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Explainer: False positives in Covid-19 tests and the risk of errors as Singapore moves towards mass testing

SINGAPORE — As Singapore grows its Covid-19 testing capacity from 8,000 tests a day now to reach the daily target of 40,000 in future, the risks of straining the country’s lab capacity grows as well, experts in the field cautioned.

Among the false positive cases was a male radiographer who had been at the Singapore Expo community care facility. Two other cases — a female healthcare volunteer and a male nurse who were also working at the facility — were ambiguous and later verified to be negative upon re-testing.

Among the false positive cases was a male radiographer who had been at the Singapore Expo community care facility. Two other cases — a female healthcare volunteer and a male nurse who were also working at the facility — were ambiguous and later verified to be negative upon re-testing.

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SINGAPORE — As Singapore grows its Covid-19 testing capacity from 8,000 tests a day now to reach the daily target of 40,000 in future, the risks of straining the country’s lab capacity grows as well, experts in the field cautioned.

On Sunday (May 10), the Ministry of Health (MOH) revealed that one laboratory testing 33 cases generated positive test results but these were later found to be “false positives”. It did not name the lab involved and said that the ministry found out about it as part of its regular review of lab tests.

No false negatives were found, the ministry added.

“MOH has taken immediate actions to rectify the situation. The laboratory has stopped all tests and is working to resolve the calibration issue.”

The impact of this stoppage was felt the next day, with 486 new cases reported on Monday compared with 876 on Sunday, with MOH saying that it was partly due to the reduced testing.

How does the Covid-19 lab test, which has a typical accuracy of 95 per cent or more, produce errors? TODAY reached out to two test kit manufacturers to find out.

HOW DO COVID-19 TESTS WORK?

So far, there are two categories of tests to detect Covid-19 cases, the experts said. 

Molecular diagnostic tests are used to detect the presence of the Sars-CoV-2 coronavirus that causes Covid-19.

The second are serologic tests, which are used to test for antibodies and detect the immune response of a person to the virus. These are useful for testing whether a person has been infected with the virus in the past.

The first method is the most common type of test used by various countries. The only available means of such tests is the reverse transcription polymerase chain reaction (RT-PCR) assay, which can produce results in a matter of hours.

Simply put, RT-PCR allows for labs to find out whether a virus is present in a swab sample. The swab may contain a tiny amount of virus, but labs can use RT-PCR to quickly multiply the genetic material in that sample to a level that allows them to detect whether the virus is present.

Methods differ, but labs detect the presence of a certain virus — Sars-CoV-2, for example — by looking for a fluorescent signal that is created when genetic material belonging to the virus exceeds a certain threshold.

Dr Charlie Lee, head of research and development and senior vice-president of Vela Diagnostics, said that this is a powerful method with many applications in molecular biology and medical diagnostics.

HOW DO FALSE RESULTS OCCUR?

Studies have shown that RT-PCR is a highly sensitive test because a small amount of the virus can be amplified to a degree that allows it to be measured. 

One report from the American Society for Microbiology Covid-19 International Summit stated that if the test shows a positive result, it is most likely to be correct unless the sample was contaminated.

So how do false positives occur, such as in the 33 cases in Singapore?

MOH said that the errors were due to “an apparatus calibration issue” for one of the test kits at the lab.

It added that it has taken immediate actions to rectify the situation. “The laboratory has stopped all tests and is working to resolve the calibration issue.”

Dr Johnson Ng, founder and chief executive of JN Medsys, told TODAY that any error along the RT-PCR process — whether it is the nasal swab collection, extraction of genetic material from the sample and detection of the virus material — can result in errors.

Asked about calibration issues, Dr Ng explained that the test kits have to be performed using a PCR instrument that requires calibration.

In short, the instrument measures the degree of fluorescence that indicates whether the virus is present or not, and has to be calibrated to know what the background signal is.

“Otherwise it may wrongly detect an increase in signal intensity when there isn't any,” Dr Ng said, adding that the instrument could also be faulty.

Dr Lee agreed that sub-optimal instrument settings is a common cause of error. 

He added that other causes can also lead to false positives. A common mistake is due to contaminated samples, since a contaminated sample of someone without Covid-19 would be amplified and falsely detected as positive by the highly sensitive RT-PCR test.

The design of the test kit itself may also be flawed — if the kits are not designed to be specific to Sars-CoV-2 and could also detect other similar viruses such as influenza, then false positives could also happen, Dr Lee added.

In response to queries, Associate Professor Raymond Lin, Director of the National Public Health Laboratory, reiterated that miscalibrations do not mean that the test kits are faulty.

“The kits are tested with different machines for which it is approved, and the laboratory needs to ensure that calibration is done for the components of each test kit,” he added. 

WHAT ABOUT ‘EQUIVOCAL’ RESULTS?

A spokesperson from Woodlands Health Campus, which manages the medical team at Singapore Expo and Max Atria, said that of the 33 false cases so far, one was identified to be a 43-year-old male radiographer who had been at the community care facility.

On Sunday, MOH said that there were another two cases that had been “equivocal” and were later verified to be negative upon re-testing.

Woodlands Health Campus said that the two cases are a female healthcare volunteer, 52, and a male nurse, 20, who were both also working at the Singapore Expo.

“All three cases have been discharged and are resting at home,” the spokesperson said.

Dr Leong Hoe Nam, an infectious diseases specialist at Rophi Clinic in Mount Elizabeth Novena Hospital, said that equivocal results are basically “weak positives”, or test findings that are borderline cases.

“PCR amplifies whatever genetic content there is in the reaction. Even with just one copy of the genetic material, or (if it is) a needle in the haystack, you can get a positive result,” he said.

Dr Lee from Vela Diagnostics explained that different RT-PCR tests have different sensitivities and performance characteristics, known as the limit of detection. 

This could contribute to the occurrence of borderline cases — the test may not be able to robustly detect samples with viral amounts that fall below the limit of detection, he said.

SAFEGUARDS NEEDED

Dr Leong said that the possibility of flawed results highlight the importance of safeguards.

“Lab errors happen, sometimes from the kits that are faulty, sometimes because of processing errors or contamination. Hence, it is important to double-check all results.

“MOH does this by having the tests repeated by a separate laboratory after each newly diagnosed case, just like how an editor checks the work of a writer,” he said.

Re-tests for the 35 cases were done at the National Public Health Laboratory, MOH said on Sunday. The lab is located within the National Centre for Infectious Diseases (NCID).

The lab’s director, Assoc Prof Lin, said that testing for Covid-19 can be tricky, considering the difficulties at the sampling stage. He revealed that there have been some cases where the virus has infected the lungs, but cannot be detected in the back of the nose and throat where samples are usually taken.

“Sometimes the test is only positive on repeat sampling, as the illness evolves,” he said.

Assoc Prof Lin highlighted the importance of considering the symptoms present, hence individuals without symptoms are more difficult to screen for the virus. 

While PCR tests are still very reliable in identifying positive cases, there are still limitations, he noted.

“We have various ways to confirm the (PCR) tests, but it is sometimes possible to miss cases, or occasionally to have false positives,” he said. 

Dr Ng from JN Medsys said that due to the nature of the tests, it is a challenge for labs to ensure that every step of the testing process is done properly. 

“The problem here is the sheer amount of samples that the lab has to deal with every day,” he said. He did not say how many tests his lab conducts daily.

Across Singapore, the authorities test around 8,000 cases each day, up from the 2,900 a day in the initial days of the outbreak, Minister for National Development Lawrence Wong said last Friday.

Singapore will have the capability to perform 20,000 daily tests in a few weeks, and eventually reach 40,000 tests a day, he added.

Dr Leong from Rophi Clinic said that the boosted testing regimen will be required to quickly test most, if not all of the 400,000 foreign workers residing within and outside dormitories.

He said that the daily number of cases will continue to be high due to this “catch-up testing”, and estimated that it would take another month before the numbers start to fall, depending on how fast the authorities are able to actively test, isolate and treat the migrant workers.

As Singapore ramps up its testing, Dr Ng said that labs have already put in place strict processes to ensure the accuracy of results.

However, while the RT-PCR test is the most accurate test for Covid-19 for now, a 100 per cent accuracy rate will be impossible. Dr Ng said that it is already a good sign that only 35 false positives were detected out of all the tests done to date.

“I would say that the lab results are still highly reliable. However, we should be careful not to strain the labs’ capacity too much because that will give rise to potential errors,” he warned.

 

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