Abstract: A simple clinical screening (CS) tool for respiratory virus (RV) infection was introduced and evaluated in a single hematology ward, as part of a strategy to reduce nosocomial RV infection. Up to 6 clinical symptoms or signs were scored and a prede¢ned threshold score of ! 2 prompted paired nose/throat swab (NTS) collection for RV testing. The criterion standard for RV infection was positive immuno£uorescence (IF) or polymerase chain reaction (PCR) for 7 and 15 viruses, respectively. The tool was shown to be most bene¢cial at excluding infection at a threshold score of 1 ( ) at a prevalence of 22%. The tool's ability to diagnose infection was limited (positive predictive value 27% and 29% at thresholds 1 and 2). The sensitivity of IFcompared with PCR was 45% for the 7 viruses common to both, and 23% for the extended virus panel detected by PCR. An algorithm incorporating CS, paired NTS collection at a threshold of 1 symptom or sign, and sensitive testing including PCR can guide infection control measures in hospitalized hematopoietic stem cell transplant recipients.
Nosocomial respiratory virus (RV) infections after hematopoietic stem cell transplantation (HSCT) are an avoidable complication with severe clinical outcomes. For example, pneumonia has been reported in up to 75% of HSCT recipients acquiring parain£uenza virus (PIV) nosocomially, with an associated mortality of 47% (1) . Nosocomial RVoutbreaks have been reported in multiple HSCT units (1^4), with 8 outbreaks in 13 European centers over 10 years; in 6, transplant programs were halted or closed in order to contain these infections (5) .
Recommended methods to prevent nosocomial RV infection include a daily checklist for the presence of signs and symptoms of RV infection in patients beginning on the day of admission (6) , placing all patients with respiratory symp-toms in isolation and using respiratory and contact precautions when entering the room, excluding symptomatic sta¡ or visitors from the unit, and deploying sta¡ with a respiratory illness to non-patient areas (6, 7 ) . Isolation precautions can be modi¢ed when the etiology of the illness is known (7 ) . Although recommended, the diagnostic accuracy of using a daily checklist to prompt viral testing and infection control measures has not been assessed.
In this study, we developed a clinical case de¢nition for RV infection based on scoring a suite of signs and symptoms of respiratory infection. A threshold score of 2 or more triggered the collection of respiratory tract specimens for viral diagnosis. The diagnostic accuracy of the case de¢nition was evaluated in adult HSCT recipients.
All patients admitted to the hematology ward, Westmead Hospital (a university teaching hospital) in Sydney, Australia, between July 1, 2005, and September 30, 2007, were eligible for clinical screening (CS) and per-protocol testing for RV infection. This ward manages general hematology and HSCT patients, and performs 40^55 allogeneic and 15^25 autologous HSCTs annually on patients aged 16 years and above. Approval for the study was granted by the Sydney West Area Health Service Ethics Committee.
A CS score was derived from a checklist of 6 features: cough; fever 4381C in the last 24 h; sneezing, runny nose, or nasal stu⁄ness; shortness of breath; oxygen saturation o95% on room air; and crackles on chest examination documented by clinician in the patient record. One point was assigned to each positive ¢nding. Nurses were requested to perform the screen daily, and enter the CS score into a log located in the bedside chart. Paired nose and throat swabs (NTS) for viral testing were recommended for patients with scores ! 2.
The screening tool was piloted from April 2005. Hematology nurses were educated by the researcher (P. E. F.), the nurse educator, and senior nursing sta¡ on the use of the screening tool, sample collection techniques, and transport of swabs to the laboratory. Screening logs were placed in the patient's bedside charts on admission.
Clinical characteristics of episodes of respiratory illness were ascertained prospectively by direct questioning of patients and caregivers by the researcher, and observations of temperature, oxygen saturation, and physical examination ¢ndings provided by treating clinicians in the case notes, and laboratory and radiology results in electronic hospital databases. Demographic data and HSCT type and date were retrieved.
The following de¢nitions were used: Upper respiratory tract infection (URTI): Rhinorrhea, sneezing, cough, coryza, with or without fever, with a normal chest examination and absence of pulmonary in¢ltrates on radiological imaging (chest x-ray or computed tomography).
V|ral URTI: URTI with detection of RV from URT secretions collected by NTS (8^11).
Lower respiratory tract infection (LRTI, or pneumonia): Fever and hypoxia (oxygen saturationso95% on room air) or pulmonary in¢ltrates on radiological imaging (chest x-ray or computed tomography).
V|ral LRTI: LRTI with the detection of RV in bronchoalveolar lavage (BAL) specimen (bronchoscopic or nonbronchoscopic), endotracheal aspirate, or URT secretions (8^10).
Upper and lower respiratory tract infection (U&LRTI): LRTI with concurrent rhinorrhea, sneezing, or coryza.
V|ral U&LRTI: U&LRTI with detection of RV in BAL (bronchoscopic or non-bronchoscopic), endotracheal aspirate, or URTsecretions (8^10).
Combined NTS were collected using sterile swabs moistened with viral transport medium (Copan Diagnostics, Corona, California, USA). BAL was performed on patients with adequate respiratory reserve at the discretion of hematology and respiratory physicians. Intubated and ventilated patients frequently had BAL or non-bronchoscopicB AL samples collected during intubation. Initial virological testing was performed in the Respiratory V|rology Laboratory of the Centre for Infectious Diseases and Microbiology Laboratory Services, the Institute of Clinical Pathology and Medical Research,Westmead Hospital. Samples were processed for indirect immuno£uorescence (IF) using monoclonal antibodies (Chemicon Inc., Temecula, California, USA) against in£uenza A and B, PIV 1^3, respiratory syncytial virus (RSV), and adenovirus (ADV). Human metapneumovirus antisera for IF (D3DFA, Diagnostic Hybrids, Athens, Ohio, USA) were available from November 2006. IF-negative NTS samples and all BAL samples were cultured for viruses in monkey kidney cell lines (LLC-MK2\BGM) and human embryonic lung ¢broblasts (MRC5). Cultures were observed twice weekly (days 1^21) for cytopathic e¡ect. IF tests were performed during normal working hours, with results available the same day for samples reaching the laboratory by 2 PM.
Residual £uid from clinical samples was frozen ( À 301C). An aliquot was subsequently collected and stored at À 801C until transferred to the Queensland Paediatric Infectious Diseases Laboratory for polymerase chain reaction (PCR) for PIV 1^3; in£uenza A and B; RSV; human metapneumovirus (12, 13); ADV; rhinoviruses; coronaviruses OC43, 229E, NL63, and HKU1 (14, 15); polyomaviruses WU and KI (16, 17 ) ; and human bocavirus (18) .
The number of respiratory tract infection (RTI) episodes in HSCT recipients was determined by review of all clinical data. To con¢rm the validity of the CS score, a maximum possible (MP) score was calculated by reviewing the clinical information obtained on the same day that the CS score was logged. This information was gathered prospectively, with the MP score tallied retrospectively without knowledge of viral test results.
Statistical analysis was performed using SPSS version 15.0 and DAG___Stat (19) . Medians were reported with the 25th and 75th interquartile values and compared using the W|lcoxon signed rank test. Chi-squared tests were used to compare proportions. Performance of the CS score and MP score was calculated using the k measurement of agreement. Diagnostic accuracy was assessed using sensitivity, speci¢city, positive, and negative predictive values (NPV) and likelihood ratios with corresponding 95% con¢dence intervals (95% CI). The CS score was compared with combined IFand PCR testing as the criterion standard, and IF testing with PCR as the criterion standard. Two-tailed P-values are reported.
A total of 1181 respiratory specimens (1078 NTS, 100 BAL samples, and 3 sputa) were collected over the 3 -year study period from 377 patients (183 HSCTand 194 general hematology patients). HSCT recipients were predominantly male (123/183, 67%) with a median age at ¢rst sampling of 46 years (25th quartile 34, 75th quartile 55). A median of 3 NTS (1, 5) was collected from each HSCT recipient.
The Table 2 , P 5 0.003), and in RV-positive episodes (P 5 0.02).
The sensitivity and speci¢city of the CS score in samples from HSCT recipients using a threshold of 2 were 62.5% (45.8^77.3%) and 56.9% (48.4^65.2%), respectively (Table  3 ). Using a threshold of 1, the corresponding values were 85.0% (70.2^94.3%) and 34.7% (27.0^43.1%). Results were similar in patients sampled during the admission for HSCT. The diagnostic accuracy was comparable during the high in£uenza/RSV winter season in Australia (JuneŜ eptember) and the remainder of the year (data not shown). The post-test probability of RV infection in HSCT recipients was calculated for varying prevalence rates (Table 4) . Compared with the observed prevalence of 22%, a score ! 2 increased probability of infection to 29% (23^35%), and a score 0^1 reduced this probability to 15% (11^22%). The corresponding post-test probabilities using a threshold score ! 1 were 26% (23^30%) and 11% (5^20% 
This is the ¢rst study to our knowledge to con¢rm the validity and value of using a simple clinical score to trigger microbiological testing for RV infections in hematology or HSCT patients. Daily CS of HSCT recipients for symptoms or signs of viral URTI or LRTI has been recommended previously (6), but guidelines for when sampling and testing should be performed have not been developed.
The CS tool was shown to be the most useful at detecting patients without an RV infection, and limited in its ability to diagnose those with such an infection. Such a tool is bene¢cial to stratify an approach to testing for readily transmissible organisms in a highly vulnerable patient setting. It performed best at excluding RV infection with a threshold score of 1 symptom or sign at the given prevalences with an NPV of 89% (78^96%). Fewer samples were collected at a threshold of 1, and veri¢cation bias may have skewed these results. It should be noted that the screening tool quanti¢ed the number of characteristics, rather than gave weighting to speci¢c symptoms or signs, in order to detect a variety of RVs that may have di¡erent clinical features and severity.Weighting of speci¢c clinical features may improve the performance of the CS tool; this could not be further assessed in this study. The likelihood ratios for scores above and below the screening thresholds corresponded with small, but potentially important, changes in the post-test probability of infection (20) . The CS tool did not exclude infection when prevalence rates were high. In an outbreak setting with a potential prevalence of 50%, testing and infection control measures are warranted in all patients.
This study, which was designed to evaluate the CS tool in a working clinical context, revealed a signi¢cant limitation, namely, the low compliance observed with the screening tool. A score was recorded with only 36% of all respiratory samples collected and in half of these the score exceeded 2. Although the majority of all samples were taken without a logged score, NTS sampling increased 4 -fold following the introduction of the screening tool, with RV detection in 25% of samples without a logged score during episodes of RTI. This is comparable with viral detection from samples scoring ! 2 and may suggest that CS was performed but scores were not recorded.
For practical reasons, paired NTS was chosen as the sample for diagnostic testing rather than nasopharyngeal aspiration (NPA). While NPA obtains the highest viral burden from the URT (21), paired NTS have a sensitivity equivalent to NPA in children (22) . While this may not be the case in adults, owing to lower viral loads (23), sensitive molecular methods were used to optimize RVdetection. NTS cause less pain than NPA in children (24) and adults (25, 26) . NTS were used in this study because of their greater tolerability in adults (especially HSCT recipients who may have complicating factors such as mucositis), particularly as multiple specimens were collected from each patient. Additional bene¢ts of NTS include greater ease of collection by nurses, and consequent earlier collection than with NPA, which requires additional equipment and expertise (24) . RVs were detected in a greater proportion of NTS during episodes of URTI than LRTI. As URTspecimens are less sensitive for RV detection than BAL during LRTI (27) , it remains essential to consider BAL in the absence of a positive RV in this setting (7) .
Based on our data, molecular methods of identi¢cation should be included in a diagnostic algorithm to detect clinically important RVs in addition to IF and viral culture.We noted 23% of PCR-positive RVs were also positive by IF. While PCR can detect 15 individual viruses and IF only 7 of these, IF was positive for only 45% of the 7 viruses positive by PCR. The superior performance of PCR for these 7 viruses is well known, with sensitivity of IF in symptomatic children of 63^70% (28) . IF for ADV is known to be insensitive (15^30% in symptomatic children [28] ), and 0 of 10 PCR-positive samples in the current study were detected by IF. In adult HSCT recipients, the poor sensitivity of IF compared with combined IF, PCR, and viral culture in nasal wash specimens has been documented previously for in£uenza (1/3 samples), RSV (3/6), and PIV (2/18) (29) . We have con¢rmed these results with a larger sample and paired NTS specimens. In addition, more than half of all positive specimens were viruses not detectable by IF kits, showing the importance of broadening the panel for RV testing. While multiplex real-time PCR for 13 RVs is now available at a relatively low cost (30) , its use is dependent on laboratory resources and practicalities. The diagnostic accuracy of a clinical scoring system and viral testing procedures as implemented in this study can guide infection control recommendations for RV infection in HSCTand general hematology units.We recommend that with a score of 1 or more, infection control measures should be implemented and timely, sensitive, and speci¢c laboratory testing undertaken, including molecular testing. Although IF testing is potentially rapid (within a working day), it is insensitive, hence infection control measures should be maintained pending results of PCR. However, the rapid turnaround time of IFand point-of-care testing remains important to guide treatment for in£uenza and RSV.
In summary, a simple CS tool for RV infection was developed and implemented in an HSCTand general hematology ward. The tool was most e¡ective at excluding RV infection using a threshold of 1 clinical symptom or sign, and we recommend that this should trigger sampling for viral testing. We recommend that PCR be included with point-of-care testing, IF, and viral culture for comprehensive virological surveillance.