In this study, we presented data demonstrating that a strategy of pooling specimens can be used to test for the presence of influenza virus using the CDC rRT-PCR swine flu panel (24
). Our data showed that the increase in cellular material, including nucleic acid, due to pooling of multiple specimens did not affect detection of the 2009 H1N1 influenza virus by rRT-PCR (). Additionally, there was no loss of sensitivity or dilution of a positive specimen in a pool since the extraction of each pool was concentrated at the elution step. As expected, the pooling of multiple specimens positive for the 2009 A/H1N1 virus with higher concentrations of viral nucleic acid did not have an inhibitory effect on PCR, as demonstrated by the CT
values of pools being similar to those of the specimen with the lowest CT
value (). Furthermore, the presence of other respiratory viruses, such as adenovirus and parainfluenza virus, or multiple positive influenza virus specimens did not impede PCR amplification of the 2009 H1N1 influenza virus (). Finally, the results reported by three state public health laboratories testing a blinded pooling panel showed both inter- and intralaboratory reproducibility ( and ). These results suggest that NP/throat swab specimens could be pooled, extracted, and tested for influenza virus by PCR with levels of detection and reproducibility similar to those obtained by processing each specimen individually.
The MagNA Pure TNA kit is one of the extraction methods validated for the CDC rRT-PCR swine flu panel (24
). To optimize pooling of up to 10 specimens requires a larger input volume than that recommended for the MagNA Pure TNA kit (Roche Diagnostics, Indianapolis, IN). The large-volume TNA kit (Roche Diagnostics, Indianapolis, IN) allowed for processing of sample volumes up to 1 ml on the MagNA Pure LC instrument. Ten specimens were pooled, extracted, and concentrated upon elution using this kit. The sensitivity of the large-volume TNA kit was similar to that of the small-volume MagNA Pure TNA kit (). Detection of a specimen with a CT
value in the high 20s was not compromised when mixed with four or nine negative specimens ( and data not shown). This demonstrated that viral nucleic acid is not diluted in pools of negative specimens, since the final elution volume of the large-volume TNA kit is 100 μl, the same as the elution volume of the small-volume MagNA Pure TNA kit recommended by the CDC rRT-PCR flu panel protocol. Hence, essentially all nucleic acid from the large-volume TNA kit is captured and not diluted. Others have also evaluated the MagNA Pure LC large-volume TNA kit and shown acceptable sensitivity when used in conjunction with real-time PCR for detection of other viruses, such as hepatitis B and C viruses (3
The pooling strategy proposed by this study does not necessitate large structural or workflow changes for laboratories currently performing the CDC rRT-PCR flu panel or the rRT-PCR swine flu panel using the MagNA Pure LC or other instruments recommended for these two protocols. Three state public health laboratories successfully tested a blinded influenza virus pooling panel without extensive supplementary training or the purchase of additional equipment. Laboratories, however, are required to develop and validate an algorithm and workflow scheme for tracking specimens in pools.
For laboratories lacking automated extraction instrumentation, manual extraction methods, such as the QIAamp viral RNA minikit (Qiagen, Valencia, CA), one of the manual extraction methods validated for the CDC rRT-PCR flu panel, can be used to extract and concentrate pools of specimens. However, laboratories should take into consideration that manual extraction methods can be more labor intensive than automated systems. Additional studies need to be performed in order to determine whether manual extraction methods will provide similar results using a pooling protocol and be useful in surge situations.
The decision to implement a pooling protocol should take into consideration the current positivity rate. The benefit of pooling is nullified if every pool yields a positive result, which may occur during periods of high positivity rate, and thus demands subsequent testing to reassess every specimen in the pool individually. One strategy to circumvent this is to alter the size of the pool to account for the prevalence of the situation. For instance, if the positivity rate is near 10%, pools of five specimens may prove more practical than 10 specimens. Pools of one positive and four negative specimens showed similar results in terms of CT values to pooling one positive and nine negative specimens (data not shown).
This investigation did not address the potential cost effectiveness of pooling influenza virus specimens. However, pooling schemes have been shown to increase testing capacity and lower the cost per test for detection of viral infections such as Chlamydia trachomatis
, HIV, and hepatitis C (4
). These studies also optimized the pooling strategy such that sensitivity was not diminished due to dilution of positive specimens in pools of increasing size. Laboratories should be aware, however, that a negative pool result would not distinguish between a true negative and an indeterminate/inconclusive result due to poor specimen collection or handling. Theoretically, our pooling design, if applied properly, can increase testing capacity and conserve reagents during times of pandemic surveillance, surge testing, clearing of backlogs, and in limited-resource settings. In addition, this study may serve as a model for pooling specimens for diagnosis of other infectious diseases.
In summary, we have demonstrated that NP/throat swab specimens can be pooled and tested for the presence of influenza virus without sacrificing sensitivity. The pooling scheme can increase testing capacity if used in combination with an automated extraction method, such as the MagNA Pure LC instrument, and the CDC real-time PCR assay for influenza virus.