Line Immunoassay for Confirmation and Discrimination of Human T-Cell Lymphotropic Virus Infections in Inconclusive Western Blot Serum Samples from Brazil

DISCUSSION

HTLV-1- and HTLV-2-seroindeterminate WB results are prevalent worldwide, with rates fluctuating according to country and study group (geographic areas and populations where the disease is or is not endemic). Several attempts have been made to improve WB sensitivity and specificity, such as adding HTLV-1 and HTLV-2 recombinant envelope proteins and transmembrane protein to the HTLV-1 viral lysate. These include rgp46-I, rgp46-II, and GD21, the latter of which blocks the cross-reactivity of gp21 with Plasmodium falciparum in regions where malaria is endemic (22, 23). In spite of these efforts, Blot 2.4 continues to yield high rates of WB-indeterminate and/or untypeable HTLV results (4–8, 10, 11, 13–17, 21). The present study found similar results and disclosed that in populations presenting a high risk of acquiring viral infections (G1 and G2) as well in the general population (G3) of Brazil, a large number of WB-inconclusive results were detected. Several hypotheses were taken into consideration for these WB-inconclusive results, such as low HTLV-1 and HTLV-2 proviral loads, mutations in the provirus (defective particles), low-level production of viral antigens consequently leading to a low level of specific antibody production, the seroconversion period, cross-reactivity with other antigens or viruses, coinfection with HIV, and the use of antiretroviral therapy, among others (5, 12–15, 24–27).

Interestingly, the majority of WB-indeterminate results that were not confirmed by seroconversion or PCR assays were due to cross-reactivity with Gag antigens. For instance, the WB-indeterminate pattern exhibiting p19, p26, p28, p32, p36, and p53, termed the HTLV-1 Gag indeterminate profile (HGIP), has been detected in epidemiological studies, mostly in Cameroon and in the Caribbean, but was not associated with true HTLV-1 infection (23, 24). This pattern was not frequently described in Brazilian WB-indeterminate samples (5, 13, 15, 26) and was also not observed in any samples analyzed in the present study. Although two samples here presented an incomplete HGIP pattern (without p32 and p36 bands), only the p19-I/II band was detected by the LIA. Thus, these samples were considered HTLV negative according to the manufacturer’s criteria for the LIA. Corroborating the LIA result, the same profile (only the p19-I/II band) was detected when we analyzed samples from five patients who attended HTLV outpatient clinics in São Paulo during the years 2000 to 2006, which exhibited p19, p26, p28, and p36 WB bands (5).

Moreover, we conducted a retrospective analysis of 108 well-characterized blood samples from patients from São Paulo (tested by two serological assays [WB and LIA] and two molecular assays [qPCR for pol and PCR-RFLP analysis for tax]) and confirmed a series of more than 10 HTLV-1 and 10 HTLV-2 samples as being positive by both WB and LIA criteria, and they were concordant. In addition, we revised data for two samples that were HTLV-1 and -2 positive by WB, one of which was confirmed to be HTLV-1 and HTLV-2 positive by the LIA and molecular assays (15). Additionally, we tested two other HTLV-1 and -2 WB-positive samples by the LIA, and only HTLV-1 was confirmed, taking into account the intensity of the discriminatory bands (p19-I, gp46-I, and gp46-II). Of note, the WB-indeterminate profile (strong GD21 and p28 bands) described in Pygmies and Bantus living in the southern Cameroonian rainforest (28) has not been detected in any study conducted in São Paulo (5, 13, 15) and here.

Although PCR assays presented lower sensitivity than WB in detecting true HTLV-1/2 infection in HIV-HTLV-coinfected individuals in São Paulo, Brazil, the molecular assays were able to confirm and discriminate between HTLV-1 and HTLV-2 in some WB-indeterminate and HTLV-untypeable cases, indicating that both serological and molecular assays are useful for HTLV diagnosis (13, 15). Due to the presence of large numbers of WB-indeterminate samples, coupled with the high cost of obtaining WB assays and LIAs in Brazil, we recently proposed an algorithm that employs qPCR to confirm HTLV infection, followed by testing any PCR-negative samples by WB or an LIA. This strategy was shown to reduce costs and improve the diagnostic accuracy of HTLV-1/2 detection (13, 15). Nonetheless, due to highly divergent socioeconomic conditions among different regions in Brazil, in laboratories without the means to perform molecular assays, high-performance serological testing presents an acceptable alternative.

Some studies of HTLV diagnosis conducted in blood donors in Latin America (considered an area where HTLV-1/2 is endemic) have reported differing numbers of WB-indeterminate samples, which were subsequently confirmed to be positive by PCR (29–31). Also, in blood donors from another area where the disease is endemic, northeast Iran, WB-indeterminate samples were found to be positive by PCR, and the most prevalent WB bands presented various combinations of rgp46-I, GD21, and gp21 (32).

In corroboration with these findings, the majority of serum samples here that presented WB patterns of GD21 and/or rgp46-I or -II plus p19 or p24 were subsequently confirmed to be positive by the LIA. These types of WB patterns were observed in G1, and PCR assays demonstrated HTLV-1 or HTLV-2 positivity (data not shown) (15). In addition, the majority of serum samples that presented only Gag bands in the WB analysis were negative for HTLV-1/2 infection by the LIA. Of note, one blood sample from G1 that showed a faint GD21 band in the WB analysis tested negative for HTLV-1/2 by both the LIA and PCR. Another serum sample presenting a GD21, rgp46-I, and rgp46-II WB pattern was found to be negative by the LIA; unfortunately, this sample could not be analyzed by PCR because only serum was sent to the laboratory for analysis. However, retesting of this serum sample by WB and the LIA confirmed the discrepant results. It is interesting to note that in serum samples (n = 14) that tested HTLV untypeable by the LIA, PCR confirmed the presence of HTLV-1 in 5 samples and HTLV-2 in another 2 samples from G1 (data not shown), emphasizing the need for employing molecular assays to confirm an HTLV diagnosis in patients with HIV-HTLV coinfection.

In G2, the majority of WB-indeterminate patterns presented either GD21 alone or this protein in association with one Gag or envelope band. The LIA confirmed the presence of HTLV-2 in 11/21 (52.4%) of the WB-indeterminate samples. The high number of HTLV-2-positive samples in G2 leads us to suppose that these patients acquired HBV and HCV, as well HTLV-1/2 and HIV, at the same time, probably by the parenteral route and prior to the time when serological testing for HIV and HBV (1989), and subsequently for HTLV and HCV (1993), became mandatory in blood banks throughout Brazil; in addition, intravenous drug addiction was more frequent in this country, as previously described (18–20). Corroborating this hypothesis, older age and male sex predominated in G2. Regarding the lack of WB detection of truly HTLV-2-infected samples, we have described this difficulty since 2000 (8, 13, 15) and hypothesized that the rgp46-II (K55) present in the WB strip is not as sensitive for the detection antibodies to the HTLV-2 strains that circulate in Brazil (HTLV-2a subtype, variant 2c) (33). This seems not to be the case for the gp46-II present in the LIA strip.

Concerning the two WB-indeterminate samples in G2 with negative HTLV results by the LIA, both presented reactivity for GD21 in the WB analysis, and one of the samples showed a faint band. Curiously, the LIA demonstrates the best performance in this group of patients, with 20/24 (83.3%) of HTLV-positive samples being detected among the WB-inconclusive samples. Unfortunately, only plasma/serum samples from these patients were available for analysis, which did not allow the use of PCR to perform a comparative analysis of serological and molecular results. Nonetheless, associations between HTLV-1/2 and hepatitis B and C have been reported in several studies conducted in Brazil and elsewhere (18–20, 24, 29, 34).

The WB-inconclusive patterns in G3 were quite different from those in the other groups analyzed. Several of the HTLV-untypeable samples demonstrated the presence of almost all bands corresponding to the HTLV-1 viral lysate, without reactivity to rgp46-I, and six of nine were subsequently confirmed to be HTLV-1 positive by the LIA. Twelve out of the 25 WB-inconclusive samples that could be submitted for PCR (9 WB indeterminate and 3 HTLV indeterminate), 11 were confirmed to be HTLV-1 infected, 6 of which were HTLV untypeable by LIAs (data not shown). In addition, the serum samples that tested negative by LIAs presented three different WB-indeterminate patterns: (i) GD21, (ii) p24 (faint band), and (iii) rgp46-II (faint band). Only one of these samples could be tested by PCR and presented an HTLV-negative result (data not shown). In summary, the samples in G3 were confirmed to be positive for HTLV-1 or HTLV but not HTLV-2 infection. This finding could be partially related to the ethnic origin of the included individuals (African descendants), the lack of HIV infection in this group, and the characteristics of the patients seen at HTLV outpatient clinics in Salvador, BA (35).

Of note, the reasons described above to explain WB-inconclusive results could also be applied to the PCR-negative results in truly HTLV-1/2-infected individuals, including the low proviral loads in HIV/AIDS patients undergoing antiretroviral therapy in G1 and, in some cases, in G2 (13–15); the observation that HTLV-2 infection is known to show low proviral loads (11, 12, 25); the presence of defective provirus not detected by the primers employed in the PCR assays (27); and infection with other viruses, such as HTLV-3 or HTLV-4, which can be detected only by using specific primers (36, 37).

It is noteworthy that since the discovery of HTLV-3 and HTLV-4 in central Africa (36, 37), studies have been conducted to ascertain whether assays that are commercially available and employed for HTLV-1/2 diagnosis are able to detect these new HTLVs (38–40). The results obtained confirmed that HTLV-1/2 screening assays are sensitive for the detection of antibodies to HTLV-3 and HTLV-4 (38) and disclosed discordant and misclassified results in relation to confirmatory serological assays (WB and LIA) (36–41).

In fact, we could not rule out HTLV-3 and HTLV-4 infections in Brazil, since populations in central Africa migrated from Africa and Australia to the American continent before Asiatic population migration, and their descendants, such as the Amerindians, could maintain such viruses or spread them to the general population, which could justify the frequent presence of WB-indeterminate results in Amerindians, as previously described (42).

In relation to the LIA, although it presented the best performance in detecting HTLV-1 and HTLV-2, we could not exclude misclassified positive results, as occurred in HTLV-3- and HTLV-4-infected individuals, who could be erroneously diagnosed as being infected with HTLV-2 (36–38).

Of note, despite the fact that the LIA demonstrated better performance than WB in the serological diagnosis of both HTLV-1 and HTLV-2, additional considerations are warranted for both assays. With respect to WB, we consider the lack of an ability to score the intensity of a positive band to be a problem, since it is not known when a faint band should be considered truly positive. The criteria (band profiles) established by the manufacturer to confirm HTLV-1 and HTLV-2 infections in WB assays are excessively stringent and deserve a review. Taking into account the results obtained here, we suggest that samples presenting only one Gag band (p19 or p24) plus GD21 and rgp46-I or rgp-46-II should be considered HTLV-1 and HTLV-2 positive, respectively, since samples that demonstrated p24, GD21, and rgp46-I bands were confirmed to be HTLV-1 positive by the LIA and PCR. In contrast, we detected true HTLV-2 positivity in samples that showed p19, GD21, and rgp46-II bands. In addition, when Gag bands were undetectable but both envelope bands (GD21 and rgp46-I or rgp46-II) were present, it was impossible to rule out true HTLV-1 or HTLV-2 infection, since seroconversion could be taking place. Indeed, when all bands showed reactivity to HTLV-1 viral lysate antigens, even in the absence of rgp46-I, it was possible to confirm HTLV-1 infection.

In conclusion, the LIA was shown to be the best serological test for confirming HTLV-1 and HTLV-2 infections, regardless of whether individuals were HTLV monoinfected or coinfected. We further highlight the need to review some WB criteria based on our results and those reported previously by others. It remains to be determined whether the superior performance of the LIA was due to the less stringent criteria employed than for WB. Further studies are necessary to confirm these results in a variety of risk populations from Brazil and elsewhere.