Journal of Clinical Microbiology, May 1999, p. 1324-1328, Vol. 37, No. 5
0095-1137/99/$04.00+0
Evaluation of the INNO-LIA HTLV I/II Assay for Confirmation
of Human T-Cell Leukemia Virus-Reactive Sera in Blood
Bank Donations
Ester C.
Sabino,1,*
Maan
Zrein,2
Carlos P.
Taborda,1
Marcia M.
Otani,1
Gabriela
Ribeiro-Dos-Santos,1 and
Amadeo
Sáez-Alquézar1
Fundação Pró-Sangue,
São Paulo, Brazil,1 and
Innogenetics N.V., Ghent, B-9052, Belgium2
Received 28 July 1998/Returned for modification 4 September
1998/Accepted 17 November 1998
 |
ABSTRACT |
We have evaluated a new serological confirmatory test (INNO-LIA
HTLV I/II Ab [INNO-LIA]) for human T-cell leukemia virus (HTLV) using
a large collection of samples from Brazilian blood donors (São
Paulo region) and compared the results with those obtained by Western
blotting (WB) tests (WB2.3 and WB2.4). Blood donations were initially
screened by enzyme-linked immunosorbent assays (ELISAs) based on viral
lysates, and repeatedly reactive samples were further tested by WB2.3.
When available, samples were also tested by PCR, two additional ELISAs
based on recombinant antigens (recombinant ELISAs), a new-generation WB
assay (WB2.4), and the INNO-LIA. Of the 18,169 samples tested, 292 (1.61%) were repeatedly reactive in the ELISAs (viral lysate based)
and were further tested by WB2.3; 97 were positive (19 that were typed
as HTLV type I [HTLV-I], 12 that were typed as HTLV type II
[HTLV-II], and 66 that were nontypeable), 17 were negative, and 178 had indeterminate results. Of the samples with indeterminate results,
172 were tested by INNO-LIA, which could resolve 153 samples as
negative. Regarding the positive samples, WB2.3 and INNO-LIA produced
concordant results for all HTLV-I-positive samples, whereas for HTLV-II
they agreed for 10 of 12 samples; the 2 samples with discordant results
were considered to be positive for HTLV-II by WB with WB2.3 but
negative for HTLV-II by INNO-LIA and the two recombinant ELISAs.
Furthermore, of the 66 nontypeable samples, 60 underwent testing by
INNO-LIA; 54 turned out to be negative by the latter test as well as by recombinant ELISAs. In conclusion, the new serological confirmatory assay for HTLV (INNO-LIA HTLV I/II Ab) resolved the results for the
majority of the indeterminate and positive-untypeable samples frequently observed by WB assays.
| |
INTRODUCTION |
The spread of the human T-cell
lymphotropic viruses (HTLV; type I [HTLV-I] and type II
[HTLV-II]) from known regions of endemicity to other parts of the
globe has led public health authorities in many countries to institute
routine screening procedures for these retroviruses. This is the case
in Brazil, where nationwide blood bank screening for HTLV became
mandatory in 1993. The prevalence of HTLV-I and HTLV-II in
Brazil varies regionally (1, 6); prevalence rates may reach
as high as 1.35% among blood donors and 35.2% among intravenous drug
users in the northeast of the country, and HTLV-II infections are
frequently found among certain Amerindian tribes (3, 5).
A major problem with the mass screening of blood for HTLV
antibodies has been the unacceptably high rate of false reactivity associated with commercial HTLV enzyme-linked immunosorbent assay (ELISA) kits (9). The rate of discarded units subsequent to HTLV screening reached levels of 2.5% when testing first began in
1991. Unfortunately, confirmatory tests have hitherto offered only
limited help in solving this problem. Indeed, the Western blotting (WB) confirmatory technology often gives rise to complex reactivity patterns, frequently rendering results inconclusive due to
the presence of nonspecific bands. This makes counseling for
ELISA-reactive blood donors even more complex and often requires the collection of a second sample for repetition of ELISA and WB.
Recently, a new HTLV confirmatory assay (INNO-LIA HTLV I/II
Ab [INNO-LIA]; Innogenetics, Ghent, Belgium) appeared to be
useful in resolving the results for samples with indeterminate results by WB for well-defined HTLV sera (13). This prompted us to
compare the new test with a commercial WB assay with a large number of blood donations reactive for HTLV by ELISA screened routinely at our
blood bank. As newer-generation ELISA and WB kits became available, we
also subjected the repeatedly reactive sera to further investigations.
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FIG. 1.
Layout of INNO-LIA strips. The antigen lines are
compared to the scoring lines to provide a relative intensity for each
line. If a sample is confirmed to be positive, according to the
criteria presented in Materials and Methods and Fig. 2, HTLV type
determination can be obtained by comparing the relative intensities of
the antigen lines in the discrimination area. Line 1, antigen gag p19;
line 2, gag p24; line 3, env gp46; line 4, env gp21; line 5, gag p19-1;
line 6, env gp46-1; line 7, env gp46-2.
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|
| |
MATERIALS AND METHODS |
Samples.
At the Fundação Pró-Sangue
Hemocentro de São Paulo, a total of 18,169 blood donors were
screened by ELISA in June 1995 for HTLV infection. Of these, 3,158 were
tested with the Hemobio HTLV I/II kit (Embrabio, São Paulo,
Brazil) and 15,011 were tested with the Vironostika HTLV-I kit
(Organon Teknika, Boxtel, The Netherlands); both kits are based on HTLV
lysates. All initially reactive samples were further tested by ELISA in
duplicate. Samples that reacted repeatedly (n = 292)
were additionally characterized by WB (HTLV blot 2.3 [WB2.3];
Diagnostic Biotechnology, Singapore). The available samples
(n = 279) were then tested by the new INNO-LIA and by
two new-generation HTLV ELISAs (see below). Whenever cells from a
donation unit were available, further testing by an in-house PCR was
performed (n = 230).
The screening assays (two ELISAs) were performed in duplicate, with
results expressed as a mean value of the ratio (sample signal/cutoff).
Additional screening assays.
When sufficient repeatedly
reactive sera were available, additional testing by the HTLV ELISA from
Murex Diagnostics (Dartford, United Kingdom) (n = 273)
and by Ortho Diagnostic System (Raritan, N.J.) (n = 256) was performed. Both kits are newer-generation assays that use
recombinant proteins and selected synthetic peptides as HTLV antigens.
Due to the more limited availability of serum, the additional assays
were performed only once and the results are expressed as a ratio. A
sample was considered reactive if the ratio was equal to or greater
than 1. A ratio of 0.8 was considered "grey-zone reactivity,"
which is generally considered to be an indication for repeating the test.
Confirmatory assays.
Originally, WB2.3 was used to
characterize all repeatedly reactive samples. The interpretation of the
results obtained by this WB was done according to the manufacturer's
criteria. Some samples with a positive nontypeable WB pattern
were also submitted to further testing with the HTLV WB version 2.4 (WB2.4) from Diagnostic Biotechnology (n = 57). This
new generation of the WB assay uses a truncated HTLV-I recombinant
gp21 (rgp21) protein which resolves most of the samples falsely
reactive for rgp21 with the previous version of the WB assay, WB2.3.
Samples were also tested by INNO-LIA (n = 279), and the
interpretation criteria were supplied by the manufacturer. The INNO-LIA kit uses recombinant antigens and synthetic peptides derived from both
HTLV-I and HTLV-II protein sequences. In addition to these HTLV
antigens, control lines are used for semiquantitative evaluation of the
results, as well as for sample addition and reagent controls. The
layout of the strips is shown in Fig. 1.
The assay procedure can be summarized as follows: serum or plasma
samples were diluted 1:100 and were incubated at room temperature (25°C) overnight; this was followed by three washing steps with washing buffer before the addition of an alkaline phosphatase anti-human immunoglobulin conjugate. The three washing steps were performed again, followed by the addition of a chromogen. Color development was then stopped with an appropriate stop solution. Following the visual interpretation protocol, after color development, each line was compared to the control lines, and the intensities were
scored as follows: 0 (
), no response or an intensity less than that
of the cutoff line; 0.5 (±), intensity equal to that of the cutoff
line; 1 (+), intensity between those of the cutoff line and the 1+
control line; 2 (++), intensity between those of the 1+ control line
and the 3+ control line; 3 (+++), intensity equal to that of the 3+
control line; 4 (++++), intensity greater than that of the 3+ control line.
A sample was classified as positive if it reacted with at least one
envelope antigen (gp21 or gp46) and one gag antigen (p19 or
p24). Alternatively, two reactive envelope antigens (gp21 and gp46)
also indicated a positive sample. When an isolated band or no reaction
appeared, the sample was considered negative. When two gag
antigens were reactive, the sample was considered indeterminate. Discrimination between HTLV-I and HTLV-II was indicated by the corresponding specific antigens present on the same strip (Fig. 2).
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FIG. 2.
Criteria for interpreting the results obtained with the
INNO-LIA strips; reactivity to none, one, or more of the specified
antigens is interpreted as negative, indeterminate, or positive, as
assigned. The positive samples are discriminated as HTLV-I or
HTLV-II depending on the intensity of the color of the strips;
e.g., if the env gp46-1 plus gag p19-1 bands are more intense than the
env gp46-2 band, the sample is typed as HTLV-I.
|
|
Finally, blood units from donors repeatedly reactive for HTLV were used
to obtain peripheral blood mononuclear cells for PCR. For erythrocyte
lysis, 10 ml of blood was mixed with 10 ml of 0.4% saponin and 0.5%
NaCl, and the mixture was centrifuged at 2,000 × g for
10 min. The peripheral blood mononuclear cell pellet was washed twice
with 0.9% NaCl and resuspended in 100 µl of lysis buffer (10 mM
Tris-buffer [pH 8.3], 0.5% Nonidet P-40, 0.5% Tween 20) and
digested with 40 µg of proteinase K at 65°C for 2 h. The DNA
was purified by phenol-chloroform extraction. All samples were
subjected to PCR amplification for two HTLV genes: pol and tax. If a discordant result was obtained, PCR was repeated
in duplicate for both sets of primers; for the pol gene, 1 µg of DNA was amplified in one round with primers SK110 and SK111
(7) in a reaction mixture containing 2.0 mM
MgCl2 and 0.2 mM each primer. The PCR product was detected
by liquid hybridization with probe SK112 for HTLV-I and probe SK188
for HTLV-II; for detection of the tax gene, DNA was
amplified by a nested PCR with SK43 and SK44 in the first round and
TAX1 (5'-GTGTTTGGCGATTGTGTACA-3') and
TAX2 (5'-CCATCGATGGGGTCCCA-3') in the second
round in a reaction mixture containing 2.5 mM MgCl2 and 0.2 mM each primer. The PCR product was analyzed upon electrophoresis on a
1% agarose gel.
| |
RESULTS |
Screening.
The screening of 18,169 blood donations for HTLV by
viral lysate-based ELISAs resulted in 292 (1.6%) samples repeatedly
reactive for HTLV-I or HTLV-II. Subsequent confirmatory testing
by analysis with WB2.3 indicated the following: 31 serum samples
(10.6%) were typed as HTLV-I or HTLV-II, 66 serum samples
(22.6%) were HTLV positive but nontypeable, 178 serum samples (60.9%)
were indeterminate, with various patterns, and 17 serum samples (5.9%)
were negative (Table 1; Fig.
3). If only the results of WB2.3 were
taken into consideration, the prevalence of HTLV antibodies was 0.53%
(97 of 18,169). However, of the 66 HTLV-positive but nontypeable
samples, 57 could be retested with a later version of the WB kit
(WB2.4) with a truncated rgp21 (GD21), which is less prone to
nonspecific reactivity (8). By testing with WB2.4,
only 5 of 57 samples remained positive and were still
nontypeable. The results for the remaining 52 samples became
indeterminate by losing their reactivity to the rgp21 antigen
originally present in WB2.3.
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FIG. 3.
Blood donor samples (the numbers of samples are given in
parentheses) were tested by the various assays, as specified (see
descriptions in Materials and Methods); the numbers of samples with the
indicated results by WB with WB2.3 and WB2.4 (positive, indeterminate,
or negative) are also given in parentheses. EBB, Embrabio; EIA,
ELISA.
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|
PCR.
Amplification of the pol and tax
genes was performed for 230 of the 292 ELISA-reactive samples. The
results are summarized in Table 1 (see also Fig. 3). Of the 31 samples
typed with WB2.3 (19 HTLV-I and 12 HTLV-II), 25 were analyzed
by PCR; only 21 (84%) were positive (both tax and
pol genes were amplified). The 143 WB-indeterminate
samples were also tested by PCR, and none was amplified with both
sets of primers; 3 samples were positive only by the tax
PCR. Another tax-positive pol-negative sample was
found among the 66 samples nontypeable but positive by WB. These
samples were not considered to be positive. Unfortunately, a second
fresh sample could not be obtained from these donors to confirm the PCR
results in terms of sensitivity. The remaining positive but nontypeable or indeterminate samples were all negative by PCR.
INNO-LIA versus WB2.3.
Of the 292 samples tested with WB2.3,
279 could be further investigated by INNO-LIA (Table
2; Fig. 3); 54 of 60 WB2.3-positive but
nontypeable samples and 153 of 172 WB2.3-indeterminate samples were
resolved as negative by INNO-LIA. Both WB and INNO-LIA had the
same results for 19 HTLV-I-positive and 10 of 12 HTLV-II-positive samples. The two remaining
HTLV-II-positive samples were negative by both recombinant ELISAs
and were indeterminate by WB with WB2.4.
INNO-LIA testing resulted in the detection of seven additional
HTLV-I-positive samples that were either positive (n = 1) or indeterminate (n = 6) by WB with WB2.3. Two
samples were HTLV-II positive by INNO-LIA, while they were
indeterminate by WB with WB2.3 and WB2.4. Finally, 12 samples were
positive but nontypeable by INNO-LIA; of these, 4 were nontypeable but
positive and 8 were indeterminate by WB with WB2.3. The reactivity
patterns of the 21 samples with discrepant results are presented in
Table 3; of these, 13 samples (samples
255, 1270, 314, 256, 274, 297, 419, 426, 432, 460, 1091, 1100, and
1175) were positive by more than one ELISA, with 6 of them also being
positive by one of the WBs. Two samples that were borderline by the two
ELISAs were either positive by the WB (sample 1164) or PCR positive
only for the tax gene (sample 404).
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TABLE 3.
Detailed analysis of reactivity patterns of samples with
discrepant results by screening and confirmatory assays
|
|
| |
DISCUSSION |
Screening for HTLV by viral lysate-based ELISAs in Brazil has
hitherto led to a high rate of discarding of units of blood. The
majority of the samples repeatedly reactive by ELISA indeed have
indeterminate results upon complete investigation by supplemental tests such as WB or are negative as determined by INNO-LIA and PCR.
Although the new version of the WB assay, WB2.4, has substantially decreased the high rate of false-positive results usually obtained by
WB with WB2.3 (4, 8), a large number of indeterminate results are still observed due to reactivity with bands other than GD21.
All WB-indeterminate and nontypeable but positive samples were negative
by PCR with both sets of primers (Table 1), in agreement with other
studies that have shown a low percentage of HTLV infections among
individuals whose samples have indeterminate results by WB and these
reactivity patterns (10, 11). Four samples were positive
only for tax primers. Unfortunately, a second sample could not be obtained from these donors. It is possible that they truly
represent infected persons because HTLV infections have been found in
samples with low levels of reactivity by serological tests
(12). However, in the absence of a second sample to confirm the initial PCR results, one should be cautious about the overall interpretation of the serological test results for those samples.
In Table 3 we presented the reactivity patterns for 21 discrepant
samples that tested positive by INNO-LIA. Those that tested negative by
PCR and the two recombinant ELISAs (n = 12) were
probably from noninfected individuals. However, since none of the PCR
or recombinant ELISAs showed a fully optimized sensitivity, it is not
possible to reach a final conclusion about these samples. Although nine
samples were found to be negative by PCR, they were positive by at
least one of the new-generation ELISAs and/or by one of the WB
versions, indicating possible infection.
Nevertheless, if upon prospective study the samples with nontypeable
but positive patterns by INNO-LIA represented false-reactive samples, more stringent criteria for Brazilian samples could be validated. For instance, in the confirmation module of the
INNO-LIA, when the gp21 antigen was not reacting, we observed that
truly positive samples reacted at least with the three other antigens (p19, p24, and gp46), while potentially false-reactive samples showed
limited reactivities to only two of these antigens. This additional
stringency criterion would resolve the results for most of the
PCR-negative and INNO-LIA-positive samples. A prospective study by a
standardized PCR method can help in further validation of stringency
criteria in terms of both sensitivity and specificity. We believe that
more stringent criteria might be necessary in tropical areas like
Brazil, where a high frequency of nonspecific reactions to HTLV
antigens is often reported (2). The screening for infections
with virtually all HTLV variants may be compromised by omitting
reactivities to the gag antigens. Thus, the samples with
indeterminate or positive but nontypeable results by INNO-LIA represent a suitable and focused target in attempts to
isolate new hypothetical HTLV strains.
In conclusion, the sensitivity of INNO-LIA was high in that it could
detect all samples reactive by all assays. The two samples positive for HTLV-II by WB with WB2.3 but negative by
INNO-LIA proved to be falsely typed on the basis of WB2.4
and recombinant ELISA results. INNO-LIA eliminates the
majority of the samples with indeterminate results by WB (153 of
172) and positive but nontypeable samples (54 of 60). Furthermore, the
typing capability is more accurate by INNO-LIA than by WB.
| |
ACKNOWLEDGMENT |
We thank Fred Shapiro for critical review of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
Fundação Pró-Sangue, Av. Dr. Enéas
de Carvalho Aguiar, 155, 1° And.-Bloco 04, C.E.P. 05403-000, São Paulo, Brazil. Phone: 55-1130615544. Fax: 55-112808317. E-mail: sabinoec{at}usp.br.
| |
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Journal of Clinical Microbiology, May 1999, p. 1324-1328, Vol. 37, No. 5
0095-1137/99/$04.00+0
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Abstract
Introduction
