Risk factors for Haemophilus influenzae and pneumococcal respiratory tract colonization in CVID
Authors: Federica Pulvirenti, Romina Camilli, Maria Giufrè, Cinzia Milito, Fernanda Pimentel de Araujo, Fabiola Mancini, Rita Cardines, Alessandra Ciervo, Annalisa Pantosti, Marina Cerquetti, Isabella Quinti
Journal: Journal of Allergy and Clinical Immunology (2018)

Disease-specific studies focused on infection risk in common variable immune deficiencies (CVIDs) are needed to define strategies for controlling respiratory infections predominantly due to bacteria such as Streptococcus pneumoniae and Haemophilus influenzae.1Bonilla F.A. Barlan I. Chapel H. Costa-Carvalho B.T. Cunningham-Rundles C. de la Morena M.T. et al.International Consensus Document (ICON): common variable immunodeficiency disorders.J Allergy Clin Immunol Pract. 2016; 1: 38-59Abstract Full Text Full Text PDF Scopus (481) Google Scholar Little information is available on the rate of airway bacterial carriage and its consequence in hypogammaglobulinemias. Despite IgG replacement, recurrent respiratory infections are common in CVID, possibly leading to chronic lung damage2Quinti I. Soresina A. Guerra A. Rondelli R. Spadaro G. Agostini C. et al.IPINet Investigators. Effectiveness of immunoglobulin replacement therapy on clinical outcome in patients with primary antibody deficiencies: results from a multicenter prospective cohort study.J Clin Immunol. 2011; 31: 315-322Crossref PubMed Scopus (215) Google Scholar and poor quality of life.3Quinti I. Pulvirenti F. Giannantoni P. Hajjar J. Canter D.L. Milito C. et al.Development and initial validation of a questionnaire to measure health-related quality of life of adults with common variable immune deficiency: the CVID_QoL Questionnaire.J Allergy Clin Immunol Pract. 2016; 4: 1169-1179.e4Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar Thus, patients are often prescribed antibiotics and/or long-term antimicrobial prophylactic regimens. Several regimens are used including rotation or periodically changing antibiotics.4Kuruvilla M. de la Morena M.T. Antibiotic prophylaxis in primary immune deficiency disorders.J Allergy Clin Immunol Pract. 2013; 1: 573-582Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar However, antibiotics influence antimicrobial resistance among airway microbiota. In a recent meta-analysis on patients with chronic lung diseases, 30% of S pneumoniae showed resistance to macrolides.5Cameron E.J. McSharry C. Chaudhuri R. Farrow S. Thomson N.C. Long-term macrolide treatment of chronic inflammatory airway disease: risks, benefits and future developments.Clin Exp Allergy. 2012; 42: 1302-1312Crossref PubMed Scopus (64) Google Scholar In this observational longitudinal study, we investigated the rate of nasopharyngeal and oropharyngeal mucosal carriage of S pneumoniae and H influenzae identified by cultural methods and real-time PCR (RT-PCR), the antimicrobial susceptibility patterns, and the antibiotic usage in adult patients with CVID (http://esid.org/Working-Parties/Registry/Diagnosis-criteria). In addition, we verified whether colonization would be associated with risk for respiratory infections. Exclusion criteria for swabs collection included acute respiratory infections and antibiotic treatment in the month preceding the swabs collection except for antibiotic prophylaxis. Rate of respiratory infections, clinical data, and days of antibiotic usage in the 6 months before swabs collection were recorded. After swabs collection, respiratory infections were recorded for an additional 6 months. Sample collection and identification of S pneumoniae and H influenzae were performed according to the World Health Organization standard procedures by cultural and molecular methods on nasopharyngeal and oropharyngeal swabs collected on the same day. For both bacterial species, minimum inhibitory concentrations of antimicrobial agents were determined. The interpretative criteria were based on European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints (http://www.eucast.org/clinical breakpoints/) (see the Methods section in this article's Online Repository at www.jacionline.org). The 12-month study included 93 patients with CVID (mean age, 49.4 ± 14.4 years; range, 18-77) with a 1:1 male to female ratio. Participants were on IgG replacement at a monthly cumulative dose of 400 mg/kg. Mean IgG trough levels at the study time were 688.8 ± 156.0 mg/dL. Nineteen patients were exposed to children younger than 6 years at home or through work. Thirty-four (41%) patients had bronchiectasis and 9 (9.6%) were on chronic antibiotic prophylaxis. The carriage prevalence by culture was 10.8% and 26.9% for S pneumoniae and H influenzae, respectively, regardless of the isolation site (see Table E1 in this article's Online Repository at www.jacionline.org). Four percent of CVID were cocolonized by both S pneumoniae and H influenzae. In line with other pneumococcal carriage studies, after the long-term use of glycoconjugate vaccines, we found only 20% of the 13 pneumococcal isolates belonging to vaccine serotypes6Flasche S. Van Hoek A.J. Sheasby E. Waight P. Andrews N. Sheppard C. et al.Effect of pneumococcal conjugate vaccination on serotype-specific carriage and invasive disease in England: a cross-sectional study.PLoS Med. 2011; 8: e1001017Crossref PubMed Scopus (218) Google Scholar: 10A and 19A (2 isolates each), 6A, 12F, 23A, 35F, and 37 (1 isolate each); 4 isolates were nontypeable. All 36 H influenzae isolates were nontypeable, confirming the absolute prevalence of this serotype, especially among respiratory tract isolates, in the present Hib vaccination era (see Table E2 in this article's Online Repository at www.jacionline.org). Compared with culture, RT-PCR allowed identifying a higher carriage rate of Spneumoniae and/or H influenzae (10.8% vs 52.7%, P < .0001 and 26.9% vs 39.8%, P = .043, respectively). In the RT-PCR assay, the cycle threshold value was significantly lower in samples found positive by culture compared with those positive by RT-PCR, suggesting that the former samples had a higher S pneumoniae and/or H influenzae bacterial load. By RT-PCR the percentage of CVID cocolonized carriers significantly increased (4% vs 24%; P = .0001). H influenzae/S pneumoniae carriers identified by only RT-PCR had a milder immunological defect than culture-positive patients (IgA, 15.8 ± 3.2 vs 2.7 ± 0.9 mg/dL, P = .0001; IgM, 22.2 ± 4.4 vs 9.9 ± 4.1, P = .030). Risk factors related to colonization were initially identified by binary logistic regression. Low IgM and low IgA serum levels were found to be risk factors for S pneumoniae and H influenzae colonization. Colonization by culture was associated with IgM serum levels of less than 5 mg/dL (odds ratio [OR], 3.7; 95% CI, 0.86-15.99; P = .05; OR, 2.5; 95% CI, 0.95-6.45; P = .05, respectively) and with IgA serum levels of less than 7 mg/dL (OR, 5.7; 95% CI, 0.69-47.17; P = .05; OR, 8.9; 95% CI, 1.92-40.84; P = .001, respectively). Of note, patients cocolonized had particularly low IgA and IgM serum levels (1.3 ± 0.7 mg/dL and 1.7 ± 1.2 mg/dL, respectively). This confirmed data on patients with hyper-IgM syndrome and healthy subjects where IgM antibodies have a distinct anti–H influenzae activity in the prevention of respiratory tract bacterial colonization.7Micol R. Kayal S. Mahlaoui N. Beauté J. Brosselin P. Dudoit Y. et al.Protective effect of IgM against colonization of the respiratory tract by nontypeable Haemophilus influenzae in patients with hypogammaglobulinemia.J Allergy Clin Immunol. 2012; 129: 770-777Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 8Choi J. Nix E.B. Gaultier G.N. Cox A.D. McCready W. Ulanova M. Naturally occurring bactericidal antibodies specific for Haemophilus influenzae lipooligosaccharide are present in healthy adult individuals.Vaccine. 2015; 33: 1941-1947Crossref PubMed Scopus (6) Google Scholar, 9Barra A. Dagan R. Preud'homme J.L. Bajart A. Danve B. Fritzell B. Characterization of the serum antibody response induced by Haemophilus influenzae type b tetanus protein-conjugate vaccine in infants receiving a DTP-combined vaccine from 2 months of age.Vaccine. 1993; 11: 1003-1006Crossref PubMed Scopus (20) Google Scholar Other potential risk factors such as age, previous respiratory infections, bronchiectasis, chronic lung diseases, IgG trough level of less than 600 mg/dL, and defects in other immunological parameters were not associated with colonization. Antibiotic use and long-term prophylaxis in the previous 6 months were not associated with H influenzae and S pneumoniae colonization. However, participants exposed to young children were more likely to be colonized by H influenzae (OR, 5.9; 95% CI, 2.00-13.36; P = .001), but not by S pneumoniae. Multivariate analysis confirmed that IgA serum levels of less than 7 mg/dL (OR, 5.4; 95% CI, 1.08-26.70; P = .040) and the presence of children younger than 6 years in the house/workplace (OR, 4.5; 95% CI, 1.37-14.50; P = .013) had a direct effect on H influenzae colonization (Table I).Table IRisk factors for S pneumoniae and H influenzae colonization as detected by culturen (%)Univariate analysisMultivariate analysisS pneumoniaeH influenzaeH influenzaeOR95% CIP valueOR95% CIP valueOR95% CIP valueSex (female)47 (51.1)0.950.25-3.54NS0.700.27-1.70NS———Age < 35 y12 (12.9)3.520.77-16.10NS1.430.39-5.23NS———IgM < 5 mg/dL34 (38.2)3.710.86-15.99.052.480.95-6.44.051.630.55-4.79NSIgA < 7 mg/dL58 (65.2)5.690.69-47.17.058.861.92-40.84.0015.361.08-26.7.040IgG trough level <600 mg/dL8 (8.6)0.600.16-2.35NS0.710.26-1.82NS———Exposition to children19 (20.4)0.420.05-3.38NS5.892.00-17.36.0014.451.37-14.50.013URTI in the previous 6 mo21 (25.0)1.740.45-6.73NS0.930.34-2.58NS———LRTI in the previous 6 mo32 (38.5)1.860.50-6.96NS1.540.60-3.92NS———CLD49 (53.3)0.550.14-2.09NS0.930.37-2.34NS———Bronchiectasis34 (36.5)1.080.27-4.37NS0.820.31-2.18NS———Use of antibiotics in the previous 6 mo47 (50.5)0.980.26-3.63NS0.870.35-2.18NS——— Amoxicillin/Clavulanic acid21 (24.4)1.200.22-6.57NS0.970.30-3.17NS——— Macrolides13 (15.1)1.970.35-11.15NS1.210.33-4.50NS———Antibiotic prophylaxis9 (9.7)1.040.12-9.31NS0.750.15-3.91NS———CD3+CD4+ <400 cell/mm322 (31.4)3.410.69-16.76NS0.810.25-2.66NS———CD19+ <8%36 (51.4)1.670.37-7.59NS0.690.23-2.01NS———CD27+IGD−IgM− <3%30 (56.6)1.030.21-5.11NS2.080.60-7.19NS———CLD, Chronic lung disease; LRTI, lower respiratory tract infection; NS, not significant; URTI, upper respiratory tract infection. Open table in a new tab CLD, Chronic lung disease; LRTI, lower respiratory tract infection; NS, not significant; URTI, upper respiratory tract infection. Carriers identified by culture, but not by RT-PCR, had a higher risk of upper respiratory tract infection but not lower respiratory tract infection within 3 months of swabs collection (hazard ratio, 3.0; 95% CI, 1.0-9.20; Log-rank test P = .05), with the highest levels of significance recorded within 60 days (hazard ratio, 4.1; 95% CI, 1.70-10.0; Log-rank test P = .002; Fig 1). Thus, the lack of a clinical correlation with RT-PCR data did not allow to suggest to routinely adopt RT-PCR in the colonization assessment of patients with primary antibody deficiencies. The actual impossibility to replace IgA and IgM at the mucosal level implied the need to consider additional therapeutic interventions in primary antibody deficiencies. such as the use of antibiotic treatment or prophylaxis. However, a major concern related to the use of antibiotic therapy or prophylaxis is that it exerts selective pressure on airway microbiota, facilitating the emergence of antibiotic resistance. Our study added information on rates of antibiotic resistance in S pneumoniae and H influenzae isolates in CVID. As shown in Table E2, only 3 of 13 pneumococcal isolates were susceptible to all antibiotics tested (penicillin, ceftriaxone, erythromycin, clindamycin, levofloxacin). In particular, 9 of 13 pneumococcal isolates were nonsusceptible to penicillin. However, only 1 isolate was fully resistant, whereas the others showed low-level penicillin resistance, which is considered potentially overcome by high-dose penicillin in case of respiratory infections. Differently, 19 of 36 H influenzae isolates were susceptible to all antibiotics tested. Ampicillin and azithromycin resistance was the most frequently detected: 9 of 36 isolates were resistant to ampicillin and 10 of 36 were resistant to azithromycin. Although the EUCAST breakpoint tables (Version 8, 2018) no longer report clinical breakpoints of macrolide for H influenzae in respiratory infections, it indicates the epidemiological cutoffs to detect isolates with acquired macrolide resistance. In the 6 months preceding the swabs collection, beta-lactams were the most frequently administered antibiotic (22 patients), followed by macrolides (14 patients). Carriers of susceptible strains were never treated by beta-lactams and/or macrolides, whereas carriers of strains nonsusceptible to beta-lactams and macrolides were treated for a mean of 14.4 ± 6.6 days (P = .05). In conclusion, in CVID very low IgA serum level is a risk factor for carriage and colonization acts as a bacteria reservoir and as a risk factor for respiratory complications. We thank our patients and their families and the Jeffrey Modell Foundation for the continuous support to our center. Nasopharyngeal and oropharyngeal swabs were collected once for each patient and transferred to the laboratory within 4 hours of collection. Aliquots of STGG medium were streaked on 5% sheep blood Columbia agar plates supplemented with 5 μg/mL gentamycin to isolate S pneumoniae and on chocolate blood agar containing Vitox, bacitracin, vancomycin, and amphotericin B (Haemophilus Selective Agar, Oxoid Ltd, Basingstoke, Hampshire, UK) to selectively culture H influenzae. Colonies resembling S pneumoniae or H influenzae were further identified by standard microbiological tests. S pneumoniae isolates were serotyped by the Quellung reaction using commercially available antisera (Statens Serum Institut, Copenhagen, Denmark). The capsular genotype of H influenzae isolates was determined by PCR, as previously reported.E1Falla T.J. Crook D.W.M. Brophy L.N. Maskell D. Kroll J.S. Moxon E.R. PCR for capsular typing of Haemophilus influenzae.J Clin Microbiol. 1994; 32: 2382-2386Crossref PubMed Google Scholar For molecular detection of S pneumoniae and H influenzae in nasopharyngeal and oropharyngeal specimens, genomic DNA was extracted from STGG broths using QIAmp DNA Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. S pneumoniae was detected by RT-PCR targeting the lytA gene and using primers and probes previously described.E2Carvalho M.D.G. Tondella M.L. McCaustland K. Weidlich L. McGee L. Mayer L.W. et al.Evaluation and improvement of real-time PCR assays targeting lytA, ply, and psaA genes for detection of pneumococcal DNA.J Clin Microbiol. 2007; 45: 2460-2466Crossref PubMed Scopus (391) Google Scholar For H influenzae, RT-PCR was carried out targeting the hpd gene with primers hpdF822 and hpdR952 and following conditions previously reported.E3Wang X. Mair R. Hatcher C. Theodore M.J. Edmond K. Wu H.M. et al.Detection of bacterial pathogens in Mongolia meningitis surveillance with a new real-time PCR assay to detect Haemophilus influenzae.Int J Med Microbiol. 2011; 301: 303-309Crossref PubMed Scopus (88) Google Scholar For both bacterial species, minimum inhibitory concentrations (MICs) of antimicrobial agents were determined by E-test (Etest, bioMérieux, Durham, NC). The interpretative criteria were based on EUCAST breakpoints (http://www.eucast.org/clinical breakpoints/). The antimicrobial agents tested in MIC determination were penicillin, ceftriaxone, erythromycin, clindamycin, and levofloxacin for S pneumoniae isolates and ampicillin, amoxicillin-clavulanic acid, azithromycin, cefotaxime, ciprofloxacin, and meropenem for H influenzae isolates. Patients' demographic characteristics and clinical characteristics were expressed by mean ± SD or by frequencies, where appropriate. The outcome measures in this analysis were colonization by S pneumoniae and colonization by H influenzae identified by traditional culture and by RT-PCR. Clinical and immunological characteristics were investigated to reveal the relationships between the variable and each outcome measure. Comparison of continuous variables was performed by the unpaired t test. We calculated differences in frequencies between groups using Fisher exact test. To determine which variables to consider for building the multivariate logistic regression model for H influenzae and/or S pneumoniae, a binary logistic regression was run. Variables with a P value of less than .25 were considered for the multivariate logistic regression model. The backwards stepwise selection method was used to build the multivariate model. We computed the pooled ORs for end points with 95% CI. Cutoff points for age, CD19+CD27+IgD−IgM− frequency, IgM serum levels, and IgA serum levels were based on the highest sensitivity value plus the specificity identified by the receiver-operating characteristics curve method curve between S pneumoniae and/or H influenzae colonized and not colonized patients. Infections-survival rates were estimated by using the Kaplan-Meier method. The statistical significance was set at the conventional level of P < .05. All statistical analyses were performed using the statistical package STATA 11 (StataCorp LP, College Station, Tex).Table E1S pneumoniae and H influenzae colonization by culture and RT-PCR in CVIDSpecies/isolation siteCVID (n = 93), n (%)Culture+RT-PCR+S pneumoniae Nasopharynx and/or oropharynx10 (10.8)49 (52.7) Nasopharynx8 (8.6)32 (34.4) Oropharynx6 (6.5)35 (37.6)H influenzae Nasopharynx and/or oropharynx25 (26.9)37 (39.8) Nasopharynx22 (23.7)30 (32.3) Oropharynx14 (15.1)27 (29.0) Open table in a new tab Table E2Serotype and antibiotic susceptibility of S pneumoniae and H influenzae isolates from nasopharynx and oropharynx in patients with CVIDSpneumoniae serotypeSitenPG R/ITX IEM RCL RLV RH influenzae serotypeSitenAM RXL RCT RAZ RCI RMP R6A∗S pneumoniae isolate fully resistant to penicillin.NP1YesYesYesYesNoNTHiNP6NoNoNoYesNoNo10ANP1NoNoNoNoNoNTHiNP4YesNoNoNoNoNo12FNP1NoNoNoNoNoNTHiNP1YesNoNoYesNoNo19ANP1YesNoYesYesNoNTHiNP1YesYesNoNoYesNo23ANP1NoNoYesYesNoNTHiNP10NoNoNoNoNoNo35FNP1YesNoNoNoNoNTHiOP2YesNoNoNoNoNoNTNP1YesNoYesYesYesNTHiOP2NoNoNoYesNoNoNTNP1YesNoYesNoNoNTHiOP1YesNoNoYesNoNo10AOP1NoNoNoNoNoNTHiNP9NoNoNoNoNoNo19AOP1YesNoYesYesNo37OP1YesYesYesNoNoNTOP1YesYesYesYesYesNTOP1YesNoYesNoNoTotal151031072369101010AM, Ampicillin; AZ, azithromycin; CI, ciprofloxacin; CL, clindamycin; CT, cefotaxime; EM, erythromycin; I, intermediate; LV, levofloxacin; MP, meropenem; NP, nasopharynx; NT, nontypeable; NTHi, nontypeable H inlfuenzae; OP, oropharynx; PG, penicillin; R, resistant; TX, ceftriaxone; XL, amoxicillin-clavulanic acid.∗ S pneumoniae isolate fully resistant to penicillin. Open table in a new tab AM, Ampicillin; AZ, azithromycin; CI, ciprofloxacin; CL, clindamycin; CT, cefotaxime; EM, erythromycin; I, intermediate; LV, levofloxacin; MP, meropenem; NP, nasopharynx; NT, nontypeable; NTHi, nontypeable H inlfuenzae; OP, oropharynx; PG, penicillin; R, resistant; TX, ceftriaxone; XL, amoxicillin-clavulanic acid.