Clinical course of community-acquired respiratory syncytial virus pneumonia in newborns hospitalized in neonatal intensive care unit

Bulgular: PCR esas alındığında, RSV tanısında HAT’ın duyarlılığı %90, özgüllüğü %78.5 idi. RSV pnömonili hastalardan dördünde atriyal septal defekt (ASD) saptandı ve hepsi mekanik ventilasyon desteği gerektirdi. Birinci gün Downes skoru intravenöz sıvı gereksinimi (p= 0.001, r= 0.48), toplam oksijen desteği (p= 0.000, r= 0.63), tekrar beslenmeye başlama süresinin uzunluğu (p= 0.001, r= 0.46) ile doğru orantılıydı. Kan pH düzeyi ile Downes skoru negatif ilişkiliydi (p= 0.031, r= 0.46). İkinci gün Downes skoru ASD’li hastalarda ASD olmayanlara göre daha yüksekti (3.8 ± 2.6 vs. 2 ± 1.1, p= 0.01). Hastanede yatış süresini etkileyen en önemli faktör ikinci gün Downes skoru idi (p= 0.02, OR: 1.9, %95 GA (1.1-3.2)]. Tüm hastalar sağlıklı olarak taburcu edildi.


INTRODUCTION
Respiratory syncytial virus (RSV) is a major cause of pneumonia in infants and young children worldwide (1)(2)(3).The clinical differentiation of bacterial from viral (RSV) pneumonia is very difficult.Therefore, a rapid and reliable diagnosis is essential for further clinical management, e.g., isolation or cohortation measures, withholding of antibiotic treatment.Laboratory diagnosis by cell culture and viral serology is usually necessary to identify the etiologic agent, although the final results of RSV isolation by tissue culture usually require several days.Rapid antigen test (RAT) results are available in 15 minutes (4).Multiplex real time polymerase chain reaction (RT-PCR) is a highly sensitive method for diagnosis of viral infection and has been used successfully in children with RSV.The multiplex RT-PCR has a sensitivity for RSV of 90% and a specificity of 99.2% using virus culture as the gold standard (5,6).Because of the higher costs and longer turnaround time of the molecular tests, as compared to the antigen-detection tests, some laboratories use antigen detection to screen incoming clinical samples and only perform a more sensitive molecular test on the negative samples (7).Although there are many studies investigating predictive values of RAT for RSV in children and adults (8)(9)(10), there are limited data on this issue in newborns (11,12).Furthermore, to our 3 knowledge there is no study using Downes' score in assessment of disease severity in newborns with RSV pneumonia.Therefore, in this study, we aimed to define predictive values of RAT for RSV in newborns.The factors affecting the clinical course of community-acquired RSV pneumonia in hospitalized newborns were also evaluated.

Inclusion and Exclusion Criteria
The newborns younger than 30 days and attended to the outpatient clinics with complaints of respiratory symptoms were assessed by the physicians and hospitalized in our NICU if required.During the study period, a total of 54 newborns hospitalized in NICU with signs and symptoms of pneumonia were included in the study.Exclusion criteria were presence of severe congenital malformations, intrauterine infections, and parents unwilling to give informed consent.

Demographic and Clinical Characteristics
Detailed history was taken from the parents of the newborns with sign and symptoms of pneumonia.Demographic and clinical characteristics were recorded to previously prepared study forms.Diagnosis of pneumonia was made using standard clinical, laboratory and radiological criteria.The clinical findings were apnea, tachypnea, poor feeding, abdominal distention, jaundice, emesis, respiratory distress, and circulatory collapse.Ventilator-dependent infants might have increased oxygen and ventilator requirements or purulent tracheal secretions.Blood white blood cell count, serum C-reactive protein (CRP) values, blood culture, Gram staining and culture of tracheal aspirates could help diagnosis.Bilateral alveolar densities with air bronchograms were defined as characteristic in pneumonia.Bronchiolitis was considered if there were mild symptoms of an upper respiratory tract infection at the beginning of the disease and then it progressed to cough, wheeze, onset of dyspnea, increased respiratory rate and retractions of the respiratory muscles, indicating lower respiratory tract involvement.Although there were no definite criteria, clues to a diagnosis of RSV pneumonia might be the finding of crackles, respiratory distress and dullness to percussion (13)(14)(15).At the first and second days of the admission, the severity of the respiratory distress was assessed by Downes' sco-re which is a comprehensive scoring system and can be applied to any gestational age and condition (Table 1) (16).A score of > 6 is indicative of impending respiratory failure.Appropriate supportive treatment including oxygen, fluid, resuscitation and inotropic agents were administered.Antibiotic and/or antiviral medications were started according to advice of in charge consultant neonatologist.Even though the use of antibiotics for all infants with bronchiolitis is not thought to be helpful, their use in a selected subset who may also have bacterial pneumonia may be indicated.A progressively increasing FiO 2 requirement to maintain a saturation of 90-92% in a preterm and 94-96% in a term baby is a sensitive indicator of the severity and progress of distress.We used clinical deterioration, blood culture and/or tracheal aspirate positivity in combination with chest radiograph, temperature abnormality, high blood leucocyte count and serum CRP values to make decisions for treatment with antibiotics (17).Coagulase negative staphylococcus generally were considered to be contaminants, except when two positive blood cultures and/or evidence of a true infection based on clinical course and laboratory data existed.
The variables which may affect the clinical course were recorded.

Laboratory Evaluation
Whole blood count, arterial blood gases, CRP, and blood cultures were taken from all subjects.Pulmonary Xray was performed.All the patients were evaluated for congenital heart diseases by echocardiography.Specimen collection A total of 54 nasopharyngeal aspirates from newborns with clinical signs of pneumonia were taken.The specimens were taken within the first 72 hours after admission.Nasopharyngeal aspirates from both nostrils were combined in a standardized manner in NaCl 0.9% without any additives by using a sterile suction trap.The secretions were immediately transported to the laboratory and either analyzed immediately (RAT, during working hours) or stored at +4°C for a maximum of 24 h until further processing (RAT, after working hours).According to the manufacturer's instructions, specimens (nasal washes and aspirates) can 237 Tuberk Toraks 2013; 61(3): 235-244

RESULTS
A total of 44 newborns with community-acquired pneumonia were included the study.All the 15 patients were healthy previously.

Diagnostic Results for RAT and Multiplex RT-PCR
Forty-four specimens were examined by both RAT for RSV and multiplex RT-PCR.Thirty (68.2%) samples were positive for RAT, 27 (90%, 27/30) samples of which were positive for PCR testing.By RAT for RSV 14 (31.8%)samples were negative.Eleven (78.6%, 11/14) of these specimens were negative for multiplex RT-PCR.According to the study results, RAT has a sensitivity of 90% and a specificity of 78.5%.Positive and negative predictivity were calculated to be 90% and 78.6%, respectively.Positive likelihood ratio was 4.09 with a low negative likelihood ratio (0.12) (Table 2).
During the study period, nosocomial pneumonia developed in five of the patients who were hospitalized in NI-  2).Regarding the results the highest RSV incidence (n= 18) was on January.

Comparison of Multiplex RT-PCR RSV Positive Patients According to Downes' Score
Further analyses were performed on 30 patients whose RSV positivity was confirmed by multiplex RT-PCR and their data were compared according to Downes' score.There was no significant difference between the two groups according to demographic characteristics except gestational age (Table 3).Demographic and clinical characteristics of the multiplex RT-PCR positive patients according to Downes' score are seen on Table 3  Among RSV positive patients, antibiotic treatment was started in 18 with severe clinical findings, high CRP levels, and positive blood cultures.Only supportive treatment was given to twelve of the patients.Oseltamivir was started in four patients, who needed mechanical ventilation, until multiplex RT-PCR results were obtained.

Clinical Course of Multiplex RT-PCR RSV Positive Patients According to Downes' Score
There were significant differences between the infants with low and high Downes' scores according to intravenous fluid requirement, time of tachpnea, time to reenteral feeding, and total day of oxygen support.Length of hospital stay was also longer in infants with high Downes' score, although not significantly (Table 5).The first and second day Downes' scores were higher in infants requiring mechanical ventilation 5.

Complications of Multiplex RT-PCR RSV Positive Patients
When evaluated for encountered complications during follow-up in NICU, seconder bacterial pneumonia developed in 2 (6.6%) patients.Sinus tachycardia was detected in another 1 (4.3%) patient at the 5 th of admission; there was no fever or finding supporting myocarditis.Apneic convulsion was observed in 1 (3.3%) infant; any metabolic abnormalities were not detected and cranial imaging studies were normal.Diarrhea developed in 3 (10.0%)infants; Rotavirus was shown in 2 (6.7%) of them.Conjunctivitis was observed in 2 (6.7%) patients; conjunctival cultures were negative.All the infants discharged from the hospital with a good health.No problems were observed in any of the patients at 6 th month visit.The patients with ASD have been followed-up by department of pediatric cardiology.

DISCUSSION
We found that RAT showed a high sensitivity and thus proved to be satisfactory to exclude RSV etiology in a newborn with pneumonia confirms a RSV diagnosis.Overall specificity, however, was nearly high, making it acceptable to confirm a RSV diagnosis.In a recent study performed on 3691 newborn subjects, it was demonstrated that RAT has high sensitivity (90.3%) and specificity (88.2%) for the diagnosis of RSV respiratory tract disease (11).In this study, we used the rapid IC test and showed that the sensitivity and specificity of RAT were 90% and 78.5%, respectively.We think that rapid confirmation or elimination of RSV would facilitate prompt and adequate measures to restrict transmission of the virus in a NICU containing high-risk infants.
It was suggested that because of the limited antibody response and reduced concentration of passively acquired maternal antibodies in neonates, and having smaller airways preterm neonates were highly susceptible to RSV (18).During the study period, prematurity rate was 16.6% among infants with community-acquired RSV pneumonia.It is not clear how breast-feeding reduces the risk of RSV infection.Although recent immunological studies have tried to define the mucosal and/or systemic mechanisms of protection against respiratory infections during the early months of life, more studies are required to identify which elements modify the evolution of disease (19).Interestingly, 70% of our patients with RSV pneumonia were breastfed.Cough, rhinorrhea and fever were reported to be the most common symptoms, and respiratory distress and apnea were serious complications frequently seen in young infants caused by RSV bronchiolitis and pneumonia (20).In our study, cough, nasal congestion, grunting, vomiting, nasal discharge and cyanosis were the major clinical findings, while hyperinflation, atelectasis, and infiltration were the most common radiological findings observed in the RSV infected infants, consistent with previous data.It was reported that lower respiratory distress with wheezing caused by RSV was commonly found in children less than two years old (21).Rales were heard in all of our study patients with RSV pneumonia.It can be said that RSV infection should be suspected in the presence of such symptoms during the RSV season.Premature infants in their first six months, children with underlying cardiac or pulmonary disease in their first two years, immune-compromised children (particularly transplant patients), and healthy infants younger than six weeks of age are at the highest risk of severe RSV infection.These children are likely to have prolonged hospital stays, and are more likely to require admission to the NICU and to need mechanical ventilation.The absence of a vaccine to prevent RSV infection narrows preventive measures to a combination of public health advice and the use of passive immunization with palivizumab, the only licensed product available for prevention of RSV lower respiratory tract disease in high-risk infants and children during the RSV season (22).In our practice, palivizumab prophylaxis has been performed on high-risk infants during the winter season.According to recommendations of American Academy of Pediatrics (AAP), congenital heart diseases requiring palivizumab prophylaxis include cyanotic heart diseases, moderate to severe pulmonary hypertension, and congestive heart failure requiring medication (19).In the current study, all the patients were evaluated by echocardiography and secundum ASD was shown in seven of them.The four had RSV infection and required mechanical ventilation.Pulmonary hypertension and cardio-pulmonary insufficiency developed in these infants.We think that ASD might negatively affect the course of the disease with increased viral load in lungs due to increased pulmonary blood flow.Although it may be difficult to say palivizumab prophylaxis in infants with ASD because to small number of the cases, but it might be recommended that the patients with severe RSV infection should be evaluated by echocardiography.Previous studies have shown that bacteriemia or serious bacterial infection was present in some children admitted with RSV infection (23).Blood cultures were considered to be positive in 6.6% of our patients with RSV infection.Extrapulmonary manifestations are common in children with severe RSV infection, suggesting RSV may infect organs other than the lung.Life-threatening extrapulmonary manifestations of RSV infection include central apneas, seizures, status epilepticus, ventricular tachycardia and fibrillation, heart block and pericardial tamponade and can be detected by adequate monitoring (24).Although underlying metabolic abnormalities such as hypoglycemia, and/or hyponatremia may cause seizure, the pathophysiological mechanism of RSVrelated seizures remains unclear (25).Detection of RSV in the cerebrospinal fluid has also supported a direct invasion of the central nervous system in RSV disease.Apnea developed in 23.3% of our patients.One had apneic convulsion and she had no any metabolic or intracranial abnormalities.Presumably, the arrhythmias may be secondary to RSV pericarditis, myocarditis, or some other infection of the heart.The possibility that the cardiac arrhythmias may have been caused by one of the cardiotropic viruses infecting the child at the same time as RSV has also been reported (26).In our study, sinus tachycardia not related to myocarditis, cardiac insufficiency or anemia developed in two patients.The other extra pulmonary manifestations of RSV infection include hyponatremia, hepatitis hypothermia, rash, thrombocytopenia and conjunctivitis.Conjunctivitis was found in two of our patients and there was no bacterial grew in conjunctival cultures.Severe RSV infections resulting in acute respiratory distress syndrome with a mortality rate of 40-70% have been reported in the literature (27).The statistical significant association between RSV infection and days of hospitalization and oxygen therapy confirms that this infection is cause of severity illness and that prompt recognition of the diagnosis that include early treatment with oxygen therapy helped to improve the clinical state of the children.The severity of respiratory distress may be assessed by Silverman-Anderson Score and Downes' Score (16,28).While the Silverman Anderson Retraction Score is more suited for preterms with RDS, the Downes' score is more comprehensive and can be applied to any gestational age and condition.Scoring should be done at half hourly intervals and a chart maintained to determine progress.In a study, Downes' score > 7 or FiO 2 > 50% after 15-20 minutes of CPAP were found to be predictive of CPAP failure (29).Pillai et al. defined persistent/worsening respiratory distress if Silverman score ≥ 4 addition to other criteria (30).In our study, objective assessment of disease severity was made using Downes' score.Downes' scores of the patients were higher among patients requiring mechanical ventilation.The length of hospital stay was longer in infants with higher Downes' score and having longer oxygen support.We speculate that Downes' score is prognostic in evaluation and follow-up of patients with RSV pneumonia.The treatment and care for RSV depend on the assessment of the severity of respiratory compromise and of respiratory exhaustion with decreased respiratory effort.In addition, the consequences of respiratory compromise, in particular the inability to feed and drink, determine the management of these infants.Additional risk factors and preexisting illness (e.g., chronic lung disease of prematurity or history of reactive-airway disease) also influence disease management.Among RSV positive patients, antibiotic treatment was started in 18 with severe clinical findings, high CRP levels, and positive blood cultures.Ribavirin is an antiviral drug that is very effective against RSV in vitro and is licensed for use by inhalation for severe RSV bronchiolitis.However, due to teratogenic side effects, ribavirin cannot be administered as an aerosol in the presence of pregnant women (e.g., medical staff).
A further problem with antiviral drugs is the timing of application; for example, anti-influenza virus drugs need to be applied during the first 48 hours of illness to be effective (31).A Cochrane review found that published reports of trials of ribavirin lack the power to provide reliable estimates of its effects but suggested that ribavirin may reduce the duration of mechanical ventilation and hospitalization (32).RSV is spread by infected respiratory secretions (33).To control the spread of RSV in NICU during a community outbreak of RSV infection isolation of all infants with respiratory disease is generally impossible.Utilizing six infection control procedures (isolation of infected infants, handwashing between infants, change of gowns between caring for infants, cohorting of staff to infants, isolation of high risk in-fants, limitations of visitors) would reduce nosocomial infection rate.During the RSV season, we implemented control procedures including isolation of the positive cases, contact precautions, limitation of visitors and closure to elective admissions.These measures were effective and an outbreak did not develop in our NICU.RSV was detected in only two patients among hospitalized infants.
To our knowledge, this is the first study using Downes' score in evaluating newborns with RSV pneumonia.However, our study has a limitation; this study reports the results of only one tertiary care medical center in Turkey.A comprehensive study including local clinics, regional hospitals and medical centers would provide more details of RSV infections in Turkey.As conclusion, RAT is sensitive and specific in detecting RSV infections in newborns.Physicians may use Downes' score for evaluation of disease severity in infants with RSV pneumonia and subsequently target preventive and monitoring strategies toward newborns at high risk.ASD may increase the disease severity in RSV pneumonia.
was conducted on patients attending outpatient clinics of Department of Neonatology and hospitalized in Neonatal Intensive Care Unit (NICU) of Dr. Sami Ulus Maternity and Children Training and Research Hospital, Ankara, Turkey from November 2010 to April 2011.The study was approved by Local Ethics Committee.

Table 1 . Downes' score.
Platinium Taq mix, 12.5 mL of 2X Master mix, and 4 mL nuclease-free water.RT-PCR amplification conditions were as follows; reverse transcription at 50°C for 30 minutes, Taq inhibitor activation 95°C for 2 minutes and 45 cycles at 95°C for 15 seconds, 55°C for 30 seconds.Chicago ILL, USA) was used for statistical analysis.Kolmogorov Smirnov test was used to analyze the distribution of data.Data were expressed as the arithmetic mean ± standard deviation.Differences between two groups were tested using Mann Whitney U test.Chi-square tests were performed for categorical variables.Spearman test was used to analyze correlation between variables.Sensitivity, specificity, (14)g.The presence of a positive control line with a positive test line was considered a positive result.All tests were performed by the primary investigator (B.A).Each box of Respi-Strip was checked with a positive control included in the box before being used in patients.The rapid test was evaluated before knowing the PCR result.Multiplex RT-PCR ELISARespiratory system viruses including RSV A and B, rhinovirus, influenza A and B, coronavirus, H1N1, human metapneumovirus, parainfluenza, and adenovirus were investigated by multiplex RT-PCR.For multiplex RT-PCR, the oligonucleotide primers were designed to amplify the nucleoprotein (N) and phosphoprotein (P) genes of RSV, because they are highly conserved and are regions of the RSV genome which allow subgrouping of RSV strains into A and B types(14).The RT-PCR was performed with "in-house" RT-PCR protocol provided by Center for Disease Control (CDC, USA), using 2x buffer and SuperScript III Platinum ® One-Step Quantitative RT-PCR System (Invitrogen; CA, USA) with Stratagene Mx3005P (Strategene; California, USA) RT-PCR machine.The 25 mL PCR 22 mixture contained 5 mL of extracted RNA, 1 mL each of forward and reverse primers, 1 mL probe, 0.5 mL Su-perScript III RT/and positive and negative predictive values of RAT for RSV was calculated.Positive and negative likelihood ratios were also given.Factors affecting the longer hospital stay in RSV pneumonia were investigated by multivariate logistic regression analysis.The level of significance was set at 5% in all comparisons.

Table 2 . RAT and multiplex RT-PCR results of the patients.
n= 1, 3.3%) grew in blood cultures of RSV positive infants.Streptococcus viridians grew in the tracheal aspirate of 1 (3.3%) patient with high Downes's score.

Table 3 . Demographic and clinical characteristics of the multiplex RT-PCR RSV positive patients according to Downes' score. Downes' score ≤ 6 Downes' score > 6 (n= 24) (n= 6) p
week, present of ASD, second day Downes' score were evaluated by multivariate analysis.It was found that longer hospital stay was related to higher second day Downes' score (p= 0.02, OR: 1.9, CI 95% (1.1-3.2).The first day Downes' score was positively correlated with time of intravenous fluid requirement (p= 0.001,