key: cord-0919326-mqlm7cji
authors: Omata, Masao; Hirotsu, Yosuke; Sugiura, Hiroki; Maejima, Makoto; Nagakubo, Yuki; Amemiya, Kenji; Hayakawa, Miyoko; Tsutsui, Toshiharu; Kakizaki, Yumiko; Mochizuki, Hitoshi; Miyashita, Yoshihiro
title: The dynamic change of antibody index against Covid-19 is a powerful diagnostic tool for the early phase of the infection and salvage PCR assay errors
date: 2021-01-05
journal: J Microbiol Immunol Infect
DOI: 10.1016/j.jmii.2020.12.009
sha: 574db62f606403e960c08b032aff9d4ebdf14823
doc_id: 919326
cord_uid: mqlm7cji

Background Currently, PCR assay is a golden standard for diagnosis of Covid-19. However, it needs nasopharyngeal swabs, expensive instruments and expertise. It even causes PCR errors. Methods We validated the antibody assay (Roche) in 36 followed patients and 1,879 controls (medical staffs). Results Of 1,879 medical staffs, only two (0.11%) were positive by Cut off Index (COI;1.0) (mean±SD, 0.094±0.047). Thirty six patients were composed of three groups; Group A,4 from Diamond Princess cruise ship, Group B, 2 infected in Africa, and Group C, 30 infected in Japan. PCR assays were conducted at outside laboratories before and repeated in house after hospitalized. Of 36 at admission, positive antibody was seen in 4/4 from the ship, 0/2 from Africa, and 5/30 from Japan. Two from Africa showed the increase of COI and became positive on days 8 and 13. Thirty Japanese was divided in two groups, e.g., 23 showed dynamic increase of COI up to 84.4 within 3 days while active virus replication present (Group C). In remaining 7 (7/30, 23%) (Group C’), no rise of antibody nor positive in house PCR assays, indicative of false positive results of PCR at the beginning. Conclusion This antibody testing has a wide dynamic ranges of COI and, thus, could be utilized in the early infection phase. This may also compliment and even help to avoid possible PCR errors. Therefore, this can serve as a powerful diagnostic tool, needed in the frontline of the clinic and hospitals.

The number of the individuals infected by Covid-19 virus may reach over 10 million at the 47 end of June, 2020 1,2 . The diagnostic tool for this infection includes PCR assay as the only 48 measure employed worldwide 3 . However, PCR assay is time consuming, expensive, 49 cumbersome, and need some expertise 4 . In particular, PCR error causes tremendous 50

confusions. The preventive measures were required for people involved including the family, 51 office workers, hospital staff and close contacts, if PCR assay were reported positive. 52

However, PCR assay is still a golden standard to deicide the prevention and choice of 53 treatment modality 5,6 . Other modalities of the antigen-antibody assay is about to be 54 developed 7 . However, the knowledge about these antigen and antibody assay is still limited. 55

In this communication, we report the developed antibody assay that we employed may 56 become a very powerful diagnostic tool not only for patients in the convalescent phase but in 57 the early phase of this infection. This can even be easily available at clinics and hospitals. 58

Comparison was made to the golden standard PCR assays in serial swab samples and the 59 time course of the development of the antibodies in our Japanese patients along with 60 patients from outside (Diamond Princess cruise ship and Africa). 61 62

Methods 63

Patients and Controls 64

Group A. 4 patients from Diamond Princess 65

On February 11 th , 2020, we were asked from the government to see patients from the 66

Diamond Princess. We are 150km west of Tokyo, in the city of Kofu, where 400,000 people 67 are living. 68

No infected patients by Covid-19 virus had existed till then. However, we are the 600 bed 69 medical center of the district, with emergency helicopters and intensive care units. We are 70 prepared to have all the patients in critical conditions. We received the patient on February 71 11 th , one couple of Americans, husband and wife. At the beginning, husband walked in but in 72 a few days, needed ventilator. However, no improvement of blood gas was seen. On 73

February 19 th , our emergency team did a bronchoscope drainage to take the build-up 74 plaque out of the major bronchial tree. After that, blood gas dramatically improved. In the 75 meantime, the infected wife developed massive brain hemorrhage. Decompensating 76 surgery was done for this wife and they both went back to the homeland safely in the end of 77

April, after 2.5 months hospital stay. If they passed away, they could be the 1 st American. 78

The other two from Diamond Princess were one Chinese and one Japanese passenger 79 who went home uneventfully by the end of February. 80

During the hospital stays of four patients, we took 11 blood samples and 19 81 nasopharyngeal swabs.

In March, one couple (pregnant wife and husband) back from Africa were transferred from 84 other hospital. They were obviously infected in Africa. Their clinical course was uneventful 85

and discharged in 2 weeks. During their hospital stay, we took 12 serial blood samples and 86 15 nasopharyngeal swabs for PCR assay. 87

Group C and C'. 30 from Japan, Yamanashi 88

After the experience on 6 aforementioned patients from outside with the very stressful 89 daily practices and some relief for whole hospital staff, we hoped this will not happen in 90

Japan. However, number of infected patients incrementally increased in our country from 91 the beginning of the April and reached the height on April 12 th 7 . Furthermore, we are now 92 facing the "2nd wave" by the end of October, 2020. Antibody test and COI (Cut off Index) 117

To screen the presence of antibody against SARS-CoV-2, serum were tested by using 118

the Elecsys Anti SARS CoV 2 (S300) RUO (Roche Diagnostics, Basal, Switzerland) on 119 cobas 8000 automated platform. Recombinant protein representing the nucleocapsid (N) 120

antigen was used for the determination of antibodies. This assay utilizes the 121 electrochemiluminescence immunoassay (ECLIA) principle 8 . 122

In brief, biotinylated recombinant antigen and ruthenium-labeled recombinant antigen 123 form a sandwich complex. After addition of streptavidin-coated microparticles, sandwich 124

complexes are magnetically captured onto the surface of the electrode, then induces 125 chemiluminescent emission.

Samples with a Cut off Index (COI; signal sample/cut-off) <1.0 were considered as 127 negative, those with a COI≥1.0 were considered as reactive (positive). 128

Viral nucleic acid extraction 130

We collected nasopharyngeal swabs with cotton swabs and universal transport media 131 (Copan, Murrieta, CA). Total nucleic acid was automatically isolated using the MagMax 132 J o u r n a l P r e -p r o o f Viral/Pathogen Nucleic Acid Isolation Kit (Thermo Fisher Scientific, Waltham, MA 133

) on automated machine KingFisher Duo Prime as previously described 9 . 134

Briefly, we added 200 µL of universal transport media, 5 µL of Proteinase K, 265 μL 135

Binding Solution, 10 μL Total Nucleic Acid Binding Beads, 0.5 mL Wash Buffer, and 1 mL or 136 0.5 mL of 80% Ethanol to each well of a Deep-well 96-well plate. 70 μL of Elution solution 137

was added to Elution Strip. Total nucleic acids were stored at −80 °C. 138 139

The protocol was designed by the National Institute of Infectious Diseases (NIID), 141

Japan 10 . To detect SARS-CoV-2, we performed one-step RT-PCR according to the NIID 142 protocol (version 2.7).

The reaction mixture comprised 5 μL of 4× TaqMan Coralville, IA) 11 . 150

The RT-qPCR assays were conducted on a StepOnePlus Real-Time PCR 151

Systems machine (Thermo Fisher Scientific) with the following cycling conditions: 50°C for 5 152 min for reverse transcription, 95°C for 20 s, and 45 cycles of 95°C for 3 s and 60°C for 30 s.

The threshold line was set at 0.2. The Ct value was assigned to each PCR reaction and the 154 amplification curve was visually assessed. 155

156

This study protocol was approved by the Institutional Review Board of our hospital. 158

Results 160

In all four patients, PCR assays were conducted at, and turned out positive and directly 162 transferred from the Diamond Princess cruise ship. Exact dates of infection were not known, 163 but all were positive for the antibody on admission with rise of Cut off Index (COI) of 164 antibody (Fig. 1) . Initial COI of four cases were 37.9, 1.67, 56.4, and 26.8, in cases #1, #2, 165 #3, and #4, respectively ( Fig. 1, Group A) . In case #2 which showed the lowest COI, we 166

tested the change of COI in 12 serial samples from day one to day 40. The COI markedly 167

increased to 95.4 on day 40 ( Fig. 1, Group A) . 168

A couple of pregnant wife (#5) and husband (#6) living in Africa came back to Japan for 170 preparing delivery (Fig. 1, Group B ). They were transferred to our hospital because we have 171 neonatal intensive care facility. PCR assays were repeatedly positive, but the antibody 172 response was not reactive on admission (0.0782 and 0.0815), and sluggish in increase of 173 COI but turned positive on day eight (COI: 1.15) and on day 14 (COI: 1.15), respectively ( Fig.  174 1, Group B). 175

Twenty three Japanese patients (Group C) 176

Aforementioned six patients were infected outside Japan. From the end of March, 2020, 177 30 patients from our area were admitted to our hospital.

Of the 30 patients, 23 (#7 to #29) showed very brisk antibody response after admission 179

( Fig.1 , Group C and Table 1 ). We serially checked the change of COI in 194 blood samples 180 from the 23 patients (Group C). Although 18 patients with "negative" tests at admission by 181

pre-defined criteria of COI above 1.0, steady increase of COI were noted in all cases 182 afterwards ( Fig.1 , Group C and Table 1 ). 183

For these 23 patients during the hospital stays, we took nasopharyngeal swabs 248 184 times and performed PCR assays (average 10.8 times per patient, ranges 4 to 27). Of 185 particular interest, 26 (96%) patients showed steady increase of antibody level while PCR 186 assay demonstrated active virus replication within 10 days ( Fig.2 and Table 1 ).

In contrast to the 23 patients with brisk antibody response, seven Japanese patients (#30 189

to #36) stayed negative for the antibody with very low COI (0.074 to 0.096) throughout and 190 never became positive ( Fig. 1 , Group C' and Table 1 ). All these patients were admitted to 191 our hospital with positive PCR assay results at outside laboratories. After admission, we 192 conducted in house PCR assays for the seven patients 28 times (range, 2 to 7 times). None 193 of them yielded positive results. Thus, in these cases, no evidence of Covid-19 infection was 194

obtained.

The Ct values of PCR assays in these seven cases which we obtained from the outside 196 laboratories were 40, 39, 41, 37, 39, 37 and 36 in cases #30, 31, 32, 33, 34, 35 and 36, 197 respectively (Table 1) . These results suggest the antibody test elucidates the presence or 198

absence of Covid-19 infection and compliment the results of PCR assays and salvage the 199 possible errors.

To further ensure possible PCR errors in these 7 cases (Group C'), we compared Ct 202 values of PCR assays and COI of the antibody between 23 cases (Group C) and 7 (Group 203 C'). We obtained the information on Ct values of PCR assays from outside laboratories 204

where the presence of Covid-19 infection was first diagnosed by their PCR assays.

Of the 30 Japanese cases (Groups C and C'), in 23 cases (#7 to #29) the Ct values of 206 PCR ranged from 12 to 33 (mean±SD, 21.7±5.1), suggesting high virus load. All these 207 cases were proven positive by our repeated in house PCR assays. In addition, the newly 208

introduced antibody assay revealed dynamic change of the indices in serum samples (Fig.  209 2). In contrast, 7 in Group C' (#30 to #36) which had basically no antibody response, showed 210

Ct values ranging from 36 to 41 (mean±SD, 38.4±1.8) ( Figure 3 and Table 1 ). There 211

were statistically significant differences of Ct values between Group C and Group C' 212 (p=4.1x10 -9 ) ( Figure 3 ).

We determined the COI in 1,879 medical staffs as a control. Of these, COI was less than 215

1.0 in 1,877 (99.89%). Only two (0.11%) medical staffs had over 1.0 COI (1.220 and 1.100 216 COI, respectively). However, the COI did not show the increase by the second test (1.180 217 and 1.080, respectively) and PCR tests were negative in these two control.

These results clearly indicate the Ct values of PCR should be carefully defined to give 219 plus minus results because it is the golden standard of Covid-19 infection. Our data clearly 220

indicate that Ct values of PCR above 36 could be indicative false positive results. However, 221

now the antibody assay may salvage or cover the defects of errors intrinsic to PCR assay 222 223

Discussion 224

Early diagnosis of the virus infection, especially COIVD-19 is urgently needed. We are 225

in frontline of the patients care in a medical center in Japan. Not only the patients care, we 226 also have to prevent our hospital staff from the infection 12 . If you include in and out patients 227

altogether, 3,000 to 4,000 people in our hospital are at the very high risk of the infection at 228 any time.

We have conducted in house PCR assay in our hospital more than 2,000 cases in the 230 past 2 months 4,11,13,14 . We have made a swab taking team with full protection for Covid-19 15 . 231

Thus, by combining of swab sampling and PCR assay team working hard 24 hours a day, 232

we performed PCR assay for all the patients before hospitalization and tried to maintain our 233 ordinary hospital function. In addition, we performed PCR assays for our staff to protect 234 them from invisible agent 15 . Fortunately, we started genome analysis center (GAC) 6 years 235 ago in our hospital and some of the member had been involved in molecular biological 236 studies of viruses [16] [17] [18] chromatography assay only showed simultaneous elevation of the both antibodies at the 244 same time (data not shown). Also, for this newly introduced antibody assay (Roche) against 245

Covid-19 in the current study, we anticipated this can be used mainly for patients in 246

convalescent phase. However, as was shown, the index, COI, of the antibody had a very 247 wide dynamic range from 0.08 to over 80, e.g., nearly 1,000 times difference. As a control 248 group, we took blood samples from medical staffs. Their COI was extremely low (0.094) and 249 their ranges very narrow (0.047 SD). 250

If you see the Cut of Index (COI) of the antibody assay, it gave us the very early sign of 251 the infection. At the time of the admission, COI of the antibody was elevated in almost all the 252 cases (Groups A and C). In a few cases, COI of the antibody is below pre-set level of 1.0, 253 but immediately after their values steady increased and reached above the cut of level 254

within 10 days (Figs. 1, Group B ). 255

The current study contained different groups of patients; 4 from Diamond Princess cruise 256

ship who were obviously infected from the end of January, during Wuhan epidemics (Group 257

A) 19 and 23 from Japan who were infected in April (Group C). In these two groups, the 258 antibody response is remarkably brisk to the antigens employed in this assay system, 259

whereas the antibody response was present but took time in the couple of pregnant wife and 260

husband from Africa (Fig. 1, Group B) At least, people have to realize this method is a semiquantitative assay and not simply 279

giving black and white. Our present results clearly indicate high Ct values of PCR could be a 280

"hint" to suspect false positive results (Figure 3 ). If PCR needed 36 or more cycles to see the 281

"band", we need to confirm whether they are true positive results. However, laboratories are 282 obliged, at least in Japan, to give plus and minus answers by predefined indices. Our data 283 certainly indicate PCR cycles over certain numbers have a risk of "false" positive and we 284 advocate setting the gray zone between black and white. Because false positive results, in 285 particular, of Covid-19 infection may provoke enormous amount of not only the medical but 286 also social 'confusions', sometime even infringement of privacy 23, 24 . 287

This communications may provide the information on the utility at the antibody against 288

Covid-19. It can be utilized as a very powerful, convenient tool and give the comfort to the 289 medical staff by this assay. This methods can even be employed at the Emergency 290

Department waiting for 10 minutes and get the useful result whether they are not having any 291 sign of the infection or the possibility of the ongoing current infection by measuring Cut off 292

Index of the antibody. This method could be employed not only for epidemiological mass 293 survey but also in the frontline hospital and clinics. However, obviously the limitation of this 294 study includes the antibody assay alone cannot be utilized as diagnostic tool in countries 295

where many are already infected and antibody prevalence is much higher than Japan 25-27 . 296

In addition to antibody assay, convenient antigen-antibody assay may be needed as was 297 employed in other virus infection 17,18 . 298

We are currently testing the amino antigen detection method. patients from Africa, Group C; 23 Japanese patients who were diagnosed PCR positive at 388 outside laboratories before transferred to our hospital and re-confirmed by our in house PCR 389

assay, Group C'; 7 Japanese patients diagnosed positive at outside laboratory with Ct 390

(threshold cycle values 36 to 41), but could not be re-confirmed by our laboratory after 391 hospitalization, 392

Serial COI (Cut off Index) was shown from day 0 to day 50 in the upper panel and COI from 393 day 0 to day 10 (expanded) in the lower panel. 

A Novel Coronavirus from Patients with Pneumonia in 315

World Health Organization, Coronavirus disease (COVID-2019) situation reports

World Health Organization