This dataset underlies AfriDx D6.7 report on Clinical study of NAT in COVID-19.

Summary of Project

The AfriDx Project comprises nucleic acid testing (NAT) for COVID-19 using the PATHPOD system and compared with the RT-qPCR as the gold standard. KNUST, KCCR and NMIMR received PATHPOD and cartridges from the DTU (see D2.2). The cartridges for the testing were shipped in two (2) batches. Training on PATHPOD usage was done and used for testing covid-19 samples. Data obtained was compared with the gold standard RT-PCR.

The overall the testing against RT-LAMP was circa 60% sensitive, but the specificity dropped from 80.0% in the first batch to 24% in the second batch. Analysis of the factors causing the drop in specificity is on-going.

In the first batch a total of 1,947 tests were conducted, with 531 positive and 1416 negative results, producing 302 samples returning a false positive (FP) 133 samples giving a false negative (FN). The data showed that the FNs could be related to the copy number of virus in the sample, with a strong correlation with RT-PCR C_T value and true positive/false negative LAMP outcome. The second batch of nucleic acid testing recorded a total of 1,612 test for N-gene in Ghana with 1208 positive and 404 negative tests. This showed an exceptionally high occurrence of false positive test results: 1134 (76.2%) samples returned a false positive (FP) compared with the RT-PCR and 49 (3.9%) samples gave a false negative (FN). Nevertheless, the correlation with C_T remained, suggesting that the PATHPOD was functioning correctly and that the results were revealing some contamination or deterioration of reagents or sample.

Some initial analysis of the raw data from PATHPOD revealed some characteristics of sample and/or reagent contamination as well as the outcome of a poorly sealed cartridge, that would result in an erroneous signal. Further analysis is needed to fully understand any design modifications that might be beneficial. The impact of shipping and storage on the cartridges also has potential impact and it is particularly noteworthy that the second batch of testing, using cartridges from the same manufacturing run as the first batch, performed less well.

Methodology

The PATHPOD equipment was placed on a clean and flat surface and switch on with the knob located at the back of equipment to turn on the equipment. The oropharyngeal sample in 300ul of PBS was heated at 95 ⁰C for 5min to inactivate virus. The master mix room table was disinfected with suitable disinfectant against DNA/RNA contamination. Wearing appropriate gloves, the Pathpod cartridge was removed from the refrigerator and allow 15-30 min at room temperature for the cartridge to acclimatize, and place in the chip holder. The attached temporary sealer film covering the wells was removed and discarded.

After short vortex of sample, 6 µl of sample and/or controls were added directly into the center of the well. the yellow paper from the PCR film was removed and placed over the film chip. The film was sealed properly to the chip using a soft roller ready for processing in the PATHPOD system.

Using the Pathpod keyboard, sample ID was entered and COV assay was selected. The start bottom was pressed to heat the machine, thereafter the cartridge was inserted and start bottom was pressed again to run the program. When the assay was completed, result was read from both the screen and the LEDs next to the keyboard. Ensuring that the position on the machine matches the position on the chip. The following interpretation was inferred as results:

GREEN LIGHT: NEGATIVE BLINKING RED LIGHT: POSITIVE YELLOW LIGHT: RE-TEST THE SAMPLE

Datasets

Dataset	Description
PATHPOD ID 25.zip	Raw data from all runs on PATHPOD Device ID 25
PATHPOD ID 26.zip	Raw data from all runs on PATHPOD Device ID 26
PATHPOD ID 30.zip	Raw data from all runs on PATHPOD Device ID 30
AfridX evaluation data_KNUST.V2.xlsx	Comparison data for RT-PCR and PATHPOD performed at KNUST
ALL tests_for DTU. V2.xlsx	Comparison data for RT-PCR and PATHPOD performed at NMIMR

This dataset underlies AfriDx D6.7 report on Clinical study of NAT in COVID-19.

Summary of Project

The AfriDx Project comprises nucleic acid testing (NAT) for COVID-19 using the PATHPOD system and compared with the RT-qPCR as the gold standard. KNUST, KCCR and NMIMR received PATHPOD and cartridges from the DTU (see D2.2). The cartridges for the testing were shipped in two (2) batches. Training on PATHPOD usage was done and used for testing covid-19 samples. Data obtained was compared with the gold standard RT-PCR.

The overall the testing against RT-LAMP was circa 60% sensitive, but the specificity dropped from 80.0% in the first batch to 24% in the second batch. Analysis of the factors causing the drop in specificity is on-going.

In the first batch a total of 1,947 tests were conducted, with 531 positive and 1416 negative results, producing 302 samples returning a false positive (FP) 133 samples giving a false negative (FN). The data showed that the FNs could be related to the copy number of virus in the sample, with a strong correlation with RT-PCR C_T value and true positive/false negative LAMP outcome. The second batch of nucleic acid testing recorded a total of 1,612 test for N-gene in Ghana with 1208 positive and 404 negative tests. This showed an exceptionally high occurrence of false positive test results: 1134 (76.2%) samples returned a false positive (FP) compared with the RT-PCR and 49 (3.9%) samples gave a false negative (FN). Nevertheless, the correlation with C_T remained, suggesting that the PATHPOD was functioning correctly and that the results were revealing some contamination or deterioration of reagents or sample.

Some initial analysis of the raw data from PATHPOD revealed some characteristics of sample and/or reagent contamination as well as the outcome of a poorly sealed cartridge, that would result in an erroneous signal. Further analysis is needed to fully understand any design modifications that might be beneficial. The impact of shipping and storage on the cartridges also has potential impact and it is particularly noteworthy that the second batch of testing, using cartridges from the same manufacturing run as the first batch, performed less well.

Methodology

The PATHPOD equipment was placed on a clean and flat surface and switch on with the knob located at the back of equipment to turn on the equipment. The oropharyngeal sample in 300ul of PBS was heated at 95 ⁰C for 5min to inactivate virus. The master mix room table was disinfected with suitable disinfectant against DNA/RNA contamination. Wearing appropriate gloves, the Pathpod cartridge was removed from the refrigerator and allow 15-30 min at room temperature for the cartridge to acclimatize, and place in the chip holder. The attached temporary sealer film covering the wells was removed and discarded.

After short vortex of sample, 6 µl of sample and/or controls were added directly into the center of the well. the yellow paper from the PCR film was removed and placed over the film chip. The film was sealed properly to the chip using a soft roller ready for processing in the PATHPOD system.

Using the Pathpod keyboard, sample ID was entered and COV assay was selected. The start bottom was pressed to heat the machine, thereafter the cartridge was inserted and start bottom was pressed again to run the program. When the assay was completed, result was read from both the screen and the LEDs next to the keyboard. Ensuring that the position on the machine matches the position on the chip. The following interpretation was inferred as results:

GREEN LIGHT: NEGATIVE BLINKING RED LIGHT: POSITIVE YELLOW LIGHT: RE-TEST THE SAMPLE

Datasets

First Batch Data:

Dataset	Description
PATHPOD ID 25.zip	Raw data from all runs on PATHPOD Device ID 25 in Batch 1
PATHPOD ID 26.zip	Raw data from all runs on PATHPOD Device ID 26 in Batch 1
PATHPOD ID 30.zip	Raw data from all runs on PATHPOD Device ID 30 in Batch 1
AfridX evaluation data_KNUST.V2.xlsx	Comparison data for RT-PCR and PATHPOD performed at KNUST
ALL tests_for DTU. V2.xlsx	Comparison data for RT-PCR and PATHPOD performed at NMIMR

Second Batch Data:

Dataset	Description
PATHPOD ID 25 - BATCH 2.zip	Raw data from all runs on PATHPOD Device ID 25 in Batch 2
PATHPOD ID 26 - BATCH 2.zip	Raw data from all runs on PATHPOD Device ID 26 in Batch 2
AFRIDx DATA SECOND BATCH.xlsx	Comparison data for RT-PCR and PATHPOD Second Batch