Pitfalls in the Diagnosis of Pandemic (Novel) A/H1N Influenza. Kamaljit Singh, M.D., D(ABMM), Shawn Vasoo, M.D., Jane Stevens, MT (ASCP), Paul

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1 JCM Accepts, published online ahead of print on 1 February 0 J. Clin. Microbiol. doi:./jcm.0-0 Copyright 0, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 Pitfalls in the Diagnosis of Pandemic (Novel) A/H1N1 00 Influenza Kamaljit Singh, M.D., D(ABMM), Shawn Vasoo, M.D., Jane Stevens, MT (ASCP), Paul Schreckenberger, Ph.D, D(ABMM), Gordon Trenholme, M.D., Kamaljit Singh, M.D. Rush University Medical Center 1 W Congress Parkway Chicago, IL 01 kamalsinghs@hotmail.com Shawn Vasoo, M.D. Rush University Medical Center, 1 W Congress Parkway Chicago, IL 01 Vasoo.shawn@gmail.com Jane Stevens, MT (ASCP) Rush University Medical Center 1 W Congress Parkway Chicago, IL 01 Jane_M_Stevens@rush.edu Paul Schreckenberger, Ph.D, D(ABMM) Loyola Medical Center S First Avenue Maywood, IL 01 Downloaded from on December, 01 by guest 1

2 Gordon Trenholme, M.D. Rush University Medical Center 1 W Congress Parkway Chicago, IL 01 Gordon_M_Trenholme@rush.edu Address for correspondence : Kamaljit Singh, Rush University Medical Center, 1 W Congress Parkway, Suite Jelke Building, Chicago, IL 01 kamalsinghs@hotmail.com or Kamaljit_Singh@rush.edu ph: 1-1, fax: 1- Conflicts of Interest: Shawn Vasoo: None; Kamaljit Singh: Visitors Speakers Bureau, Wyeth Scientific Advisory Board, Quidel Corporation, San Diego, CA; Gordon Trenholme: None; Jane Stevens: none; Paul Schreckenberger: none Downloaded from on December, 01 by guest

3 Abstract: We report on patients with negative RT-PCR for Pandemic (H1N1) 00 using nasopharyngeal swabs but positive RT-PCR on bronchoalveolar lavage (BAL). Patients with an influenza-like illness and severe pneumonia with negative influenza RT-PCR on nasopharyngeal swabs should undergo further testing of specimens from the lower respiratory tract. Laboratory diagnosis of influenza is important for treatment, surveillance, infection control, chemoprophylaxis and monitoring of resistance. Rapid and sensitive nucleic acid amplification tests have widely replaced virus isolation as the reference standard (). However, despite improvements in sensitivity and specificity, results of these tests need to be interpreted in conjunction with the history and clinical findings of the patient. False negatives may occur because of low virus quantities, poorly collected specimens, inappropriate handling or delayed transport and technical reasons such as presence of viral inhibitors. Herein, we report our experience with cases seen at Rush University Medical Center (RUMC), Chicago in which testing of nasopharyngeal swabs were negative for Pandemic (H1N1) 00 influenza virus but positive on bronchoalveolar lavage (BAL) (Table1). Patient 1 A 0-year-old male with asthma was admitted to RUMC on July 00 with a day history of fevers to. C, chills, productive cough with mild hemoptysis, and diarrhea for 1 day. He was tachycardic with a SpO of % on room air (RA) and negative chest X-ray (CXR). Levofloxacin was empirically started. A polyester tipped nasopharyngeal swab (Puritan Medical Products, Guildford, ME) in MRT transport media (Remel, Downloaded from on December, 01 by guest

4 Lenexa, KS) was collected on admission and tested negative for respiratory viruses by Luminex xtag RVP RT-PCR (Luminex, Austin, TX) and Centers for Disease Control and Prevention (CDC) Novel A/H1N1 RT-PCR performed at the Illinois Department of Public Health (IDPH). The patient had a chest CT scan that showed bilateral upper lobe confluent airspace opacities with multiple small lung cysts and scattered micronodules. Bronchoscopy was performed on July which found copious thick clear secretions and scattered hyperemia and airway wall edema throughout both lungs. The BAL was positive for Pandemic (H1N1) 00 influenza by both Luminex and CDC RT-PCR. The patient improved clinically and was discharged without antiviral treatment. Patient A -year-old female who was1 weeks pregnant was admitted to RUMC on 1 October 00 with 1 week of fevers, cough and myalgias. She had a past history of asthma and thrombophilia secondary to a methylenetetrahydofolate reductase (MTHFR) mutation, with previous miscarriages and was on enoxaparin for deep venous thrombosis. She was tachycardic with a SpO of % on RA. CXR showed a left lower lobe infiltrate and she was started on ceftriaxone, azithromycin and oseltamivir. Chest CT scan showed multilobar pneumonia. Nasopharyngeal swab collected on admission was negative by Luminex and CDC RT-PCR. The patient s condition gradually deteriorated with nausea, vomiting, diarrhea and worsening hypoxia requiring intubation on hospital day. Bronchoscopy done that day showed diffuse airway petechiae. The BAL was positive for Pandemic (H1N1) 00 influenza by Luminex and CDC RT-PCR. The patient was started on IV zanamivir but her condition worsened and ECMO was initiated on October. The patient expired on 0 October. Patient Downloaded from on December, 01 by guest

5 A -year-old male with obstructive sleep apnea, complex partial seizures and partial right-frontal lobectomy was admitted to RUMC on November 00 for increased seizure frequency and 1 day of fevers to. C. He also reported shortness of breath but had normal SpO of % on RA with negative CXR. Nasopharyngeal swab collected on admission was negative by Luminex and CDC RT-PCR. The patient s year old son was also reportedly ill with fever and cough. The patient had intermittent low-grade fevers and a dry cough during his hospitalization with fluctuating SpO from mid-0 s to low 0 s. A chest CT done on November showed mild bibasilar ground glass opacities. Because of persistent desaturations CXR was repeated on November and now showed new bilateral infiltrates. There were no other patients or staff with Pandemic (H1N1) 00 influenza on the same medical floor. On November, the patient was intubated because of worsening hypoxemia and a bronchoscopy and repeat nasopharyngeal swab were performed. The repeat nasopharyngeal swab was again negative for Pandemic (H1N1) 00 influenza by Luminex and CDC RT-PCR but the BAL was positive by both assays. The patient was extubated on 1 November and discharged home after completion of a day course of peramivir. The 00 H1N1 Influenza A pandemic has posed a number of unexpected challenges and many unanswered questions for the diagnostic microbiology laboratory. One of the fundamental issues that remains unresolved is what is the best specimen for diagnosis of influenza. Published opinions range widely regarding the diagnostic sensitivities of nasal aspirates versus washes or swabs, regular versus flocked swabs or combined nasal and throat versus a single nasopharyngeal sample (1,,, ). Part of the difficulty in interpreting these studies include the use of different study populations, employment of a wide variety of sampling techniques, use of different diagnostic tests such as immunofluorescence, culture and nucleic acid amplifcation tests and selection of Downloaded from on December, 01 by guest

6 different groups of respiratory viruses for testing. Current CDC guidelines for laboratory diagnosis of Pandemic (H1N1) 00 virus recommend the collection of either nasopharyngeal swab, nasal aspirate or a combined nasopharyngeal and oropharyngeal swab (). However, there are few data on the viral kinetics of Pandemic (H1N1) 00 in humans and consequently the optimal body site for testing. Our report highlights the potential for false negative results with RT-PCR performed using nasopharyngeal swabs in patients with Pandemic (H1N1) 00 virus infection. Potential reasons for this include false negative RT-PCR due to technical reasons or inadequate sampling from poorly collected nasopharyngeal swabs. However, all of our in-house RT-PCR results concurred with the CDC RT-PCR assay making it unlikely that these were false negative RT-PCR results. In addition, patient had a second negative nasopharyngeal swab collected simultaneously with the BAL specimen which argues against poor sampling as a likely explanation. While it remains unclear if the use of nasal aspirates may improve diagnostic yield for Pandemic (H1N1) 00 influenza compared to nasopharyngeal swabs, we are aware of an unpublished case at Loyola Medical Center, Chicago in which a -year-old patient with severe influenza pneumonia had a negative nasal aspirate RT-PCR on admission but was later positive for Pandemic (H1N1) 00 influenza on BAL by both ProFlu+ (Gen-Probe Inc., San Diego) and CDC RT-PCR (personal communication, Paul Schreckenberger, PhD). An alternative plausible reason for our findings is that there is more extensive viral replication in the columnar epithelial cells of the lower respiratory tract compared to the squamous epithelial cells of the upper respiratory tract. In a study of ferrets infected with seasonal and Pandemic (H1N1) 00 viruses, Munster V J et al. reported that while seasonal A/H1N1 virus replication was confined to the nasal cavity, Pandemic (H1N1) Downloaded from on December, 01 by guest

7 virus also replicated in the trachea, bronchi and bronchioles (). Childs et al. found that compared to seasonal human H1N1 virus and triple reassortant swine H1N1 virus, the Pandemic (H1N1) 00 virus is able to bind efficiently to both α- and α--linked sialyl glucan receptors (). Binding to α--linked receptors is associated with the ability of influenza viruses to cause disease in the lower respiratory tract, where there are a greater proportion of α- compared to α--linked sialyl glucans (). A recent report by, Blyth et al. found that of 1 patients with severe Pandemic (H1N1) 00 influenza requiring intensive care unit admission and mechanical ventilation had negative RT-PCR specimens from the upper respiratory tract but positive bronchoscopic specimens (). Similarly, all of our patients had evidence of lower respiratory tract disease and of patients had severe influenza pneumonia requiring intensive care unit admission and mechanical ventilation. These findings suggest that patients with an influenza-like illness and pneumonia with negative nasal aspirate or swab RT-PCR and no other explanation for their illness should undergo lower respiratory tract sampling for influenza RT-PCR. Establishing the diagnosis of influenza in hospitalized patients is important both for treatment and infection control purposes and may reduce orders for further diagnostic tests. Downloaded from on December, 01 by guest

8 References: 1. Abu-Diab A, Azzeh M, Ghneim R, Ghneim R, Zoughbi M, Turkuman S, Rishmawi N, Issa AE, Siriani I, Dauodi R, Kattan R, Hindiyeh MY. Comparison between Pernasal Flocked Swabs and Nasopharyngeal Aspirates for Detection of Common Respiratory Viruses in Samples from Children. J Clin Microbiol 00; : 1-1. Blyth CC, Iredell JR, Dwyer DE. Rapid Test Sensitivity for Novel Swine-Origin Influenza A (H1N1) Virus in Humans. N Engl J Med Accessed November 0, 00. DOI:./NEJMc000. Chan KH, Peiris JS, Lim W, Nicholls JM, Chiu SS. Comparison of nasopharyngeal flocked swabs and aspirates for rapid diagnosis of respiratory viruses in children. J Clin Virol 00; : -. Childs R A, Palma A S, Wharton S et al. Receptor bindings specificity of pandemic influenza A (H1N1) 00 virus determined by carbohydrate microarray. Nature Biotech 00; (): -. Centers for Disease Control and Prevention (CDC). Interim Guidance of Specimen Collection, Processing and Testing for Patients with Suspected Novel Influenza A (H1N1) Virus Infection. Accessed on 1 November, 00. Lieberman D, Lieberman D, Shimoni A, Keren-Naus A, Steinberg R, Shemer- Avni Y.. Identification of Respiratory Viruses in Adults: Nasopharyngeal versus Oropharyngeal Sampling. J Clin Microbiol 00; : - Downloaded from on December, 01 by guest

9 Moore C, Corden S, Sinha J, Jones R. Dry cotton or flocked respiratory swabs as a simple collection technique for the molecular detection of respiratory viruses using real-time NASBA. J Virol Methods 00;1: -. Munster VJ, de Wit E, van den Brand JM, Herfst S, Schrauwen EJ, Bestebroer TM, van de Vijver D, Boucher CA, Koopmans M, Rimmelzwaan GF, Kuiken T, Osterhaus AD, Fouchier RA.. Pathogenesis and Transmission of Swine-Origin 00 A (H1N1) Virus in Ferrets. Science. 00 ;:1-. Petric M, Comanor L, Petti CA. Role of The Laboratory in Diagnosis of Influenza during Seasonal Epidemic and Potential Pandemics. J Infect Dis 00:1:S- 1. Shinya K, Ebina M, Yamada S, Ono M, Kasai N, Kawaoka Y. Avian flu: influenza virus receptors in the human airway. Nature 00; 0:- Downloaded from on December, 01 by guest

10 Table 1. Characteristics of patients with positive BAL for Pandemic 00 (H1N1) Influenza Patient no. Age/ Sex Underlying conditions* Admit date/ Diagnosis Date and result of nasopharyngeal swab/aspirate RT-PCR# Date and result of BAL RT- PCR Radiographic findings ICU Admission Mechanical ventilation or ECMO (days) Antiviral Treatment Length of stay/outcome 1 0/M Asthma July /F Pregnancy Asthma MTHFR mutation* Deep Venous Thrombosis /M Complex partial seizures Obstructive sleep apnea Obesity Hyperlipidemia Viral Syndrome 1 Oct Pneumonia Nov Seizures July/ Negative July/ Positive 1 Oct/ Negative 0 Oct/ Positive Nov/ Negative Nov/ Positive Bilateral Upper lobe opacities Left lower lobe pneumonia Bibasilar opacities No No No days/ Survived Yes Yes Mechanical ventilation () ECMO ( ) Mechanical ventilation () Oseltamivir/ Zanamivir IV Oseltamivir/ Peramivir IV 1 days/ Deceased 1 days/ Survived Downloaded from MTHFR = Methylenetetrahydrofolate reductase # RT-PCR = real-time polymerase chain reaction BAL = Bronchoalveolar lavage ECMO = Extracorporeal membrane oxygenation on December, 01 by guest 1