Pooling Nasopharyngeal/Throat Swab Specimens To Increase Testing Capacity For Influenza Viruses By Pcr

Pooling Nasopharyngeal/Throat Swab Specimens To Increase Testing Capacity for Influenza Viruses by PCR

Real-time PCR methodology can be applied to rapidly and accurately detect influenza viruses. During times of surge testing or enhanced pandemic surveillance, public health laboratories (PHLs) may experience overwhelming demand for testing, evenwhile the prevalence of positive specimensremains low. To improve laboratory capacity and testing efficiency during surges, we evaluated whether nasopharyngeal (NP)/throat swab specimens can be pooled and tested for the presence of the 2009 H1N1 influenza virus without a reduction in sensitivity. Pools of 10 specimens were extracted and concentrated upon elution on the MagNA Pure LC instrument, and real-time PCR was performed on the AppliedBiosystems 7500 Fast platform, using the CDC swine influenza virus real-time RT-PCR detection panel (rRT-PCR swine flu panel). Specimens in positive pools were singly reextracted and retested by PCR to identify individual positive samples. Initial studies showed that spiking a pool of nine negative specimens (100
l each) or 900
l of virus transport medium with 100
l of a positive clinical specimencaused no loss of sensitivity by rRT-PCR testing. Pools containing either multiple positive specimens or specimens positive for other respiratory viruses also showed no negative effect on crossing threshold (CT) values. To test the robustness of the pooling protocol, a panel of 50 blinded samples was sent to three PHLs and tested in five pools of 10. All PHLs correctly identified the positivespecimens.This study demonstrates the feasibility of using a pooling strategy to increase capacity and conserve resources during surge testing and periods of enhanced influenza surveillance when the prevalence is low.

Reverse transcription–real-time PCR (rRT-PCR) is an important diagnostic tool for detection of influenza virus in public health laboratories. The Centers for Disease Control andPrevention (CDC) human influenza virus rRT-PCR detection and characterization panel (rRT-PCR flu panel) was cleared by the U.S. Food and Drug Administration (FDA) for diagnosing human influenza virus infections and H5N1 viruses (6) in September 2008. Likewise, the CDC real-time RT-PCR protocol for detection and characterization of swine influenza virus (rRT-PCR swine flu panel) (24) was authorized by theFDA under an emergency use authorization (EUA) to diagnose 2009 H1N1 influenza virus infections (7) in April 2009 and subsequently cleared by the FDA in June 2010. A combination of the seasonal flu panel with the swine flu panel was cleared by the FDA in September 2011 (8). The use of either protocol allows for rapid detection of influenza viruses from clinical specimens. However, during aninfluenza outbreak or pandemic, test workloads in public health laboratories may rapidly outstrip testing capacity, and reagent shortages may occur. The recent outbreak of a novel influenza A/H1N1 virus (2009 H1N1) (1, 2) is a clear example. At the start of the pandemic, there was a high volume of specimens that required testing but a low prevalence of the 2009 H1N1 strain. One possible way to increasetesting capacity and conserve reagents is to pool specimens prior to RNA extraction, concentrate the RNA, test the pools, and subsequently retest single specimens from positive pools to identify the positive specimens.

Pooling specimens to increase efficiency of testing and cost effectiveness is not unprecedented. Testing specimens in pools has been used to detect infections such as humanimmunodeficiency virus (HIV) and the hepatitis B and C viruses (5, 18). Blood banks worldwide are able to screen millions of blood donations by implementing the minipool nucleic acid amplification technology (NAT) testing method to detect transfusion-transmissible viruses (9, 14, 18, 20, 21, 23). The size of the minipools differs from country to country, but each sample in a positive pool is...