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Journal of Clinical Microbiology, November 2001, p. 3877-3882, Vol. 39, No. 11
Department of Sexually Transmitted Diseases,
Windeyer Institute of Medical Sciences, Royal Free and University
College Medical School, University College London,
London,1 and Molecular Infectious
Diseases Group, Department of Paediatrics, Weatherall Institute of
Molecular Medicine, University of Oxford,
Oxford,2 United Kingdom
Received 25 June 2001/Returned for modification 30 July
2001/Accepted 12 August 2001
The possible transmission of Pneumocystis carinii f.
sp. hominis from patients with P. carinii
pneumonia to asymptomatic health care workers (HCW), with or without
occupational exposure to human immunodeficiency virus
(HIV)-infected patients with P. carinii pneumonia, was
examined. HCW in a specialist inpatient HIV-AIDS facility and a
control group in the general medical-respiratory service in the same
hospital provided induced sputum and/or nasal rinse samples, which were
analyzed for the presence of P. carinii f. sp.
hominis DNA by using DNA amplification (at the gene
encoding the mitochondrial large subunit rRNA [mt LSU rRNA]).
P. carinii f. sp. hominis DNA was
detected in some HCW samples; those with the closest occupational
contact were more likely to have detectable P.
carinii DNA. P. carinii DNA was detected in one
HCW who carried out bronchoscopy over a 2-year period. P.
carinii-positive samples were genotyped by using DNA sequence
variations at the internal transcribed spacer (ITS) regions of the
nuclear rRNA operon, along with bronchoalveolar lavage samples from
patients with P. carinii pneumonia hospitalized at the
same time. Genotyping identified 31 different P. carinii
f. sp. hominis ITS genotypes, 26 of which were found in
the patient samples. Five of the eight ITS genotypes detected in HCW
samples were not observed in the patient samples. The results suggested
that HCW in close occupational contact with patients who had P.
carinii pneumonia may have become colonized with P.
carinii. Carriage was asymptomatic and did not result in the
development of clinical disease.
The fungal pathogen
Pneumocystis carinii f. sp. hominis is primarily
associated with pneumonia in the profoundly immunocompromised, e.g.,
those with human immunodeficiency virus (HIV) infection, and also in
patients undergoing organ transplantation or chemotherapy for
malignant disease. Recent data suggest that exposure to P. carinii f. sp. hominis is frequent and that
reinfection with different isolates of P. carinii f. sp.
hominis commonly occurs (14, 15, 17, 35).
Little is known about the life cycle of the fungus; the reservoir of
infectious P. carinii f. sp. hominis has not yet been elucidated nor have the modes of transmission of the infection.
It is now widely accepted that the P. carinii organisms that
infect each mammalian species are host specific and that the infection
in humans is not acquired from an animal reservoir (32, 39). Airborne acquisition of P. carinii infection has
been demonstrated by using the rat model (13). The
existence of airborne P. carinii organisms has been
supported by the identification of P. carinii DNA in samples
of airborne spores from rural environments (38), from
animal facilities housing immunosuppressed rats with P. carinii pneumonia, and in hospital inpatient and outpatient rooms
used for treating patients with P. carinii pneumonia
(1, 2, 16, 29).
P. carinii has been found in low numbers in the lungs of
patients who did not have pneumonia, both HIV-infected individuals and
patients who were only mildly immunocompromised (4, 9, 27, 28,
31), suggesting that these persons may act as asymptomatic carriers of P. carinii. In a study on the transmission
of P. carinii using the mouse model of the infection,
it was shown that immunocompetent BALB/c mice that were carrying
subclinical levels of P. carinii were able to transmit
the infection by the airborne route to highly susceptible, uninfected
SCID mice (7). However, the routes of transmission of
P. carinii f. sp. hominis remain unclear.
In this study the possible transmission of P. carinii
f. sp. hominis from patients with P. carinii
pneumonia to health care workers (HCW) who were in contact with these
patients, was examined. A search was carried out for the presence of
P. carinii f. sp. hominis DNA in
respiratory tract samples from HCW who were in contact with
P. carinii-infected patients and from a control group who did not have occupational contact with this patient group. Positive
samples were genotyped at the internal transcribed spacer (ITS)
regions, along with samples from P. carinii-infected patients with whom the HCW were in contact.
Samples.
27 HCW (19 female) were prospectively studied; 18 had been working for >3 months in a specialist HIV-AIDS inpatient care
facility at the Middlesex Hospital site of UCL Hospitals, London,
United Kingdom, and 9 worked exclusively in the general medical and
respiratory services that were based at a geographically different site
within the hospital and had no occupational contact with the HIV-AIDS inpatient facility. Each HCW provided a hypertonic saline-induced sputum sample; some also provided a nasal rinse sample at the same
time. Some HCW provided induced sputum samples on separate occasions;
one HCW gave six samples. In total, samples were obtained on 36 occasions from the 27 HCW over a 2-year period. Informed consent was
obtained from all subjects, and the guidelines of the Middlesex
Hospital Local Research Ethics Committee were followed in the conduct
of this research.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.11.3877-3882.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Pneumocystis carinii f. sp. hominis
DNA in Immunocompetent Health Care Workers in Contact with Patients
with P. carinii Pneumonia
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
20°C, within 5 min of collection) prior to analysis. In addition,
as a further precaution, the nebulizer was washed with soapy water and
sterilized by immersion in 3% glutaraldehyde between each saline
induction, and single-use disposable tubing and mouthpieces were used
for each procedure. Nasal rinse samples were obtained as follows. While
the HCW was standing erect and breathing through the mouth, he or she
occluded the right nostril. Sterile normal saline (10 ml) was slowly
instilled into the left nostril by using a 10-ml sterile single-use
syringe for 20 to 30 s. Refluxing saline was caught in a sterile
universal container which was held against the upper lip below the
nostril. The sample was sealed immediately and processed as for the
induced sputum samples.
Respiratory samples (bronchoscopic alveolar lavage [BAL] fluid in 35 patients and induced sputum in 1 patient) were obtained from 36 HIV
type 1 antibody-positive persons admitted with P. carinii pneumonia during the same time period as the HCW provided samples. All patients had typical clinical presentations, response to
specific anti-Pneumocystis therapy and positive results from methenamine silver staining of BAL fluid or induced sputum. At the time
of study patients with suspected or confirmed P. carinii pneumonia were cared for on an open ward. Bronchoscopy and
sputum induction were carried out in a dedicated room which shared
ventilation with the rest of the ward. Bronchoscopists and staff
supervising sputum induction did not wear HEPA filter masks. Thus, a
total of 72 respiratory samples from 63 persons were examined.
Detection of P. carinii f. sp. hominis by DNA amplification. DNA extraction of the samples was carried out as previously described (35, 36). Detection of P. carinii was carried out by using a nested PCR at the gene encoding the mitochondrial large subunit rRNA (mt LSU rRNA), with primers pAZ102-H (5'-GTGTACGTTGCAAAGTACTC-3') and pAZ102-E (5'-GATGGCTGTTTCCAAGCCCA-3') in the first-round amplification, followed by pAZ102-X (5'-GTGAAATACAAATCGGACTAGG-3') and pAZ102-Y (TCACTTAATATTAATTGGGGAGC-3') in the second-round amplification as previously described (34, 38, 42). Taq DNA polymerase (Promega, Southampton, United Kingdom) was used throughout the study. Negative controls were included in each experiment, in both DNA extraction and amplification, to monitor for possible contamination. DNA extraction and PCR were performed in a laminar flow cabinet and disposable tips, tubes, and reagent aliquots were used to avoid contamination. A sample of P. carinii f. sp. hominis DNA, obtained from a patient with histologically confirmed P. carinii pneumonia, was used as a positive control in each experiment.
P. carinii f. sp. hominis ITS genotyping. DNA amplification at the ITS regions was performed by using PCR with primer pair ITSF3 (5'-CTGCGGAAGGATCATTAGAAA-3') and ITS2R3 (5'-GATTTGAGATTAAAATTCTTG-3') (35). In some samples, nested PCR was performed to achieve higher levels of sensitivity, by using primer pair N18SF (5'-GGTCTTCGGACTGGCAGC-3') and N26SRX (5'-TTACTAAGGGAATCCTTGTTA-3') in the first-round amplification, and ITSF3 and ITS2R3 in the second-round amplification (36). Amplification products were cloned into the plasmid vector pGEM T-Easy (Promega, United Kingdom), and the sequence of four clones was determined by using the Big Dye terminator cycle sequencing kit and an ABI377 DNA sequencer, running the data collection software version 2.1 (Applied Biosystems, Warrington, United Kingdom). Sequence data analysis was performed by using Chromas 1.62 software (Technelysium Pty., Ltd.) and the University of Wisconsin Genetics Computer Group software, version 10.1 (Genetics Computer Group, Madison, Wis.). An ITS genotype was assigned to each sequence as previously described (34-36).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Presence of P. carinii f. sp.
hominis DNA in respiratory samples from health care
workers.
It was hypothesized that immunocompetent HCW who were in
contact with patients with P. carinii pneumonia may
become transiently colonized with P. carinii and that
they may act as transient carriers of the infection. DNA amplification,
using nested PCR at the mt LSU rRNA, was used to search for
P. carinii f. sp. hominis DNA in the HCW
respiratory samples. This method has been shown to be highly sensitive
and specific (5, 10, 23, 36, 40, 41). P. carinii f. sp. hominis DNA was detected in 11 samples, of which 10 were from HCW who were in contact with P. carinii-infected patients and 1 was from the control group (Table
1). Seven positive samples were from two
HCW, both of whom were sampled on more than one occasion. Both HCW were
physicians (doctor 1 and doctor 10), who performed BAL on patients with
P. carinii pneumonia and were therefore in direct
contact with P. carinii-infected individuals. In
contrast, no P. carinii f. sp. hominis DNA
was detected in samples from two control group HCW (doctor 4 and doctor
5) who performed BAL on general medical-respiratory patients. Despite rigorously attempting to exclude any immunosuppressed HCW from the
study, one contact group HCW (nurse 7) had occult malignancy at the
time of providing a respiratory sample and so was potentially immunosuppressed; a diagnosis of carcinoma was made shortly afterward. During follow-up, with a median duration of 98 months (range, 15 months
[the nurse with occult lung cancer] to 113 months), no HCW developed
P. carinii pneumonia. In summary, the fraction of
contact HCW who had detectable P. carinii f. sp.
hominis DNA was about twice as high (4/17 = 0.24, nurse
7 excluded) as the fraction of non-contact HCW (1/9 = 0.11).
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Genotyping of P. carinii f. sp.
hominis from infected patients and contact HCW.
In
order to investigate whether transmission of P. carinii
was taking place between infected patients and contact HCW, ITS genotyping was used to examine the types of P. carinii
f. sp. hominis in samples from contact HCW and P. carinii-infected patients who were hospitalized at the time of
sampling (Table 2). ITS genotyping was
carried out on 36 samples from patients with P. carinii
pneumonia, as previously described (10, 35, 36). ITS
genotyping was attempted on the 11 positive respiratory samples from
HCW shown to contain P. carinii f. sp.
hominis DNA. Amplification at the ITS locus was only
successful on five samples, from two different HCW (doctor 1 and doctor
10), both of whom performed BAL on patients with P. carinii pneumonia. It was not possible to amplify the sample from
the HCW in the control group at the ITS locus. This reflected the fact
that the amount of P. carinii DNA in this type of
noninvasive respiratory tract sample was considerably lower than in BAL
samples from patients with P. carinii pneumonia. In
addition, detection of P. carinii f. sp.
hominis by nested PCR at the ITS regions is less sensitive
than at the mt LSU rRNA (21, 36). This is because the
mitochondrial genome is present in multiple copies within each
P. carinii organism, whereas there is only one copy of
the ITS regions (8, 26, 33).
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A4a9, B1a7,
B1a10,
B7a3, and
B7a10were not observed in
any of the patient samples. Other differences between ITS genotypes in
patient samples and HCW samples were also observed, e.g., there was a
higher frequency of ITS1 "A" type in the HCW samples.
P. carinii f. sp. hominis
carriage in asymptomatic immune-competent HCW.
Two of the six HCW
who were positive for P. carinii DNA were
physicians who peformed BAL on patients with P. carinii
pneumonia. Positive samples in one HCW were obtained over a period of
27 months, and a total of 7 different ITS genotypes were observed (Table 3). During the study period, this
HCW had no intercurrent illnesses and received no immunosuppressive
drugs and no antimicrobials active against P. carinii.
In two samples from these HCW, no ITS genotyping was possible, despite
detection of P. carinii f. sp. hominis DNA
using PCR at the mt LSU rRNA, indicating that these samples contained
very small quantities of P. carinii.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study P. carinii DNA was found in respiratory tract samples of HCW, the majority of whom had occupational contact with HIV-infected patients with P. carinii pneumonia. P. carinii DNA has not generally been found in these types of noninvasive respiratory tract samples in either immunocompromised or immunocompetent individuals without P. carinii pneumonia (30). Previous studies examining serum titers of P. carinii antibodies in HCW have shown conflicting results (20, 22), and in one of the studies P. carinii DNA was not detected in oropharyngeal washings from contact HCW (22), but this may be explained by the use of a single round rather than nested PCR.
The data from this study support the hypothesis that immunodeficient patients with P. carinii pneumonia may exhale P. carinii into the environment, where it may be inhaled by HCW who are occupationally in close proximity, and this may lead to asymptomatic carriage. The samples used in this study were collected in the early 1990s. Since then, management protocols in this institution have been changed because of concerns about nosocomial transmission of tuberculosis, and currently HIV-infected patients with any respiratory symptoms, including those with suspected or confirmed P. carinii pneumonia, are nursed in side rooms with negative-pressure ventilation. Staff performing bronchoscopy and/or supervising sputum induction wear HEPA filter masks, and these procedures are carried out in a room which has negative-pressure ventilation.
Analysis of the ITS genotypes of P. carinii isolated from the HCW and the P. carinii-infected patients who were hospitalizsed at the time of sampling resulted in a complex picture with a large number of ITS genotypes being observed in both groups. One ITS genotype, B1a3, was found in both groups, suggesting the possibility of transmission of P. carinii. B1a3 is a common genotype, accounting for 20% of all patient episodes in this study and being the most frequently observed genotype in some other studies (18). Five of the genotypes were only found in HCW samples, suggesting that P. carinii genotypes that cause disease in immunocompromised patients may be different from those which are carried asymptomatically by immunocompetent HCW.
Analysis of the samples from one HCW (doctor 1), who was sampled repeatedly, showed the presence of P. carinii DNA over a period of 27 months. Laboratory tests confirmed that this HCW was immunocompetent. The presence of P. carinii DNA could be accounted for by a number of explanations, including transient carriage of P. carinii f. sp. hominis, constituting a dynamic situation in which organisms were cleared by the immunocompetent host followed by reinfection from (i) a P. carinii-infected patient with clinical and laboratory confirmed pneumonia; (ii) an immunocompromised HIV-infected patient, asymptomatic but colonized with P. carinii; and (iii) an asymptomatic HCW who was not part of the study who may have been immunocompetent or immunosuppressed and colonized with P. carinii. Another explanation is the possible long-term asymptomatic carriage of P. carinii f. sp. hominis in the HCW (doctor 1).
These preliminary data add support to the hypothesis that P. carinii may be transmitted from a P. carinii-infected patient to an immunocompetent host, an observation that has previously been shown in the mouse model of the infection. Transmission of P. carinii organisms from immunocompetent BALB/c mice, transiently parasitized with P. carinii organisms after close contact with P. carinii-infected SCID mice, to P. carinii-free SCID mice has been demonstrated (7). Contact for only 1 day with an infected SCID mouse was sufficient to allow transfer of organisms to the asymptomatic carrier and then transmission to a susceptible recipient.
The circulation of P. carinii between infected and susceptible hosts has been suggested not only from studies in animal models but also in human infection. Recent studies investigating mutations in the gene encoding dihydropteroate synthase have shown a correlation not only with prior sulfur prophylaxis or treatment but also with geographical location, indicating transmission either directly or through a common environmental source (3, 12). In addition, in a study examining P. carinii transmission, P. carinii DNA was found in nasopharyngeal samples from the mother, doctor, and nurse caring for an HIV-negative child with P. carinii pneumonia but not in 30 control hospital staff members who did not have contact with the infected patient (37). Furthermore, geographic clustering of cases of P. carinii pneumonia among HIV-infected patients has been suggested from studies using analysis of zip code zones (6, 25).
The data in this study suggest a complex picture of circulation of P. carinii between immunosuppressed and immunocompetent individuals, in the former leading to clinical pneumonia and in the latter to leading to asymptomatic carriage. The results suggest that there are at least two different routes of transmission of P. carinii: (i) from immunocompromised P. carinii-infected patients to immunocompromised susceptible individuals and (ii) from immunocompromised infected individuals to immunocompetent individuals who are in situations of very close contact. Our results do not exclude the possibility of other transmission routes, such as transmission from asymptomatic carriers of P. carinii to immunocompromised patients, or the possibility of exposure to exogenous environmental reservoirs of infectious organisms. Further studies will clarify this complex picture and help to elucidate the routes of transmission and carriage of P. carinii f. sp. hominis.
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ACKNOWLEDGMENTS |
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This research was supported by the Royal Society (A.E.W.), the Wellcome Trust (A.E.W.), the Medical Research Council (H.E.A.), the Camden and Islington Community Health Services (NHS) Trust (R.F.M.), and the fifth Framework Programme of the European Commission (contract number QLK2-CT-2000-01369).
We thank the HCW and patients who participated in the study and Lynden Guiver for technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Molecular Infectious Diseases Group, Department of Paediatrics, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom. Phone: 44-1865-222344. Fax: 44-1865-222626. E-mail: wakefiel{at}molbiol.ox.ac.uk.
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Abstract
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Materials and Methods
Results
Discussion
References
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Journal of Clinical Microbiology, November 2001, p. 3877-3882, Vol. 39, No. 11
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.11.3877-3882.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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