Abstract
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.
MATERIALS AND METHODS
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.
In order to exclude an effect from underlying immunosuppression, HCW were asked not to participate in the study if they had any of the following exclusion criteria: (i) if they were receiving inhaled, topical, or systemic corticosteroids or other immunosuppressive therapy; (ii) if they had diabetes mellitus, some chronic pulmonary disease (including bronchiectasis, cystic fibrosis, chronic bronchitis, or asthma), or allergic rhinitis or sinusitis; (iii) if they were pregnant; (iv) if they knew or suspected that they might have HIV infection; (v) if they had a current or past history of malignancy; or (vi) if they had experienced an upper or lower respiratory tract infection within the 6 weeks prior to participation in the study. At the time of providing each sample, all HCW were asymptomatic for respiratory disease.
Induced sputum samples were obtained by HCW inhaling 20 to 30 ml of 2.7% saline (Kendall Laboratories, Basingstoke, United Kingdom) using an ultrasonic nebulizer (Ultraneb 99m; DeVilbliss, Feltham, United Kingdom) as previously described (24, 40). In order to avoid contamination, samples were collected in sterile universal containers, sealed immediately, and double bagged before freezing (at −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).
RESULTS
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).
TABLE 1.
Detection of P. carinii f. sp. hominis DNA in respiratory samples from HCW
| Sample | Date of sample (mo.yr) | P. carinii DNA detected | HCWa | Occupational contact with P. carinii- infected patients |
|---|---|---|---|---|
| E1 | 12.1991 | No | Physio 1-1 | Yes |
| E2 | 12.1991 | Yes | Doctor 1-1 | Yes |
| E3 | 12.1991 | No | Doctor 2-1 | Yes |
| E4 | 12.1991 | No | Doctor 3 | Yes |
| E7 | 12.1991 | No | Nurse 1 | Yes |
| E11 | 12.1991 | No | Doctor 7-1 | Yes |
| E32 | 3.1992 | No | Physio 1-2∗ | Yes |
| E33 | 3.1992 | Yes | Doctor 1-2∗ | Yes |
| E43 | 3.1993 | Yes | Doctor 1-3∗ | Yes |
| E49 | 3.1993 | No | Nurse 2 | Yes |
| E57 | 3.1993 | No | Doctor 8 | Yes |
| E58 | 3.1993 | No | Nurse 4 | Yes |
| E60 | 3.1993 | Yes | Secretary | Yes |
| E61 | 4.1993 | Yes | Nurse 5 | Yes |
| E62 | 4.1993 | No | Nurse 6 | Yes |
| E63 | 4.1993 | Yes | Nurse 7† | Yes |
| E66 | 4.1993 | No | Physio 2 | Yes |
| E75 | 4.1993 | No | Doctor 7-2∗ | Yes |
| E76 | 4.1993 | No | Doctor 9 | Yes |
| E78 | 4.1993 | No | Doctor 2-2∗ | Yes |
| E81 | 9.1993 | No | Nurse 10 | Yes |
| E86 | 9.1993 | No | Nurse 11 | Yes |
| E88 | 9.1993 | Yes | Doctor 1-4∗ | Yes |
| E94 | 11.1993 | Yes | Doctor 10-1 | Yes |
| E97 | 1.1994 | Yes | Doctor 10-2∗ | Yes |
| E101 | 3.1994 | Yes | Doctor 1-5∗ | Yes |
| E116 | 7.1994 | No | Doctor 1-6∗ | Yes |
| E5 | 12.1991 | No | Doctor 4 | No |
| E6 | 12.1991 | No | Doctor 5 | No |
| E10 | 12.1991 | No | Doctor 6 | No |
| E56 | 3.1993 | No | Nurse 3 | No |
| E67 | 4.1993 | No | Lung function technician | No |
| E68 | 4.1993 | No | Pharmacist | No |
| E73 | 4.1993 | No | Nurse 8 | No |
| E74 | 4.1993 | Yes | Nurse 9 | No |
| E79 | 4.1993 | No | Social worker | No |
Symbols: †, immunocompromised; ∗, repeat sample. Physio, physiotherapist.
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).
TABLE 2.
ITS genotypes of P. carinii f. sp. hominis isolated from patients with P. carinii pneumonia and asymptomatic HCW
| Date of sample (day.mo.yr) | Patients with P. carinii pneumonia
|
HCW
|
||
|---|---|---|---|---|
| Sample code | ITS genotype | Sample | ITS genotype | |
| 24.5.91 | D74 | A2c1 | ||
| 26.9.91 | D122 | Ca3 | ||
| 16.10.91 | D129 | B1a4 B1e1 | ||
| 22.11.91 | D138 | B1a3 B1c1, B1b1 | ||
| 17.12.91 | Doctor 1-1 | B1a3 A4a3 B1a10 A4a9 | ||
| 6.1.92 | D148 | B1a3 Ca3 | ||
| 29.5.92 | D204 | B2a1 B2a2 | ||
| 22.7.92 | D222 | B1d1 B2a1 | ||
| 29.7.92 | D224 | B2a1 | ||
| 2.9.92 | D229 | B1a3 | ||
| 4.11.92 | D253 | B1a3 B1c1 | ||
| 27.11.92 | D262 | B2a1 | ||
| 5.2.93 | D283 | B2a1 | ||
| 12.2.93 | D288 | B1b1 B2a1 | ||
| 19.2.93 | D289 | B1b2 | ||
| 19.2.93 | D290 | A3a1 B1a1 B2a1 B2a4 | ||
| 24.2.93 | D295 | B2a1 | ||
| 24.2.93 | D296 | B2a8 B2a1 | ||
| 19.3.93 | D298 | B1d1 B2a1 | ||
| 22.3.93 | Doctor 1-3 | A4a3 B7a3 | ||
| 19.4.93 | D303 | B2a5 | ||
| 5.5.93 | D310 | B1a1 B1a3 B1e1 B1e2 B1e3 B2a1 B2a2 B2a3 B5a3 | ||
| 9.6.93 | D321 | B1b2, B2a1, | ||
| 15.6.93 | D328 | B2a1 | ||
| 15.6.93 | D329 | B2a1 | ||
| 15.7.93 | H27 | B1b1 | ||
| 6.8.93 | D342 | B2a1 | ||
| 8.9.93 | D347 | B2a1 | ||
| 23.9.93 | Doctor 1-4 | B7a10 | ||
| 29.9.93 | D349 | B1a1 B1a2 B1a3 B2a1 B2a3 B4a3 | ||
| 4.10.93 | D351 | B1b1 | ||
| 6.10.93 | D352 | A4a3 | ||
| 18.11.93 | Doctor 10-1 | A1c1 | ||
| 5.1.94 | D386 | A2b2 A2c1 B1b2 B3b2 | ||
| 16.3.94 | D410 | B1a3 | ||
| 23.3.94 | Doctor 1-5 | B1a7 B1a10 B7a10 | ||
| 25.3.94 | D413 | B1a3 | ||
| 3.6.94 | D438 | B2a1 | ||
| 6.7.94 | D445 | B2a1 | ||
| 5.10.94 | D474 | B1b2 | ||
| 26.10.94 | D482 | B2a1 | ||
In this study, 31 ITS genotypes of P. carinii f. sp. hominis were identified, of which 6 genotypes had not previously been reported. A total of 42 ITS genotypes have been described by using this method of typing (10, 35, 36), results that are comparable to the large number of types found in other studies (11, 18, 19). In the patient samples a total of 26 different P. carinii f. sp. hominis ITS genotypes were found. More than one ITS genotype was observed in 16 of 36 (44%) patient samples. Type B2a1 was the most common type, found in 20 of 36 (55%) samples, as a single infection in 11 of 36 (30%) and as a mixed infection in 9 of 36 (25%). No samples from HCW had type B2a1. Eight different ITS genotypes were found in the HCW samples, and more than 1 ITS type was observed in three of five (60%) samples. Interestingly, five of the eight ITS genotypes, identified in the HCW samples—A4a9, B1a7, B1a10, B7a3, and B7a10—were 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.
TABLE 3.
P. carinii f. sp. hominis DNA detection and ITS genotypes in two asymptomatic HCW who performed bronchoscopies on patients with P. carinii pneumonia
| Sample | Date of sample (day. mo. yr) |
P. carinii DNAa
|
ITS genotype | |
|---|---|---|---|---|
| mt LSU rRNA | ITS | |||
| Doctor 1 | ||||
| 1-1 | 17.12.91 | + | + | B1a3 A4a3 B1a10 A4a9 |
| 1-2 | 6.3.92 | + | − | |
| 1-3 | 22.3.93 | + | + | A4a3 B7a3 |
| 1-4 | 23.9.93 | + | + | B7a10 |
| 1-5 | 23.3.94 | + | + | B1a7 B1a10 B7a10 |
| 1-6 | 15.7.94 | − | − | |
| Doctor 10 | ||||
| 10-1 | 18.11.93 | + | + | A1c1 |
| 10-2 | 24.1.94 | + | − | |
+, detected; −, not detected.
DISCUSSION
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.
ACKNOWLEDGMENTS
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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