Why Many People are Negative Despite Covid-19 Symptoms?

Many people with Covid-19 symptoms have been testing negative. There are several likely reasons, such as mutations of the virus escaping detection, quality of sampling and testing, and efficacy of RT-PCR kits.

As India struggles to cope with the unprecedented demand for RT-PCR tests, reports suggest that up to 20% symptomatic Covid-19 patients are testing negative.

This apparent trend of false negative results may deny serious patients hospital admission and critical care while letting asymptomatic ones move around and spread the virus. The trend has prompted experts, including AIIMS director Dr Randeep Guleria, to recommend Covid-19 treatment for everyone showing classic symptoms irrespective of RT-PCR results.

Why Negatives Test Results?

RT-PCR tests, considered the gold standard in the diagnosis of Covid-19, are not meant to be perfect. The minimum sensitivity (ability to detect positives) demanded by the Indian Council of Medical Research (ICMR) for validating an RT-PCR test is 95%. That means up to 5% false negative results are expected.

While anecdotal reports suggest that the share of false negative results is on the rise across India, there is no study yet to back this.

In theory, four broad factors determine the accuracy of an RT-PCR test: the viral load in the person, the quality of sample collection and processing, the efficacy of the test kit itself, and also the benchmark for test interpretation.


Typically, Covid-19 manifests adequately by the fifth day of an infection cycle. Tested any sooner post exposure, an infected person may turn out Covid-negative. This may not be a major factor behind the recent false negative trend as certain Covid-19 mutants apparently show early symptoms.

Also Read: The New Coronavirus Symptoms Day By Day You Need To Observe

A number of symptomatic patients who tested false negative in RT-PCR assays have subsequently been confirmed Covid-positive in Bronchoalveolar Lavage (BAL) tests that collect samples from the lower respiratory tract through a bronchoscope. This has led to conjectures that certain mutants bypass the upper respiratory tract to target the lung — a scenario that may deny an RT-PCR test, based on sample collected from the nasal cavity and throat, the viral load required for an accurate result.


The number of Covid test labs in India increased from 14 in February 2020 to more than 2,400 in April 2021. Such a rapid expansion required approving hundreds of labs for RT-PCR testing and training thousands of technicians in a hurry.

As a safeguard, ICMR in July 2020 listed 30 Quality Control labs to check on all the Covid labs it approved. Another 8 QC labs have been added since. However, sources in multiple labs across three states said “only a few inspections of facilities and instruments” took place.

“Ideally, a mix of positive and negative samples should be picked up at random from every lab periodically for re-testing. Was that ever done? Yes. Was that done consistently? No,” said a scientist with an ICMR institute who is not authorised to speak to the media.

Approached for periodic lab monitoring and quality compliance data since July 2020, ICMR and a number of QC labs remained silent.


Tests can go wrong at various stages — from bad sample collection and storage to faulty extraction and amplification. All RT-PCR kits include an internal control (IC) to safeguard against a scenario when no RNA is extracted/amplified, leading to a false negative. The IC can be exogenous or endogenous. It is exogenous when an artificial RNA template molecule is added to each sample before RNA extraction. The test is considered void when the synthetic RNA is not detected post-extraction and a re-test is prescribed. And, an endogenous control uses a human ‘house-keeping’ gene present in the sample; its non-detection after the RNA extraction procedure invalidates the test.

“A human house-keeping gene also ensures the sample quality… But over 75% RT-PCR kits in the Indian market use the cheaper exogenous internal controls,” said a Mumbai-based molecular biologist.

ICMR’s April 2020 SOP on RT-PCR held that exogenous control for extraction “can be ignored as it will not reflect the quality of the sample collected”. It recommended “separate RNase P or any other human house-keeping gene… should be run parallel in a separate tube… (to) check both the quality of sample collected” and the extraction procedure.

In May 2020, the mega tender floated by ICMR/HLL for RT-PCR kits specified similar requirements. A number of manufacturers argued different companies use different internal controls including exogenous ones which were approved by regulatory agencies such as FDA and CEIVD, and that adverse selection based on house-keeping genes would discourage fair competition. Eventually, the tender was revised to include both endogenous and exogenous options for internal control.


A RTR-PCR test targets specific area/s of one or more viral genes to detect its presence. A test may return false negative results if a mutation occurs in the segment of the genome assessed. Tests that use multiple genetic targets, like the ones in use in India, are less likely to be fooled by mutations.

Regulators such as the US FDA routinely monitor the impact of mutations on RT-PCR tests While experts such as Dr Rakesh Mishra, director of Hyderabad’s Centre for Cellular and Molecular Biology (CCMB), feel ICMR should initiate a similar exercise, it may not be easy to monitor real-time over 200 RT-PCR kits approved in India against emerging mutations.

For one, no manufacturer has (or will) made public the detailed sequences its tests target. “While submitting for validation to ICMR, they only mention the target genes and not the primer sequence,” said a senior researcher with the Health Ministry.

Until now, though, there is ground to feel fairly confident even in the absence of any real-time efficacy check. “So far, Covid-19 variants have mainly S gene (spike protein) mutations. Majority of kits here use conserved (unchanged) regions of targets,” said Dr Shanta Dutta, director of National Institute of Cholera and Enteric Diseases (NICED), Kolkata.

Reduction in Testing Price

In one year, the price of the RT-PCR kit fell from over Rs 1,100 to under Rs 40. Since import duty exemption was withdrawn in October 2020, most foreign brands left the Indian market due to the additional 15% cost burden. A number of Indian brands, too, have been refusing to sell below Rs 100. Many have found dedicated corporate clientele who do not mind paying a little extra to ensure quality. Some are even offering multiple PCR kits at different price levels.

While nobody says it on record, many question the quality of the cheaper competition. The counterview is that every test kit available in the market meets ICMR’s validation benchmarks and that any price war ultimately benefits the consumer.

Not everyone is convinced, though. “The corresponding drop in the cost of the lab test is from Rs 4,500 to Rs 800. Besides, does one visiting her local lab for a Covid test know which kit is being used? How many labs will spend Rs 100 when they can get away with using a Rs-40 kit?” said one of the first Indian manufacturers of Covid PCR kits.

This article was originally posted at: Indian Express

Leave a Reply

Your email address will not be published.

New Coronavirus Symptoms Day By Day Next post The New Coronavirus Symptoms Day By Day You Need To Observe