Diagnosis

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By Bernd Sebastian Kamps
& Christian Hoffmann

Diagnosis

Rapid identification and isolation of infected individuals is crucial. Diagnosis is made using clinical, laboratory and radiological features. As symptoms and radiological findings of COVID-19 are non-specific, SARS-CoV-2 infection has to be confirmed by nucleic acid-based polymerase chain reaction (PCR), amplifying a specific genetic sequence in the virus. Within a few days after the first cases were published, a validated diagnostic workflow for SARS-CoV-2 was presented (Corman 2020), demonstrating the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks.

There is an interim guidance for laboratory testing for coronavirus disease (COVID-19) suspected human cases, published by WHO on March 19, 2020 (WHO 2020). Several comprehensive up-to-date reviews of laboratory techniques in diagnosing SARS-CoV-2 have been published recently (Chen 2020, Löffelholz 2020).

In settings with limited resources, no testing capacity should be wasted. Importantly, patients should only be tested if a positive test results in imperative action. This is not the case in the following examples:

  • Young people who had contact with an infected person a few days earlier, have mild or moderate symptoms and live alone. They do not need PCR testing, even if they get fever. They’ll remain in home quarantine, on sick leave if necessary, at least 14 days after the onset of symptoms. A test would only be useful to clarify whether they can work in a hospital or other health care facilities after quarantine. Some authorities require at least one negative test (nasopharyngeal) before starting work again (in addition to at least 48 hours of being symptom-free).
  • A couple returning from an epidemic hotspot and feel a slight scratch in their throats. As they should remain in quarantine anyway, again, no testing is needed.
  • A family of four with typical COVID-19 symptoms. Testing only one (symptomatic) person is sufficient. If the test is positive, it is not necessary to test the other household contacts – as long as they stay at home.

These decisions are not easy to commnicate, particularly to fearful and worried patients.

In other situations, however, a test must be immediately carried out and repeated if necessary, especially for medical professionals with symptoms, but also, for example, in nursing homes, in order to detect an outbreak as quickly as possible.

Even though there are constantly updated recommendations by authorities and institutions of the country’s health system about who should be tested by whom and when: they are constantly changing and have to be constantly adapted to the local epidemiological situation. With decreasing infection rates and increasing test capacities, more patients will certainly be able to be tested in the future, and the indication for a test will be expanded.

Specimen collection

SARS-CoV-2 can be detected in different tissues and body fluids. In a study on 1,070 specimens collected from 205 patients with COVID-19, bronchoalveolar lavage fluid specimens showed the highest positive rates (14 of 15; 93%), followed by sputum (72 of 104; 72%), nasal swabs (5 of 8; 63%), fibrobronchoscopy brush biopsy (6 of 13; 46%), pharyngeal swabs (126 of 398; 32%), feces (44 of 153; 29%), and blood (3 of 307; 1%). None of the 72 urine specimens tested positive (Wang 2020). The virus was also not found in the vaginal fluid of 10 women with COVID-19 (Saito 2020). It was also not found in sperm and breast milk (Song 2020, Scorzolini 2020). On rare occasions, however, the virus may be detected in tears and conjunctival secretions (Xia 2020).

Samples can also be taken from sputum (if producible), endotracheal aspirate, or bronchoalveolar lavage. It is likely that lower respiratory samples are more sensitive than nasopharyngeal swabs. Especially in seriously ill patients, there is often more virus in the lower than in the upper respiratory tract (Huang 2020). However, there is always a high risk of “aerosolization” and thus the risk that staff members become infected.

However, viral replication of SARS-CoV-2 is very high in upper respiratory tract tissues which is in contrast to SARS-CoV (Wölfel 2020). According to WHO, respiratory material for PCR should be collected from upper respiratory specimens (nasopharyngeal and oropharyngeal swab or wash) in ambulatory patients (WHO 2020). It is preferred to collect specimens from both nasopharyngeal and oropharyngeal swabs which can be combined in the same tube.

Nasopharyngeal swabs – practical issues

It is important to carry out the swab correctly. Both nasopharynx and oropharyngeal swabs have a number of error options that all can lead to false negative results. In addition, protective measures must be taken in order not to endanger the examiner. Every swab carries a high risk of infection! Respiratory protection, protective glasses, gowns and gloves are required. The correct putting on and taking off of the protective clothing should be practiced! Many mistakes occur even when a protective mask is removed.

For the smear, the patient should sit on a chair and put his head slightly back. The examiner should stand at a slightly offset position in order to avoid a possible cough drop. Tell the patient that it can be uncomfortable for a short time. Swabs should be used that are suitable for virus detection and have the most flexible plastic shaft possible. Wooden sticks can inactivate viruses and pose a high risk of injury. The swab should be held between thumb and forefinger, like a pencil, so the end should not touch anything. The posterior wall of the nasopharynx is often reached after 5-7 cm, indicated by a slight resistance. “Nose popules” is not enough! Touching the teeth and tongue should be avoided when taking a throat swab; the swab should be removed from the back wall, directly next to the uvula. Caution with the gag reflex! There is a wealth of practical videos on the internet for the correct execution of the swabs. After appropriate instruction, many patients can perform the swabs themselves.

We have established swabs for patients who are able to do this (most of them!) at home. A courier with the tubes is sent directly to the patient’s home, and the courier places the tubes in front of the door. Direct contact between patient and courier should be avoided. The swab tubes should not be touched by the courier (either put them directly in a bag or collect them with an inverted bag) and should be brought back directly (no mailing!). This requires prior, precise instruction, but is usually quite feasible.

The swabs can be stored dry or in a small amount of NaCl solution; if necessary, this should be clarified with the laboratory beforehand. Quick PCR examination is important, preferably on the same day if possible. Heat is not favorable. In a small study, samples were inactivated by incubation in a water bath at 56°C for 30 minutes. 7/15 samples with low viral values ​converted to false negative. Longer storage also led to false negative results (Pan 2020).

Lower respiratory specimens may include sputum (if produced) and/or endotracheal aspirate or bronchoalveolar lavage in patients with more severe respiratory disease. However, a high risk of aerosolization should be considered (adhere strictly to infection prevention and control procedures). Additional clinical specimens may be collected as COVID-19 virus has been detected in blood and stool (see below).

Gathering specimens from nasopharyngeal and throat swabs can cause discomfort for patients and put health-care workers at risk. The virus is present in saliva and several studies have shown that posterior oropharyngeal (deep throat) saliva samples are feasible and more acceptable to patients and healthcare workers (To 2020, Yu 2020). Throat washing may be used for monitoring due to its non-invasiveness and reliability. Throat washing was harvested by asking patients to oscillate over the posterior pharyngeal wall with 20 ml sterile normal saline. After 5-10 seconds, they spit out the normal saline from their throat to a sterile container. In 24 paired throat washings and nasopharyngeal swabs specimens, the positive testing rate of throat washing was much higher than that of swabs (Guo 2020).

Although no cases of transmission via fecal-oral route have yet been reported, there is also increasing evidence that SARS-CoV-2 is actively replicating in the gastrointestinal tract. A larger study from Zhuhai, China showed prolonged presence of SARS-CoV-2 viral RNA in fecal samples. Of the 41 (55%) of 74 patients with fecal samples that were positive for SARS-CoV-2 RNA, respiratory samples remained positive for SARS-CoV-2 RNA for a mean of 16.7 days and fecal samples remained positive for a mean of 27.9 days after first symptom onset (Wu 2020). In 22/133 patients, SARS–CoV-2 was still detected in the sputum or feces (up to 39 and 13 days, respectively) after pharyngeal swabs became negative (Chen 2020). These studies have raised concerns about whether patients with negative pharyngeal swabs are truly virus-free, or sampling of additional body sites is needed. However, the clinical relevance of these finding remains unclear and there is one study that did not detect infectious virus from stool samples, despite having high virus RNA concentrations (Wölfel 2020).

SARS-CoV-2 is rarely detected in blood (Wang 2020, Wölfel 2020). What about transmission risk associated with transfusions? In a screening study of 2,430 blood donations in Wuhan, plasma samples were found positive for viral RNA from 4 asymptomatic donors (Kwon 2020). Another study from Korea found seven asymptomatic blood donors who were later identified as COVID-19 confirmed cases. None of 9 recipients of platelets or red blood cell transfusions tested positive for SARS-CoV-2 RNA. Transfusion transmission of SARS-CoV-2 was considered to be unlikely (Chang 2020). As with feces, it remains unclear whether detectable RNA in the blood signifies infectivity.

PCR

Several different qPCR-based detection kits are available as labs worldwide have customized their PCR tests for SARS-CoV-2, using different primers targeting different sections of the virus’s genetic sequence. A review of different assays and diagnostic devices was recently published (Löffelholz 2020). A protocol for real-time (RT)-PCR assays for the detection of SARS-CoV-2 for two RdRp targets (IP2 and IP4) is described at https://www.who.int/docs/default-source/coronaviruse/real-time-rt-pcr-assays-for-the-detection-of-sars-cov-2-institut-pasteur-paris.pdf?sfvrsn=3662fcb6_2

Novel real-time RT-PCR assays targeting the RNA-dependent RNA polymerase (RdRp)/helicase, spike and nucleocapsid genes of SARS-CoV-2 may help to improve the laboratory diagnosis of COVID-19. Compared to the reported RdRp-P2 assay which is used in most European laboratories, these assays do not cross-react with SARS-CoV in cell culture and may be more sensitive and specific (Chan 2020).

If not, the limits of detection of six commercial kits differ substantially (up to 16-fold difference), with the poorest limits likely leading to false-negative results when RT–PCR were used to detect SARS-CoV-2 infection (Wang 2020). According to the authors, manufacturers should analyze the existing problems according to the clinical application and further improve their products.

Qualitative PCR

A qualitative PCR (“positive or negative”) is usually sufficient in routine diagnostics. Quantification of viral RNA is currently (still) only of academic interest. False positive results are rare. The main problem of any qualitative PCR is above all the false negative results. They have many causes. Incorrect smears are particularly common, but laboratory errors also occur.

Several studies have shown that asymptomatic patients also have positive PCR results and can transmit the virus (Bai 2020, Cereda 2020, Rothe 2020). Viral shedding may begin 2 to 3 days before the appearance of the first symptoms. Analyzing a total of 414 throat swabs in 94 patients, the highest viral load in throat swabs was found at the time of symptom onset. Infectiousness started from 2.3 days (95% CI, 0.8–3.0 days) before symptom onset and peaked at 0.7 days before symptom onset (He 2020). Infectiousness was estimated to decline quickly within 7 days.

In a cohort of 113 symptomatic patients, the median duration of detection of SARS-CoV-2 RNA was 17 days (interquartiles 13-22 days), measured from the onset of the disease. In some patients, the PCR was positive even longer: male gender and a severe course (invasive mechanical ventilation) were independent risk factors for prolonged shedding (Xu 2020).

Recent reports from patients have repeatedly gained much media attraction, showing positive results after repeated negative PCR and clinical recovery (Lan 2020, Xiao 2020, Yuan 2020). These studies raise the question of re-activation or re-infection of COVID-19 (see below, clinical chapter). Currently, the results are much more likely due to methodological problems (Li 2020). At low virus levels, especially during the last days of an infection, the viral load can fluctuate and sometimes be detectable, sometimes not (Wölfel 2020). Reactivation, and also a rapid reinfection would be very unusual for coronaviruses.

Quantification of viral load

Several studies have evaluated the SARS-CoV-2 viral load in different specimens. In a small prospective study, the viral load in nasal and throat swabs obtained from 17 symptomatic patients was analyzed in relation to day-of-onset of any symptoms (Zou 2020). Of note, the viral load detected in the asymptomatic patients was similar to that in the symptomatic patients, which suggests the transmission potential of asymptomatic or minimally symptomatic patients.

In another study on 82 infected individuals, the viral loads in throat swab and sputum samples peaked at around 5–6 days after symptom onset, ranging from around 79,900 copies/ml in the throat to 752,000 copies per mL in sputum (Pan 2020). In a study on oropharyngeal saliva samples, unlike SARS, patients with COVID-19 had the highest viral load near presentation, which could account for the fast-spreading nature of this epidemic (To 2020). The median viral load in posterior oropharyngeal saliva or other respiratory specimens at presentation was 5.2 log10 copies per mL (IQR 4.1-7.0) in this study. In a total of 323 samples from 76 patients, the average viral load in sputum (17,429 copies/test) was significantly higher than in throat swabs (2,552 copies) and nasal swabs (651 copies). Viral load was higher in the early and progressive stages than in the recovery stage (Yu 2020). According to a recently published study, viral shedding may already begin 2-3 days before the appearance of the first symptoms and the infectiousness profile may more closely resemble that of influenza than of SARS (He 2020).

Higher viral loads might be associated with severe clinical outcomes. In a study evaluating serial samples from 21 mild and 10 severe cases (Liu 2020), mild cases were found to have an early viral clearance, with 90% of these patients repeatedly testing negative on RT-PCR by day 10 post-onset. By contrast, all severe cases still tested positive at or beyond day 10 post-onset. However, large and prospective trials are needed to evaluate the role of SARS-CoV-2 viral load as a marker for assessing disease severity and prognosis.

Diagnosis in the setting of shortage of PCR test kits

There is no doubt that the overall goal must be to detect as many infections as possible. However, in many countries, a shortage of supply test kits does not meet the need of a growing infected population. Thus, pooled samples are often used to save material. Several samples are examined together. Only when such a pooled sample is positive, will the samples be examined individually.

Some studies have also investigated whether the diagnosis in high prevalence periods and countries cannot be made without PCR detection if necessary. A large retrospective case-control study from Singapore has evaluated predictors for SARS-CoV-2 infection, using exposure risk factors, demographic variables, clinical findings and clinical test results (Sun 2020). Even in the absence of exposure risk factors and/or radiologic evidence of pneumonia, clinical findings and tests can identify subjects at high risk of COVID-19. Low leukocytes, low lymphocytes, higher body temperature, higher respiratory rate, gastrointestinal symptoms and decreased sputum production were strongly associated with a positive SARS-CoV-2 test. However, those preliminary prediction models are sensitive to the local epidemiological context and phase of the global outbreak. They make only sense during times of high incidence. In other words: if I see a patient during the peak of an epidemic, presenting with fever, cough, shortness of breath and lymphopenia, I can be almost sure that this patient suffers from COVID-19. During phases, when the incidence is lower, these models do not make sense. There is no doubt that the nucleic acid test or genetic sequencing serves as the gold standard method for confirmation of infection. Whenever PCR is available, PCR should be performed.

Serology

Detection of past viral infections by looking for antibodies an infected person has produced will be among the most important goals in the fight against the COVID-19 pandemic (Brief review: Petherick 2020). Antibody testing is multipurpose: these serological assays are of critical importance to determine seroprevalence, previous exposure and identify highly reactive human donors for the generation of convalescent serum as therapeutic. They will also support contact tracing and screening of health care workers to identify those who are already immune. How many people really got infected, in how many did the virus escape the PCR diagnosis, and for what reasons, how many patients are asymptomatic, and what is the real mortality rate in a defined population? Only with comprehensive serology testing (and well-planned epidemiological studies) will we be able to answer these questions and reduce the ubiquitous undisclosed number in the current calculations. Several investigations are already underway in a wide variety of locations worldwide.

But, outside clinical studies: who should be tested now? Testing actually makes no sense for patients with a previous, proven COVID-19 disease. However, it can still be done if, for example, you want to validate a test. In addition to those involved in health care or working in other professions with a high risk of transmission, such testing can also be useful in order to identify possible contact persons retrospectively.

Several groups are working towards these tests (Amanat 2020), some of them are already commercially available.

Antibody testing usually focuses on antigens (proteins). In the case of SARS-CoV-2, different Enzyme-Linked Immunosorbent Assay (ELISA) kits based on recombinant nucleocapsid protein and spike protein are used (Löffelholz 2020). The SARS-CoV-2 spike protein seems to be the best target. However, which part of the spike protein to use is less obvious and there is a lot hanging on the uniqueness of the spike protein. The more unique it is, the lower the odds of cross-reactivity with other coronaviruses—false positives resulting from immunity to other coronaviruses. Cross reactivity to other coronaviruses can be challenging. So called confirmation tests (usually neutralization tests) can be used to reduce false positive testings.

Even with a very high specificity of 99% and above, especially in low-prevalence areas, the informative value is limited and a high rate of false positive tests can be assumed. An example: With a specificity of 99%, it is expected that one test out of 100 is positive. In a high prevalence setting, this is less relevant. However, if a person is tested in a low prevalence setting, the likelihood that a positive test is really positive (the positive predictive value, i.e. the number of really positive tests divided by the number of all positive tests) is low. In a population with a given prevalence of 1%, the predictive value would only be 50%! Current estimates from Iceland, a well-defined but unselected population, still have shown a relatively constant rate of around 0.8% in March 2020 (Gudbjartsson 2020). Even in apparently more severely affected countries, the infection rates are only slightly higher. If we assume an infection number of 133,800 (April 17) for Germany, a country with one of the world’s largest number of infections, and assume that the number of undetected infections is about 5 times as high, then the prevalence in Germany is overall still below 1%. Almost every hundredth is infected, every second positive test would be false positive, even with a specificity of 99%. General antibody screening in the population will therefore produce a fairly high rate of false positive tests.

Of note, serologic responses to coronaviruses are only transient. Antibodies to other human, seasonal coronaviruses may disappear even after a few months. Preliminary data suggest that the profile of antibodies to SARS-CoV-2 is similar to SARS-CoV (Xiao 2020). For SARS-CoV, antibodies were not detected within the first 7 days of illness, but IgG titre increased dramatically on day 15, reaching a peak on day 60, and remained high until day 180 from when it declined gradually until day 720. IgM was detected on day 15 and rapidly reached a peak, then declined gradually until it was undetectable on day 180 (Mo 2006). As with other viruses, IgM antibodies occur somewhat earlier than IgG antibodies which are more specific. IgA antibodies are relatively sensitive but less specific (Okba 2020).

The first larger study on the host humoral response against SARS-CoV-2 has shown that humoral response to SARS-CoV-2 can aid to the diagnosis of COVID-19, including subclinical cases (Guo 2020). In this study, IgA, IgM and IgG response using an ELISA-based assay on the recombinant viral nucleocapsid protein was analyzed in 208 plasma samples from 82 confirmed and 58 probable cases (Guo 2020). The median duration of IgM and IgA antibody detection were 5 days (IQR 3-6), while IgG was detected on day 14 (IQR 10-18) after symptom onset, with a positive rate of 85.4%, 92.7% and 77.9% respectively. The detection efficiency by IgM ELISA was higher than that of PCR after 5.5 days of onset of symptoms. In another study of 173 patients, the seroconversion rates (median time) for IgM and IgG were 82.7% (12 days) and 64.7% (14 days), respectively. A higher titer of antibodies was independently associated with severe diseases (Zhao 2020). It will be seen in the coming months how the human antibody response to SARS-CoV-2 evolves over time and how this response and titres correlate with immunity. It is also conceivable that in some patients (e.g. those with immunodeficiency), the antibody response remains reduced.

Radiology

Computed tomography

Computed tomography (CT) can play a role in both diagnosing and assessment of disease extent and follow-up. Chest CT has a relatively high sensitivity for diagnosis of COVID-19 (Ai 2020, Fang 2020). However, around half of patients may have a normal CT during the first 1-2 days after symptom onset (Bernheim 2020). On the other hand, it became clear very early in the current pandemic that a considerable proportion of subclinical patients (scans done before symptom onset) may already have pathological CT findings (Chan 2020, Shi 2020). In some of these patients showing pathological CT findings evident for pneumonia PCR in nasopharyngeal swabs was still negative (Xu 2020). On the other hand, half of the patients who later develop CT morphologically visible pneumonia can still have a normal CT in the first 1-2 days after the symptoms appear (Bernheim 2020).

However, one should not overestimate the value of chest CT. The recommendation by some Chinese researchers to include CT as an integral part in the diagnosis of COVID-19 has led to harsh criticism, especially from experts in Western countries. The Chinese studies have been exposed to significant errors and shortcomings. In view of the high effort and also because of the risk of infection for the staff, many experts strictly reject the general CT screening in SARS-CoV-2 infected patients or in those with suspicion (Hope 2020, Raptis 2020). According to the recommendation of the British Radiology Society, which made attempts to incorporate CT into diagnostic algorithms for COVID-19 diagnostics, the value of CT remains unclear – even if a PCR is negative or not available (Nair 2020, Rodrigues 2020). A chest CT should only be performed if complications or differential diagnoses are considered (Raptis 2020).

In blinded studies, radiologists from China and the United States have attempted to differentiate COVID-19 pneumonia from other viral pneumonia. The specificity was quite high, the sensitivity nuch lower (Bai 2020). A recent metaanalysis found a high sensitivity but low specificity (Kim 2020). The sensitivity of CT was affected by the distribution of disease severity, the proportion of patients with comorbidities, and the proportion of asymptomatic patients. In areas with low prevalence, chest CT had a low positive predictive value (1.5-30.7%).

If pathological, images usually show bilateral involvement, with multiple patchy or ground-glass opacities (GGO) with subpleural distribution in multiple bilateral lobes. Lesions may display significant overlap with those of SARS and MERS (Hosseiny 2020).

A systematic review of imaging findings in 919 patients found bilateral multilobar GGO with a peripheral or posterior distribution, mainly in the lower lobes and less frequently within the right middle lobe as the most common feature (Salehi 2020). In this review, atypical initial imaging presentation of consolidative opacities superimposed on GGO were found in a smaller number of cases, mainly in the elderly population. Septal thickening, bronchiectasis, pleural thickening, and subpleural involvement were less common, mainly in the later stages of the disease. Pleural effusion, pericardial effusion, lymphadenopathy, cavitation, CT halo sign, and pneumothorax were uncommon (Salehi 2020).

The  evolution  of  the  disease  on  CT  is  not  well  understood. However, with a longer time after the onset of symptoms, CT findings are more frequent, including consolidation, bilateral and peripheral disease, greater total lung involvement, linear opacities, “crazy-paving” pattern and the “reverse halo” sign (Bernheim 2020). Some experts have proposed that imaging can be sorted into four different phases (Li 2020). In the early phase, multiple small patchy shadows and interstitial changes emerge. In the progressive phase, the lesions increase and enlarge, developing into multiple GGOs as well as infiltrating consolidation in both lungs. In the severe phase, massive pulmonary consolidations and “white lungs” are seen, but pleural effusion is rare. In the dissipative phase, the GGOs and pulmonary consolidations were completely absorbed, and the lesions began to change into fibrosis.

In a longitudinal study analyzing 366 serial CT scans in 90 patients with COVID-19 pneumonia, the extent of lung abnormalities progressed rapidly and peaked during illness days 6-11 (Wang 2020). The predominant pattern of abnormalities after symptom onset in this study was ground-glass opacity (45-62%). As pneumonia progresses, areas of lesions enlarge and developed into diffuse consolidations in both lungs within a few days (Guan 2020).

Most patients discharged had residual disease on final CT scans (Wang 2020). Studies with longer follow-up are needed to evaluate long-term or permanent lung damage including fibrosis, as is seen with SARS and MERS infections. Pulmonary fibrosis is expected to be the main factor leading to pulmonary dysfunction and decline of quality of life in COVID-19 survivors after recovery. More research is needed into the correlation of CT findings with clinical severity and progression, the predictive value of baseline CT or temporal changes for disease outcome, and the sequelae of acute lung injury induced by COVID-19 (Lee 2020).

Of note, chest CT is not recommended in all COVID-19 patients, especially in those who are well enough to be sent home or those with only short symptomatic times (< 2 days). In case of COVID-19, a large number of patients with infection or suspected infection swarm into the hospital. Consequently, the examination workload of the radiology department increases sharply. Because the transmission route of SARS-CoV-2 is through respiratory droplets and close contact transmission, unnecessary CT scan should be avoided. An overview of the prevention and control of the COVID-19 epidemic in the radiology department is given by An et al.

Ultrasound and PET

Some experts have postulated that lung ultrasound (LUS) may be helpful, since it can allow the concomitant execution of clinical examination and lung imaging at the bedside by the same doctor (Buonsenso 2020, Soldati 2020). Potential advantages of LUS include portability, bedside evaluation, safety and possibility of repeating the examination during follow-up. Experience especially from Italy with lung ultrasound as a bedside tool has improved evaluation of lung involvement, and may also reduce the use of chest x-rays and CT. A point scoring system is employed by region and ultrasound pattern (Vetrugno 2020). However, the diagnostic and prognostic role of LUS in COVID-19 is uncertain.

Whether there is any potential clinical utility of other imaging techniques such as 18F-FDG PET/CT imaging in the differential diagnosis of complex cases also remains unclear (Deng 2020, Qui 2020).

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