Chlamydia pneumoniae: a new opportunistic infectious agent in AIDS?

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1 ORIGINAL ARTICLE Chlamydia pneumoniae: a new opportunistic infectious agent in AIDS? Rosa Monno 1, Emilia Leone 1, Paolo Maggi 2, Giovanni Buccoliero 2, Maria A. valenza 1 and Gioacchino Angarano 2 1Institute ofmedical Microbiology, and 2Clinic ofinfectious Diseases, University ofbari, Bari, Italy Objective: To determine the incidence of Chlamydia pneumoniae respiratory tract infection in HIV-positive or AIDS patients. Methods: Serum samples from 82 HIV-positive patients with fever and respiratory symptoms were evaluated using microimmunofluorescence assay to detect C. pneumoniae-specific IgG and IgM antibodies. Results: Twenty patients were found to have IgG antibodies to C. pneumoniae at titers ranging between 1: 16 and 1: Seven of the patients had evidence of acute C. pneumoniae infection (a fourfold rise in the titer of IgG antibody, or a single IgG titer of ~1 :512, or a single IgM titer ~1: 16). Five were diagnosed as having pneumonia and two bronchitis. No co-infection with other respiratory tract pathogens was found. Conclusions: Results of this study indicate that C. pneumoniae may playa role in the etiology of respiratory tract infections in HIV-positive patients; this fact should affect empirical antibiotic prescription. Key words: Chlamydia pneumoniae, AIDS, HIV, respiratory infection. INTRODUCTION Chlamydia pneumoniae represents a new addition to the list of microbial pathogens. This microorganism causes human respiratory infections including pneumonia, bronchitis and pharyngitis [1]. In addition, chronic C. pneumoniae infection may be associated with both chronic ischemic heart disease and acute myocardial infarction [2J. Meningoencephalitis, the Guillain-Barre syndrome, arthritis and erythema nodosum have also been associated with C. pneumoniae infection [3-6]. Serologic studies indicate that C. pneumoniae is one of the most widespread infectious agents, and epidemiologic studies have indicated a worldwide distribution of this microorganism [7]. Pneumonia is a major problem among patients with AIDS [8J. Therefore, it seemed reasonable to test Corresponding author and reprint requests: Rosa Monno, Istituto di Microbiologia Medica, Policlinico, P.G. Cesare, Bari, Italy Tel: Fax: Revised version accepted 3 October 1996 AIDS patients with pneumonia for the presence of C. pneumoniae infection. In fact, an outbreak of C. pneumoniae infection has been reported in a group of15 of26 former injection-drug users living together in the community [9]. There have been two further reports of C. pneumoniae infection in HIV-positive subjects [10,11]. In an effort to understand better the importance of C. pneumoniae infection in HIV-positive patients in our area, we have analyzed serum samples from HIV-positive and AIDS subjects since 1992 for antibody by a microimmunofluorescence technique. MATERIALS AND METHODS Test population The group ofpatients consisted of82 subjects (55 males and 27 females) examined at the Clinic of Infectious Disease, Policlinico, Bari, Italy. The age of the patients ranged from 18 to 60 years, with a mean age of 32. All patients were HIV seropositive, as detected by ELISA and confirmed by immunoblotting. HIV-positive subjects were classified according to the Center for Disease Control criteria as IV B (5), IV C1(31), IV C2 (1), IV D (6); 39 were asymptomatic. A diagnosis of 187

2 188 Clinical Microbiology and Infection, Volume 3 Number 2, April 1997 community-acquired pneumonia and/or bronchitis was based on clinical signs and symptoms (fever, cough, dyspnea, abnormal breathing sounds), and radiologic findings. Routine blood tests, sputum and/or bronchoalveolar lavage cultures and evaluation of Pneumocystis carinii, cytomegalovirus (CMV) and Mycobacterium spp. were also performed in symptomatic patients. Sample collection Peripheral blood samples were collected by antecubital venepuncture. Serum samples were separated by centrifugation, aliquoted and stored at - 20 C before being tested. Paired serum samples were obtained from 28 patients and single samples from the remaining 54 patients. Serologic studies Serologic testing for C. pneumoniae antibody in the immunoglobulin G and M (IgG and IgM) classes was performed by the microimmunofluorescence (MIF) method. This method uses formalinized purified elementary bodies of C. pneumoniae strain TW-183 as antigen (Washington Research Foundation, USA). The antigen spots are placed on the microscope slide. For IgG antibody measurements, twofold dilutions were incubated at 37 C for 30 min with the antigen; slides were washed and incubated with antihuman IgG antibody conjugates (Fluoline-G, biomerieux Italia; Rome, Italy) for 30 min at 37 C. For IgM antibody measurements, diluted sera were incubated overnight at +4 C with the antigens; slides were washed and incubated with antihuman IgM antibody conjugates (Fluoline M; biomerieux Italia, Rome, Italy) for 30 min at 37 C. Positive IgM antibodies were confirmed after absorption with rheumatoid factor (RF) absorbent (biomerieux Italia, Rome, Italy) to remove unspecific reactivity. Serologic evidence ofacute infection with C. pneumoniae was defined as a fourfold or greater rise in the titer of IgG or IgM antibody, or a single IgM titer of ;::: 1:16, or a single IgG titer of ;::: 512. Sera from patients positive for C. pneumoniae were also tested for total antibody to Chlamydia trachomatis and for IgG antibody to Chlamydia psittaci by an indirect immunofluorescence test (C. trachomatis spot IF; biomerieux Italia, Rome, Italy; C. psittaci spot IF, biomerieux Italia, Rome, Italy). Serum samples were also tested for the presence of specific antibodies to Mycoplasma pneumoniae (Mycoplasma pneumoniae IgG, Mycoplasma pneumoniae IgM, Bayer Diagnostici, Italy), and Legionella pneumophila (Legionella indirect fluorescent test, Zeus Scientific Inc., Raritan, NJ, USA). Serology for Coxiella burnetii and the most common viruses was not routinely carried out. RESULTS A total of 82 HIV-positive patients observed over a period of 4 years from October 1992 to September 1995 were tested, of whom 20 (24.4%) were found positive for C. pneumoniae IgG at titers ranging between 1: 16 and 1: Two of the patients had Table 1 Data of AIDS patients with current C. pneumoniae infection C. pneumoniae serology Risk factors CD4+ cell Site of radiographic Patient Agelsex for HIV count Clinical diagnosis changes 19G 19M Treatment 40/M Heterosexual 9/mrn 3 Bronchitis 1024 Neg. Ci, 1+A' acquisition E 2 371M Drug user 16/mrn 3 Pneumonia Interstitial right 512 Neg. E Pleuritis middle lobe 3 381M Homosexual 321mrn 3 Pneumonia Interstitial right 1024 b Neg. E acquisition Pleuritis, KS middle lobe 512' 4 191M Hemophilia 9/mm 3 Pneumonia Interstitial left 16 b Neg. E lower lobe 128' 5 331M Drug user 7/mrn 3 Bronchitis 512 Neg. SXT' C 6 341M Drug user 27/mm 3 Pneun10nia Alveolar and interstitial C right middle-basal lobe 7 36/F Heterosexual 65/rnm 3 Pneumonia Alveolar right 16 b Neg. l' acquisition lower lobe 64' C 'Antibiotic therapy empirically based. b Acute serum. 'convalescent serum. A= amikacin; C = clarithromycin; Ci= ciprofloxacin; E = erythromycin; 1=imipenem; SXT=sulfamethoxazole-trimethoprim; KS=Kaposi's sarcoma.

3 Monno et al: Chlamydia pneumoniae infections in AIDS C. pneumoniae IgG antibody titers of 1: 1024, two had titers of 1:512 and two had titers of 1:128. Titers of 1:64 were found in six of the patients. Low-level titers of antibodies (1: 16 to 1: 32) were observed in eight of the patients. These antibody levels represent the highest titer obtained for each patient. Seven of the patients with C. pneumoniae antibody had evidence ofacute infection. One patient had a high titer ofigg antibody (1: 1024), two patients had an IgG titer of 1:512, and one patient had IgG antibody levels of 1: 1024 in the acute serum and 1: 512 in the convalescent serum. In another two patients a fourfold increase in IgG titer was observed (from 1: 16 to 1: 128 and from 1:16 to 1:64, respectively). Finally, one patient had an IgM titer of 1:64 and an IgG titer of 1: 128. A summary of data from the seven patients with current C. pneumoniae infection is provided in Table 1. Five were diagnosed as having pneumonia and two bronchitis. In three of the patients with current C. pneumoniae infection, interstitial changes were observed. Alveolar opacities in the right middle-basal lobe with development of bilateral interstitial opacities were observed in one case (patient 6) and in another case alveolar opacities in the right lower lobe wete found (patient 7). In two patients, pleural effusions which complicated right middle lobe pneumonia were present. No co-infection with cytomegalovirus, mycobacteria, P. carinii, L. pneumophila, Mycoplasma pneumoniae and other common pathogens was found in these seven patients. The possibility that the antibodies to C. pneumoniae resulted from cross-reactivity with other chlamydia was ruled out by testing sera positive for IgG to C. pneumoniae also for antibodies to C. trachomatis and C. psittaci. Antibodies against these microorganisms were not detected. Four patients received erythromycin 3 g/day for 14 days, and a favorable clinical response was observed in three patients. One patient (patient 2) failed to respond and died in respiratory failure. Three patients received clarithromycin 1 g/ day for 14 days; a favorable clinical response was seen in two patients. Another patient failed to respond and died (patient 6). DISCUSSION Pulmonary infection is the most commonly recognized clinical problem in AIDS. More than 80% of the opportunistic infections seen in patients with AIDS involve the lung; in fact, the lung is known to be susceptible to infections or to other insults during times of immunosuppression [8,12]. C. pneumoniae is a newly recognized third species of the genus Chlamydia [13]. Since C. pneumoniae was first reported, several publications have confirmed its importance as a new respiratory pathogen. 189 In fact, C. pneumoniae accounts for 10% of cases of community-acquired pneumonia and there is evidence that C. pneumoniae may also account for cases of nosocomial pneumonia [14,15]. In our previous study, we found that 13 of91 adult patients with respiratory tract infection had serologic evidence of recent infection with C. pneumoniae. The clinical picture was consistent with asthmatic bronchitis in three patients, whilst exacerbations of chronic obstructive pulmonary disease were found in five subjects, and pneumonia was found in the remaining five [16]. Serologic studies in several countries have shown an antibody prevalence from 40% to over 50%, suggesting that exposure to C. pneumoniae IS very common. Recently, studies on the incidence of C. pneumoniae infection in HIV-positive subjects have been reported. Bronchoalveolar lavage specimens from HIVinfected patients with pneumonia were examined by culture for the presence of C. pneumoniae. Five of 50 (10%) specimens examined were positive for C. pneumoniae, and four of these five specimens contained other pulmonary pathogens (P. carinii in two cases and Mycobacterium tuberculosis in another two). Four of the five specimens were from patients with AIDS. However, the role of C. pneumoniae in the pathologic process remains unclear, because serologic data were not available and because four of these patients recovered with antimicrobial agents (such as pentamidine or antimycobacterial agents) not effective against C. pneumoniae [10]. Another study reported that in an HIV-l-infected population, C. pneumoniae seroprevalence was found to be higher than in immunocompetent controls (26% in HIV-positive children versus 11 % in HIV-negative children; 60% in HIV-positive drug users versus 40% in HIV-negative drug users). The authors suggested that HIV-infected subjects seem to be at higher risk of developing C. pneumoniae infections and that immunodeficiency rather than lifestyle may be the risk factor for C. pneumoniae infection [17]. In another study, among 108 immunodeficient patients, 34% of whom were HIV positive, C. pneumoniae DNA was demonstrated in bronchoalveolar lavage fluid from 11 (1 %) [18]. In addition, an outbreak of C. pneumoniae infection in 26 former injection drug-users has been reported in Italy by Blasi et al. [9]. Pneumonia was the syndrome most frequently observed (in eight HIVpositive and in three HIV-negative subjects) followed by pharyngitis and flu-like syndrome. We found current C. pneumoniae infection in 7 of 82 patients studied. In the remaining 13 patients, antibodies were found at low titer, suggesting a seroprevalence of 16% in this group of patients, lower than that reported by Blasi et al. [17]. Patients with

4 190 Clinical Microbiology and Infection, Volume 3 Number 2, April 1997 AIDS are at risk for infection with a wide range of microorganisms. In fact, alteration of T-Iymphocyte function by HIV increases host susceptibility to opportunistic infections caused by mycobacteria, viruses, fungi and protozoa. Because HIV also alters Blymphocyte function, it may increase a patient's risk of bacterial infection. Many of the clinical infections observed in AIDS may be due to reactivation oflatent infections acquired earlier in life. Previously, studies showed that the prevalence of antibody to C. trachomatis [19] and C. pneumoniae [20] in HIV-positive subjects is higher than in HIV-negative subjects and controls. It is well known that the humoral response, as well as the cell-mediated immune response, plays a role in maintaining an effective defense against C. trachomatis [21], whereas few data are available on the interaction between C. pneumoniae and the immune system [22]. It appears that, among bacterial infections that may be reactivated when the cellular immune system is destroyed by HIV, chlamydia may playa role. In fact, C. pneumoniae infections in HIV-positive patients have been reported [9-11]. On the other hand, Moncada et al. have isolated C. trachomatis from brush biopsies and lung lavage in only 3 of 263 individuals with AIDS (0.5%), suggesting that this microorganism is not an important respiratory tract pathogen in this population [23]. All these findings should alert bacteriology laboratories and clinicians to investigate for the presence of C. pneumoniae infections in HIV-positive patients with lower respiratory tract infections. Furthermore, the realization that C. pneumoniae may be an etiologic agent of pneumonia and bronchitis in these patients should affect empirical antibiotic prescription. 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