Cost-Effective Pooling of DNA from Nasopharyngeal Swab Samples for Large-Scale Detection of Bacteria by Real-Time PCR

Abstract

We investigated the potential of pooling DNA from nasopharyngeal specimens to reduce the cost of real-time PCR (RT-PCR) for bacterial detection. Lyophilization is required to reconcentrate DNA. This strategy yields a high specificity (86%) and a high sensitivity (96%). We estimate that compared to individual testing, 37% fewer RT-PCR tests are needed. R eal-time PCR (RT-PCR) is an essential tool for routine diagnostics and large epidemiological studies of infectious diseases (1, 2). However, its cost remains significant and limits its use. To reduce it, samples can be pooled (3)(4)(5). This idea was suggested by Dorfman in 1943 (6) and has been used for the serological diagnosis of infectious diseases. Pooling samples is also an efficient way to screen for the nucleic acids of viruses, bacteria, or parasites (4,5,(7)(8)(9)(10)(11)(12)(13)(14)(15). However, pooling can decrease the sensitivity of assays due to the dilution of the samples, which is problematic in clinical diagnostics (16). Here, we investigated the potential of pooling DNA from clinical specimens using lyophilization to concentrate the pooled DNA and maintain the sensitivity of RT-PCR.We screened 2,380 nasopharyngeal swabs (Remel, USA) by RT-PCR to detect their rates of Streptococcus pneumoniae, Haemophilus influenzae, and Klebsiella pneumoniae carriage. DNA was extracted using a Macherey-Nagel NucleoSpin-96 kit. A total of 119 pools containing 80 l of DNA from 20 patients was frozen for 4 h at Ϫ20°C, lyophilized using a Lyovac GTZ instrument (Leybold Heraeus, France), and redissolved in 80 l of sterile water. The effectiveness of lyophilization was verified with pool controls, including one positive sample with a known threshold cycle (C T ) value for each bacterium. The specificities of the primers and probes (17, 18) were verified in silico by conducting a BLAST search in GenBank and performing RT-PCR on 10 to 15 closely related bacterial strains present in the respiratory tract (see Tables S1 and S2 in the supplemental material). RT-PCR was performed using 5 l of DNA per reaction and a 7900HT thermocycler (Applied Biosystems). Nuclease-free water was used as a negative control, and DNA from a clinical strain was used as a positive control. The cutoff C T value for positive results was Յ38. Each sample was also tested individually. The DNA extraction quality of each pool was verified by RT-PCR targeting the human beta-actin gene (19).The prevalences for the individual samples were 9% (215/ 2,380) for S. pneumoniae, 7% (169/2,380) for H. influenzae, and 4% (95/2,380) for K. pneumoniae. Among the pools tested, 69% (82/119) were positive for S. pneumoniae, 53% (63/119) were positive for H. influenzae, and 53% (63/119) were positive for K. pneumoniae (Table 1). Among them, 184/357 (52%) were truepositive results and 142/357 (40%) were true-negative results. However, 24/357 (7%) were false positives (negative individual samples), and 7/357 (2%) negative pools contained positive individual specimens, yielding false negatives (see Table S3 in the sup...

Regression analysis and variable selection for two‐stage multiple‐infection group testing data

“…Following the proof of theorem 3 in the work of Wang et al, 41 one can show that, with the optimal ( 1 , 2 ) from (8), pr( 1, =  1 , 2, =  2 ) → 1 as N → ∞. In other words, any ( 1 , 2 ) that does not lead to the correct variable selection cannot be selected by (8) when the number of individuals is large. The purpose of this subsection is to provide a shrinkage estimator of the regression coefficients, of which the sparsity pattern can help us identify the truly relevant risk factor for each infection.…”

“…Pooled testing (also known as group testing) was initially proposed to screen for syphilis among War World II American army recruits . Since this seminal work, pooling techniques have been successfully implemented to screen for many other infectious diseases, including HIV, hepatitis B virus (HBV), hepatitis C virus (HCV), influenza, and herpes . Besides disease screening, many other areas, including genetics, veterinary science, medical entomology, blood safety, and drug discovery, have also used the method of pooling.…”

Group testing regression models with dilution submodels

“…Dorfman is usually credited with its introduction, having proposed it to screen US soldiers for syphilis during World War II. Now widely regarded for its cost‐savings potential, group testing is commonly used in large‐scale sexually transmitted disease screening applications involving HIV, HBV/HCV,() chlamydia and gonorrhea, and for other infections including West Nile virus and influenza . Pooling individuals arises more generally in environmental monitoring, entomology, genetics, animal testing, drug discovery, and food safety …”

Group Testing for SARS-CoV-2: Forward to the Past?

“…Sensitivity at various group sizes is crucial for determining whether group testing is desirable and for estimating optimal group size. This can be informed by evaluating the effect of pooling one infected specimen with a varying number of uninfected specimens on the sensitivity and cycle threshold (Ct) values [18,21] and mathematical models taking into account viral load progression and dilution effects [22]. Early results from laboratory experiments suggest that a single positive SARS-CoV-2 sample can be detected in pools of up to 32 samples, with an estimated false negative rate of 10% [16].…”

“…However, because group testing potentially requires fewer tests to be performed, average time delays may actually be reduced, especially in a pandemic setting where laboratories are working at full capacity. For example, a study from France found that, compared with individual testing, pooling deoxyribonucleic acid (DNA) from nasopharyngeal specimens, and retesting positive group samples, considerably reduced both the cost and time delays of using RT-PCR for bacterial detection [21].…”