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Friday, December 23, 2016 Written by Ellen Jo Baron, Ph.D., D(ABMM), Prof. Emerita, Stanford University Director of Medical Affairs, Cepheid

Case Study Award: Cepheid Xpert® Flu Assay Results: Positive in More than One Way

Jack L. Brothers, MT(ASCP), Microbiology Technical Supervisor, works at a surprisingly beautiful hospital (Figure 1) in an unlikely place: Anchorage, Alaska. The Joint Base Elmendorf-Richardson is the largest U.S. military installation in Alaska and was created when former Elmendorf Air Force and Fort Richardson Army bases were merged in 2010.

The base medical facility has 60 inpatient beds but its larger mission is to provide medical care to over 35,000 joint service members, dependents, Veterans Administration (VA) patients, and retirees throughout Alaska. One usually does not think about VA hospitals as receiving many pediatric patients, but in this case, families of service members are a large component of the population served. For allowing us to benefit from his experience with a GeneXpert assay, Jack will receive a copy of the recently published second edition of the popular book published by ASM Press, Molecular Microbiology: Diagnostic Principles and Practice, hand signed by the two co-editors David Persing and Fred Tenover. Jack Brothers elegantly described the situation and how the Cepheid Xpert Flu assay, with final results delivered less than 2 hours from sample collection, saved one tiny patient and his family a lot of grief and potential further trauma (notably enabling the cancellation of a spinal tap). This is Jack’s case description.

The parents of a two month old male infant brought their child to the 673rd Medical Group DOD/VA Joint Venture Hospital Emergency Department late one night in March of 2012. The chief complaint was a high fever with intermittent shortness of breath and "gasping for air with a dry cough." Vital signs revealed a rectal temperature of 101.1°F, pulse of 194, respirations of 40 per minute, and O2 Saturation of 98% on room air. In addition to the tachycardia and dyspnea, the physical exam noted pharyngeal erythema, and a blanchable rash on the cheeks and chest. Blood was collected for a complete blood count (CBC) and culture, and an intravenous line was inserted. A nasal washing (using normal saline) was also obtained and sent to the laboratory for viral testing, and a chest X-ray was ordered.

The CBC was essentially normal, as was the chest X-ray. A rapid antigen detection assay for respiratory syncytial virus (RSV) was performed on the nasal washings and was negative. Rapid antigen detection assays for influenza A and influenza B performed on the same sample were also negative and were reported to the physician handling the case in the Emergency Department.

EJB comment: Luckily for the baby, Jack’s laboratory policy requires reflexing samples from some patients that are negative by the rapid influenza antigen assay to testing with the Cepheid Xpert Flu assay, designed to detect both influenza A, B, and A[2009 H1N1], as he explains further below.

Per laboratory policy, Senior Airman Jessica Green pipetted 600µl of the original nasal wash into 3ml of Universal Transport Medium (UTM), and then used 300µl of the diluted sample to set up a Cepheid Xpert Flu assay (Figure 2). The Cepheid® RT-PCR assay was "influenza A positive, no influenza B detected, no influenza A 2009 H1N1 (A/California/7/2009-like) detected", and was certified. A few minutes later, she received a call from the doctor. He wanted to make absolutely sure that he was interpreting the results correctly, and that the PCR test was indeed positive for Influenza A. Jessica explained that the rapid antigen tests can sometimes give false negative results, and that the PCR is much more sensitive and also more accurate. He thanked her, and said that there was no longer a need to do a spinal tap on the baby since they now knew the origin of his fever. The child was given Tamiflu and sent home.

The policy at the 673rd Medical Group Laboratory is to confirm the results of rapid influenza antigen tests by performing reflex PCR testing on all samples obtained from patients considered at high risk for respiratory distress, and on all samples collected from inpatients. We define high risk for influenza testing to be patients less than one year of age, patients over 65 years of age, and those with known conditions that would make them more vulnerable to viral infections, such as chronic obstructive pulmonary disease (COPD) or immune suppression. We perform reflex testing using the real-time Reverse Transcription Polymerase Chain Reaction (RT-PCR) Xpert Flu assay on a Cepheid 4-well GeneXpert instrument.

Most PCR testing requires an extraction procedure that necessitates batching of samples. In this case the Cepheid GeneXpert enabled a busy Technician to perform an accurate, sensitive RT-PCR flu assay at 2:30 in the morning, while the patient was still in the Emergency Department. Less than 2 hours elapsed from the time the sample was collected until the final PCR result was certified and sent to the provider. As a result, additional expensive testing to determine the cause of a fever of unknown origin was avoided, and the relieved parents were able to return home with an accurate diagnosis for their child in minimum time. The Alaska State Virology Laboratory in Fairbanks confirmed the Cepheid Xpert Flu result (A/H3), but those results were not available until the following week.