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Journal of Medical Virology 79:1951–1958 (2007) Respiratory Syncytial Virus Infections in Hospitalized Infants: Association Between Viral Load, Virus Subgroup, and Disease Severity Ime ` ne Fodha, 1 Astrid Vabret, 2 Leila Ghedira, 3 Hassen Seboui, 4 Slaheddine Chouchane, 3 John Dewar, 5 Neji Gueddiche, 3 Abdelhalim Trabelsi, 1 * Noureddine Boujaafar, 1 and Franc ¸ ois Freymuth 2 1 Research Unit UR06SP20, Laboratory of Bacteriology-Virology, University Hospital Sahloul, Sousse, Tunisia 2 Laboratory of Human and Molecular Virology, Caen University Hospital, Avenue Georges Cle´menceau, Caen Cedex, France 3 Paediatric Unit of Fattouma Bourguiba University Hospital, Monastir, Tunisia 4 Neonatal Unit, Farhat Hached University Hospital, Avenue Ibn El Jazzar, Sousse, Tunisia 5 MRC/Medunsa Diarrhoeal Pathogens Research Unit, University of Limpopo, Limpopo, South Africa The relationships between host factors, virus strain, viral load, and illness severity in respira- tory syncytial virus (RSV)-induced bronchiolitis are poorly defined. These relationships were evaluated prospectively in 81 previously healthy infants hospitalized with RSV bronchiolitis. Dis- ease severity was determined by the respiratory rate, the duration of hospitalization, and whether patients during their hospitalization required pediatric intensive care unit admission or mechanical ventilation. RSV typing into sub- group A and B was obtained by RT-PCR-hybrid- ization assay. The nasopharyngeal RSV viral loads were measured by real-time quantitative RT-PCR. Disease severity correlated significantly with the presence of risk factor (estimated gesta- tional age < 37 weeks and/or birth weight < 2,500 g) and with chronologic age 28 days at time of sample collection. The results revealed signifi- cant association between disease severity and nasopharyngeal RSV viral load. Analysis also showed that disease severity was not associated significantly with RSV subgroup. Thus, RSV disease severity is likely to be determined by an interplay between host and virus factors, includ- ing RSV load. J. Med. Virol. 79:1951–1958, 2007. ß 2007 Wiley-Liss, Inc. KEY WORDS: respiratory syncytial virus; dis- ease severity; RSV subgroups; viral load; risk factors INTRODUCTION Respiratory syncytial virus (RSV) is a member of the Pneumovirus genus of the family Paramyxoviridae and is the most common cause of acute lower respiratory tract infection in infants and young children [Law et al., 2002]. There is extreme variability in RSV disease severity in previously healthy infants. Approximately 50% of infants are infected during their first year of life, yet only 3% of infants < 1 year of age are hospitalized for RSV infection [Glezen et al., 1986; Boyce et al., 2000; DeVincenzo, 2004]. Of RSV-hospitalized children, only 10% require mechanical ventilation. In addition, 5 – 10% of those who require mechanical ventilation die. Fur- thermore, more than half of RSV-hospitalized infants and a large percentage of infants who require mechan- ical ventilation and even die from RSV infection are previously healthy. These striking disease severity differences cannot be explained by existing neutralizing antibody titers alone and are likely due to a combination of host and viral factors. During primary RSV infection, host factors such as young age (6 months), prematurity, low-birth weight, and chronic diseases have been associated with this severe disease [Imaz et al., 2000; Lanari et al., 2002]. On the other hand, viral factors associated with disease severity are not understood sufficiently. Published reports disagree as to whether or not there is a difference in the pathogenicity of the two RSV subgroups: several studies that compared severity of infants infected with group A and group B have not revealed significant clinical differences [McIntosh et al., 1993; Wang et al., 1995; Kneyber et al., 1996]. However, some investigators have concluded that infection with group A [Hall et al., 1990; Walsh et al., 1997; Imaz et al., *Correspondence to: Abdelhalim Trabelsi, Laboratory of Bac- teriology-Virology, University Hospital Sahloul, Sousse 4053, Tunisia. E-mail: [email protected] Accepted 21 August 2007 DOI 10.1002/jmv.21026 Published online in Wiley InterScience (www.interscience.wiley.com) ß 2007 WILEY-LISS, INC.

Respiratory syncytial virus infections in hospitalized infants: Association between viral load, virus subgroup, and disease severity

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Journal of Medical Virology 79:1951–1958 (2007)

Respiratory Syncytial Virus Infections inHospitalized Infants: Association Between ViralLoad, Virus Subgroup, and Disease Severity

Imene Fodha,1 Astrid Vabret,2 Leila Ghedira,3 Hassen Seboui,4 Slaheddine Chouchane,3

John Dewar,5 Neji Gueddiche,3 Abdelhalim Trabelsi,1* Noureddine Boujaafar,1

and Francois Freymuth2

1Research Unit UR06SP20, Laboratory of Bacteriology-Virology, University Hospital Sahloul, Sousse, Tunisia2Laboratory of Human and Molecular Virology, Caen University Hospital, Avenue Georges Clemenceau,Caen Cedex, France3Paediatric Unit of Fattouma Bourguiba University Hospital, Monastir, Tunisia4Neonatal Unit, Farhat Hached University Hospital, Avenue Ibn El Jazzar, Sousse, Tunisia5MRC/Medunsa Diarrhoeal Pathogens Research Unit, University of Limpopo, Limpopo, South Africa

The relationships between host factors, virusstrain, viral load, and illness severity in respira-tory syncytial virus (RSV)-induced bronchiolitisare poorly defined. These relationships wereevaluated prospectively in 81 previously healthyinfants hospitalized with RSV bronchiolitis. Dis-ease severity was determined by the respiratoryrate, the duration of hospitalization, and whetherpatients during their hospitalization requiredpediatric intensive care unit admission ormechanical ventilation. RSV typing into sub-group A and B was obtained by RT-PCR-hybrid-ization assay. The nasopharyngeal RSV viralloads were measured by real-time quantitativeRT-PCR. Disease severity correlated significantlywith the presence of risk factor (estimated gesta-tional age<37 weeks and/or birth weight<2,500g) and with chronologic age� 28 days at time ofsample collection. The results revealed signifi-cant association between disease severity andnasopharyngeal RSV viral load. Analysis alsoshowed that disease severity was not associatedsignificantly with RSV subgroup. Thus, RSVdisease severity is likely to be determined by aninterplay between host and virus factors, includ-ing RSV load. J. Med. Virol. 79:1951–1958,2007. � 2007 Wiley-Liss, Inc.

KEY WORDS: respiratory syncytial virus; dis-ease severity; RSV subgroups;viral load; risk factors

INTRODUCTION

Respiratory syncytial virus (RSV) is a member of thePneumovirus genus of the family Paramyxoviridae and

is the most common cause of acute lower respiratorytract infection in infants and young children [Law et al.,2002]. There is extreme variability in RSV diseaseseverity in previously healthy infants. Approximately50% of infants are infected during their first year of life,yet only 3% of infants< 1 year of age are hospitalized forRSV infection [Glezen et al., 1986; Boyce et al., 2000;DeVincenzo, 2004]. Of RSV-hospitalized children, only10% requiremechanical ventilation. In addition, 5–10%of those who require mechanical ventilation die. Fur-thermore, more than half of RSV-hospitalized infantsand a large percentage of infants who require mechan-ical ventilation and even die from RSV infection arepreviously healthy. These striking disease severitydifferences cannot be explained by existing neutralizingantibody titers alone and are likely due to a combinationof host and viral factors. During primary RSV infection,host factors such as young age (6 months), prematurity,low-birth weight, and chronic diseases have beenassociated with this severe disease [Imaz et al., 2000;Lanari et al., 2002]. On the other hand, viral factorsassociated with disease severity are not understoodsufficiently. Published reports disagree as to whether ornot there is a difference in the pathogenicity of the twoRSV subgroups: several studies that compared severityof infants infected with group A and group B have notrevealed significant clinical differences [McIntosh et al.,1993;Wang et al., 1995; Kneyber et al., 1996]. However,some investigators have concluded that infection withgroup A [Hall et al., 1990;Walsh et al., 1997; Imaz et al.,

*Correspondence to: Abdelhalim Trabelsi, Laboratory of Bac-teriology-Virology, University Hospital Sahloul, Sousse 4053,Tunisia. E-mail: [email protected]

Accepted 21 August 2007

DOI 10.1002/jmv.21026

Published online in Wiley InterScience(www.interscience.wiley.com)

� 2007 WILEY-LISS, INC.

Page 2: Respiratory syncytial virus infections in hospitalized infants: Association between viral load, virus subgroup, and disease severity

2000] orB [Hornsleth et al., 1998] can be associatedwithmore severe illness. Comparisons between viral loads interms of disease severity have also produced controver-sial results: some studies reported an associationbetween nasopharyngeal RSV viral load and diseaseseverity [Buckingham et al., 2000; DeVincenzo, 2004;DeVincenzo et al., 2005],whereas other authors failed tosupport such a conclusion [Wright et al., 2002]. A betterunderstanding of the interrelationships between riskfactors,RSVsubgroup, viral load, anddisease severity isimportant to the development of RSVvaccines and otherintervention strategies againstRSV illness. The presentstudywas performed to characterize these relationshipsin RSV-infected, hospitalized infants without immuno-deficiency or chronic disease.

MATERIAL AND METHODS

Patients

Eighty-one infants with mean age 1.9�0.3 months(range 0–12) hospitalized with a clinical diagnosis ofbronchiolitis and positive RSV immunofluorescence testwere included in the study. Bronchiolitis was defined asan acute infection of the lower airway characterized byincreased respiratory effort (>50 breaths permin and/oruse of accessory respiratory muscles), and expiratorywheezing and/or crackles. A detailed questionnaire andnasopharyngeal aspirate were obtained upon admis-sion. In order to rule out a nosocomial infection, theclinical sample was collected within the first 24 hr ofadmission. Exclusion criteria were: (1) hemodynami-cally significant congenital heart disease; (2) knownimmunodeficiency; (3) requirement for supplementaloxygen therapy for a>7 days period in the 3 monthsbefore admission for treatment of bronchopulmonarydysplasia; (4) treatment with systemic glucocorticoidswithin the 3 weeks before admission, unless adminis-tered for�24 hr before sample collection for the acuteRSV illness; (5) treatment with ribavirin during thecurrent illness; and (6) receipt of RSV-specific antibodypreparations within the 3 months before enrolment inthe study.

Clinical Data

Disease severity was determined by the respiratoryrate, the duration of hospitalization, and whetherpatients during their hospitalization required pediatricintensive careunit admission ormechanical ventilation.Patients who had increased respiratory frequency> 60breaths per min, who were hospitalized for more than7 days, who were transferred to an intensive care unitand/orwho required artificial ventilationwere classifiedas having severe disease.Patients were stratified according to their estimated

gestational age and birth weight into groups with orwithout risk factor: prematurity<37 weeks and/or low-birth weight< 2,500 g were the conditions assigned toinfants who belonged to the group with risk factor forsevere RSV infection.

RSV Typing by RT-PCR-HybridizationAssay (RT-PCR-EIA)

RNA was extracted from the nasopharyngealaspirates using the QIAmp viral RNA kit (Qiagen,Courtaboeuf, France).

Reverse-transcription-polymerase chain reaction(RT-PCR) was used to group RSV strains into subgroupA and B. This used two primers described previously[van Milaan et al., 1994]: a cDNA primer complemen-tary to positions 887 to 910 in the 1B gene and a reverseprimer complementary to positions 1,083 to 1,106 at thestart of the N gene [Fodha et al., 2004b]. For thehybridization of the amplified products, two previouslydescribed 50-biotinylated probes were used [Freymuthet al., 1995]: the probe for RSV subgroup A annealed atposition 961 to 1,001, and a probe for RSV subgroup Bannealed to a sequence within the 1B gene. Primersand probes were synthesized by the Unit of OrganicChemistry, Pasteur Institute (France). The PCR tech-nique was performed on 5 ml of the cDNA mixture astemplate by the addition of 25 ml of sterile water, 5 ml of10� PCR buffer, 5 ml of deoxynucleoside triphosphates(2mMeach), 5ml of primersP1andP2at10mMeach, and2.5U ofTaq polymerase (Perkin-Elmer Cetus,Norwalk,CT).Amplification reaction conditions consisted of 5minat 948C; followed by 40 cycles of heat denaturation for10 sec at 948C, primer annealing for 30 sec at 528C andprimer extension for 30 sec at 728C; andafinal extensionat 728C for 10 min. Measures to prevent samplecontamination were observed carefully by performingspecimen extraction and preparing cDNA and PCRproducts in separate laboratories. All PCR were runwith negative controls. Amplified products obtained byRT-PCRwere detected by a DNA enzyme immunoassaybased on the hybridization of amplified DNA with asingle-stranded DNA, 50-biotinylated probe, coated onthe wall of a microtiter plate via a streptavidin-biotinbond. The probe-DNA hybrid was detected using ananti-ds-DNA monoclonal antibody and by the additionof an enzyme tracer (anti-mouse IgG conjugated tohorse-radish peroxidase) (GEN-ETI-K DEIA, DiaSorin,Antony, France). The optimal concentration of the proberequired for the test was 0.5 ng/ml, and 20 ml of thedenatured amplicon was dispensed into each well. Theassay was performed as recommended by the manufac-turer, and an index value was defined as O.D. samplevalue/O.D. cut-off value. The assay was validated usinga set of samples of both RSV groups characterizedpreviously.

RSV Load Measurement by Real-TimeQuantitative RT-PCR

RNA standard for RSV quantitation. A 1,084-bpDNA fragment derived from theN gene of theRSV strainA2was generated by conventional RT-PCRwith primersPPN1andPPN2 [Gueudin et al., 2003]. ThePCRproductwas cloned into pCR2.1-TOPO from the TOPO TACloning kit (Invitrogen, Cergy Pontoise, France) accord-ing to themanufacturer’s instructions.The fragmentwas

J. Med. Virol. DOI 10.1002/jmv

1952 Fodha et al.

Page 3: Respiratory syncytial virus infections in hospitalized infants: Association between viral load, virus subgroup, and disease severity

cloned downstream of the T7 RNA polymerase promoter.After transcription of the plasmids, the orientation of theinsert was verified by enzymatic restriction with Xba I.The plasmids were then purified with the QiagenPlasmid Maxi Kit (Qiagen GmbH, Hilden, Germany).

To obtain transcripts of equal length, each plasmidwas cut with 5 mg of SpeI. Five ml of each plasmid wastranscribed with T7 RNA polymerase from the Ribop-robe in vitro transcription system (Promega, Madison,WI) according to the manufacturer’s instructions. Toremove all DNA, a DNAse treatment was performed.The DNA-free (Ambion Inc., Austin, TX) system wasused; this removes DNA without the need for phenol/chloroform extraction or heating. The RNA concentra-tion was estimated spectrophotometrically and theaverage of four measurements was determined.Aliquots were stored at �808C. Once an aliquot hadbeen thawed it was never refrozen. For each quantita-tion, two standard aliquots were subjected to serial10-fold dilutions in DEPC (diethylpyrocarbonate)-treated water. After DNase treatment, the removalof DNA from the RNA standard was determined bycarrying out PCR without the reverse transcriptionstep. The results showed that no amplification occurred.

Reverse transcription. The standard (5 ml) wasserially diluted and the extracts from the unknownsamples were simultaneously subjected to reverse tran-scription using the Omniscript Reverse Transcriptase kit(Qiagen GmbH, Germany). Samples were incubated at378C for 1 hr and then at 938C for 5min. RSVwas reversetranscribed usingN1 primer [Gueudin et al., 2003], whichtargetedRSVgenomicRNAinthegeneN.Astheefficiencyof the reverse transcription can vary, a plasmid-tran-scribed RNA was used to standardize the assay.

Real-time quantitative PCR. A LightCyclerinstrument (Roche Molecular Biochemicals, Molsheim,France) was used to amplify and to quantify theamplification product after each cycle. A hybridizationprobe system was used for the detection. Fluorescencewasmeasuredafter the annealing step andwasbased on

the fluorescence resonance energy. Primers N1 and N2generated a 230-bp product. The product was detectedwith the hybridization probes SN1 (labeled withfluorescein) and SN2 (labeled with red640). The ampli-fication reactionwas undertaken in a 20 ml mixture withthe LightCycler (LC)-DNAmaster hybridization probes(RocheMolecular Biochemicals, France). Hot-start PCRwas carried out; 2 ml of LC-mixwas incubatedwith 0.2 mlof the antiTaq antibody (Clontech, Saint-Germain-en-Laye, France) for 30 min at room temperature. There-after, 3.5 mM MgCl2, 0.2 mM of each primer, 0.25 mMSN1, and 0.5 mMSN2 were added. Finally, 2 ml of cDNAwas added to 18 ml of the PCR mixture in each capillarytube and amplified as follows: 958C for 2 min for onecycle, followedbydenaturationat 958C10sec, annealingat 528C for 20 sec, and extension at 728C for 15 sec for 45cycles.

Statistical Methods

Continuous variables were analyzed with the one-way analysis of variance (ANOVA) and Student’s t-test;chi-square was performed for ordinal or categoricaldata. Pearson’s correlation tests were used to determinethe relationship between severity of disease and thefollowing independent variables: risk factor category(according to estimated gestational age and birthweight); chronologic age category at the time of samplecollection (� or>28 days); gender; nasopharyngeal RSVquantity; RSV subgroup. All statistical tests wereconducted at the two-tailed 0.05 level of significance.

RESULTS

A total of 81 patients were included in the study.Demographic and clinical characteristics of the 81infants hospitalized with RSV bronchiolitis accordingto the presence of risk factor and the age are summar-ized in Table I. A higher percentage of neonates(patients�28 days’ old) presented at least one riskfactor compared with the group of older infants (44.7%

J. Med. Virol. DOI 10.1002/jmv

TABLE I. Demographic and Clinical Characteristics of 81 Infants Hospitalized With RSV Bronchiolitis According to thePresence of Risk Factor and the Age (Center Coast of Tunisia, 2005)

Number of cases; % of cases

Totalpatients(N¼ 81)

Risk factor (RF) Age

Group withRF (N¼ 28)

Group withoutRF (N¼ 53)

Neonates � 28 days(N¼ 38)

Infants> 28 days(N¼ 43)

Risk factor (estimated gestationalage< 37 weeks and/or birthweight< 2,500 g)

28 (34.6%) 28 (100%) 0 (0%) 17 (44.7%) 11 (25.6%)

Age� 28 days (neonates) 38 (46.9%) 17 (60.7%) 21 (39.6%) 38 (100%) 0 (0%)Age> 28 days 43 (53.1%) 11 (39.3%) 32 (60.4%) 0 (0%) 43 (100%)Male 46 (56.8%) 16 (57.1%) 30 (57.7%) 20 (52.6%) 26 (60.5%)Minimum and maximum duration

of hospitalization (days)2–23 3–23 2–19 3–19 2–23

Mean duration of hospitalization(days)

7.77 9.1 7.3 8.9 7.0

Hospitalization> 7 days 37 (45.7%) 19 (67.9%) 18 (34%) 24 (63.2%) 13 (30.2%)Intensive care unit admission

and/or artificial ventilation27 (33.3%) 17 (60.7%) 10 (18.9%) 22 (57.9%) 5 (11.6%)

Respiratory frequency> 60 breathsper min

32 13 (46.4%) 19 (36.5%) 19 (50.0%) 13 (30.2%)

RSV Disease, Risk Factors, and Viral Load 1953

Page 4: Respiratory syncytial virus infections in hospitalized infants: Association between viral load, virus subgroup, and disease severity

vs. 25.6%; P< 0.001). Mean duration of hospitalizationwashigheramongthepatientswith risk factor (9.1days)and the neonates (8.9 days) compared with the patientswithout risk factor (7.3 days) and older than 28 daysold (7.0 days), respectively (P¼ 0.03 in both cases). Ahigher proportion of patients with risk factor (60.7%)or� 28 days old (57.9%) required admission in apediatric intensive care unit and/or artificial ventilationcompared to patients without risk factor (18.9%) andwho were older than 28 days old (11.6%), respectively(P< 0.001). No significant difference in clinical datacould be observed according to the gender: results weresimilar between boys and girls when comparing respi-ratory rate, duration of hospitalization, and require-ment for pediatric intensive care unit admission ormechanical ventilation.RSV nasopharyngeal viral loads among the 81

patients were comprised between 610 and 3.2� 107

transcripts of RSV per ml of nasopharyngeal aspirates.Mean RSV nasopharyngeal viral loads was 2.8� 106

transcripts of RSV per ml of nasopharyngeal aspirates.Mean RSV viral loads were similar for groups with/without risk factor and to gender and comprisedbetween 2.6� 106 and 3.2� 106 transcripts of RSVper ml of nasopharyngeal aspirates. On the other hand,newborn infants�28 days old had higher mean naso-pharyngeal RSV viral loads compared to older infants(3.8� 106 vs. 1.7� 106 transcripts of RSV per ml ofnasopharyngeal aspirates).During the year 2005, among the 81 RSV strains

isolated in the Center coast of Tunisia, 11.1% wereidentified as groupARSV (N¼9), 74.1%as groupBRSV(N¼60), and 14.8% of strains remained untypeable(N¼12). Clinical characteristics and viral loads ofinfants infected according to RSV subgroup are shownin Table II. The proportion of patients with risk factorwas higherwithin those infectedwith untypeable strainof RSV. Therefore, more frequent need for pediatricintensive care unit admission and/or artificial ventila-tion was noted for that group of patients (50% vs. about30% for RSV A and B subgroup). The proportion ofinfants presenting risk factor was higher in the RSVsubgroupB-infectedpatients compared to those infectedwith subgroup A. This resulted in a higher proportion of

infants being hospitalized for more than 7 days amongRSV subgroupB-infected patients. No significant differ-ences in RSV viral loads were found between RSVsubgroups.

Characteristics of patients with severe and non-severe RSV disease among groups without and withrisk factor are shown inTable III. Irrespective of the riskfactor category, mean nasopharyngeal RSV viral loadswere always higher in patients presenting with severedisease.Whatever was the risk factor category, 74–76%of severe diseases were due to RSV B, which wasexpected as 74.1% of total isolated RSV strains belongedto subgroup B.

The relationship between the presence of risk factor(estimated gestational age< 37 weeks and/or birthweight<2,500 g) and severity of RSV disease wasinvestigated among all patients ‘group using thePearson correlation test. As results revealed significantassociation betweendisease severity and the presence ofrisk factor (P< 0.001), the relationship between variousindependent variables and severity of RSV disease wasinvestigated first among all patients and then amongthe group of patients without risk factor. In both cases,disease severity correlated significantly with twoparameters: chronologic age�28 days at the time ofsample collection (P< 0.001 in both groups) and RSVnasopharyngeal quantity (P¼0.024 and P¼ 0.027 in allpatients’ group and in the group without risk factor,respectively). Finally, analysis in both groups showedthat disease severity was not associated significantlywith RSV subgroup.

DISCUSSION

RSV has been themajor cause of low respiratory tractinfections in infants since its recognition more than40 years ago. Unfortunately, progress in understandingthe epidemiology of RSV has not been matched byconcomitant development of effective interventionstrategies. The present study was aimed at helping toidentify factors associated with increased severity ofdisease.

We selected infants at an age at which theyweremostlikely to be experiencing theirfirstRSV infection. Illness

J. Med. Virol. DOI 10.1002/jmv

TABLE II. Clinical Characteristics and Viral Loads of Infants Infected With RSV According to RSV Subgroup (Center Coast ofTunisia, 2005)

Number of cases; % of cases RSV A (N¼ 9) RSV B (N¼ 60)Untypeable RSV

(N¼ 12)

Age� 28 days (neonates) 4 (44.4%) 27 (45.0%) 7 (58.3%)Age> 28 days 5 (55.6%) 33 (55.0%) 5 (41.7%)Male 4 (44.4%) 36 (51.4%) 6 (50.0%)Risk factor (estimated gestational age< 37 weeks and/or

birth weight< 2,500 g)2 (22.2%) 20 (33.3%) 6 (50.0%)

Minimum and maximum duration of hospitalization (days) 3–17 2–23 4–19Mean duration of hospitalization (days) 7 7.89 7.87Hospitalization> 7 days 3 (33.3%) 27 (45.0%) 7 (58.3%)Intensive care unit admission and/or artificial ventilation 3 (33.3%) 18 (30.0%) 6 (50.0%)Extreme RSV nasopharyngeal quantity (RSV

transcripts/ml)6.1� 102–1.6� 107 1.9� 103–2.8� 107 2.2� 103–3.2� 107

Mean RSV nasopharyngeal quantity (RSV transcripts/ml) 4.2� 106 2.6� 106 2.9� 106

1954 Fodha et al.

Page 5: Respiratory syncytial virus infections in hospitalized infants: Association between viral load, virus subgroup, and disease severity

severity was assessed by four indirect indicators, giventhat monitoring bymeans of such objective measures asoxygen saturation and blood gases was feasible only inpatients who were very ill. The need for pediatricintensive care unit transfer or mechanical ventilationappeared a reliable severity indicator as they are onlyindicated in patients whose respiratory function isdisturbed significantly, as judged by the presence ofpronounced chest wall in-drawings, severe hyperinfla-tion, cyanosis, or apnea [Hornsleth et al., 1998; Imazet al., 2000]. The duration of hospital stay is commonlyused as a rough indicator of the severity of RSV-associated illness, although itwas not possible to controlfor inter-physician differences in the decision to dis-charge the patient from hospital [Hornsleth et al., 1998;Imaz et al., 2000]. Duration of hospital stay>7 dayswaschosen to indicatemore severe illness.A respiratory rateof>60 breaths/min on admission to hospital was shownto be associated with a greater severity of illness andwith subsequent clinical deterioration [Brooks et al.,1999].

A number of clinical factors were associated withmore-severe disease after hospitalization. As expected,prematurity and/or low-birth weight increased thelikelihood of more-severe disease. This observationsupports findings of several previous studies [Berg-strasser et al., 1998; Wright et al., 2002; Nielsen et al.,2003; Weisman, 2003; Figueras-Aloy et al., 2004; Lawet al., 2004]. In the present study, newborn infants hadmore frequent episodes of severe disease compared toolder patients. In spite of a large amount of data amongchildren of all ages, few studies have focused on theburden of disease attributed to RSV in neonates [Fodhaet al., 2004a]. Infection before 6 or even 3 months of ageis known to be associated with an increased risk forsevere illness [Kaneko et al., 2001; Nielsen et al., 2003;Papadopoulos et al., 2004;Voets et al., 2006].Age indayswas found previously to correlate inversely withduration of supplemental oxygen and need for ventila-tory support (Wright et al., 2002). Thus, it was notsurprising tonote ahigher frequency of severedisease inneonates. Nevertheless, the present study was realizedin newborns already weakened mostly by low-birth

weight and/or premature birth (44.7% of neonatespresented a risk factor vs. 25.6% of older infants), whichmay explain partially the increased severity of illness insuch a population.

This 1-year retrospective study of children with lowrespiratory tract infections and RSV diagnosis in theCentral coast of Tunisia showed that there has been apredominance of subgroup B infections in 2005(Table II). These findings agree with the previousTunisian study that reported subgroup B as the strainfound most frequently in the same region from 2000 to2002 [Fodha et al., 2004b]. In the present study, nodifferences in viral load between RSV subgroups weredetected, as demonstrated previously by DeVincenzo[2004].

Clinical severity of RSV infection is associatedcommonly with epidemiological and hosts factors butless well defined is the influence of factors such as viralstrain [Savon et al., 2006]. In the present study, theRSVsubtype was not correlated significantly with theseverity of illness. However, there is a limitation of theresults presented in the current study concerningstatistical power. Indeed, because of a relatively smallnumber of patients with RSV-A, true differences indisease severity may not be able to be demonstrated bythis study. Several studies have evaluated differences inclinical severity between subgroups A and B, butinvestigations have given varying results in differentlocations. Inabouthalf of these studies, theRSVsubtypewas not correlated significantly with severity [Hendryet al., 1986, 1987; Monto and Ohmit, 1989; Russi et al.,1989; Wilson et al., 1990; Tsutsumi et al., 1991;McIntosh et al., 1993; Wang et al., 1995; Kneyberet al., 1996; Bergstrasser et al., 1998; DeVincenzo,2004]. Other studies have found RSV-A [Hall et al.,1990;McConnochie et al., 1990;Walsh et al., 1997; Imazet al., 2000; Papadopoulos et al., 2004] or B [Hornslethet al., 1998] as the most severe pathogen; however, insome cases, this effect was lost when confounders weretaken into account in logistic regression models. Meth-odological factors could explain why those studies havefound differences in severity. For example, if thecommunity attack rate for group A viruses was greater

J. Med. Virol. DOI 10.1002/jmv

TABLE III. Baseline Characteristics of Patients With Severe and Nonsevere RSV Disease Among Groups Without and WithRisk Factor (Center Coast of Tunisia, 2005)

Patients without riskfactora (N¼ 53)

Patients with risk factora

(N¼ 28)All patients

(N¼ 81)

Severe(N¼ 27)

Nonsevere(N¼ 26)

Severe(N¼ 25)

Nonsevere(N¼ 3)

Severe(N¼ 52)

Nonsevere(N¼ 29)

Neonates� 28 days 17 (63.0%) 4 (15.4%) 15 (60.0%) 2 (66.7%) 32 (63.5%) 6 (20.7%)Infants> 28 days 10 (37.0%) 22 (84.6%) 10 (40.0%) 1 (33.3%) 20 (36.5%) 23 (79.3%)Male (N¼ 46) 13 (48.1%) 17 (65.4%) 13 (52.0%) 3 (100.0%) 26 (50.0%) 20 (69.0%)Mean RSV nasopharyngeal quantity

(RSV transcripts/ml)4.2� 106 1.0� 106 1.1� 107 2.3� 106 4.9� 106 1.7� 106

RSV subgroup A (N¼ 9) 4 (14.8%) 3 (11.5%) 2 (8.0%) 0 (0%) 6 (11.5%) 3 (10.3%)RSV subgroup B (N¼ 60) 20 (74.1%) 20 (77.0%) 19 (76.0%) 1 (33.3%) 39 (75.0%) 21 (72.4%)RSV untypeable strains (N¼ 12) 3 (11.1%) 3 (11.5%) 4 (16.0%) 2 (66.7%) 7 (13.5%) 5 (17.2%)

aEstimated gestational age<37 weeks and/or birth weight< 2,500 g.

RSV Disease, Risk Factors, and Viral Load 1955

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than group B viruses, which was often the case, a largernumber of severely ill infants might have been hospi-talized during the group A-dominant period, even if thedistribution of illness severity was identical for eachvirus subgroup. Indeed, the unique study with RSVsubgroup B as predominating strains was also the onlyone where type B infections produced more severedisease than did type A infections [Hornsleth et al.,1998]. Thus, the discrepancies in the results of differentstudies looking into RSV subtype and bronchiolitisseverity need to be further elucidated.In the present study, nasopharyngeal loads of RSV

determined after hospital admission correlated with aclinically relevant measure of disease severity, becauseRSV-infected infants with severe disease had highernasopharyngeal loads than did infants with non-severedisease. The limitations of this study must be acknowl-edged. First nasopharyngeal aspirates were collected atone time point for each patient; usually this was theafternoon after admission. It is likely, however, thatthe RSV loads measured in these aspirates are close tothe maximum nasopharyngeal RSV viral loads for eachpatient, because peak nasal RSV loads in hospitalizedinfants are usually seen shortly after admission [Hallet al., 1975]. A second limitation is that this study onlyevaluated RSV load in the upper respiratory tract,because lower respiratory tract secretions cannot beobtained reliably fromnon-intubated patients. Previousstudies have shown, however, that RSV viral load innasal washes of intubated infants closely approximatesthat found in tracheal aspirates [Malley et al., 1998,2000]. Thus, in the absence of specific antiviral therapythe nasopharyngeal RSV viral load in infants withprimary infectionmaywell be indicative of theRSVviralload in the lower respiratory tract.In this context, the results of the current study

suggest that RSV disease, although perhaps beinglargely a result of a pathogenic immune response,appears to be driven by the RSV viral load. Indeed, itis unlikely that the differences in RSV loads observed inthis study result purely from variability in the collectionof nasal specimens or performance of the real-time RT-PCR assay. Although variations in sample volume orquality could affect the measured RSV concentrations,attempts to minimize these variations by having allsamples collected by relatively few investigators whoeach used a standard method. In addition, to ensure thereproducibility of the assay, a sample of known RSVviral load was tested in parallel with each assay. Otherinvestigators also reported previously a positive associ-ation between RSV viral load in respiratory specimensand disease severity, using different virological techni-ques and different definitions of disease severity [Hallet al., 1976; Buckingham et al., 2000; DeVincenzo andBuckingham, 2002; DeVincenzo et al., 2005]. However,this association is still controversial [Wright et al., 2002;Legg et al., 2003]. Two main factors could explain thediscrepancies between studies. The first factor was thedefinition of the study population. Both studies that didnot found an association worked on a heterogeneous

group of subjects, without excluding patients who hadunderlying conditions (such as congenital heart diseaseand chronic lung disease) that would disrupt theoret-ically the putative relationship between RSV viral loadand disease severity. Because the extent and severity ofthese underlying conditions is variable, disease severityin these patients is more likely to be a function of theirunderlying disease than of their RSV load. The secondfactor involves the definition of disease severity. ForWright et al. [2002], the calculation of disease severitywas based mainly on respiratory illness scores deter-mined for up to 13 days after the initial enrollmentassessment. Unfortunately, approximately half thesubjects received RSV immune globulin infusions afterthis initial assessment but were still included in thesedisease severity calculations. RSV immunoglobulintherapy could have affected disease severity, thusconfounding any correlation between severity of diseaseand RSV load [DeVincenzo and Buckingham, 2002].Concerning the study realized by Legg et al. [2003],disease was considered as severe after the patientshowed signs of acute bronchiolitis, whereas in thecurrent study, all included patients were hospitalizedfor RSV bronchiolitis.

If nasal RSV viral load correlates with diseaseseverity, then the question of why antiviral therapyhas not been shown to result in significant clinicalbenefit to RSV-infected infants remains unanswered. Itis possible that the anti-RSV therapies tested to date donot reduce viral load sufficiently at the doses adminis-tered at the proper time in the disease course to affectsignificantly clinical outcomes. It is also conceivable thatantiviral therapy may benefit only a subset of patientswith RSV disease; further studies would be needed todefine this subset [Buckingham et al., 2000]. Alterna-tively effective therapy for RSV disease may requiretreatment strategies aimed not only at reducing viralload, but also at addressing factors intrinsic to thepatient, such as the nature of the host inflammatoryresponse to infection [Hornsleth et al., 1998; Legg et al.,2003]. In conclusion, RSV disease severity is likely to bedetermined by an interplay between host factors (e.g.,underlying conditions and nature and magnitude of theinflammatory response) and virus factors, includingRSV viral load. Further study is clearly needed in thisimportant area.

ACKNOWLEDGMENTS

We are grateful to Dr. Nesrine Zaoui for her help incollecting clinical data. We also thank the staff of thelaboratories of Virology of Caen and Sahloul UniversityHospitals for their technical help. Finally, we thank thenursing staff of the Pediatric Unit of Fattouma Bour-guiba University Hospital and the Neonatal Unit ofFarhat Hached University Hospital for assisting insample collection.

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