From the aDivision of Infectious Diseases, University Hospitals of Geneva, Geneva, Switzerland
bDivision of Pulmonary Medicine, University Hospitals of Geneva, Geneva, Switzerland
cDivision of Infectious Diseases, University Hospital of Basel, Basel, Switzerland
dDivision of Pulmonary Medicine, University Hospital of Basel, Basel, Switzerland
eDivision of Pulmonary Medicine, University Hospital of Lausanne, Lausanne, Switzerland
fDivision of Infectious Diseases, University Hospital of Zurich, Zurich, Switzerland
gCentral Laboratory of Virology, University Hospitals of Geneva, Geneva, Switzerland.
Received 9 July, 2007
Revised 12 November, 2007
Accepted 13 November, 2007
Correspondence to Jorge Garbino, MD, Division of Infectious Diseases, University Hospitals of Geneva, 24 Rue Micheli-du-Crest, 1211 Geneva 14, Switzerland. Tel: +41 22 37 29 839; fax: +41 22 37 29 832; e-mail: email@example.com
Respiratory viruses are commonly restricted to the upper respiratory tract in humans and generally result in few complications. Case series and studies in children, however, suggest that respiratory viruses might contribute to pulmonary complications and morbidity in HIV-infected patients, particularly those with low CD4 cell counts [1–7]. Although guidelines recommend vaccination to prevent influenza complications in HIV-infected patients , this is based on relatively few studies. In particular, the role of influenza compared with other respiratory viruses in this population has not been investigated in a systematic fashion and their potential clinical consequences are not established. This is related mainly to the fact that screening for respiratory viruses requires the use of costly and specialized molecular diagnostic procedures [9–11], and is also a result of the lack of specific therapy. As bronchoalveolar lavage (BAL) is a routine procedure performed to rule out opportunistic infections in HIV-infected patients with low CD4 cell counts in the presence of lower respiratory tract disease, it provides the opportunity to obtain high quality lower respiratory specimens to screen for viruses . In HIV-infected children , respiratory viruses have also been shown to contribute to bacterial pneumonia, and it can be hypothesized that the outcome of opportunistic infections such as Pneumocystis pneumonia could be worsened by co-infection with these agents. Moreover, in the HAART era, a change can be observed in the relative incidence and type of pulmonary infections .
In this study, we used a largely validated set of real-time reverse transcriptase–polymerase chain reaction (RT–PCR) assays targeting 17 different respiratory viruses [10,11] to screen every BAL specimen performed in HIV-infected patients to rule out an opportunistic event. This panel of polymerase chain reaction assays allows to screen in a sensitive manner the presence of respiratory viral infection and to take into account the recently discovered metapneumovirus, coronavirus and bocavirus. Our goal was to assess the incidence and the epidemiological pattern of respiratory viruses in HIV-infected patients and to evaluate their potential clinical impact.
Patients and study design
The study was conducted from 2003 to 2006 at the university hospitals of Geneva, Basel and Zurich in the context of the Swiss HIV Cohort Study. Study participants were HIV-infected patients aged over 16 years who underwent a BAL whatever the reason leading to the procedure. A specific case report form including a one-month follow-up was completed for each patient by the investigator in each site who was blinded to RT–PCR results.
BAL was performed following a standardized protocol: 30–50 ml sterile saline solution was instilled two to four times with the bronchoscope wedged into a subsegmental bronchus, either at the site of the radiographic abnormality or in the middle lobe in the absence of radiographic abnormalities.
All BAL specimens were initially pooled and then separated into aliquots and processed similarly for subsequent analysis. Gram stain, acridine orange, auramine, and Giemsa staining were carried out for the direct identification of bacteria, mycobacterium, fungi and parasites. Cultures for bacterial identification were inoculated under standard aerobic conditions on four different media as well as on specific media for mycobacterium detection when indicated by the clinical conditions. Pulmonary bacterial infection was considered confirmed when quantitative culture for a given bacteria yielded more than 103 cfu/ml of specimen in association with clinical and radiological symptoms; otherwise, it was not considered an infection.
Patients who underwent serial BAL procedures were considered only once if the BAL was related to the same episode and performed within one month of the previous episode; otherwise each episode was counted separately.
Frozen aliquots (−80°C) were used for viral nucleic acid identification. Real-time TaqMan RT–PCR assays for the detection of influenza A, B, and C, respiratory syncytial virus A and B, parainfluenza virus (PIV) 1, 2, 3 and 4, human rhinovirus, enterovirus, human metapneumovirus (HMpV), coronavirus OC43, 229E, NL63 and HKU1 and bocavirus were performed as previously described [10,11,13]. Viral cultures for cytomegalovirus and herpes simplex virus were performed in each participating centre.
The study protocol was approved by the ethic committees at each institution and all patients signed an informed consent form.
Between November 2003 and November 2006, we analysed 59 BAL specimens from 55 HIV-infected patients enrolled in the study. The median age was 42.5 years (range 24.3–71.2) and 42 (76%) patients were male. At enrolment, the median CD4 cell count was 55 cells/μl (range 2–650) and the median HIV viraemia was 284 077 copies/ml (range 10–4.6 × 106 RNA copies/ml). At the time of the BAL procedure, 28 cases (47.5%) were under HAART. Of these, 14 (23.7%) were on cotrimoxazole prophylaxis and one case was on ganciclovir prophylaxis. In 56 cases (95%), BAL was performed to rule out a possible opportunistic infection or pneumonia suggested by clinical symptoms and associated conditions. In the three remaining cases (5%), BAL was performed to investigate the diagnosis of a tumour-like process, a laryngeal papilloma and the origin of a haemoptysis.
Fever was present in 37 cases (62.7%) and 21 (35.6%) were treated by an antibiotic targeting a respiratory infection. Other respiratory symptoms present at the time of the BAL procedure were: cough, 25 (42.4%); dyspnoea, 20 (33.9%); sputum, 17 (28.8%); flu-like illness, six (10.2%); and rhinopharyngitis, five (8.5%). A new radiological infiltrate was present in 78% (n = 46) of cases (Table 1). Ninety per cent (n = 53) of cases were hospitalized and eight (13.6%) were on respiratory support. Among the latter patients, seven were housed in an intensive care unit (ICU) at the time of the BAL procedure. Four cases (6.8%) were treated by immunosuppressive drugs.
Eleven of 59 episodes (18.6%) were positive for at least one respiratory virus. The distribution of the different types of respiratory viruses was as follow: coronavirus OC43 in three (27.3%) cases followed by influenza A in two (18.2%). PIV 2, PIV 3, PIV 4, bocavirus, human rhinovirus A and HMpV were identified each in one case (9%). In 29 BAL specimens (49.2%), 35 microorganisms (other than respiratory viruses) were isolated and considered as pathogens with the following distribution: Pneumocystis jiroveci was the most frequent and was identified in 14 (40%) followed by Streptococcus pneumoniae, five (14.2%); Mycobacterium tuberculosis, five (14.2%); Haemophilus influenzae, three (8.6%); Pseudomonas aeruginosa, two (5.7%); Nocardia spp., two (5.7%); Moraxella catharralis, one (2.8%); Klebsiella pneumoniae, one (2.8%); Mycobacterium avium complex, one (2.8%); and Mycobacterium kansasi, one (2.8%). In addition, cytomegalovirus was isolated in 13 specimens (22%), but none was considered as a cause of pneumonia. No herpes simplex virus was isolated. When respiratory viral investigations were added to bacterial, parasites, and fungal investigations it was possible to establish a microbiological aetiology in 61% (n = 36) of cases.
Among the 11 cases positive for a respiratory virus, seven (63.6%) were positive only for a virus and in four (36.4%) cases a concomitant infection was identified (P. jiroveci, H. influenzae, M. avium complex, S. pneumoniae; Table 2). In the virus-positive cases, six (54.5%) were under HAART, three (27.3%) had a CD4 cell count of more than 200 cells/μl, and one (9%) had a viral load less than 50 copies/ml. Seventy-three per cent of patients were hospitalized at the time of BAL, 91% had been treated with antibiotics previously (27.3% had no response to antibiotics), 82% had new radiological infiltrates, and 82% a suspicion of opportunistic infection, 18.2% were hospitalized in the ICU.
In 39% of all cases (M = 23), neither bacterial or fungal microorganisms nor viruses were identified. The seasonal distribution of BAL procedures was 32 (54.2%) during the winter season and 27 (45.8%) during the summer season. Among the 11 cases positive for a respiratory virus, seven cases (63.6%) occurred during the winter season and four cases (36.4%) occurred during the summer season.
The mortality rate at one month (available for all but three cases) was 7.3% (four patients). Causes of death were respiratory failure of undetermined origin, complicated pneumocystosis, haemoptysis and septic shock secondary to pneumonia, each in one case. None of these four fatal cases had a respiratory viral infection.
This study assessed the potential role of respiratory viruses in a cohort of HIV-infected patients presenting with an acute lower respiratory tract illness. Of the 59 cases enrolled, 11 had a respiratory viral infection detected in BAL specimens, representing an incidence of 18.6% in this population requiring invasive investigations. All cases presented with respiratory tract symptoms or chest radiological abnormalities and 90% (n = 53) were hospitalized. In 50.8% (n = 30), classic microbiological investigations targeting bacterial and opportunistic pathogens remained negative. When prescribed, response to antibiotic treatment was limited. It was particularly notable that respiratory viral infections were more frequent in those in whom other microbiological investigations remained negative compared with those with a confirmed concomitant bacterial or opportunistic disease, although this difference was not statistically significant. When respiratory viruses were considered, a microbiological aetiology was able to be established in up to 61% of cases. Similar to the overall population, respiratory virus positive cases were all hospitalized, 18.2% were admitted to the ICU and most received antibiotic treatment. These observations support a role for respiratory viruses in symptom production, and suggest that screening for these viruses might avoid additional investigations or treatment in a substantial number of cases. The prospective study design, together with a systematic screening of 17 different viruses by sensitive RT–PCR assays and the use of standardized lower respiratory specimens, has ensured the best possible setting for this investigation. Of note is the fact that the rate of positivity of 18.6% compares favourably with our previous investigations in hospitalized patients, as well as with similar findings in other studies [10,11,14].
Recent screening studies targeting four different viruses in non-hospitalized HIV patients with respiratory symptoms during winter seasons identified influenza as the most frequent with a positivity rate of 40% . As expected in our study, the majority of viral infections also occurred during the winter season. Moreover, screening for all respiratory viruses independent of both clinical symptoms and season permitted a large spectrum of viral aetiology that was not only limited to the classic viruses such as influenza or respiratory syncytial virus. Indeed, coronavirus was the most frequent together with parainfluenza viruses. The latter has been reported as the most frequent in a study of HIV-infected children  and HMpV infection was demonstrated to be one of the most frequent viruses incriminated in lower respiratory tract infections in this population . The respective role of each agent will thus vary according to the assay, the population and the season studied. The design of our study limited this variability. To the best of our knowledge, coronavirus has not previously been studied in this population [12,17,18]. This illustrates that when considering respiratory viral infections in these severely immunosuppressed patients, any investigations assessing their impact should not be restricted to prototype viruses and should also target all the different family of viruses. This also limits the potential role of vaccination and antiviral interventions that are presently restricted mainly to influenza infections. In recent years, several new respiratory viruses have been identified, and only systematic screening using the most appropriate molecular tools such as in this study can assess their respective role. The respective impact of each virus will also vary according to the intensity of seasonal outbreaks during the study period. It should be noted that rhinovirus was surprisingly infrequent in our study.
Highly immunosuppressed HIV-infected patients living in the community are frequently exposed to respiratory viruses that circulate all year round. Half of the patients enrolled in the present study presented a confirmed bacterial or opportunistic disease, but it seems likely that in many cases concomitant respiratory viral infections have triggered, accelerated or worsened the clinical symptoms. Studies in HIV-infected children have shown an increased number of viral respiratory infections that promoted complications including bacterial pneumonia . It should also be considered that despite effective antiretroviral combination therapy, immune reconstitution is often incomplete and non-opportunistic infections could still occur. Impaired humoral immunity could also limit the production of neutralizing antibodies. Similarly, prophylaxis of opportunistic infections has no effect on respiratory viral infections. Our study provides an estimate of the potential impact of respiratory viruses in immunosuppressed HIV-infected patients presenting with lower respiratory tract diseases. On the basis of our results, it seems likely that respiratory viruses contribute to symptoms in a number of cases.
The authors would like to thank Patricia Suter for assistance with laboratory assays and Rosemary Sudan for editorial assistance.
The members of the Swiss HIV Cohort Study are M. Battegay, E. Bernasconi, J. Böni, HC Bucher, Ph. Bürgisser, A. Calmy, S. Cattacin, M. Cavassini, R. Dubs, M. Egger, L. Elzi, M. Fischer, M. Flepp, A. Fontana, P. Francioli (President of the SHCS, Centre Hospitalier Universitaire Vaudois, CH-1011- Lausanne), H. Furrer (Chairman of the Clinical and Laboratory Committee), C. Fux, M. Gorgievski, H. Günthard (Chairman of the Scientific Board), H. Hirsch, B. Hirschel, I. Hösli, Ch. Kahlert, L. Kaiser, U. Karrer, C. Kind, Th. Klimkait, B. Ledergerber, G. Martinetti, B. Martinez, N. Müller, D. Nadal, M. Opravil, F. Paccaud, G. Pantaleo, A. Rauch, S. Regenass, M. Rickenbach (Head of Data Center), C. Rudin (Chairman of the Mother & Child Substudy), P. Schmid, D. Schultze, J. Schüpbach, R. Speck, P. Taffé, A. Telenti, A. Trkola, P. Vernazza, R. Weber, S. Yerly.
Sponsorship: This study was supported by the Swiss National Research Foundation (grant no. 320060-101670) and the Swiss HIV Cohort Study (grant no. 457).
Conflicts of interest: None.
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