Repeatedly Negative Reverse Transcriptase-Polymerase Chain Reaction in a Clinically Suspected Case of COVID-19 in India

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a huge public health crisis. The reverse transcriptase-polymerase chain reaction (RT-PCR) is widely used as the gold standard molecular test for COVID-19 diagnosis. However, there have been concerns about false-negative results with this test.[1] We present here a case report to emphasize that even repeatedly negative RT-PCR does not rule out COVID-19, and the clinical management of patients should be based on signs and symptoms consistent with SARS-CoV-2 infection and a chest computed tomography (CT). Written informed consent was obtained from the patient. A 36-year-old male, who is a trained physician and an anesthetist, was posted in the intensive care unit of a government tertiary hospital in India to treat severely and critically ill COVID-19 patients. Each duty lasted for 7 days (6 h each), and he was on a 1-week self-quarantine between the duty periods. He was using personal protective equipment (PPE) during all of his duties and had intubated five patients while wearing PPE along with an N95 mask and face shield. He did not take hydroxychloroquine or any other drug as prophylaxis. After completing three duties, he started to develop a low-grade fever, myalgia, malaise, and occasional dry cough. Myalgia and malaise were persistent and severe, and they did not resolve with nonsteroidal anti-inflammatory drugs (NSAIDs). A nasopharyngeal swab was given for RT-PCR on the 5th and 7th day of the onset of symptoms in two different laboratories (one public and one private). Both tests came out as negative for SARS-CoV-2 nucleic acid. The RT-PCR tests in both the laboratories were performed using kits approved by the Indian Council of Medical Research (ICMR), the authority of India that regulates biomedical research in the country. His blood was tested twice (on the 5th and 13th day of symptom onset) for complete blood count, inflammatory markers, coagulation indexes, and other laboratory parameters [Table 1]. Most of these parameters were within the normal range, except for a slightly elevated creatine phosphokinase (CPK) and increased neutrophil-to-lymphocyte ratio in the second test. A chest CT scan was taken on the 9th day of the onset of symptoms. It showed a tiny subpleural ground-glass opacity in the posterior aspect of the superior segment of the left lower lobe. The CT image, read by a certified and trained radiologist, was given a COVID-19 reporting and data system (CO-RADS) score of 3 (i.e., indeterminate) on a scale of 1 (very unlikely) to 5 (very likely).[2] There was no evidence of consolidation of any lobe or mural thickening. Based on the chest CT findings, he was treated with oral methylprednisolone (8 mg twice daily) for 5 days and oral ivermectin (12 mg once daily) for 3 days, following which myalgia and malaise resolved. An ICMR-approved serology test (false positivity rate is <1%) was performed using enzyme-linked immunosorbent assay on the 19th day of the onset of symptoms, which turned out to be positive for SARS-CoV-2 IgM antibodies. Written informed consent was obtained from the patient. This case of COVID-19 emphasizes a few key issues, which are relevant to the clinical management of COVID-19 patients. First, in our patient, persistent and severe myalgia and malaise were the main symptoms, supported by elevated CPK levels. This is in line with previous observations that myalgia in COVID-19 patients can be more severe and last longer than with other viral infections.[3] Myalgia may reflect generalized inflammation and cytokine response and is a frequent symptom in up to 36% of COVID-19 patients.[3] Myalgia in our patient got resolved only with steroid therapy and ivermectin but not with routine NSAIDs. Previous reports show that myalgia may be unresponsive to conventional NSAIDs in COVID-19 patients; instead,, treatment to reduce the viral load could be more effective.[3] In line with this, the use of steroids and ivermectin in our patient possibly reduced the viral load, which may have then improved the oxygenation of erythrocytes and decreased the lactate levels in muscles,[3] thereby resolving myalgia. Second, our patient's RT-PCR was negative twice during the course of acute illness. A systematic review of 34 studies involving 12,057 COVID-19 confirmed patients showed that up to 54% of patients might have an initial false-negative RT-PCR.[1] Based on this, the authors of this review call for repeated testing in patients with suspicion of SARS-CoV-2 infection. However, in our patient, even the repeated testing on day 7 came out to be negative. Research shows that the probability of false-negative RT-PCR is around 20% on day 7.[4] Since the patient had a mild illness, it is possible that the viral shedding duration was short, and the level of viral nucleic acids was too low to be detectable on RT-PCR. Other possibilities include improper specimen sampling, improper conduct of the test, and mismatch between the RT-PCR assay and the SARS-CoV-2 genome.[1] Studies show that a chest CT has a higher sensitivity to diagnose COVID-19 than the RT-PCR test,[1] and the sensitivity improves further when these tests are performed in conjunction.[1] The CO-RADS score of our patient's chest CT was 3, which indicates that there was an acute infection, but unsure whether COVID-19 was involved.[2] While the chest CT was indeterminate of COVID-19 infection, it assisted with treatment and isolation, given the exposure status of the patient and the presence of clinical signs and symptoms consistent with SARS-CoV-2 infection. Finally, our patient tested positive for SARS-CoV-2 IgM antibodies on the 19th day of symptom onset. A Cochrane review of 57 publications (54 study cohorts with 15,976 samples) from Asia, Europe, the USA, and China concluded that the antibody tests are likely to be useful in detecting SARS-CoV-2 infection at 15 or more days of symptom onset.[5] While cross-reaction with other coronaviruses may have resulted in a false-positive serology test in our patient, it is less likely given that our patient is an healthcare professional (HCP) treating COVID-19 patients and the rate of SARS-CoV-2 infection was high in India during that time. In conclusion, our case emphasizes that when RT-PCR is repeatedly negative for SARS-CoV-2 RNA, the possibility of a false-negative result should be considered in light of the patient's exposure status, clinical signs and symptoms consistent with SARS-CoV-2 infection, and chest CT findings. Such an approach would help to isolate the patient and closely monitor their illness so that early and appropriate treatment can be initiated if required.Table 1: Key laboratory test results of the patientDeclaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

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