Jpn. J. Infect. Dis., 53, 116‑123, 2000

Original Article

Nationwide Nucleic Acid Amplification Testlng Of Hepatitis B Virus,
Hepatitis C Virus and Human Immunodeficiency Virus Type 1 forBlood Transfusion and FollowIUp Study ofNucleic Acid
AmpliflCation Positive Donors
Japanese Red Cross NAT Screenlng Research Group*(Received May 9, 2000. Accepted June 19, 2000)
StJMMARY: This study described a program for and the results of a nationwide nucleic acid amplification

testing (NAT) screening for hepatitis B virus (HBV), hepatitis C virus (HCV), and human impunodeficiency
viruStype 1 (HIV‑ 1) by multiplex reagent with a pooled system. After routine serological screenlng, this test wasused in order to be in time for blood transfusions. The Japanese Red Cross currently supplies donated blood allover Japan for blood transfusion.AsofJanuary 2000, 2,140,207 units (5,093 pools) were tested by a pool sizeof500 and 19 HBV DNAIPOSitive cases and 8 HCV RNA‑positive cases were found. Since February 2000, thepool size was switched to 50 and among 420,770 units (8,564 pools), 7 HBV DNAIPOSitive cases and I HCVRNA‑positive case were found. HIV RNA was not detected in any of the tested pools. Among the 26 HBV DNA
positives, 22 were wild type; of these, 6 (23%) had hepatitis B surface antigen (HBsAg) that was undetectable byovemight enzyme immunoassay (EIA). Except for one case, in which coexisting antibody inhibited the immune
reaction, all 1 7 casesthat were followed later showed seroconversion. In I 0 0fthese cases, HBV DNA disappeared
below the level of detection and seroconversion of IgM anti‑HBc and anti‑HBc antibody occurred during theobservationperiod. The remalnlng 4 cases were precore mutants and all had an undetectable level ofHBsAg by
EIA･ Three cases did not show IgM anti‑HBc seroconversion, which should be observed during the early stage

ofHBV infection･Asfor the HCV RNA, the followingtypes were identified: 2 genotype II (lb), 3 genotype III(2a)I and 4 gen.otypeIV (2b)･ A weak anti‑HCV positive reaction was observed in two cases and strong
seroconversion ln One Case among 4 0fthe cases that were followed. Although it is not lO0%, NAT narrows thewindow period in early‑stage infection, resulting ln an exponential reduction of the virus load that escapes

serological screenlng tests for blood destined for blood transfusions. In the case of HBV, NAT screenlng detects
HBV DNA in persistently infected individuals with extremely low levels of HBV antlgen and antibody o洗en

observed in the case of HBV mutants.

mutants or viral loadsthat are undetectable by current serologi‑

INTRODUC TION

cal screenlng me仇ods. In血e United States, the risk orgiv‑

During the period from November 1 997 to November 1 999,

1ng blood duringaninfectious windowperiod has been esti一

5.6 million serologically negative donations were tested manu‑

mated to be 1 :63,000 in血e case orHBV, 1 : 103,000 f♭r HCV;

ally by nucleic acid amplification testing (NAT) in order toand 1:641,000 for HIV‑I; there, too, the risk ofHBV is the

reduce the virus load &om source plasma for plasma derived

highest (3). Therefore, newer laboratory assays to detectproducts. We found 78 donations positive for hepatitis B virus

directly the presence of virus genome with high sensitivity
and speciflCitysuch as NAT are necessary.
Currently, testsare beingperformed based on cost consider‑(HBV) (1 :72,000), 10 for hepatitis C virus (HCV) (1 :560,000)
and 2 for human immunodeficiency virus type 1 (HIV‑1)(1 :2,800,000);仇e risk or HBV window‑period donations isations, and should be done as rapidly as possible in order tohighest among these three viruses (1). These data indicate

provide blood for transfusion of cellular components. Sacher etthat blood transfusions have become safer in contemporaryal･ (4) have noted that HCV is particularly suited for minipooI

Japan than they ever were before. The Japanese system is a

NAT because of the highlevels of virus load observed during

100% voluntary blood donation system and serologlCal

the window period. However, the effects ofHBV DNA screen‑
lng are more COmplex because HBV seems to be present inscreenlng lS Performed at nationwide Japanese Red Cross

lowtiters during the window period. However, we quantified
IIBV genomesin6 hepatitis B seroconversion panels of Boston
Biomedica, Inc. (Bridgewater, Mass., USA) during the early(JRC) blood centers by automated agglutination tests using
PK7200 (2). Residualrisk does persist due to blood donations

made duringthe window periodinearly stages of infTection. In

stage ofHBV infection; the virus growth curve showed an

addition, another risk is due to late‑stage infection with virus

exponential straight line that proceeded agalnSt the date of

donation. Doubling time was 2.0 days and log time was 6.5*Members of the Japanese Red Cross NAT Screenlng Researchdays (5). Therefわre, a highly sensitive NAT assay with anGroup are listed in the Appendix.

approprlate pool size could narrow the window period ofearly‑Stage HBV infection, thus resulting ln an exponential
reduction of the virus loadthat escapes from present screenlngS.

Corresponding author: Tsugikazu Tomono, Japanese Red Cross

plasma Fractionation Center, Koyo Build. 7th Fl. , Hamamatsu‑

cho 1‑10‑17, Minato‑kuTokyo 105‑0013, Japan. Tel: +81‑3‑3433‑

In December 1 998, a highly sensitive multiplex HBV/HCV/

71 83, Fax: +8 1‑3‑3433‑71 85, E‑mail: tomono@pfc.jrc.or.jp

HIV‑ 1 reagent (MPX) was developed for NAT for HBV, HCV

116

biotinylated probes co‑captured HBV, HCV, HIV‑1 andIntemal Control (IC) on streptavidin‑Coated paramagnetic
microparticles (I 2) using GT‑ I 2/GT‑X (Roche Diagnostics
K.K., Tokyo). Coamplification and real‑time detection of
HBV, HCV, HIVll and IC were performed by TaqMan PCR

and HIV‑1 infections, which can thus be screened at one time.
Therefore, the cost of screenlng lS reduced and the timerequired for testlng lS also less than that needed for screenlng

individual viruses. Withthe development of a rapid transporta‑tion system, computer networks,anautomatic sample poolingsystem,andanautomated viral nucleic acid detection system,(1 2). A detailed account of the procedure and its principles

we started nationwide NAT screerung of serologically negative,

was reported by Chaka lmpraim et al･ (12)･ Carryl0Ver †asvoluntarily donated blood at two NAT centers in Japan sinceprevented by excluding samples by serologlCal prescreenlng

July 1 999. After the resolution of MPX‑positive samples into

in the blood centers and by use ofAmpErase (13) in the NAT

individual vims, each JRC blood center passed on the results

to the donors and a follow‑up study of NAT‑positive cases

centers. Multiplex HBV/HCV/HIV‑ 1 reagent (Roche Diagnos‑
tics and Roche Molecular Systems, Inc., Pleasanton, Calif.,

was initiated in order to distinguish between transient orUSA) possessed a 95% detection limit f♭r HBV:25 copies/

chronicinfection. An additional goal was the improvementml, HCV:100 IU/ml and HIV‑1:60 copies/ml and was usedas the first‑step NAT screenlng reagent Without target
discrimination. Results were provided within 5‑6 h. Non‑
template controls (NTC) were employed to detemine thein the health care of voluntary blood donors.

MATERIALS AND METHODS

cutoffvalue with specifically‑designed data reduction soRware.

Serological tests for HBV, HCV and HIV‑1 infections

The results were reported as positive, negative, or inhibitory

and measurement of serum alanine aminotransferase(12). In this manner, 20,000 units were tested every day by(ALT): All voluntarily donated blood in JRC was qualified
by a questionnaire administered by the JRC blood centers

the use of limited human resources. All samples were handled

throughout Japan. These samples were screened for hepatitis

We also identified individual samples with a double‑check

B surface antigen (HBsAg) by reverse passive hemagglutina‑

SyStem･and controlled automatically by a bar‑code computer system･

Reporting NAT results to blood centers: NAT results withtion (RPHA), for anti‑HBV core antibody (anti‑HBc) byhemagglutination inhbition (HI) , and for anti‑HBsAg antibody

MPX were reported immediately by the NEC computer
network (NEC Corp., Tokyo) to each blood center and MPX‑

(anti‑HBs) by passive hemagglutination (PHA) using reagentsnegative units were released for blood transfusions or as

prepared by the JRC, as described elsewhere (6, 7). Anti‑
HCV antibody (anti‑HCV) was tested by PHA (Dainabot Co･

source plasma f♭r plasma‑derived products. When an MPX‑

Ltd. , Tokyo) or by particle agglutination (PA) (Fujirebio lnc. ,positive pool was fわund,仇e release was withheld until the

Tokyo) (8).Anti‑HIV‑ I/2 was tested by agglutination of gelatin

positive unit could be identi丘ed. A触r the MPX‑positive unit

was identified, other MPX‑negatives were released.
Later MPX‑positive units were resolved into HBV DNA
by the method ofIizuka et al. (6), HCV RNA by the method
orOkamoto et al. (14), and HIV‑1 RNAby the method or
Matsumoto et al. (15) at two JRC NAT Centers.
CharacteriZ:atioTI Of NATIPOSitive samples: HBV DNA
and HCV RNA, identified by two JRC NAT centers, Wereparticles coated with recombinant HIV‑ 1/gp4 1 , HIV 1/p24
and HIV12/gp36 (Fujirebio) (9). All of these agglutination
screenlngS Were Carried out uslng an automatic PK7200(Olympus Co. Ltd., Tokyo) and were confirmed by speciflC
inhibition tests. The endpoint of the 2‑fold dilution of test
samples was expressed as an exponent of 2n versus the end
titer of the specific inhibition test; these results of the tests aresent to the Research Laboratory ofJRC Saitama Blood Center.
Virus genomes of both viruses were quantified. Furthermore,
subtype, genotype and mutation in the precore region of HBVand the genotype ofHCV were identirled.
Samples from HBV NAT‑positive donors were further

shown in Table 3 and 4. Serum ALT levels were measured by

the method ofWroblewski and LaDue (10). All these tests
were carried out in each blood center. Serologically positive

and ALT‑elevated (>6 1 IU) Samples were excluded from NAT
SCreenlng･

Shipment: Five milliliters of each donated blood sample

tested fわr HBsAg by ovemight enzyme immunoassay (EIA)

were taken into a polyethylene terephthalate (PET) tube

by Auszyme II (Abbott Laboratories, North Chicago, ill.,containlng 7.5 mg ofEDTA. 2K and were sent to two NArcenters, in Tbkyo or in Chitose, Hokkaido. The most directUSA) for IgM‑typeanti‑HBcAb (IgMHBcAb) by Corzyme‑M

arrived within 1 day ordonation. Samples were kept at 2‑(Abbott Laboratories). Hepatitis B e antigen antibody wastested by a commercialkit (HBeAg/Ab; Institute of Immu‑
nology, Co. Ltd., Tokyo). HBV DNA was quantified by the

80C during shipplng and storage so仙at仇e viral liters could

method of Iizuka et al. (6) and sequenced to determine

route by air‑night or surface transportation was chosen. Bloodsubtype, genotype, and mutation in the precore region (16).

remain unchanged as indicated (1 1 ).

Poolimg Or Samples and NAT or pool samples by

As for samplesfrom HCV NAT‑positive donors, HCV RNA

Simultaneous MPX: For sample preparation, 0. 1 ml of every
50 serologically negative samples was harvested, and 5 ml

was quantifled by ABI PRISM 7700 (PE Applied Biosystems,
Faster City, Calif., USA) and genotype was determined bycollected is glVen the ten or 50 pool. A pool size or 500

genotype‑specific primers ( 1 7).

consisted often of each 50 pool. When the MPX‑positive
500 pool was found, 10 samples from the 50 pool were tested

RESUI.TS

separately. Individual units from the MPX‑positive 50 pool
to a pool size of 50 in order to increase the test'S sensitivity

Positive rate of HBV DNA, HCV RNA and HIV‑1 RNA
in NAT‑positive donors: From July 1999 to February 2000,

and to shorten time necessary for pooling and resolving.

2,560,977 serologlCally negative donors were screened bywere identified. Since February 2000, all tests are switched

Pooling was perf♭med uslng ALOKA automatic pooling

MPX in two NAT centers. All 54 JRC blood centers in Japansystem, APS‑NAT (Aloka Co. Ltd., Tbkyo), with a capacity

participated in this program. MPX‑positive units wereto pool 10,000 units successively in 6 h. For the preparation

resolved into either HBV DNA and HCV RNA. The resultsof specimens for NAT from pooled plasma, target‑Specificof the 500‑pool test and the 50‑pool test are shown separately

117

in Table I. HBV DNA‑ and HCV RNA‑positive cases

statistical difference between these two groups was not benumbered 26 (1/98,499) and 9 (1/284,553), respectively. No
HIVl 1 RNA was found. We were able to withholdthe releaseobserved on the x2 test (Table 1); this result is due to thevery low number of NAT‑positive cases.

of infected units from proceeding to blood transfusion. The
HBV DNA‑positive rate of the 50‑pool group (1/60,1 10) was

NAT‑positive donors:Asshown in Figure, among the 26

higher than that of the 5001POOl group (1/1 12,642), but a

HBV DNA positives, 22 were wildtypeand 4 were precore

QuantificatiorL and characteriZ:ation of HBV DNA in

Table I ･ Positive rate ofHBV DNA, HCV RNAand HIV‑1 RNA ofMPX positive donors
NAT

Participatlng

Center

Tbkyo

Blood

Pool

Centers

27

Tbta1

27

54

ofBlood

500pool50

Chitose

No.

Size

poo1

500poo1

Units

(No.

1,274,807
222,262

rested)50pool

198,5082, 140,207

50 poo1

420,770Tota1

2,560,977

HBV(3,008)(4,514)865,400

500 poo1

NAT Positive Cases

ofPools

Tested(2,085)7
3

1/182,115
1/

12

DNA

74,0871/

1/

72,11749,627

HCV

4

RNA

HIV̲

1/318,701004I/216,350

1

1/198,508(4,050)4(5,093)l;n･S･lll'/lt2.I,冒:≡;nsli;2263;;……

(8,564)(I

3,657)

26

9

n.S.=not significant by x2 test

luJSa!dou

Figure･ HBV DNA and HCV RNA copies in positive samples.
The numbers in the circles correspond to those of positive donors, shown in Tables 3‑5.

LeR column: HBV‑positive cases by ovemight ETA
Middle column: HBV‑negative cases by ovemight EIA#1 9: HBsAg‑positive and HBsAb‑positive cases#18, 22, 24, 27: Precore mutant cases, ‑0thers: Wild typecases
Right column: HCV RNA window donation
BDL: below detection limit

118

1

0
0

RNA

Table 2. Follow‑up study of HBV NAT positive donors

mutant HBV. HBV DNA contents were distributed between
2.4X105 and 2.3X 102/ml. Because our system aimed toAshorten the serological screening tlme, We used RPHA for
the HBsAg test by automatic PK 7200. We later reexamined

Wild

Type

1

Seroconversion of IgManti‑HBc, anti‑HBc8

1 7(10)*HBV DNA with anti̲HBc and anti̲HBs 1

the NAT‑positive samples by ovemightAuszyme II. Case #1 6,

つJ O 2B Precore Mutant

with a copy number of I.4X 104/ml, showed a faintly positivelgM anti‑HBc

reaction (0.064/0.060). Seventeen cases of wild type HBV

Low level of anti‑‖bc

DNA withaneven higher copy number, were ovemight EIA‑

書the number of cases dlat HBV DNA disappeared during ob駈rVationperiod.

positive except for case #19. Case #19 showed rather high

HBV DNA (1.5 X 105/ml), but had a coexisting high titer ami‑
HBc and anti‑HBs, which gave negative results for HBsAg

test by ovemight EIA. These coexistlng antibodies may

appears to be the case that all 17 wild type HBV‑infectedinterfere with the serological reactions of RPHA or EIA forcases were at an early stage of infection (i.e., in the serologi‑

the detection of HBsAg. The HBsAg‑undetectable unit with

cal window period).

both high‑titer anti‑HBc and anti‑HBs and with normal ALT
level was permissible to release for transfusion according toanti‑HBc positive sample of case #19.AsIgM anti‑HBc werethe criterion of serological screenlng before the introduction

negative and the viremia leveland high titer ofanti‑HBc (>213)

of NAT in Japan. The question remains whether or not suchcontinued, this was a case ofliBV infection, however, HBsAg

cases with high‑titeranti‑HBs are infectious, but transfusionof such units could be prevented by screenlng With NAT. Fivecases with HBV DNA contents below 5.8X 103/ml were all
EIA‑negative.
All of the four HBV precore mutants were negative for

JRC regulation, HBsAg negative units with hightiters ofanti‑
HBc are excluded from blood transfusion if anti‑HBs isundetectable, but if the anti‑HBs titer is higher than 24, theumit passes the guidelines for serological screenlng. Such umits

Wild type HBV DNA was detected in the anti‑HBs andcould not be detected because of coexistlng antibody.Asby

HBsAg by ovemight EIA. Amongthese, three was below 3.6

might be rare but can be screened out by the HBV DNA NAT･
Asfor the three HBV precore mutant cases (Table 4), case× 103/ml; however, the copy number of#27 was 3.8× 104/

contents (between 3.7‑I.4X 104/ml), as in case of#27, were#24and #1 8 showed borderline levels of and‑IiBcand negative
IgM anti‑HBc after donation; case #22 completely negative

all reactive by ovemight EIA. It is to be noticed that all four

for anti‑HBc 21 days after donation and IgM anti‑HBc wasprecore mutants were negative by EIA.

also negative. In all these cases, viremia levels did not change

ml. As six wild type cases wi血仇e same level orHBV DNA

Among the 22 wild type cases, 19 (86%) were subtypea鮎r donation, indicatingthat these three cases were not inthe

adr, genotype C, which is the predominant subtypein Japan

early stage of acute HBV infection; however, low levels of
HBV DNA existed in later stages of subclimical HBV infec‑(I 8). The subtype and genotypeoffour precore mutants weretion.

as fわllows: two adr C, one adw B, and one ayw C (#18).

Antigen expression of # I 8, ayw was discordant to genotype C

Here, Our NAT screenlng System could detect HBV DNA

by mutation, which explains why it was hardly detectable oninpersistently infected individuals with extremely low levels

Therefore, by the JRC NAT system, we were able to excludeof HBVantigenand antibodies which were often observed in
the case of HBV precore mutant infection.

19 RPHA, HI negative units out of2,140,207 (1/112,642)
totalunits bythe 500‑pool test. Seven out of420,770 (1/60,1 10)

NAT‑positive donors: HCV RNA concentration of serologl‑

units were excluded by the 50‑pool test, as shown in Table 1 ･cally undetectable NAT‑positive donors are shownin Figure

Among them, 1 0 units were undetectable by ovemight EIA･and Table 5. The concentration of HCV RNAinNAT‑positive

血e antigen assay.

QuantirICation and characterization of HCV RNA in

Four of these had precore mutants, 5 were wild type (below

donorsinthe serological window periodare high ranglng &om

5.8× 103/ml), and one was wild type with coexisting anti‑

4.3× 107‑2.7× 104, indicating a prolonged, high titer viremia

HBc and anti‑HBs.
Fouow‑up study of HBV DNA NAT‑positive donors: It is

preceding seroconversion. Two cases of genotype II (lb), 3

important toknow whether or not these NAT‑positive donors
are at the early pre‑seroconversion stage (serologicalwindow

genotype Ill (2a)and 4 genotype IV (2b) were identified･
Fouow‑up study of HCV RNA NAT‑positive donors: FourHCV RNA NAT‑positive cases were followed and describedperiod) ofHBV infection or at a late stage of chronic infectionin Table 5. One case (#101) showed high‑titer anti‑HCVwith very low levels of viremia. Moreover, Considering the

seroconversion, 201212･5 after donation. The other two cases

health care of the voluntary blood donors, we started a follow‑(#104 and #108) showed a weak anti‑HCV response 23‑28

up study of these NAT‑positive donors.

a鮎r donationand had significantly high Al:T levels. TherefoⅠ℃

Through collaboration of the corresponding blood centersthese three cases were at the early stage of HCV infection

and the courtesy and consent of the blood donors, blood wasprior to seroconversion of anti‑HCV.
In case #102, the anti‑HCV titer measured by PA was 24,

taken into 5 ml PET tubes with EDTAfrom the NATIPOSitive
donorsand sent to NAT centers. HBsAg, anti‑HBs,anti‑HBc,

which is within the threshold of negative results. When it

IgM anti‑HBc, HBeAg/anti‑HBe, and the copy number of thewas retested by PHA, the titer was 29, and thuswe obtained

HBV of each sample were measured in the Research Labora‑
tory of the JRC Saitama Blood Center.

discordant results. HCV RNA copleS Of2.7 X 104 were foundin this sample. After 87 days, the HCV RNA content slightly

We were able to follow 18 cases ofwild type HBV NATI

increased and it seemed that this was a case of persistentinfection with normal ALT levels (Table 5).

positive donors (Table 2, 3) and three cases of mutant HBV

NAT‑positive donors (Table 2, 4); IgM anti‑HBc and anti‑
HBc in I 7 wild type HBV donors (94%) Were seroconverted

DISCUSSION

after donation. Among them, HBV DNA disappeared below
detection levels in 10 cases within the follow‑upperiod. It

Blood safety as regards transfusion‑transmitted viral infeC‑

119

Table 3. Infection with wild type HBV
‑‑一一‑

G'

Age

Date
1

Sex

HBsAg

ALT

RPH

A

‥TI:･,;'∴∵‥二･.:∵ I;:.'= I:. ･ i,:I. ,･二 ≡主:I;l∴∵=,.･1 :.

つJ 4 8 8 0 0 7

1999/ 9/20

EIA

anti‑HBs
PHA

0.5/0.5

4

1

つJ9

0.021

+出十 十 出十 十 十+出十+ 十 出+吐 出 十 出十2.2

…g: 岨… 慧:dg魁鎚雌…血… o 0‑ 7腿 …慧皿… ㈹芸7.3

1999/1 I/26

3.7×

44d

<102

88d

<102
4.5

1 8

1999/12/10

つJt 51tJ93721 tJ5836n792794tJ 7 4

25

1999/12/1 3

2.7X

24

23d

<102

56d

<102

FMMMMMFM

19

2000/ 1/30

5.7

31

1999/12/10

22

adr

C

Wild

IO4

adr

C

Wild

IO4

adT

C

Wild

105

adw

C

Wild

IO3

adr

C

Wild

104

adr

C

Wild

04

adw

a

Wild

105

adr

C

Wild

adr

C

Wild

04

adr

C

Wild

104

adw

a

Wild

adr

C

Wild

adr

C

Wild

C

Wild

104

×

6.6×

26

1

<102

1999/1 1/30

27

1.1

×

7.1×108

2000/ 2/10

5.4×

32

I

01

9.7×104

1999/10/12

24

1.9×

1

2.9×104
9.8×101

1999/12/ 43036d
つJ OO tJ 2

44d

58d

26

Wild

<102

1999/ll/ 4

lOOd

25

C

X

7.9

91d

15

adr

104

3.3×10̀

4

18d

lO

Wild

<102

40d

3

C

<102

123d

23

adr

2.4×

80d

9

104

<102

1999/12/13

26d

5

Wild

<102

13d
ll

C

×

6.4X

12 431 1 21 3 12 tJ 7凸 132701

25d
21

adr

<102

85d
14

Precore

103

×

<102

26d
I 3

DNA
Genotype

<102

虹3呈墨

105d
12

×

56d

1999/10/28

HBV

Subtype

gi鐘皇

MF

7

HBe

copies/mI.:.Jl;.:l二二亨.;:.:‑.:.1.‑.;皿.tT.=I..
･二'. ･,l･･.:f=r:::M.=l:i.11∴…二.,.
<102:.;Ii:千l.I;辛1:･‑.; jl:.I1'rJ=･;〒…‥肌1.i=,I;‑ .:i;∵÷二㍗ ∵l=･一

1999/ll/10

anti‑HBc

eAg/eAb

0/0.5

/0.5

155d

8

IgM‑class::州':: :.: :I.ll:I:;77.:i州: I.言‥≡三..;こ:::. I.::.::i.:'∵5,I. I:7.I‑∵Z:lIT:.:'::1. '=.:Jd･.;:''･5,;li:. '≡羊≡:.:T=:;7‑... Tl:.f.

1 1 2 1.

106d

7

HI

2

0.022

3.6

0.025

6.0×108

×

0.223

6.9×108>2.000

4.6X106
4.3×101

72d

iA21

79d

i2B

3.7XIO]

86d

a.BBi

2.9×10】

FMM

O.028

5.3

53d

0.151

1.3×104

2000/ 2/12

0.029

6.3

14d

0.02 1

3.0×105

1999/12/18

1

2 6 5 ′0

1 1 4 4 7 1 1 2 5 ′0 0 7 7 0

X

I

×

19d

0.071

1.3×104

2000/ 2/15

0.027

3.1

20d

0.021

2.8×102

O4

1

X

04

IO2

adr

NATIPOSitive case coexisting with anti‑HBs and anti‑HBc

HBsAg

Bleeding Age Sex ALT
No.

19

二二T/"P Age
Date r 9‑
一一一一

2000/

1/26

22

M

anti‑HBs

RPHA

27

24d

0/0

2

1

EIA

0.013

1.5/I.5

9193

PHA

HI

4.5LLi4.5/2.5anti‑HBc

IgMIClass

i2i54
134

HBe

eÅg/eAb

copies/ml

0.002

iL‑

0.034

HBV

Subtype

8.8×

出=

1.6×

105

DNA
GenotypeadrC

Precore(Wild)

106

Samples unable to follow‑upa)Ceding
No･

BiTITe一一

‑p

Age

Sex

ALT

HBsAg

anti‑HBc

anti‑HBs

HBe

HBV

DNA

Age

RPHA
Oノ 5 1 tJ

MMFM

tJ O 1 1

0 5 0 0 0畑 伯 仲 畑 5

EIA

皿は慧

PHA
0 5 0 0 0畑 伯 仲 伯 5

HJ IgMIClass eAg/eAb copies/ml Subtypc Genotype Precore2

1999/10/ 1

0/0

0.020

O5

adr

C

6

1999/ll/ 9

0/0

0.028

■｣

2.7×

104

adr

C

16

1999/12/23

0/0

0.032

十｣

1.4×

104

adr

C

20

2000/ 1/30

0/0

0.027

‑/‑

adr

C

3 tJ 3 ′05222+underline: indicates positive result.

120

1/1

3.4

X

1

5.8×

101

WWWW

dddd

Table 4. Infection with precore mutant HBVBIeeding∩.̲▲.̲

Date

Age

sex

"ロー

Vー

HBsAg
"帆

ALT

畑花畑 ′ー伯 仲伯 仲

02000/ 1/11

56

EIA

PHA

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 03 20̲ 0 0糾 03 5 1

025 15 00 0

1 1 2 1 4 4 1 8 0 ′0 4 0 5 2

18

anti‑HBs

RPHA

021 15 00 0

2000/ 2/ 5‥㌫≡

畑e!ハ ′ー伯 仲伯 仲55

0

SI地肌肌㈹055

anti‑HBc

IgM‑class

M

25d
22

HI

5

M

2000/ 1/20

F

HBV

十

4.0× 102｢ト

2.0× 102

0.025

｢/‑

4.9 × 102

0.023

‑/‑

0.022

｢｣

3.6×

10'

Genotype

Precoreadr

C

Mutantayw

C

Mutantadr

C

MutantB

Mutant3.0× lo‡

‑/‑

2.3

‑/‑

0.016

DNA

Subtypc

0.027

0.016

25

HBe

copies/ml

0.035

0.019

21d
27

eÅg/eAbX

IO2

3.1 X 102■｣

3.8× 104

adw+underline: indicates positive result.

Table 5. Infection with HCVN｡.巳空ding

Age

Date

101

1999/ 9/21

Sex

‥b

52

ALT

y

anti ‑ H C V(2N)ーYnM

1 2 つJ 2 1 8 2 507 1 2 0 9 9 0 4 5

119d

PHA

PA

一l･315一一

一2 4 9 9

0 RU tJ 2 4

O 2 5 X X X 111 0 0 0】J '‑ ヽJ

.D 1Hrnu▼▲ I‑I

(1

5 5

1 1

150d
lO2

1999/10/12∠U 7 00

1 つJ 4

FMM

87d
lO4

1999/12/15

一 lL 5

23d
lO8

5

つJ

2000/ 1/20

28d

Samples unable to follow‑up

Not BlaetTing Age Sex AL.
つJ 2 1 2 1｣ つJ l ′‑U 5 4

FMMMM

つJ ′0 0ノ 0 2

anti ‑ HC V(2N)P HA FA

copies/m I Genotype

103

1999/ll/ 4

1 05

1 999/1 2/22

3.1 ×107106

2000/ I/ 8

1.1×107107

2000/ 1/21

1.4× 106

III (2a)109

2000/ 2/15

6.5× 105

IV (2b)12 15

5.7× 10̀

ⅠⅠⅠ (2a)IV(2b)ⅠⅠⅠ (2a)is focused on HBV, HCV and HIV‑ 1 infections.As for plasma

carry‑Over of high‑titer virus loads to NAT; 2) immediateshipping of test samples &om each blood center to NAT centersderivatives, clinical studies have verified a lack of transmis‑

by airnight or surface transport all over Japan; 3) an automatedsion of these three viruses, since effective virus removal andr!Ooling system excluding seropositives; 4) automated extrac‑

inactivation procedures have been introduced for serologicallytlOn, amplification,and simultaneous detection with a highlytion is of great concem. Currently much worldwide attention

screened source plasma (19, 20). However, residual risk

sensitive MPX; 5) a computerized reporting system connected

remains due to blood transfusions from individuals withto each blood center. Multiplex reagent‑positive units wereserologlCally undetectable levels ofviremia; such cases are

thereby excluded from availabihty for blood transfusions as

due to pre‑seroconversion at an early stage of infection or towell as from source plasma for plasma‑derived products.

viral infectionwith immunological mutants. NAT screenlng Of

Later, multiple NATIPOSitive samples were resolved into HBV,

these virus complements cu汀ent SerOloglCal screenlng by

HCV and HIV by individual NAT. The results were thendirect measurement of viral genomes with high sensitivity. ･This

reported to the donors.

results in narrowlng the window period, as well as detecting

By the JRC NAT system, 26 HBV NAT‑positives were

immunologlCal viral mutants. Therefore, NAT screenlng lS

detected from 2,560,977 sero10glCally negative voluntaryurgently needed to minimize residual risk, especially in the

blood donors, and those units were excluded from bloodcase ofblood transfusions. NAT should be done as rapidly astransfusion or source plasma.As for the serological HBsAg

possible in order to be ready for transfusions of cellularcomponents. Based on our experience of separate HBV, HCV

tests (Figure), overnight EIA or the chemiluminescencemethod are probably more sensitive than RPHA. However,and HIV‑1 NAT of 5.6 million serologlCally negative units asprescreenlng must be done as rapidly as possible fbr仇e blooda source plasma for plasma‑derived products started in

to be available for transfusion of cellular components, andNovember 1997 by manual procedures, we set up the JRC
NAT screenlng System for blood transfusion to be completed

RPHA has been ca汀ied out in 54 blood centers nationwide.

within 36‑50 h after donation (21) since July 1999.

Asdescribed above, the system consists of 1) rapid ser0‑

high titervirus loads to the NAT. Therefore, automatedagglutination tests uslng PK7200 are currently the most

10gical prescreening by automated agglutination tests to avoid

practically suitable. Actually, Since we started HBsAg screenmg

The main pu叩OSe OrpreSCreenlng lS tO avoid ca汀y‑OVer Of

121

Tokyo and Chitose, andthe JRC blood centers all over Japan.

by RPHA and anti‑HBc screening alone by HI (2, 6, 7), no
fuluminant hepatitis has been reported. SerologlCal screenlng
orHBsAg and antibody is an indirect measurement or HBV
itself. In cases of early‑stage HBV infection, the ratio of

We gratefully acknowledge the guidance and support of the
JRC President, Mr. S. Fujimori; the Executive Counselor, Dr.
T. Kusakari; the Director General, Mr. K. Otake in the JRC
Blood Services Department and Professor M. Mayumi, Jichi
Medical School. We are grateful to Ms. N. Arai for prepara‑

HBsAg versus semm HBV DNA is lower (22). In cases wi仙HBV‑mutant infection, HBsAg is often undetectable byserological tests. It should be noted that five wildtype HBVtion or the manuscript.

cases with a lower level ofvims load (#1, 20, 21, 23, 26),
four precore mutants (#18, 22, 24, 27), and one wild type

APPENDIX

HBV concomitant with both anti‑HBs and anti‑HBc (#1 9)
Were all negative when tested by overnight EIA.
A clinical follow‑up study was carried out on 18 cases ofwild type HBVinfections. Case #19 wasanexception, in whichanti‑HBV antibody might have interfered with the serologicalMurozuka2, Hiroyuki Emura2, Nobuko Suma2, Takeshi

reaction. All other 17 cases showed seroconversion oHgM

Tanaka3, Hitoshi Ohnuma3, Kimihiro Kanemitsu4, Hiroyuki

anti‑HBc and anti‑HBc by HI test, indicatlng that they wereinearly stages of primary HBV infection. In 10 of these latter

Kawakami5, Takashi Kawaguchi6, shinji Yuasa3, Kazuo

The partlCIPantS in this project are aS follows:

Hisao Yugi Iizukal , Retsuji Yamanakal , Masaki Miyamotol ,Shuhei Satohl , seiji Nakahiral , Tsugikazu Tomono2, Takashi

Murokawa4, Kiyoshi Minegishi4, Yoshifumi Iijima4, Keizou

cases, HBV DNA disappeared during me obseⅣation period.

Ozawa7, Mitsuo Hirose8, Junkichi Yanai9, Hisashi MitolO,

If negative conversion of HBV DNA is observed by further

Kouichi Kitajimal I , Michio Oka1 2, Masahide Sasaki13, Yutaka

follow‑up study, the donors may be able to reenter into the

Fujinaga14, Yoshiaki Maeda15, Tsutomu Hidaki16, shinblood donation group.

On the contrary, in the three cases with precore mutant

Chiyoda17, Toshimitsu Shingu1 8, chaka lmpraim19, Elizabeth
Dragon19, shigeru Tamatsukuri20, Toru Hirose20, Kimio

HBV infection, seroconversion of lgM anti‑HBc was not

Kurashima20, Tomohiko Olm021 , Masaaki Hibino21 , Masaaki

observed and two of these cases showed continued extremely

Takeda2㌧ Hiroaki Niwayama21 , Kusuya Nishioka4

low levels ofHBV DNA and anti‑HBc. Thus, the JRC NAT
system detected HBV DNA with undetectable levels of HBV

1Japanese Red Cross (JRC) Tokyo NAT Center, 2JRC

antlgen and antibody; this was obseⅣed in cases with HBV

Plasma Fractionation Center, 3saitama Blood Center, 4JRC

mutants, which might not be in an early stage of HBV infec‑

Headquarters Blood Services Deparhent, 5Akita Blood Center,
6Ibaraki Blood Center, 7Aichi Blood Center, 8Gifu Bloodtion. Some reports have suggested a possible linkage ofprecore

mutant‑HBV andfulminant hepatitis B in Japan (23‑25) and
Israel (26). Most of them may be screened out by sole high‑Center, 90saka Blood Center, 10Hyogo Blood Center,

titer anti‑HBc sPreTing (6)･ In Japan, the purpose of sole

Blood Center, 14Ehime Blood Center, 15Fukuoka Blood Center,. 10kayama Blood Center, 12Tottori Blood Center, 13Yamaguchi

160ita Blood Center, 17Nagasaki Blood Center, 18Miyazaki

anti‑HBc screenlng lS tO detect HBV viremia with low levels

Blood Center, 19Roche Molecular Systems Inc., Pleasanton,of HBs antigenemia in late stages of infection and not in
window periods of early‑stage infection. It is to exclude precore

CA, USA, 20Roche Diagnostics KK, Tokyo, 21ALOKA CO.
LTD, Tokyo.

or core promoter mutants of HBV potentially linked to fulmi‑
nant hepatitis Band is no longer a surrogate marker of HCV

infection. Mutant HBV infection with undetectable levels of

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Jpn. J. Infect･ Dis., 53, 2000

AUTHORIS CORRECTION

Original Article

Nationwide Nucleic Acid AmpliflCation Testlng Of Hepatitis B Virus,
Hepatitis C Virus and Human Immunodeficiency Virus Type 1 forBlood Transfusion and Follow‑Up Study ofNucleic Acid
AmpliflCation Positive Donors
Japanese Red Cross NAT Screenlng Research Group*

Volume 53, no. 3, pl16‑123, 2000: Page 117, column I, line 27, "Fujirebio Inc., Tokyo" should read "Ortho
Chemical Diagnostics KK, TbkyoM.