. found that 2 of
50 colonized patients developed PcP within 6 weeks [10].
Conversely, Davis et al. found that none of 172 colonized
patients developed PcP during a similar follow-up period
[11]. However, further studies are required to ascertain the
805
real risk of progression from colonization to PcP in HIVpositive patients.
Additionally, evidence that P. jirovecii colonization
could itself be harmful has been described. Several authors have demonstrated that Pneumocystis colonization
is associated with airway and systemic inflammation. In
addition, it was found in a recent study of nonhuman primates infected with the simian immunodeficiency virus that
persistent Pneumocystis colonization led to the development of COPD [3,16,17]. A potential association between
P. jirovecii colonization and chronic lung disease in HIVpositive patients has been debated by some authors and
warrants further investigation [18].
No reports on P. jirovecii colonization among HIVinfected individuals in Latin America have been published
[2,19]. In the present study, we investigate the prevalence
and factors associated with P. jirovecii colonization in HIVinfected patients treated at Hospital de Clı́nicas de Porto
Alegre, a tertiary hospital in Southern Brazil.
Materials and methods
In this cross-sectional study, we assessed all adult patients
(aged ≥18 years) diagnosed with HIV infection who were
admitted for various reasons to Hospital de Clı́nicas de
Porto Alegre (Rio Grande do Sul, Brazil) between August
2012 and December 2012.
Oropharyngeal wash samples were obtained from each
patient on the first day of hospitalization. The following
clinical and demographic data were collected by means
of specific questionnaires: age; gender; current ART—
chemoprophylaxis for PcP or corticosteroid therapy; current smoking habits; COPD; previous episodes of PcP; and
absolute CD4 cell count (cells per microliter).
Patients were classified as having COPD if such a diagnosis was present in their medical records. ART was defined
as the use of a combination of highly active antiviral drugs
for the treatment of HIV infection [20]. PcP chemoprophylaxis was considered to be the use of a drug with antiPneumocystis efficacy to prevent the development of PcP,
for which TMP-SMX is the most widely used agent for this
purpose [21]. The CD4 count considered for analysis was
either the measurement obtained at study admission or any
measurement obtained within the last 6 months if the patient was stable with regard to HIV treatment. Patients were
stratified into the following CD4 count (cells per microliter)
groups: ≤100, 101–200, 201–350, and >350.
HIV-positive patients with suspected PcP, as determined
clinically by the attending physician at the time of hospital
admission, were excluded from the study. Patients unable to
provide specimens due to altered levels of consciousness (eg,
somnolence) were also not included. The hospital’s ethics
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committee approved the investigation, and all patients provided written informed consent for participation.
Oropharyngeal washing is a simple, noninvasive sample
collection method that consists of gargling with 10 ml of
normal saline solution for 1 min and collecting the resulting
expectorate in a sterile container [22].
Pneumocystis jirovecii was detected by analyzing
oropharyngeal samples with nested PCR amplification of
the Pneumocystis mtLSUrRNA gene. After sample digestion with proteinase K at 56o C, DNA was extracted from
the samples using a commercial kit (QIAamp DNA mini
kit; Qiagen, Hilden, Germany), and the gene encoding mtLSUrRNA was amplified. A two-step protocol was used for
nested PCR [22]. In brief, this consisted of an initial amplification round, for which the external primers pAZ102-E
(5’-GAT GGC TGT TTC CAA GCC CA-3’) and pAZ102H (5’-GTG TAC GTT GCA AAG TAC TC-3’) were
used, yielding a 346-bp fragment. The primers pAZ102-X
(5’-GTG AAA TAC AAA TCG GAC TAG G-3’) and
pAZ102-Y (5’-TCA CTT AAT ATT AAT TGG GGA GC3’) were used in a second round of amplification to yield
a 260-bp product. Both amplification rounds included 40
cycles of amplification. The PCR products were analyzed
by electrophoresis on a 1.5% agarose gel that contained
ethidium bromide, and the bands were visualized under ultraviolet light. To prevent false-positive results due to contamination, pipette tips with filters were used at all stages.
DNA extraction, reaction mixture preparation, PCR amplification, and detection were performed in different areas of
the laboratory. In addition, a positive control was included
in each reaction. To detect any cross-contamination, all
PCR steps were performed with a negative control of sterile
water. All experiments were repeated at least twice.
Statistical analysis was performed using SPSS software
(version 20.0; SPSS, Chicago, IL, USA). The χ 2 test or Fisher
exact test was used for qualitative variables. The Student t
test or Mann-Whitney U test was applied for quantitative
variables, and the variance test was used to confirm a normal distribution. Pearson χ 2 test was also used to compare
the different CD4 count groups according to colonization.
A P value < 0.05 was considered statistically significant.
Results
Sixty-seven HIV-positive adults were admitted to Hospital
de Clı́nicas de Porto Alegre for different reasons between
August 2012 and December 2012. Of these patients, five
were excluded due to clinical presentation suggestive of
PcP and four due to altered levels of consciousness that
prevented oropharyngeal sample collection. In all patients
with clinical suspicion of PcP, the diagnosis was later
confirmed by detection of P. jirovecii in BAL fluid by
Medical Mycology, 2014, Vol. 52, No. 8
microscopic examination of Grocott methenamine-silver–
stained material.
Thus, 58 patients were examined for P. jirovecii colonization, of which 10 with AIDS had opportunistic infections other than PcP, 8 had tuberculosis, 5 were diagnosed
as having bacterial pneumonia, 9 had various other major infections, 7 had cancer, 6 had cardiovascular diseases,
2 had anemia, and the remaining 11 had other medical
conditions.
The mean (standard deviation) age of the patients included in the study was 41.7 ± 12.9 years, and 28 patients
(48.2%) were male. Thirty-nine patients (67.2%) reported
current TMP-SMX prophylaxis, and 23 (39.6%) were on
ART. The median (range) CD4 cell count was 184 cells/µl
(2–1048). Only two patients (3.4%) reported current use of
corticosteroids, seven (12%) experienced one or more past
episodes of PcP, and a diagnosis of COPD was identified in
two patients (3.4%).
Pneumocystis jirovecii colonization was detected by
nested PCR in oropharyngeal samples in 26 (44.8%) of the
58 patients tested. Table 1 presents the clinical and demographic characteristics of patients according to colonization
status.
We identified a strong association between CD4 count
≤200 cells/µl and P. jirovecii colonization (P < 0.001) using the Pearson χ 2 test. Figure 1 presents the distribution
of individuals according to stratified CD4 count and the
frequency of colonized individuals in each CD4 cell count
group.
Discussion
In this study, we identified a high prevalence of P. jirovecii
colonization (44.8%) among HIV-infected patients admitted to a hospital in southern Brazil. Furthermore, colonization was more frequent among individuals with a CD4
count ≤200 cells/µl. These findings are similar to those
from previous studies and indicate that severely immunosuppressed patients, for example, HIV-positive individuals,
are more susceptible to colonization [10,11,14].
Our knowledge of the epidemiology of P. jirovecii infections has expanded greatly with the advent of molecular
biology methods in recent years. Several studies using PCRbased techniques suggest that P. jirovecii is a Pneumocystis
species that multiplies only in the human lung, that is, humans serve as the P. jirovecii reservoir. Therefore, the reservoir and source of infection for this microorganism appear
to be composed of PcP patients and individuals colonized
by the fungus [3,19].
Advances in molecular research have also shown that
P. jirovecii is very likely transmitted from person to person via the airborne route [23]. This form of transmission
Pereira et al.
807
Table 1. Characteristics of 58 human immunodeficiency virus–infected patients according to Pneumocystis jirovecii colonization
status.
Characteristic
Colonized patients
n = 26
Noncolonized patients
n = 32
P value
Male sex, no. (%)
Age in years, mean ± standard deviation
Antiretroviral therapy, no. (%)
CD4 count, median cells/µl (range)
Anti-Pneumocystis prophylaxis, no. (%)
Previous Pneumocystis pneumonia, no. (%)
Corticosteroid use, no. (%)
Chronic obstructive pulmonary disease diagnosis, no. (%)
Active smoker, no. (%)
13 (50)
41.2 ± 14.5
15 (57.7)
85 (2–355)
12 (46.1)
3 (11.5)
0 (0.0)
0 (0.0)
9 (34.6)
15 (46.8)
42·2 ± 11.7
24 (75.0)
256 (6–1048)
11 (34.3)
4 (12.5)
2 (6.2)
2 (6.2)
14 (43.7)
0.999a
0.776b
0.260c
<0.001d ∗
0.415c
0.999c
0.495c
0.497c
0.588c
χ 2 test with Yates correction.
Student t test.
c
Fischer exact test.
d
Mann-Whitney U test.
∗
Statistically significant.
a
b
Figure 1. Pneumocystis jirovecii colonization in 58 human immunodeficiency virus–infected patients, stratified by CD4 cell count (cells per microliter).
was documented in a report of a PcP cluster occurring in
renal transplant patients. Molecular analysis indicates that
patients in this cluster were infected with microorganisms
that shared similar genotypic features [24].
Recent evidence supports the hypothesis, demonstrated
in animal models, that individuals who are merely colonized by P. jirovecii can transmit it to other individuals, some of whom may be susceptible to developing PcP
[15]. In humans, molecular evidence of colonized individuals as potential sources of P. jirovecii infections has
been described. One report discussed the case of a sixmonth-old infant who developed PcP after contact with
her grandparents who had COPD and rheumatoid arthritis
and were colonized by the fungus [25]. Another study analyzed colonized individuals who came into contact with
patients in a PcP cluster in which the genotypic char-
acterization and analysis of the timing of contact suggest that the colonized individuals were responsible for
transmission of the organism among patients of this PcP
cluster [26].
The results of the present study show a high frequency
of P. jirovecii colonization among hospitalized HIV-positive
individuals, mainly in patients with severe immunosuppression. However, from an epidemiological point of view, it
is important to note that HIV-positive patients who have a
CD4 count >350 are able to harbor P. jirovecii.
Therefore, HIV-positive patients, irrespective of CD4
count, may constitute a significant portion of the reservoir
and source of P. jirovecii infection in the hospital setting.
Currently, the US Centers for Disease Control and Prevention do not recommend respiratory isolation for inpatients with PcP and do not provide any guidance regarding
808
P. jirovecii colonization [27]. However, recent evidence
on the transmission of this pathogen may lead experts to
consider introducing strategies to prevent the spread of
P. jirovecii in the nosocomial environment [28].
In the present study, we used oropharyngeal washing, a
noninvasive clinical sampling method, to assess P. jirovecii
colonization in HIV-positive patients. In immunocompetent patients, nested PCR is a highly sensitive (95.6%) technique for detecting colonization in oropharyngeal washings
as compared with BAL fluids and induced sputum samples
[22]. Other investigators have used oropharyngeal washings to assess colonization in several populations, including patients with cystic fibrosis, those receiving immunosuppressants, and healthy individuals [6,8,29]. Our results
demonstrate the utility of this type of clinical specimen for
identifying P. jirovecii colonization among HIV-infected
patients.
A previous study of P. jirovecii colonization in a developing country showed a very low prevalence rate, that is,
only 6% of HIV-positive subjects living in Uganda were
colonized. According to the authors, these data suggest a
limited reservoir for P. jirovecii in this population, potentially explaining the low incidence of PcP in sub-Saharan
Africa [13]. However, more recent studies carried out in
Cameroon show a rate of P. jirovecii colonization that is
comparable to that found in our study [14]. Further investigation is needed to better define the epidemiology of
Pneumocystis colonization in developing countries.
In conclusion, we reveal a high prevalence of P. jirovecii
colonization among HIV-positive patients admitted to a tertiary referral hospital in southern Brazil. Colonization was
most frequent among patients with severe immunosuppression (eg, CD4 count ≤200 cells/µl), which is consistent with
previous reports. These findings suggest that HIV-infected
individuals constitute a major reservoir and source of infection for this microorganism.
Acknowledgments
This work was supported by the Hospital de Clinicas de Porto
Alegre (Fundo de Incentivo à Pesquisa, 12–0282) and the Red
Iberoamericana sobre Pneumocystosis (212RT0450) in the framework of the Ibero-American Programme for Science, Technology and
Development (Ciencia y Tecnologı́a para el Desarrollo).
Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and the writing of the paper.
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