key: cord-0987242-kkxylcm0
authors: Caramello, Valeria; Macciotta, Alessandra; De Salve, Alessandro Vincenzo; Mussa, Alessandro; De Leo, Anna Maria; Bar, Fabrizio; Panno, Donata; Nota, Fabio; Ling, Camilla Yu Gi; Solitro, Federica; Ricceri, Fulvio; Sacerdote, Carlotta; Boccuzzi, Adriana
title: False negative RT-PCR tests in COVID-19 patients: an epidemiological analysis of 302 patients
date: 2021-09-22
journal: Public Health
DOI: 10.1016/j.puhe.2021.09.010
sha: 2e58dc41bf976579b1138eb339079efaf5318ddc
doc_id: 987242
cord_uid: kkxylcm0

Background Patients who arrive at the Emergency Department (ED) with Coronavirus disease (COVID-19), who test negative at the first Real Time-Polymerase Chain Reaction (RT-PCR), represent a clinical challenge. Objectives To evaluate if the clinical manifestation at presentation, the laboratory and imaging results, and the prognosis of COVID-19 differ in patients who tested negative at the first RT-PCR compared with those who tested positive. Furthermore, to evaluate if comorbid conditions patient-related or the period of arrival are associated with negative testing. Study design We retrospectively collected clinical data of patients who accessed the ED from 1st March to 15th May 2020. Methods We compared clinical variables, comorbid conditions and clinical outcomes in the two groups by univariate analysis and logistic regression. Results Patients who tested negative at the first RT-PCR showed a higher prevalence of cardiopathy, immunosuppression and diabetes, as well as a higher leukocyte and lower lymphocyte counts compared with patients who tested positive. A bilateral interstitial syndrome and a typical pattern at Computed Tomography scan were prevalent in the test-negative group. Test-negative patients were more likely to be admitted to the hospital but less likely to need admission in a high level of care ward. The false-negative rate increased from March to May. Conclusion False-negative RT-PCR COVID-19 patients present a similar spectrum of symptoms compared to positive cohort, but more comorbidities. Imaging helps to identify them. True positives had a higher risk of serious complications.

After the Coronavirus disease (COVID-19) outbreak, the broad clinical spectrum of symptoms and severity of this new syndrome has been widely described 1 in patients confirmed to have the disease by SARS-CoV-2 RNA detection by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) in upper airways swab [2] [3] [4] . At the very beginning of the pandemic, Xie et al. described the case of five "false-negative" patients 5 detected by chest Computed Tomography (CT) Thereafter, many other similar reports occurred [6] [7] [8] [9] , mostly diagnosed by typical findings at chest CT [6] [7] [8] [9] . The "threat" of false-negative worried many authors [10] [11] , but the best diagnostic strategy is still controversial [11] [12] [13] and the management of these cases raises some pragmatic questions about isolation, infection control and prognosis [10] [11] . The rate of patients presenting with COVID-19-like syndromes with a negative RT-PCR swab was estimated from 2 to 30% [10] [11] [12] [13] [14] [15] [16] [17] , but this measure could be underestimated due to the lack of an alternative gold-standard and because these subjects are often excluded from trials 3 . Technical pre-analytical and analytical issues 1, 2, [13] [14] [15] , genetic diversity and viral load kinetics at different anatomical sites 9,16,17 may cause a false-negative RT-PCR result. In this study, we aimed to evaluate if the clinical manifestation at presentation, the laboratory and imaging results, and the prognosis of COVID-19 are different in patients diagnosed as COVID -19, who tested negative at the first RT-PCR, in comparison with those who tested positive.

Furthermore, we evaluated if comorbid conditions patient-related or the period of arrival can influence the risk of negative testing at the first swab.

Turin province was the second most affected area in Italy during the "first wave" of the pandemic.

All patients admitted at the Emergency Department (ED) of XXX Hospital (YYY, ZZZ) with a suspected case of COVID-19 from the 1 st of March to May 15 th 2020, were retrospectively evaluated. Patients had consented to data collection for public health and research purposes at arrival and the Institutional Ethics Committee approved the study (n° 5796, 15/04/2020).

Patients who tested positive for SARS-CoV-2 RT-PCR in nasal swabs were included. Patients who tested negative for SARS-CoV-2 RT-PCR at first nasal swab were included if admitted to an isolation room in COVID-19 wards or discharged at home in isolation with a clinical diagnosis of and fulfilled at least one epidemiological criteria or clinical criteria (Supplementary Table 1) .

Test-negative COVID-19 patients were admitted to a special COVID ward area with isolation rooms to avoid the in-hospital spread of infection, and RT-PCR was repeated at 24 and 72h after the first swab or a lower airways specimen was obtained. The presence of antibodies at a serological test after discharge was also recorded. After chart review by three experienced researchers, we excluded patients who were diagnosed otherwise at the end of the hospital stay.

For all the patients we collected demographic data and comorbid conditions, symptoms at presentation, time from symptom onset, results of laboratory testing and results of imaging.

We categorized radiologic findings according to literature classification [18] [19] [20] The presence of an irregular pleural line at lung ultrasound or CT scan was also considered.

J o u r n a l P r e -p r o o f

We collected data about respiratory support (continue positive airway pressure (CPAP), noninvasive mechanical ventilation and invasive mechanical ventilation), admission or transfer to a higher level of care ward (Intensive Care Unit (ICU) or High Dependency Unit (HDU)), length of stay in the higher intensity of care wards, in-hospital mortality and total length of hospital stay.

Data were described as medians and interquartile ranges for quantitative variables and as absolute frequencies and percentages for categorical variables. Based on the non-normal distribution of the data assessed by the Shapiro-Wilks test, univariate comparisons were performed using the Wilcoxon sum rank test, in the group of positives to RT-PCR versus negative to RT-PCR. For categorical variables, we used the chi-square test or Fishers' exact test when the assumptions for conducting a chi-square test were not met. Similarly, differences in outcomes (inhospital mortality, ICU or HDU admission, need for respiratory support and length of stay) were evaluated in the two groups. Moreover, multivariable logistic regression was performed to investigate confounding factors in the observed associations. Odds ratios (ORs) and 95% confidence interval (95%CI) of disease severity, comorbid conditions and period of arrival on the result of the first RT-PCR were estimated, adjusted by age and sex. Finally, we run a sensitivity analysis in patients with moderate, severe, and critical COVID-19 (according to NIH classification 21 ) to reduce possible biases due to the inclusion of asymptomatic patients and patients without pneumonia only in the test-positive group. All tests were two-sided and a p-value < 0.05 was considered significant. Analyses were performed using R version 3. wards; 73 test-positive and four test-negative patients were discharged in isolation at home.

In this cohort, 86 patients overall reported to potentially have had a high-risk exposure to COVID-19 (16 were nursing home residents, 10 had close contact with a COVID-19 case, 41 were health workers, 18 reported a recent hospitalization or recurrently acceded outpatients' services (i.e. dialysis, oncology, day hospital, etc)). The presence of these epidemiological criteria was reported more frequently by test-negative patients than by test-positive patients. Clinical features of J o u r n a l P r e -p r o o f COVID-19 were present in most patients but were significantly higher in test-negative patients (65(99%) and 212(89%) respectively, p=0.023). Symptom's prevalence was similar in both groups except for gastrointestinal symptoms more reported by test-positive patients (39(16%) vs 4(6%), p=0.03). Comparison of demographic data, epidemiological criteria, clinical data and comorbid conditions in patients grouped by the first RT-PCR result is detailed in Table 1 . Cardiac diseases, neurological chronic conditions, lung cancer, diabetes and immunosuppression were more represented in the test-negative group than in the test-positive one. Patients who tested positive at a subsequent swab (n=16) showed no significant differences in comorbid conditions compared with those who tested persistently negative (n=50).

Laboratory tests at arrival showed that test-positive patients had a lower level of white blood cells In Table 2 , compared results of laboratory tests, imaging and COVID-19 severity according to NIH classification 21 in test-positive and test-negative patients are reported. A LUS pattern of bilateral interstitial syndrome was prevalent in test-negative patients, followed by consolidation, whereas consolidation and unilateral interstitial syndrome prevailed in test-positive patients followed by bilateral interstitial syndrome. Interestingly, 27% of test-positive patients had a normal LUS pattern. Non-specific findings prevailed in test-positive patients at CXR, whereas interstitial J o u r n a l P r e -p r o o f syndrome was prevalent in test-negative patients. Eighty percent of test-positive patients had a typical CT scan compared to 58% of test-negative patients who showed a higher rate of undetermined and atypical findings (p=0,012). In the test-positive group we observed a prevalence of asymptomatic and mild cases, by NIH COVID-19 severity classification. A moderate and severe illness prevailed in the test-negative group. The number of critical cases was significantly higher in the test-positive group. The sensitivity analysis in the subgroup of patients with moderate, severe and critical illness is described in Supplementary Table 2 : the comparison of the variables in this subgroup confirmed the findings obtained in the whole cohort.

Outcomes are described in Table 3 . The need for respiratory support was similar in the two groups. We observed that test-negative patients were hospitalized mostly in general wards and the admission in ICU or HDU was higher in test-positive patients, but mortality was similar.

Test-positive and test-negative patients showed up differently over the weeks (p<0.001): more than half of the test-negative patients were admitted after April 

In this study, we aimed to characterize a subgroup of COVID-19 patients, who tested negative at the first RT-PCR and represent nearly 20% of our sample, in accordance with previous studies 3, 12, 22 . Interestingly, laboratory confirmation of the disease by further tests (microbiological and serological tests) was achieved only in nearly half of patients who tested negative at the first RT-PCR.

To define these cases, we used careful evaluation of clinical records of the entire hospital stay by three experienced physicians, which led to exclusion of patients with a possible alternative diagnosis. This approach makes us confident of the inclusion in our cohort only of patients who were clinically COVID-19 and "false-negative" at RT-PCR at the first swab. This comprehensive clinical approach was previously applied in case reports or case series only 5, 7, 8, 14, 15, [23] [24] [25] [26] [28] [29] .

Observational studies, instead, usually have considered as false-negative patients only those who turned positive afterward 12, 16 or who showed a typical pattern at CT 15 respiratory symptoms with a differential diagnosis (a higher prevalence of smokers and COPD), they did not intend to compare false negative to RT-PCR cases with the confirmed COVID-19 cases In our cohort, test-negative patients showed a higher prevalence of very typical patterns at LUS and CXR when compared with test-positive patients: this evidence remains when restricting the analysis to patients presenting with pneumonia (moderate, severe, or critical illness according to NIH classification 21 ). We agree with other authors on the use of imaging to achieve a proper detection of cases 3,5-9 and acknowledge that having signs of interstitial pneumonia at LUS was prevalent in test-negative patients and could guide case definition. Similarly, we found that epidemiological factors in the history of false-negative patients were more represented.

When evaluating outcomes, corrected by severity, test-positive and test-negative patients showed a similar need for respiratory support and mortality rate. Nevertheless, the test-positive group showed a higher rate of admission in ICU/HDU, whereas most test-negative patients were admitted to general wards. We might hypothesize that test-positive patients had a higher viral load, which was seen to be related to more severe disease and a higher rate of detection by RT-PCR 14, 17 , but this explanation needs further validation.

Regarding possible individual characteristics that could influence a false-negative result at RT-PCR, immunosuppression, as well as other comorbid conditions, like cardiopathy and metabolic conditions, were associated with false-negative results at RT-PCR. To our knowledge, this is the first study to attempt to evaluate the effect of comorbid conditions on the possibility of having a negative swab. Other authors previously described merely case series 23, 25 or evaluated possible effects of comorbid conditions on viral clearances 26, 28 . The logistic regression confirmed that patients with more comorbid conditions had an increased risk of testing negative at the first RT-PCR.

The timing of the assay from symptom onset is known to influence the rate of RT-PCR positivity 15, 16, 29 , but in our cohort was similar among test-negative and test-positive patients. As Lippi et al. 13 , we assumed that technical issues in swab collection, transport and analysis could have reduced the efficiency of swab tests in the first phase of the pandemic because of the overload of laboratories. On the contrary, we observed an increase in false-negative in the late phase of the pandemic, which may be explained by a better clinical understanding of this illness.

Moreover, in the latter phase of pandemics in Piedmont, we admitted mainly older patients, often affected by multiple comorbid conditions 27 , who are at higher risk of testing negative. This trend is consistent with our findings that false-negative RT-PCR COVID-19 patients present more comorbidities compared to the positive cohort and, in line with a previous meta-analysis, that older age may affect RT-PCR sensitivity 18, 28 .

As other authors 2,23 we described RT-PCR negative cases both during the first weeks of the first wave, when patients showed typical phenotypes, and later when we observed many atypical cases in older patients with a great burden of comorbid conditions 23, 24, 27 . The concurrence of atypical imaging patterns and clinical courses with negative microbiological results was challenging for the emergency physician in the later phase of the first Italian pandemic wave. We suggest that in patients with associated comorbid conditions, a comprehensive evaluation of laboratory results (leukopenia) and imaging (LUS and CT) may help to define cases that could be misdiagnosed by RT-PCR alone.

Our findings must be interpreted bearing in mind that the data were collected at the very beginning of the pandemic crisis: in a similar context of uncertainties, both clinical experience and diagnostic performance of laboratory tests were rapidly evolving. In the following months, organizational and technological improvements have reduced many pre-analytical and analytical issues reducing the rate of false-negative RT-PCR test. Thanks to the shared observations on false-J o u r n a l P r e -p r o o f negative cases our colleagues were more confident in using lung ultrasound and in interpreting clinical, laboratory and imaging findings and in case of any discordant result we implemented the diagnostic algorithm using antigenic testing, the evaluation of IgM antibodies and the interpretation of Cycle threshold value for RT-PCR to achieve diagnostic confirmation.

It would be of interest to confirm with further studies if the improvement in highly sensitive and specific tests or a combination of the newly available assays has reduced the incidence of "falsenegative" at first RT-PCR during the subsequent pandemic waves.

To the best of our knowledge, this is the first study to compare symptoms, comorbid conditions Abbreviations: PO2 = oxygen partial pressure, PCO2=partial pressure of carbon dioxide, P/F ratio PO2/ fraction of inspired oxygen (FiO2), wbc= white blood cells, LDH= lactate dehidrogenases, CT = computed tomography 

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