The COVID-19 pandemic has increased the workload on testing laboratories manifold due to the highly transmissible nature of the virus, necessitating the early detection of cases [12]. An effective method to limit the spread of infection is to break the transmission chain and isolate positive patients early [13]. However, screening individuals who harbor the virus, irrespective of disease, requires more testing. This increased demand for early diagnosis puts a tremendous burden on testing laboratories. Historically, the pooling strategy has proven a feasible, reliable, and effective method for efficiently testing many samples in resource-limited setup [14]. Various studies on the application of pooled testing to clinical samples during the low prevalence of disease have shown the efficacy of this strategy in the mass screening of suspected patients. Cardoso et al. has reported successful nucleic acid amplification testing (NAT) of pooled samples for screening HBV, HCV, and HIV in blood donors [15]. Rogers G et al. have shown the feasibility of pooling for screening of Chlamydia trachomatis in pregnant women [16]. Van T et al. successfully reported the accuracy of pooled testing for the influenza virus by PCR and its potential application in routine testing [17]. Bharti AR et al. has similarly validated the pool testing strategy for malaria PCR on serum samples [18]. In the COVID-19 pandemic, Barak N et al have reported the successful application of the pooling strategy in SARS CoV-2 screening and surveillance from Israel in the early days of the pandemic [19]. Reducing testing time helps in early clinical diagnosis, triage, and treatment of patients [13, 20,]. Given this, we planned to evaluate the validity of the pooled testing strategy advised by the ICMR for the COVID-19 RT-PCR test.
In our study, sensitivity, specificity, and negative and positive predictive values were 95% for the pooling method compared to individual testing. The negative predictive value (NPV) was found to be 99%, which is of utmost importance when applying the pooling strategy to mass testing. Salazar A has similarly reported an NPP of 99.79% for pooled testing of SARS-CoV-2 conducted in Spain [21]. Excellent NPP in our study indicates the pooling method's usefulness in filtering out true negative samples, which constitute the bulk of the sample load during mass screening. The pooling method also showed a high positive predictive value (97.3%). The viral nucleic acid undergoes n times dilution in a pool with n number of samples (e.g., a positive viral RNA will undergo 5 times dilution in a pool of 5 samples in our study). Subsequently, there is an increase of x in the CT value of the sample (x is calculated as 2x = n, where n is the number of samples in a pool). This sometimes results in the observed CT value of the pool exceeding the cut-off value of the PCR cycle [19]. A slightly lower positive predictive value may be attributable to the dilution of RNA. This factor should be carefully considered while applying the pooling strategy in diagnostic laboratories.
The average difference in CT values of the E, ORF, and RdRp genes observed in our study was 2.49 [(2.159–2.821) at 95% CI], 2.62 [(2.254–2.986) at 95% CI], and 2.28 [(1.780–2.820) at 95% CI], respectively. This difference is to the expected difference in CT value arising from the dilution of samples while preparing a pool of 5 samples. In our study, pools of 5 samples were prepared, which resulted in an expected increase in the CT value of pools by 2.32 (22.32 = 5).
However, we also observed that sometimes there was a decrease in the CT value of the pool compared to individual samples. A study by More S in Oklahoma, USA, has also reported similar findings [1]. We believe this may result from human factors, which result in variations of PCR master mix preparation and dilution affecting the PCR cycle when different lab technologists perform the PCR. Dilution of inhibitory factors when a pool is prepared may also contribute. Various other studies have found similar results with varying pooled sample sizes [19, 22, 23]. Overall, the pooling strategy advised by the ICMR was validated in our study.
Various strategies for pooling have been described in the literature [24]. Pooling can be done at the source, where a pooled sample is made by collecting and dipping swabs from various patients into a single VTM. Although this is the most cost-effective method, the drawback of this method is that only pooled samples can be tested. If the individual patient has to be tested, it requires repeat sampling. Therefore, this method is useful for population studies or epidemiological surveys but cannot be used for individual patient diagnoses. Another method available is the pooling of extracted nucleic acids after extraction. Although this method allows for individual and pooled sample testing, it has a negligible influence on saving resources as the extraction process is unaffected [14]. In our study, pooling was applied to individual VTM samples during the extraction process in the laboratory. This method allows the testing of pooled as well as individual samples. However, the drawback of any pooling method is the decrease in detection sensitivity due to the dilution of samples and nucleic acids [15]. Thus, there is always a tradeoff between the acceptable decrease in the sensitivity of detection and the availability of resources.
After validation, the strategy was applied in the laboratory to routine testing during the low prevalence phase of the pandemic to evaluate its cost-effectiveness. A 65% reduction in tests was observed when pooling was applied to samples received routinely for Covid-19 testing. Various other studies have reported a 40–99% reduction in tests when a pooling strategy was applied in their setup for Covid-19 testing [14]. This is a significant cost-saving measure considering India is a resource-limited country with a large population, where wide-scale testing for COVID-19 requires prudent utilization of resources. The Safdarjung Hospital is a public hospital where all microbiological investigations are provided free of charge to the patient.
Further, the national capital territory (NCT) government had mandated a cost cap on COVID-19 RT-PCR testing at INR 800 (about USD 9.82). The overall cost saved over 3 months and 10 days was INR 8,917,000 (or USD 1094.08). The wide-scale testing performed for COVID-19 in our country with a large population calls for the economical utilization of resources. The limited number of staff available in the laboratory had to be efficiently managed during the increased workload of the pandemic. The maximum number of RNA extractions cumulatively performed using manual and automated setup was 140. A minimum of 2–3 PCR runs were required to process approximately 200 samples daily. This resulted in a turnaround time of 48 hours to report all samples received in one day. However, applying the pooling strategy led to faster reporting of results, reducing TAT from 48 hours to 24 hours. This led to faster reporting of tests, as well as requiring fewer technologists to run the tests.