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PCR Failures Raise Novel Influenza Pandemic Concerns
Recombinomics Commentary 19:30
December 25, 2011

On October 22, a respiratory specimen from the patient was positive for influenza at the hospital. On October 28, the virus was identified by real-time, reverse transcription–polymerase chain reaction (rRT-PCR) testing at the Indiana State Public Health Laboratory as an inconclusive influenza A virus, consistent with results seen with other recent A(H3N2)v infections. On October 31, genome sequencing at CDC confirmed the virus as A(H3N2)v with the M gene from the A(H1N1)pdm09 virus, similar to the viruses identified in the other cases of human infection in the United States since August 2011.

On November 21, a respiratory specimen was collected. Rapid diagnostic tests conducted by the hospital were negative for influenza and respiratory syncytial virus, but influenza A was identified by an alternative rRT-PCR at the hospital. The specimen was forwarded to the West Virginia Office of Laboratory Services, where it was identified as a suspected influenza A (H3N2)v virus. Subsequent genome sequencing conducted at CDC confirmed the virus as A(H3N2)v with the M gene from the A(H1N1)pdm09 virus.

A respiratory specimen obtained from the second child on December 7 was inconclusive by rRT-PCR at the West Virginia Office of Laboratory Services; however, the specimen was confirmed as influenza A (H3N2)v with the M gene from the A(H1N1)pdm09 virus via genome sequencing at CDC.

The above comments from the CDC December 23 early release MMWR describe three additional trH3N2 cases, including the two from the daycare center cluster in Mineral County, West Virginia.  The Indiana veterinarian (59M – A/Indiana/10/2011) was inconclusive in the CDC PCR test, as was the second case in West Virginia (3M – A/West Virginia/07.2011).  The first West Virginia case (1F – A/West Virginia/06/2011), was initially negative on a rapid test, but a suspect trH3N2 case using the PCR kit.  These descriptions highlight the technical challenges related to the detection and correct diagnosis of these cases using PCR testing in the absence of follow-up sequencing.

The above data compliments reports on the detection of the earlier pediatric H2N2pdm11 cases which were reported after the September 2 early release MMWR, which requested samples from cases with swine exposure (9F - A/Pennsylvania/10/2011, 9F- A/Pennsylvania/11/2011, 8M – A/Maine/06/2011, 8M - A/Maine/07/2011).  This request extended the CDC program that looks for novel influenza in cases with ILI symptoms in the off season, when seasonal flu levels are low due to conditions that limit transmission. 

During the off season, lab testing of influenza cases is limited.  The second Pennsylvania case listed above (PA/11/11) was identified at the University of Pittsburgh Medical Center.  In the off season, the CDC PCR test kit (FLUNAT) is not used.  Instead another PCR panel (RVPNAT), which detects influenza A, seasonal flu, and other respiratory viruses is used, and that kit identified PA/11/11 as influenza positive, but seasonal H3 and H1 negative (unsubtypable).

However, few samples tested at state or local labs are forwarded to the CDC, leading to a heavy sample bias, especially after the CDC requests samples from ILI cases with swine exposure.  Thus, samples that are inconclusive or weakly positive for seasonal H3 will not be forwarded, especially if cases are mild, like the second case in West Virginia, who did not seek medical treatment and recovered.

The detection of trH3N2 in patients with swine exposure in the off period, or early in the flu season, is associated with the type of samples tested and sequenced by the CDC.  In West Virginia only two influenza cases have been reported this season, and these were the two trH3n2 cases from the Mineral County daycare center.  Similarly, Maine has only reported two cases this season, and these were the two H3N2pdm11 cases with “swine exposure”.

Thus, in the off season, or early in the flu season, most of the samples from children under 10 that were sequenced by the CDC were novel cases, with “swine exposure” in the off season, and more recently without swine exposure, but in cases like WV/07/11, linked to a confirmed trH3N2 case.

Although the CDC continues to site trH3n2 in swine, there have been no reported matches in the 150 swine sequences submitted prior to the most recent sample from New York,
A/swine/NY/A01104005/2011, which was a match for the first 10 H3N2pdm11 cases in 2011.  Similarly, there are no matches for the two isolates from the West Virginia cluster.

However, the failure of the PCR test to conclusively identify trH3N2 via a positive result with the H1N1pdm09 NP target, raises concerns that the increased surveillance of ILI cases case, especially in children under the age of 10, will yield false negatives if not associated with CDC sequencing.  Examples of cases that should be sequneced include those from outbreaks of pneumonia like Shelby County, Indiana or Erie County, Pennsylvania.

In the 2011/2012 season, 8 of the 9 such sequences have been novel cases (H3N2pdm11, trH3N2, trH1N2), raising concerns that these novel viruses are widespread in children under the age of 10, and an aggressive sequencing program in this target population is long overdue.

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