key: cord-0823109-np78ybqs
authors: Luchsinger, Vivian; Piedra, Pedro A.; Ruiz, Mauricio; Zunino, Enna; Martínez, María Angélica; Machado, Clarisse; Fasce, Rodrigo; Ulloa, María Teresa; Fink, Maria Cristina; Lara, Pamela; Avendaño, Luis F.
title: Role of Neutralizing Antibodies in Adults With Community-Acquired Pneumonia by Respiratory Syncytial Virus
date: 2012-04-01
journal: Clin Infect Dis
DOI: 10.1093/cid/cir955
sha: fa28199c70227aa7189f847a1ee386cd240fdade
doc_id: 823109
cord_uid: np78ybqs

Background. Respiratory syncytial virus (RSV) has been implicated in the etiology of adult community-acquired pneumonia (CAP). We investigated RSV infection in Chilean adults with CAP using direct viral detection, real-time reverse-transcription polymerase chain reaction (rtRT-PCR), and serology (microneutralization assay). Methods. RSV, other respiratory viruses, and bacteria were studied by conventional and molecular techniques in adults aged ≥18 years presenting with CAP to the healthcare facilities in Santiago, Chile from February 2005 through December 2007. Results. All 356 adults with CAP enrolled had an acute blood sample collected at enrollment, and 184 had a convalescent blood sample. RSV was detected in 48 cases (13.4%). Immunofluorescence assay and viral isolation each detected only 1 infection (0.2%), whereas rtRT-PCR was positive in 32 (8.9%) cases and serology was positive in 20 (10.8%) cases. CAP clinical characteristics were similar in RSV-infected and non-RSV-infected cases. RSV-specific geometric mean serum-neutralizing antibody titer (GMST) was significantly lower at admission in the 48 RSV-infected cases compared with 308 non-RSV-infected adults (GMST in log(2): RSV/A 8.1 vs 8.9, and RSV/B 9.3 vs 10.4; P < .02). Conclusions. RSV infection is frequent in Chilean adults with CAP. Microneutralization assay was as sensitive as rtRT-PCR in detecting RSV infection and is a good adjunct assay for diagnostic research. High RSV-specific serum-neutralizing antibody levels were associated with protection against common and severe infection. The development of a vaccine could prevent RSV-related CAP in adults.

Background. Respiratory syncytial virus (RSV) has been implicated in the etiology of adult communityacquired pneumonia (CAP). We investigated RSV infection in Chilean adults with CAP using direct viral detection, real-time reverse-transcription polymerase chain reaction (rtRT-PCR), and serology (microneutralization assay).

Methods. RSV, other respiratory viruses, and bacteria were studied by conventional and molecular techniques in adults aged $18 years presenting with CAP to the healthcare facilities in Santiago, Chile from February 2005 through December 2007.

Results. All 356 adults with CAP enrolled had an acute blood sample collected at enrollment, and 184 had a convalescent blood sample. RSV was detected in 48 cases (13.4%). Immunofluorescence assay and viral isolation each detected only 1 infection (0.2%), whereas rtRT-PCR was positive in 32 (8.9%) cases and serology was positive in 20 (10.8%) cases. CAP clinical characteristics were similar in RSV-infected and non-RSV-infected cases. RSV-specific geometric mean serum-neutralizing antibody titer (GMST) was significantly lower at admission in the 48 RSV-infected cases compared with 308 non-RSV-infected adults (GMST in log 2 : RSV/A 8.1 vs 8.9, and RSV/B 9.3 vs 10.4; P , .02).

Conclusions. RSV infection is frequent in Chilean adults with CAP. Microneutralization assay was as sensitive as rtRT-PCR in detecting RSV infection and is a good adjunct assay for diagnostic research. High RSV-specific serum-neutralizing antibody levels were associated with protection against common and severe infection. The development of a vaccine could prevent RSV-related CAP in adults.

Community-acquired pneumonia (CAP) is a relevant worldwide cause of morbidity and mortality [1] , and bacteria have been recognized as the main etiological agent. However, advances in diagnostic techniques have shown the potential role of viruses [2, 3] . Respiratory syncytial virus (RSV)-the main respiratory pathogen in infants [4] -has been associated with severe respiratory illness in adults [4] [5] [6] [7] , causing between 2% and 9% of adult CAP throughout the year and up to 15% during the winter months [4] . Currently, no clinical features of CAP are characteristic of viral pneumonia. Detection of RSV infection in adults is hampered by low viral shedding, and therefore the most sensitive method should be used for diagnosis [4, 8] . Polymerase chain reaction (PCR) has been shown to be more sensitive than viral isolation (VI) and immunofluorescence assay (IFA) for viral detection in adults [6, 9] ; however, in some studies, PCR has not significantly increased the diagnostic serology yield [6, 7, 9, 10] . Serology for diagnosis of respiratory viruses is a sensitive technique that is primarily used in research [11] . A $4-fold in antibody titer in paired samples is necessary to confirm a recent infection. Although the rise in detection of virus-specific neutralizing antibodies is sensitive and provides relevant information [4] , few studies of CAP have used microneutralization for RSV diagnosis [12] [13] [14] . The aim of this study was to compare standard and molecular viral diagnostic techniques with microneutralization assay for the detection of RSV infection in adults presenting with CAP in Santiago, Chile.

We prospectively enrolled patients $18 years of age presenting with CAP in 2 hospitals (Hospital Clínico Universidad de Chile and Hospital Lucio Có rdova) in Santiago, Chile, between February 2005 and December 2007, covering 3 respiratory viral seasons. The study was approved by the university and institutional review boards, and all adults provided informed consent to participate in the study. CAP was defined by the presence of acute respiratory symptoms for ,1 week and a chest radiograph displaying new pulmonary infiltrates. Exclusion criteria included immunodeficient conditions (ie, human immunodeficiency virus, active treatment for cancer, organ transplant, immunosuppressive therapy) and hospitalization within 30 days preceding enrollment. Clinical information at admission and hospital course were extracted from hospital charts of all patients and entered in a computer database. Patient severity was assessed during the first 48 hours after enrollment according to the pneumonia severity index described by Fine et al [15] .

In addition to routine laboratory tests (complete blood cell count, biochemistry panel, oxygen saturation) at enrollment, all persons had collection of urine and blood for bacterial culture, respiratory secretion for viral and bacterial diagnostics, and an acute serum sample for serology. Sputum, nasopharyngeal aspirate, or bronchoalveolar lavage fluids were obtained depending on the condition of the patient and immediately transported on ice to the laboratory. Aliquots for different diagnostic assays were prepared and stored at 280°C for later testing. Participants were contacted after 4-6 weeks for clinical follow-up and convalescent sera collection. Sera were processed immediately and stored at 220°C. Respiratory secretions were inoculated onto HEp-2 cells for VI [16] . IFA and cultures were performed on all samples as described elsewhere using monoclonal antibodies (kindly provided by L. Anderson, Centers for Disease Control and Prevention, Atlanta, Georgia) and commercial conjugated antisera (Sigma) [16] . For real-time reverse-transcription (rtRT) PCR, samples were treated with the guanidinium thiocyanate-phenol-chloroform method for RNA extraction [17] . Complementary DNA (cDNA) was synthesized with 5 lL of RNA (sample) and 0.52 lM F gene primer (F844: 5#-TGTCTAACTATTTGAACA-3#) for 1 hour at 37°C, followed by 5 minutes at 95°C in a PerkinElmer gene AmpPCR System 2400. A fragment of N gene was amplified by rtPCR with 10 lM (each) N1 and N2 primers [18] , 1X Master SYBR Green I (Roche), and 2 lL of cDNA. The reaction was carried out for 10 minutes at 94°C and then for 50 sequential cycles at 94°C for 10 seconds, 58°C for 5 seconds, and 72°C for 30 seconds in a LightCycler 1.5 instrument (Roche). A negative (water) and 2 positive controls (RNA from RSV working pool) were included in each assay. Heatinactivated sera were tested by microneutralization assays for RSV/A/Tracy (A2-like virus) and RSV/B/18537 to measure RSV/A and RSV/B-specific neutralizing antibodies as described previously [12] .

VI in HEp-2 and MDCK cells and IFA was used for the detection of adenovirus, influenza A and B viruses (FLU), and parainfluenza viruses types 1-3 as described previously [16, 19] . Human metapneumovirus (hMPV), coronavirus, and picornavirus (PV) were detected by rtRT-PCR by amplifying fragments of the N gene, the conserved region of the replicase 1a gene, and the 5#-noncoding conserved region, respectively, as described previously [20] [21] [22] . The 390-base pair fragment of PV was purified and sequenced using the kit ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction in the automated sequencer ABI model 377 (Applied Biosystems). Serum antibodies to hMPV subgroups A and B were detected by microneutralization and enzyme-linked immunosorbent assays [23] . Hemagglutination inhibition testing for influenza antibody was performed as described previously [24] . Seroconversion RSV, FLU, and hMPV infection were defined by a $4-fold rise of antibody titer between convalescent and acute paired sera.

Sputum samples displaying .25 leukocytes and ,10 epithelial cells per 1003 power field after Gram staining were processed according to standard techniques [25] . For Legionella, isolation samples were inoculated onto buffered charcoal yeast extract and GVPC media (Oxoid) and incubated for up to 10 days. Urinary antigens for Streptococcus pneumoniae and Legionella were detected using immunochromatographic tests (Binax NOW, Portland, Oregon). Chlamydia pneumoniae was detected using primers HL and HR for nested PCR amplifying a chromosomal DNA segment (PstI 474) [26] . PCR for Mycoplasma pneumoniae and Legionella was performed as described previously [27, 28] . Serum antibodies against C. pneumoniae and M. pneumoniae were detected by commercial IFA kits (SeroFIA, Sayvon, Israel and Zeus, respectively). Immunoglobulin M (IgM) $1:16 or a $4-fold rise in immunoglobulin G (IgG) titer between paired sera was regarded as an acute infection. Legionella serology was performed using a latex test kit (Legionella Oxoid).

Analysis was performed using Z-test for categorical data and the t test and Mann-Whitney rank-sum test for continuous variables, such as geometric mean serum-neutralizing antibody titer (GMST) in log 2 . Logistic regression was used to determine whether RSV/A and RSV/B GMSTs were associated with the likelihood of not having an RSV infection or a hospitalization. Odds ratios (ORs) with 95% confidence intervals (CIs) were determined for RSV/A or RSV/B GMSTs. The level of significance was set at P , .05. Data were analyzed using SigmaStat software.

A total of 356 adults with CAP were enrolled; 330 (92.7%) were admitted to hospital, 83 (23.3%) required intensive care unit (ICU) support, 26 (7.3%) were outpatients, and 28 (7.9%) died between hospital admission and 30 days after discharge. The study population consisted of 166 (46.6%) women and 190 (53.4%) men. The mean age was 63 years (range, 18-94 years): 31.2% were 18-49 years old, 21.3% were 50-64 years old, and 47.5% were $65 years old. One or more predefined comorbidity was identified in 182 (53.5%) patients, cardiac disease being the most prevalent (18.2%); 78 (22.9%) had $2 comorbidities. One hundred twenty-nine adults (37.8%) were smokers, 72 adults (21.2%) had received antimicrobial therapy before hospital admission, and 84 (24.9%) had respiratory failure. A Fine score was determined in 341 adults with CAP: class 1 in 77 (22.6%), class 2 in 69 (20.2%), class 3 in 69 (20.2%), class 4 in 84 (24.6%), and class 5 in 42 (12.4%).

Overall, RSV infection was established for 48 of 356 (13.4%) adults with CAP. The characteristics of the patients are summarized in Table 1 . No significant differences in demographic, clinical characteristics (Tables 1 and 2) , and routine laboratory tests were observed between adults with and without RSVrelated CAP. Likewise, clinical outcome was similar between both groups (Table 3 ), except that adults infected with RSV comprised a higher proportion of cases with disease progression on chest radiograph compared with noninfected adults (14% vs 4.8%; P 5 .04).

IFA and cell culture identified 2 (0.2%) RSV infections, 1 by each technique. RSV was detected by rtRT-PCR in 32 (8.9%) adults, whereas serology diagnosed RSV infection in 20 of 184 (10.8%) adults with paired sera. Sensitivity of rtRT-PCR and microneutralization assays were similar (P 5 .5), and both were significantly better than IFA or VI (P , .001). Among the 48 adults infected with RSV, 2 (4.1%) were detected by conventional methodology (IFA and cell culture) and 32 (66.6%) by rtRT-PCR. A $4-fold rise in RSV-specific serum-neutralizing antibody titer occurred in 20 of 35 RSV-infected adults with paired sera: 6 by RSV/A assay, 9 by RSV/B assay, and 5 by both microneutralization assays. Paired serologic study was available in 19 adults with RSV-positive rtRT-PCR, and the results were concordant in only 4 cases (Table 4) .

Comparing the 26 cases detected by rtRT-PCR with the 20 patients who experienced RSV seroconversion, clinical characteristics and laboratory parameters were similar; the duration of symptoms before admission was also similar (median, 3.5 vs 

All 356 adults with CAP had neutralizing antibodies to RSV/A and RSV/B. The acute serum samples were obtained on the day of admission in 35.3% of cases and during the first week after illness onset in 66% (range, 1-22 days; median, 6.67 days). The GMSTs to RSV/A and RSV/B during the acute illness phase were significantly lower in adults with RSV infection compared with non-RSV-infected adults (8.1 6 2.2 vs 9.3 6 1.6 and 8.9 6 1.9 vs 10.4 6 1.9 for RSV/A and RSV/B, respectively; P # .028). These significant differences also remained when the analysis was limited to cases with paired sera (Table 5) .

Logistic regression analysis detected a protective role of RSV-specific serum-neutralizing antibodies against Previously reported minimal protective threshold titers for neutralizing antibodies to RSV A and B ($6.0 log 2 and $8.0 log 2 , respectively) against RSV-related hospitalization [13] were used to evaluate the risk for RSV-related CAP. A significantly higher proportion of adults with RSV-related CAP had RSV/A-specific GMST ,6.0 log 2 compared with those with non-RSV-related (Table 6) .

This prospective study of CAP in adults used a comprehensive diagnostic approach in identifying the etiologic agents in Santiago, Chile. A relevant proportion of adults with CAP were hospitalized and some died, indicating that our findings are biased toward the spectrum of severe disease. RSV was identified in 48 of 356 patients (13.4%). Because RSV detection in this population has varied from 0% to 14% [4, 5] , depending mainly on the diagnostic method used, the high frequency found can be explained by application of a sensitive molecular diagnostic method and RSV-specific serology. Here, rtRT-PCR showed significantly better diagnostic performance than did IFA and viral culture methods. It is the preferred diagnostic test for identifying RSV in adults with CAP who are expected to shed low amounts of virus [4] . However, rtRT-PCR did not identify a significant proportion of RSV-infected adults (16 of 20) who were diagnosed by serology. In agreement with Falsey et al [11] , these findings highlight the need to combine rtRT-PCR with serology to optimize the diagnosis of RSV infection in adults.

Age and comorbidities appear to influence the diagnostic sensitivity of rtRT-PCR and serology. It is well known that age may influence virus-specific antibody response [4, 29] . Serology gave a higher yield than did rtRT-PCR in the detection of RSV in healthy adults with CAP (14.1% vs 5.7%; P 5 .04); this group was significantly younger than adults with comorbidities (median age, 48 vs 72 years; P , .001). Also, the 20 patients with seroconversion had a trend toward being younger compared with 15 nonseroresponders (45% vs 26.7% ,40 years; P 5 .4). In addition, the presence of comorbidity showed a similar tendency, being less frequent among the seroresponders (31% vs 66%; P 5 .09). It has been documented that underlying disease represents a significant risk factor for severe RSV illness [14] . This inability to develop or to sustain an adequate RSV-specific neutralizing antibody response could also contribute to the pathogenesis of the disease. Delay in the evaluation of CAP may hinder the diagnosis of a viral pathogen because the virus may no longer be present and because of the difficulty in demonstrating a seroconversion between an acute and convalescent blood sample. Falsey et al [11] have suggested that young, healthy individuals may rapidly clear RSV infection, reducing the viral shedding period. Thus, a delay in the collection of a respiratory sample could explain a lower yield by rtRT-PCR than by serology in RSV-infected adults without preexisting conditions. Nevertheless, in our study, the delay in the sample collection was similar for adults with or without comorbidity (median, 5 days; P 5 .3); young people tended to consult earlier than adults .50 years of age (median, 4 and 5 days, respectively; P 5 .05); and the sensitivity of rtRT-PCR was similar between age groups. On the other hand, 40.7% (11 of 27) of cases with positive rtRT-PCR and 27.8% of seroresponders (P 5 .4) had $7 days of symptoms at the time of sample collection. This emphasizes the need of getting specimens for specific diagnosis despite late consultation.

In the 20 cases in which serology identified RSV infection, the explanation could be that lower antibody titers in acute phase sera could facilitate detecting a seroconversion; however, the mean antibody titer was similar (P 5 .07) among 20 cases with seroconversion and 164 nonseroresponders. In addition, the neutralizing antibody titers were comparable in patients with RSV-positive rtRT-PCR compared with seroresponders ( Table 5) .

The fact that approximately 10%-30% of adults with documented respiratory viral infection do not have a seroconversion could partially explain the absence of serologic response in patients with positive rtRT-PCR [8] . Also, having in place the appropriate rtRT-PCR procedures and quality controls to reduce contamination reduces the likelihood of false-positive rtRT-PCR results.

Low serum-neutralizing antibody titers against RSV have been associated with severe RSV disease in children and adults [13, 14] . In our study, serum-neutralizing antibody titers to RSV/A and RSV/B were significantly lower in RSV-infected adults compared with those not infected (RSV/A: 8.1 log 2 vs 8.9 log 2 ; RSV/B: 9.3 log 2 vs 10.4 log 2 , respectively; P , .02). Likewise, subjects with titers below the minimal protective threshold titer against RSV hospitalization reported by Piedra et al [13] (,6.0 log 2 and ,8.0 log 2 for RSV/A and B, respectively) were detected more frequently in adults with RSV-related CAP than in those with non-RSV CAP (RSV/A: 17% vs 2%, P 5 .02; RSV/B: 18% vs 6%, P 5 .03). Furthermore, logistic regression analysis showed a protective role of higher levels of RSV/A-and RSV/B-specific serum-neutralizing antibody against RSV-related CAP and admission to ICU. Thus, a RSV vaccine for increasing neutralizing antibodies titers should Table 5 . Geometric Mean Serum Antibody Titers for Respiratory Syncytial Virus (RSV) Subgroups A and B in Adults With RSV-Related and Non-RSV-Related Community-Acquired Pneumonia in Santiago, Chile, 2005 Chile, -2007 reduce hospitalization and death associated with RSV-related CAP in adults.

The epidemiology of RSV-related CAP in adults in Santiago, Chile, mirrors the months when the RSV outbreak occurs in Chile [16] , mainly from May through August. Although the majority (62.5%) of RSV-related CAP in adults occurred during the RSV season, many cases were also detected outside this period, stressing the importance of considering RSV etiology even when the outbreak has ended.

In conclusion, RSV infection is frequent in adults with CAP in Santiago, Chile. Sensitive complementary methodologies for detecting RSV should be included in the laboratory tests for identifying the etiologic agents of pneumonia in adults. Molecular diagnostics such as rtRT-PCR is one such test that should be implemented in clinical viral laboratories to improve the detection of respiratory viral pathogens. In addition, serologic assays, especially the microneutralization test, complement molecular diagnostics. Newer, faster, and easier serologic methods, such as flow cytometry-based assay to detect RSV-specific neutralizing antibody [30] , could facilitate the use of serology as a reliable diagnostic method. A safe and effective RSV vaccine would also be beneficial for the adult population. 

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Acknowledgments. We thank Dr María Luisa Garmendia for excellent statistical advice and Cristian Moreno for helpful technical assistance.Financial support. This work was supported by the Fondo Nacional de Ciencia y Tecnología (grant number 1050734) and Fondo Nacional de Investigació n en Salud (grant number SA04 I 2084).Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

c GMST to RSV/A in convalescent serum of patients with seroconversion was significantly higher than GMST to RSV/A in patients without seroconversion (P 5 .02, t test). d GMST to RSV/A in convalescent serum of patients with RT-PCR(1) and seroconversion was significantly higher than GMST to RSV/A in patients with RT-PCR(1) without seroconversion (P 5 .00, t test). e GMST to RSV/B in convalescent serum of patients with RT-PCR(1) and seroconversion was significantly higher than GMST to RSV/B in patients with RT-PCR(1) without seroconversion (P 5 .00, t test).