Abstract
Viruses and bacteria in bronchoalveolar lavage fluids, protected specimen brush samples, and bronchial biopsies from 14 patients with primary hypogammaglobulinemia (11 patients with common variable immunodeficiency [CVID] and three patients with X-linked agammaglobulinemia [XLA]) were analyzed. At the time of the study, the patients had no signs of acute respiratory infections, and no antibiotics were administered. In addition to routine bacterial and viral cultures, polymerase chain reaction tests were used for the detection of adenovirus, cytomegalovirus (CMV), herpes simplex virus 1, enterovirus, rhinovirus, Borrelia burgdorferi, Chlamydia pneumoniae, Legionella spp., Mycoplasma pneumoniae, Pneumocystis carinii, and Ureaplasma urealyticum. Viruses (four adenoviruses, one CMV, and one rhinovirus) were detected in four of the 11 (36%) CVID patients. No viruses were found in the three patients with XLA or in 13 control patients. Bacteria from the lower respiratory tract were detected in nine of the 14 (64%) patients with hypogammaglobulinemia and three of the 13 (23%) control patients. Haemophilus influenzae was the most prevalent bacterium (43%) in the hypogammaglobulinemia patients. The study shows that patients with CVID harbor viral and bacterial infections in the lower respiratory tract, which may predispose to the development of changes in the respiratory tract.
Patients with primary hypogammaglobulinemia experience recurrent respiratory tract infections. The most common respiratory pathogens found in these patients are encapsulated bacteria such as Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis. In general, no increased susceptibility to respiratory viruses has been reported. The use of regular parenteral immunoglobulin substitution therapy has been thought to reduce the occurrence of pulmonary complications, but bronchiectasis and fibrosis are still common complications in patients with primary hypogammaglobulinemia (1, 2).
In order to detect latent or chronic viral and bacterial infections in the lower respiratory tract, we used modern techniques to identify the responsible pathogens in bronchoalveolar lavage fluid (BALF), protected specimen brush (PSB) samples, and bronchial biopsies of 14 patients with primary hypogammaglobulinemia.
The study was conducted in 1995 and 1996, and included 14 patients (six women and eight men; median age 35 yr, age range 7 to 69 yr) with primary hypogammaglobulinemia who were being treated at Turku University Hospital. Three of the patients were children. Eleven of the 14 patients were diagnosed as having common variable immunodeficiency (CVID) and three patients as having X-linked agammaglobulinemia (XLA) according to the diagnostic criteria of the World Health Organization (WHO) immunodeficiency group (2). CVID was characterized by decreased serum immunoglobulin (Ig) levels, defective in vitro antibody formation (3, 4) and exclusion of other known causes of humoral immune defects. The diagnosis of XLA was based on early onset, very low serum Ig concentrations (IgG < 2 g/L), male sex, and a lack of circulating mature B lymphocytes in the peripheral blood (4). In two XLA patients, gene analysis of Bruton tyrosine kinase (Btk) was done by Professor M. Vihinen (University of Tampere, Finland) to confirm the diagnosis of XLA. All patients had also low or undetectable IgM and IgA concentrations at the time of diagnosis (Table 1).
| Patient No. Sex (age, yr), Dx | Duration of Symptoms Before Dx (yr ) | Age at the Time of Dx (yr ) | Serum IgG at Dx (g/L) | Respiratory Symptoms before Dx | Replacement Therapy | Serum IgG at the Time of the Study (g/L) | FEV1(L; % pred ) | FVC (L; % pred ) | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1, M (66), CVID | 14 | 54 | 2.0 | Pneumonia | IVIG | 8.4 | 2.12 (65%) | 2.91 (74%) | ||||||||
| 2, M (36), CVID | 4 | 34 | 2.0 | Recurrent sinusitis, pneumonia | IVIG | 5.7 | 3.68 (79%) | 4.78 (84%) | ||||||||
| 3, F (36), CVID | 4 | 22 | 2.8 | CMS, pneumonia | IVIG | 6.4 | 3.4 (93%) | 4.5 (100%) | ||||||||
| 4, F (51), CVID | 2 | 35 | 1.3 | ROM, recurrent sinusitis, pneumonia | IMIG | 8.0 | NA | NA | ||||||||
| 5, F (52), CVID | 8 | 45 | 3.9 | Bronchitis | SCIG | 5.0 | 3.1 (103%) | 4.4 (118%) | ||||||||
| 6, F (9), CVID | < 1 | 5 | 1.7 | Pneumonia | IVIG | 5.1 | NA | NA | ||||||||
| 7, M (41), CVID | 1 | 28 | 1.0 | ROM, pneumonia | IVIG | 5.3 | 2.68 (61%) | 5.42 (100%) | ||||||||
| 8, M (56), CVID | 25 | 46 | 1.2 | CMS, bronchitis, pneumonia | IVIG | 4.9 | 2.88 (74%) | 4.63 (95%) | ||||||||
| 9, F (69), CVID | 4 | 59 | 2.5 | CMS | IVIG | 6.0 | 2.08 (89%) | 3.03 (86%) | ||||||||
| 10, F (38), CVID | 1 | 23 | 2.8 | CMS, ROM, bronchitis, pneumonia | IVIG | 4.9 | 3.00 (108%) | 3.5 (106%) | ||||||||
| 11, M (14), CVID | < 1 | 1 | 1.5 | None | IVIG | 6.5 | NA | NA | ||||||||
| 12, M (22), XLA | < 1 | 1 | < 1.5 | None | IVIG | 7.0 | 7.28 (139%) | 7.56 (126%) | ||||||||
| 13, M (31), XLA | 5 | 5 | < 1.5 | ROM, pneumonia, bronchitis | IVIG | 5.0 | 4.88 (98%) | 5.5 (92) | ||||||||
| 14, M (7), XLA | < 1 | 1 | < 1.5 | Pneumonia | IVIG | 7.4 | NA | NA |
All patients were receiving regular immunoglobulin substitution therapy. Preinfusion serum IgG levels were higher than 4.5 g/L in all patients. In all but one patient, pulmonary disease had been diagnosed previously through high-resolution computed tomography (HRCT). Bronchiectasis was present in eight patients and fibrosis in five patients. None of the patients was receiving prophylactic antibiotic therapy. The patients had no signs of acute respiratory infections at the time of the study, and they had not received antibiotic treatment within 2 wk before the study.
Control samples were collected from 13 patients (eight women and five men), in whom bronchoscopy was done for diagnostic purposes at the Department of Pulmonary Diseases of the Turku University Hospital. These patients' median age was 54 yr (range: 34 to 74 yr). The control patients had no signs of acute respiratory infection. The diagnoses of control patients were sarcoidosis (two patients), prolonged cough (four patients), asbestosis (two patients), asthma (one patient), chronic bronchitis (one patient), pulmonary adenocarcinoma (two patients), and exudative pleuritis (one patient). The study was approved by the Joint Commission on Ethics of the Turku University Hospital and the University of Turku. Informed consent was obtained from all patients and control subjects.
All adult patients and control subjects underwent bronchoscopy with a flexible fiberoptic bronchoscope (BF-IT3; Olympus Optical, Tokyo, Japan) under topical anesthesia, and three children underwent bronchoscopy with a stiff bronchoscope under combined anesthesia.
After bronchoscopic exploration of the lower airways, bronchial brushing was done with the PSB system (Microbiology Specimen Brush; Microvasive Boston Scientific Corporation, Boston, MA) before bronchoalveolar lavage (BAL), in order to avoid any contamination from the upper airways. After the brushing, the brush was transected with sterile scissors and inserted into a sterile transport tube with 1 ml of sterile Ringer's solution. BAL was done in all patients and control subjects after the brushing. For BAL, the bronchoscope was wedged into the right middle lobe segmental bronchus and 100 to 160 ml saline was injected. The fluid was gently aspirated by controlled suction. The BALF was collected in sterile tubes to be transported on ice and cultured on the same day. Bronchial biopsy samples were taken from the right middle lobe and the right upper lobe carina of adult patients. No bronchial biopsy samples were taken from the three children in the study. Biopsy specimens for polymerase chain reaction (PCR) tests were collected in Tissue Tec gel and frozen in liquid nitrogen.
Bacterial cultures were done according to conventional routine procedures. Mycoplasma pneumoniae was cultured with the Pneumofast-kit (International Mycoplasma, Signes, France).
Virus cultures were done according to standard roller-tube methods in LLC-Mk2, HeLa Ohio, and A549 cells, and in human foreskin fibroblasts. The cultures were observed for cytopathogenic effects for 2 wk. Positive cultures were tested with a time-resolved fluoroimmunoassay for adenovirus (5), parainfluenza virus types 1, 2, and 3, and respiratory syncytial virus (RSV) antigens (6). Rhinoviruses were identified by their acid lability. In addition, a plate culture method with immunoperoxidase staining was used for isolation of cytomegalovirus (CMV) (7), herpes simplex virus (HSV) types 1 and 2 (8), and influenza A and B viruses (9).
Total nucleic acids were isolated from BALF and bronchial biopsy samples by proteinase-K (0.5 μg/μl) and sodium dodecyl sulfate (SDS) digestion (0.2%) at 37° C, followed by phenol–chloroform extraction and ethanol precipitation. The pellets were dissolved in nuclease-free water, incubated at 56° C for 15 min, and stored at −70° C.
BALF was examined with PCRs for the following pathogens: Borrelia burgdorferi, Chlamydia pneumoniae, Legionella spp., mycobacteria, Mycoplasma pneumoniae, Pneumocystis carinii, and CMV. Bronchial biopsy samples were examined with a PCR or reverse transcription (RT)–PCR for the following pathogens: adenovirus, CMV, enterovirus, HSV-1, rhinovirus, and Ureaplasma urealyticum.
PCR tests were done according to methods described previously (Table 2). Table 2 shows the target genes, primers, probes, amplicons, annealing temperatures, and number of cycles used in the PCR tests.
| PCR Test | Target Gene | Primers (Probe) | Amplicons, No. Cycles, Annealing Temperature | Reference | ||||
|---|---|---|---|---|---|---|---|---|
| Borrelia burgdorferi | Flagellin | WK1, FL7 | 497 bp, 40, 50° C | He et al., 1994 (36) | ||||
| Chlamydia pneumoniae | 16S | Cpn-A, Cpn-B (1311-40) | 463 bp, 35, 55° C | Gaydos et al., 1992 (37) | ||||
| Legionella spp. | 5S | L5SL9, L5SR93 (15S-1p) | 104 bp, 35, 55° C | Mahbubani et al., 1990 (38) | ||||
| Mycobacteria | 32-kD | MV1, MV2 | 423 bp, 30, 55° C | Soini et al., 1992 (39) | ||||
| Mycoplasma pneumoniae | M. pneumoniae, genomic sequence | MP5-1, MP5-2 (MP5-4) | 144 bp, 35, 55° C | Bernet et al., 1989 (40) Skakni et al., 1992 (41) | ||||
| Pneumocystis carinii | LSU | pAZ102-E, pAZ102-H (pAZ102-L2) | 346 bp, 35, 55° C | Wakefield et al., 1990 (42) | ||||
| Ureaplasma urealyticum | 16S | U2-5, U2-3 (U2p) | 196 bp, 45, 60° C | Aaltonen et al., 1997 (43) | ||||
| Adenovirus | Ad2 hexon gene | Primer 1, primer 2 (probe A) | 161 bp, 30, 61° C | Hierholzer et al., 1993 (44) | ||||
| Cytomegalovirus | MIE gene | CMVPRI1, CMVPRI2 (cmvpri.eco, cmvpri.hind) | 434 bp, 40, 65° C | Demmler et al., 1988 (45) | ||||
| Entero- and rhinovirus reverse transcription (RT)-PCR | 5′ Noncoding region protein | Primer3+, primer 4− (probe E) | 120 bp, 40, 53° C | Santti et al., 1997 (46) | ||||
| Herpes simplex virus-1 | Glycoprotein D gene | gD-1(HS1D-1), (gD-I, [HS1D-5]) | 221 bp, 41, 55° C | Aurelius et al., 1991 (47) |
Of the PCR products, 10 to 20 μl were analyzed by electrophoresis in 1.5 to 2% agarose gels containing ethidium bromide (1 μg/ml), with the results visualized and photographed under ultraviolet (UV) illumination. Nucleic acids from the gels were transferred onto nylon membranes (GeneScreen Plus; DuPont, Boston, MA) and the specificity of the signals was confirmed by hybridization with digoxigenin– deoxyuridine triphosphate (dUTP)-labeled specific probes (Boehringer Mannheim, Mannheim, Germany). The digoxigenin-labeled probes were detected with alkaline phosphatase-conjugated antidigoxigenin Fab fragments and chemiluminescent substrate disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate (CSPD) analysis (Boehringer) according to the manufacturer's instructions.
Bacteria and/or viruses were found in 11 of the 14 (79%) patients with hypogammaglobulinemia and in three of the 13 (23%) control patients (Table 3).
| Patient No. (Diagnosis) | BALF (cfu/ml ) | PSB (cfu/ml ) | Virus PCR of Bronchus Biopsies | BALF Cell Counts and Differential | Pulmonary Changes | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| 1 (CVID) | Negative | Negative | Negative | Cell count 182 × 106/L: macrophages 93%, lymphocytes 5%, neutrophils 2%, squamous cell metaplasia | Bronchiectasis | |||||
| 2 (CVID) | Mixed flora (103) | Streptococcus viridans (5 × 106), nonhemolytic S. (106), Fusobacteria (103 ), gram-positive rod (2 × 107 ), anaerobic gram-positive rod (103) | Negative | Cell count 695 × 106/L: macrophages 68%, lymphocytes 30%, neutrophils 2% | Emphysema | |||||
| 3 (CVID) | Negative | Negative | Negative | Cell count 135 × 106/L: macrophages 68%, lymphocytes 30%, neutrophils 2% | Bronchiectasis | |||||
| 4 (CVID) | Negative | Negative | CMV, adenovirus | Cell count 116 × 106/L: macrophages 86%, lymphocytes 11%, neutrophils 2%, eosinophils 1% | Fibrosis | |||||
| 5 (CVID) | Negative | Negative | Adenovirus | Cell count 84 × 106/L: macrophages 54%, lymphocytes 45%, neutrophils 1%, squamous cell metaplasia | Fibrosis | |||||
| 6 (CVID) | Haemophilus influenzae (5 × 103 ), Streptococcus viridans (103 ) | Negative | Negative | NA | Bronchiectasis | |||||
| 7 (CVID) | Haemophilus influenzae (104), Streptococcus viridans (7 × 103 ), Neisseria spp. (103) Rhinovirus | Haemophilus influenzae (5 × 107), Streptococcus viridans (3 × 106 ) | Adenovirus | Cell count 419 × 106/L: macrophages 54%, neutrophils 46% | Bronchiectasis | |||||
| 8 (CVID) | Negative | Moraxella catarrhalis (3 × 107) | Adenovirus | Cell count 90 × 106/L: macrophages 44%, lymphocytes 45%, neutrophils 10%, eosinophils 1% | Bronchiectasis | |||||
| 9 (CVID) | Haemophilus influenzae (3 × 104 ), mixed flora (3 × 103 ), anaerobic gram-positive rod (2 × 103) | Haemophilus influenzae (108) | Negative | Cell count 129 × 106/L: macrophages 41%, lymphocytes 14%, neutrophils 46% | Bronchiectasis | |||||
| 10 (CVID) | Gram-positive rod (103 ) | Negative | Negative | Cell count 210 × 106/L: macrophages 24%, lymphocytes 70%, neutrophils 6%, squamous cell metaplasia | Bronchiectasis | |||||
| 11 (CVID) | Negative | Negative | Negative | NA | No changes | |||||
| 12 (XLA) | Nonhemolytic streptococci (103), Haemophilus influenzae (3 × 103) | Mixed flora (3 × 106) | Negative | Cell count 152 × 106/L: macrophages 65%, lymphocytes 26%, neutrophils 8%, eosinophils 1% | Bronchiectasis | |||||
| 13 (XLA) | Haemophilus influenzae (103 ), Veillonella (103) | Haemophilus influenzae (106), β-hemolytic streptococci (non A, C, G) (2 × 106), Porphyromanas (106 ) | Negative | Cell count 146 × 106/L: macrophages 87%, lymphocytes 12%, neutrophils 1% | Bronchiectasis | |||||
| 14 (XLA) | Haemophilus influenzae (105) | Negative | Negative | NA | Bronchiectasis | |||||
| Control No. 1 (fibrosis) | β-Hemolytic streptococci group C (103) | Negative | Negative | Cell count 140 × 106/L: macrophages 94%, lymphocytes 5%, neutrophils 1% | Fibrosis | |||||
| Control No. 2 (prolonged cough) | Haemophilus influenzae(103) | Gram-negative rod (103) | Negative | Cell count 205 × 106/L: macrophages 79%, lymphocytes 14%, neutrophils 5%, eosinophils 1%, mast cells 1% | No changes | |||||
| Control No. 3 (pulmonary adenoca) | Streptococcus viridans (2 × 103 ), gram-positive rod (5 × 103) | Neisseria spp. (103) | Negative | Cell count 95 × 106/L: macrophages 62%, lymphocytes 30%, neutrophils 1%, eosinophils 5%, mast cells 2% | Pulmonary cancer |
Viruses were found in the samples of four (29%) patients with primary hypogammaglobulinemia. Adenoviruses were found through PCR in the bronchial biopsy samples of all four of these patients. In addition, 2 yr earlier we had cultured adenovirus in feces of another two patients of the study group at clinical follow-up. Among the adenovirus-positive patients, CMV was detected through PCR in the biopsy sample of one patient, and rhinovirus was cultured from the BALF sample of another patient. Thus, two of the four patients with primary hypogammaglobulinemia and a viral infection had a dual viral infection. No viruses were found in the samples of the 13 control patients.
Bacteria from the lower respiratory tract were found in nine of the 14 (64%) patients with hypogammaglobulinemia (Table 3). Bacterial growth was observed in BALF samples of eight patients when concentrations > 103 cfu/ml were used as a cutoff point. H. influenzae was detected in six samples. All H. influenzae strains were resistant to erythromycin, and one was also resistant to trimethoprim–sulfamethoxazole. All BALF specimens were negative for bacteria with PCR analyses. PSB specimens showed bacterial growth in six patients. Bacteria were detected in both BALF and PSB samples from five patients. Bacterial growth was observed in three of the 13 (23%) control patients.
Cytologic studies of BALF showed neutrophilia in three and lymphocytosis in eight samples from patients with hypogammaglobulinemia (Table 2). None of the patients had a history of alveolitis or sarcoidosis, which are the most common causes of lymphocytosis. Squamous cell metaplasia was detected in four patients. None of these patients was a smoker.
Bronchiectasis and pulmonary fibrosis are the most common complications in patients with primary hypogammaglobulinemia (10, 11), and they are more common and more severe in patients with CVID than in patients with XLA (12). In our study, 12 of 14 patients with primary hypogammaglobulinemia had bronchiectasis or fibrosis detected by HRCT, which is consistent with the results of Curtin and colleagues (10). To our knowledge, no previous studies have reported on the presence of viruses in the lower respiratory tract of patients with primary hypogammaglobulinemia. On the other hand, the presence of bacteria has been documented (13).
In our study, adenovirus was detected in four of the 11 (36%) CVID patients and in none of the control patients. Two additional patients with CVID had a history of fecal adenovirus excretion detected by culture. Patients with primary hypogammaglobulinemia have previously been shown to be susceptible to enterovirus and herpes virus infections while normally resistant to other viral infections (14, 15). Our findings raise the question of whether patients with CVID are at greater risk of adenovirus infection than immunocompetent hosts. Adenovirus infections have been shown to be common among immunocompromised patients, occurring in 8 to 18% of liver transplant patients and in 12% of renal transplant patients (16). Pediatric bone marrow transplant patients and patients with human immunodeficiency virus (HIV) infection also appear to be vulnerable to adenovirus infection (17, 18).
The role of adenovirus in the development of chronic bronchial changes has been documented. It is well established that adenoviruses, especially types 7 and 21, predispose to the development of bronchiectasis in children (19-21). Adenovirus DNA is also frequently found in the lungs of cigarette smokers with chronic obstructive pulmonary disease (22). However, Hogg and colleagues (23) failed to find adenovirus with in situ hybridization of lung tissue samples obtained during recovery from an acute adenovirus pneumonia or from follicular bronchiectasis, suggesting that either the adenovirus disappears from the lung or is present in a latent form. The early E1A region of adenovirus enhances the inflammatory reaction in the epithelium during infection with the virus (20, 22), and chronic inflammation is considered crucial in the development of bronchiectasis, while damaging the epithelium with proteinases and reactive oxidative agents (24, 25). Adenovirus is known to cause latent and persistent infections by different mechanisms. The virus has several mechanisms for resisting antiviral responses of the host. Proteins encoded by the E3 region protect infected cells from the lytic effects of cytotoxic T lymphocytes and tumor necrosis factor (26). Immunodeficiency may predispose to the persistence of virus infection. Serial cultures or PCR data have, however, been required to confirm persistent lower respiratory tract adenovirus infection in our patients.
Rhinovirus was found in this study in the BALF of one patient through culture, and CMV was found in a bronchial biopsy sample from one patient through PCR. Rhinovirus is considered an upper respiratory tract pathogen, but recent findings suggest that rhinovirus may also invade the lower respiratory tract (27). This is in agreement with our results. CMV is commonly found in BALF samples of immunocompromised patients without any clinical significance (28, 29). Our patient, however, developed CMV pleuritis after 6 mo, and died of disseminated CMV infection. The patient had a history of Hodgkin's lymphoma, but she had not received immunosuppressive medication for 5 yr before the study. This case suggests that persistent CMV may become active in a patient with CVID and may require initiation of antiviral medication when the virus is detected.
In addition to being marked by β-cell dysfunction, CVID is associated with defects in T cell function. A subgroup of patients with CVID shows impaired proliferative responses in vitro to antigens and mitogens, and produces reduced levels of interleukins -2, -4, and -5 and of mRNA for interferon-γ (30). Recent studies have shown signaling impairments in CVID patients' peripheral blood T cells (31, 32). Some patients with CVID show abnormally low numbers of CD4+ T cells. Three of our patients with CVID had low (< 0.75) CD4+/CD8+ ratios (4). Two of these patients had evidence of virus infection. Disorders in T cell function and abnormally low numbers of CD4+ cells may increase the susceptibility of CVID patients to viral infections (15).
H. influenzae was the most common bacterium that we found in the lower respiratory tract of patients with primary hypogammaglobulinemia. The bacterium was found in higher concentrations in PSB samples than in BALF samples, ruling out the possibility of contamination from the upper respiratory tract. Our finding is consistent with a previous report of Samuelson and colleagues (13) that in CVID patients H. influenzae was the most common bacterium isolated during exacerbations, and that patients with CVID are prone to permanent colonization with the same strain of H. influenzae. Likewise, H. influenzae is one of the most common bacteria found in the lower respiratory tract of immunocompetent patients with bronchiectasis (33, 34).
Cytologic study of BALF from patients with primary hypogammaglobulinemia showed neutrophilia in three of the nine patients with bacterial findings, supporting infection in bronchi. The high prevalence of lymphocytosis (55%) in hypogammaglobulinemia patients was interesting, since none of the patients had a history of alveolitis or sarcoidosis, which are the most common conditions related to lymphocytosis in BALF.
In conclusion, the present study showed that some patients with CVID have viral lower respiratory tract infections. Adenovirus infection may be a contributory factor in the development of bronchial damage in CVID patients. Both viruses and bacteria were found without acute respiratory symptoms. Viral infection may have synergistic effects with bacterial infection (35). The eradication of pathogens from the lower airways may require higher dosages of intravenous immunoglobulin and appropriate antibiotics than usual and, if available, appropriate antiviral agents.
Supported by grants from the Maud Kuistila Foundation, the Pediatric Research Foundation, the Finnish Antituberculosis Association Foundation, and the Academy of Finland.
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