A novel high-performance rapid screening test for the detection of total HTLV-I and HTLV-II antibodies in HTLV-I/II infected patients

Abstract

Rapid diagnosis of human T-cell lymphotropic virus (HTLV) type-I and -II infections are essential for timely and cost-effective disease interventions. MP Diagnostics ASSURE HTLV-I/II Rapid Test was developed for the rapid detection of anti-HTLV-I/II antibodies in patients’ serum, plasma, and whole blood specimens. ASSURE HTLV-I/II Rapid Test employed MP Biomedicals’ proprietary HTLV-I/II Trifusion recombinant antigen conjugated with gold nanoparticles and HTLV-I / HTLV-II recombinant antigens immobilized on the nitrocellulose membrane to detect total HTLV-I and HTLV-II antibodies. The overall performance of the ASSURE HTLV-I/II Rapid Test was found to be 99.42% sensitivity (95% Confidence Interval, 98.32–99.88%) and 100% specificity (95% Confidence Interval, 99.58–100.00%) in the tested clinical samples, including a total of 518 HTLV-I/II positive specimens (396 HTLV-I infection, 97 HTLV-II infection and 25 HTLV-I/II dual infection) and 872 HTLV negative clinical specimens consisting of 691 healthy donor samples, 116 potentially cross-reactive samples, and 65 samples with interfering substances. The ASSURE HTLV-I/II Rapid Test can effectively be deployed as a screening tool in any prevalence studies, blood banks or organ transplant centres.

Background

More than 20 million infections with oncogenic retrovirus Human T-cell lymphotropic virus (HTLV) type-1 and type-2 were approximately present worldwide. HTLV primarily spreads through various means, including transmission from mother to child, between sexual partners, breastfeeding, sharing needles, sexual contact, and through contaminated blood components [1,2,3]. HTLV-I is known as the etiological agent of adult T-cell leukemia/lymphoma (ATLL), HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and HTLV-associated uveitis [2, 4]. While the causal connections between HTLV-II and leukemia or lymphoma are not as clear as those with HTLV-1, HTLV-II infection has also been associated with HAM and other neurologic abnormalities although it is reportedly less pathogenic than HTLV-I [5, 6].

Studies of the geographic distribution of HTLV-I infection reveal that the virus is highly prevalent in Japan, Africa, the Caribbean islands, and South America [7, 8]. Many countries considered non-endemic for HTLV have limited screening efforts. The World Health Organization (WHO) is contemplating potential revisions to HTLV infection testing strategies in regions with low endemicity, particularly for organ donation purposes. In such areas, a test with lower sensitivity might be considered acceptable for organ donation if the primary aim is to achieve a rapid turnaround time [9].

The serological test has been routinely used for the diagnosis of HTLV infection. Typically, HTLV infection is confirmed with two or even three assays. Current practice usually involves two rounds of screening tests with enzyme immunoassays and confirmed with either a Western blot, line immunoassay, or nucleic acid test (NAT) [10]. In 2016, FDA approved the MP Diagnostics HTLV Blot 2.4 test as the first confirmatory assay for HTLV antibodies. This test uses a combination of recombinant HTLV-I/II proteins and native HTLV-I viral lysate to capture the antibodies specific to HTLV-I/II [11].

To enhance the screening effort and facilitate the containment of HTLV infection, this study aims to develop a screening rapid test for detecting HTLV-I and HTLV-II antibodies in human blood samples and evaluate its effectiveness. The samples under examination primarily originated from healthy donors, individuals with HTLV-I/II infections, specimens with cross-reactivity, and specimens with substances that could potentially interfere with the test results.

Materials and methods

Clinical samples

In MP Biomedical laboratory, a total of 401 HTLV-I/II positive plasma specimens (280 HTLV-I, 96 HTLV-II and 25 HTLV-I/II dual infection), 872 clinical specimens including 691 serum/plasma samples from healthy donors, 116 various cross-reactivity samples, and 65 interference substances samples were used in this study. In Pitié Salpêtrière Hospital (Paris, France), 116 HTLV-I and 1 HTLV-II positive samples from 117 patients were included for the retrospective part of sensitivity. Each sample was processed as previously described [12].

Potentially, cross-reactive serum/plasma specimens were collected from the individuals with anti-Candida albicans IgG, anti-Chlamydia trachomatis IgG/IgM, anti-Dengue IgG/IgM, anti-Hepatitis A virus (HAV) IgG/IgM, anti-Hepatitis B virus (HBV) IgG/IgM, anti-Hepatitis E virus (HEV) IgM, anti-Helicobacter pylori IgG, anti-Herpes Simplex Virus IgG/IgM (HSV) IgG/IgM, anti-Measles IgG, influenza vaccine recipient, anti-Toxoplasma IgG/IgM, anti-Tick-Borne Encephalitis Virus IgG (TBEV) IgG, anti-B19 Parvovirus IgG/IgM, anti-Rubella IgG, anti-Syphilis IgG/IgM, anti-Tuberculosis (TB) IgG, anti-Hepatitis C Virus (HCV) IgG, anti-Yellow fever virus post-immunization IgG, and anti-Malaria IgG/IgM.

In addition, 12 types of specimens with different potentially interfering substances were obtained from diabetes patients, individuals with high ethanol levels, specimens displaying hemolysis, icteric specimens, those with high lipid content, elevated bilirubin levels, increased total protein levels, specimens containing Rheumatoid Factor (RF), patients with multiple transfusions, Antinuclear Antibody (ANA) carriers, those with homozygous sickle-cell anemia, and individuals with autoimmune hepatitis, together with a 4-way anticoagulant matched set, consisting of a serum sample and its corresponding plasma samples with lithium heparin, sodium citrate, and EDTA, respectively. These samples were purchased from commercial sources (Access Biologicals, US; Precisions, US; AbBaltis, UK; SeraCare Life Science, US; SLR Research Corporation, US; ABRI, US; Boca Biolistic, US; Advanced Bioresources, US; and Trina Bioreactives, Switzerland).

Antigen Conjugation to Nanoparticles

HTLV-I/II Trifusion recombinant antigen MGK (MP Biomedicals Asia Pacific Pte. Ltd., Singapore) was conjugated to gold nanoparticles (MP Biomedicals Asia Pacific Pte. Ltd., Singapore) according to the manufacturer’s instructions. MGK is a recombinant trifusion antigen of human T-Lymphotropic virus Types I and II (HTLV-I and HTLV-II) that include the HTLV-I antigen fragments of MTA-1 and GD21 as well as the HTLV-II antigen fragment of K55.

Briefly, the HTLV-I/II Trifusion recombinant antigen was diluted to 0.2 mg/mL in dilution buffer before use. To establish the protective concentration of the conjugant via gold colloid, conduct a coefficient variable isotherm titration. The desirable amount of colloidal gold was pretreated with 5% Glutaraldehyde for 5 min with stirring. The antigen was then added to the gold solution with vigorous stirring and incubated for 10 min. The 10% bovine serum albumin (BSA) was used to block the unbound surface of gold nanoparticles. The mixture was then centrifuged at 12,000 rpm for 30 min. Carefully aspirate the supernatant. The pellet was washed and resuspended with the desired amount of gold conjugate buffer. Filter the conjugate using a 0.22 μm filter and add conjugate storage buffer to the gold suspension accordingly.

Test Principle of ASSURE HTLV-I/II rapid test

ASSURE HTLV-I/II Rapid Test is a lateral immunochromatographic assay for the rapid in vitro qualitative detection of total antibodies to HTLV-I/II in human serum, plasma, finger-pricked whole blood, or whole blood with anticoagulants. The presence of total antibodies to HTLV-I/II is detected by the colloidal gold labelled with HTLV-I/II recombinant Trifusion antigen (Fig. 1).

Fig. 1

ASSURE HTLV-I/II Rapid Test device showing both negative results for HTLV-I/II antibodies as the test line (T) is absent (a), and positive result for HTLV-I/II total antibodies as the test line (T) is present (b). In both devices, the control lines (C) were present, indicating the results were valid

Assay procedure of ASSURE HTLV-I/II rapid test (for serum/ plasma/ whole blood specimen)

The assay procedure of ASSURE HTLV-I/II Rapid Test was shown in Fig. 2.

Fig. 2

Schematics of test procedure of ASSURE HTLV-I/II Rapid Test. (A) Add 10 µL of the specimen into the sample well. (B) Add 3 drops of chase buffer into the sample well. As the test begins to work, the dark red solution will move across the result window in the center of the test device. (C) Interpret the test result from 15 to 20 min. Do not read the result after 20 min

Test device assembly

A quantity of 0.5 mg/mL of HTLV-I and HTLV-II recombinant antigens (Fapon, China), and 0.5 mg/mL of biotinylated-BSA were stripped on the nitrocellulose membrane (Sartorius Stedim Biotech, Germany) for the test and control lines, respectively. The stripped membranes were air-dried at 60 °C and blocked with blocking buffer and air-dried at 37 °C. The membranes were then stored in a dry cabinet (≤ 18%) before use. The colloidal gold nanoparticles labelled with either HTLV-I/II Trifusion recombinant antigen or goat anti-biotin were mixed and stripped on the glass fiber (KinBio, China). The stripped gold conjugates’ pads were air-dried and stored in a dry cabinet before use.

The gold conjugate pads were then assembled with nitrocellulose membrane, sample pad with plasma separation function, and absorbent pad on the adhesive backing (Nupore Filtration Systems, India).

Analytical Sensitivity Study/ Limit of detection (LoD)

The analytical sensitivity study was performed on one (1) HTLV-I and one (1) HTLV-II positive samples purchased from commercial suppliers (both HTLV-I and HTLV-II tested positive on MP Diagnostics HTLV-I/II ELISA 4.0 and genotypes were characterized by FDA-approved MP Diagnostics HTLV Blot 2.4). The limit of detection of the ASSURE HTLV-I/II Rapid Test on one (1) HTLV-I and one (1) HTLV-II was determined in parallel with MP Diagnostics HTLV-I/II ELISA 4.0. The two-fold serial dilutions of the HTLV-I and HTLV-II positive samples were prepared and subjected to ASSURE HTLV-I/II Rapid Test and MP Diagnostics HTLV-I/II ELISA 4.0 for comparison study.

Diagnostic performance analysis

The sensitivity, specificity, positive predictive value, negative predictive value, and inter-rater agreement (Kappa) of ASSURE HTLV-I/II Rapid Test were analysed using the MedCalc Software (MedCalc Software Ltd., Ostend, Belgium). The study was conducted at MP Biomedicals Asia Pacific (Singapore) and Laboratoire de Virologie Hôpitaux Universitaires La Pitié Salpêtrière-Charles Foix (Paris, France), independently. Commercial samples were tested in Singapore and 117 HTLV positive samples well characterized by EIA platforms were tested in France. The discordant results were resolved by HTLV confirmatory test, MP Diagnostics HTLV Blot 2.4 (MP Biomedicals, France).

Results

A total of 401 HTLV-positive serum and plasma samples were purchased from commercial suppliers (25 HTLV-I/II positive samples tested positive on Abbott PRISM, 360 positive samples characterized by FDA-approved MP Diagnostics HTLV Blot 2.4, and 16 positive samples certified by MP Diagnostics HTLV-I/II ELISA 4.0), along with 872 negative serum and plasma samples (691 healthy donors, 181 cross-reactive and interfering substances samples). A third-party validation study on 117 HTLV positive samples was carried out in Paris independently.

The collected samples were tested with the newly developed ASSURE HTLV-I/II Rapid Test. As shown in Table 1a, the overall diagnostic performance of the ASSURE HTLV-I/II Rapid Test was 99.42% sensitivity (95% Confidence Interval, 98.32–99.88%) and 100% specificity (95% Confidence Interval, 99.58–100.00%). The Kappa value of 0.99538 indicated a nearly perfect agreement between the ASSURE HTLV-I/II Rapid Test and the reference HTLV assays. The diagnostic performance of ASSURE HTLV-I/II Rapid Test in each HTLV category, including HTLV-I, HTLV-II and HTLV-I/II, was separately analysed in Table 1b.

Table 1a Diagnostic performance of ASSURE HTLV-I/II Rapid Test

Table 1b Diagnostic performance of ASSURE HTLV-I/II Rapid Test in each HTLV category

To further investigate the performance of the ASSURE HTLV-I/II Rapid Test on specimens with low titers, an analytical sensitivity study was conducted using one HTLV-I positive sample and one HTLV-II positive sample. As illustrated in Table 2, the limit of detection for the ASSURE HTLV-I/II Rapid Test was determined and compared with that of the MP Diagnostics HTLV-I/II ELISA 4.0. The results indicate that the ASSURE HTLV-I/II Rapid Test detected the HTLV-I positive specimen at a dilution of 1:2000 and the HTLV-II positive specimen at a dilution of 1:256. In comparison, the dilution endpoints determined by the MP Diagnostics HTLV-I/II ELISA 4.0 were also 1:2000 for HTLV-I and 1:256 for HTLV-II samples. Therefore, the analytical sensitivity of the ASSURE HTLV-I/II Rapid Test was comparable to that of the MP Diagnostics HTLV-I/II ELISA 4.0 for detecting HTLV-I/II antibodies.

Table 2 Analytical Sensitivity/ limit of detection (LoD) of ASSURE HTLV-I/II Rapid Test

The cross-reactivity of the test line of the ASSURE HTLV-I/II Rapid Test to the serum and plasma samples reactive with anti-Candida albicans IgG, anti-Chlamydia trachomatis IgG/IgM, anti-Helicobacter pylori IgG, anti-Dengue IgG/IgM, anti-Hepatitis A virus IgG/IgM, anti-Hepatitis B virus IgG/IgM, anti-Hepatitis E virus IgM, anti-HIV-I/II IgG/IgM, anti-Herpes Simplex Virus (HSV) IgG/IgM, anti-Measles IgG, influenza vaccine recipient, anti-Toxoplasma IgG/IgM, anti-Tick-Borne Encephalitis Virus (TBEV) IgG, anti-B19 Parvovirus IgG/IgM, anti-Rubella IgG, anti-Syphilis IgG/IgM, anti-Tuberculosis (TB) IgG, anti-Hepatitis C virus (HCV) IgG, Yellow Fever Virus post-immunization IgG, and anti-Malaria IgG/IgM was also examined, and the results showed that the specificity of ASSURE HTLV-I/II Rapid Test on the tested cross-reactive samples was 100% (Table 3).

Table 3 Analytical specificity performance on potential cross-reactivity for ASSURE HTLV-I/II Rapid Test

Furthermore, based on the evaluation results on 12 different types of potentially interfering substances, it is found that none of them interfered with the negative results on ASSURE HTLV-I/II Rapid Test (Table 4). In addition, the potential interference of common-used anti-coagulants was evaluated using a 4-way anticoagulant matched set, consisting of a serum sample and its corresponding plasma samples with lithium heparin, sodium citrate, and EDTA, respectively. The plasma samples yielded the same negative results as the serum sample, indicating no interfering effect of lithium heparin, sodium citrate, and EDTA on the negative results of ASSURE HTLV-I/II Rapid Test.

Table 4 Analytical specificity performance on specimens with interfering substances for ASSURE HTLV-I/II Rapid Test for HTLV-I/II

Discussion

Seroprevalence data for HTLV has primarily relied on established endemic regions, including Japan, Oceania, the Middle East, the Caribbean, and parts of South America and Africa. However, there is a shortage of reliable estimates from less densely populated countries and regions. The current figures may underestimate the actual infection rates of HTLV infection. Additionally, the situation is further complicated by the fact that a variety of laboratory-based methods for screening and diagnosing HTLV-I infection have been utilized over the past several years, which hinders the systematic estimation of the HTLV endemic population, particularly in regions with limited resources.

Based on the current HTLV testing algorithm to diagnose HTLV infection, usually two EIAs and a Western blot or line immunoassay [9]. In cases where interpreting serological data poses challenges, NAT tests may also be employed. Notably, there is a deficiency in readily available testing technologies for HTLV infection even in developed countries like Japan. In addition, given the absence of a vaccine and the limited options for the treatment of HTLV-related illnesses, fast and efficient identification of infected individuals in either endemic or non-endemic regions would be critical. Although the current screening and diagnostic approaches for HTLV infection have proven reasonably effective, there are no available tests beyond regular laboratory settings, posing a challenge to seroprevalence assessment and endemic surveillance. Therefore, there is a substantial need for the development of low-cost, swift, and non-laboratory-based tests for HTLV screening test that can be easily implemented on a global scale.

To accommodate the demand, the ASSURE HTLV-I/-II Rapid Test provides a rapid, inexpensive, and point of care diagnostic tools for screening HTLV-I/-II infections. With compatibility to various types of specimens (venous whole blood, finger-prick whole blood, serum, and plasma), it can give rapid results across all levels of the health care system without requiring the separation of serum/plasma from whole blood. Compared to the prevailing methodologies, such as the EIA platform, the ASSURE HTLV-I/II Rapid Test offers a novel, efficient, and quick solution for HTLV screening. It requires minimal equipment and skilled operators, underscoring its significance in enhancing the comprehensive and effective control of HTLV infection and transmission. In addition, the immunochromatographic rapid tests are generally stable at room temperature with a long shelf life, typically 2 years, and with minimal storage and transportation requirements because of its robustness to environmental conditions. This is particularly beneficial in remote or resource-limited settings.

The clinical assessment of the well-characterized serum/plasma samples from commercial suppliers and a third-party validation demonstrated that the ASSURE HTLV-I/II Rapid Test is accurate with a sensitivity of 99.42% and a specificity of 100%. The analytical and clinical findings underscore the test’s remarkable sensitivity and specificity, offering the potential for a more cost-effective and streamlined approach to HTLV infection screening.

There were three HLTV positive samples that were negative with the ASSURE HTLV-I/II Rapid Test. These three samples were characterized by EIA platforms, and two of them had the lowest reading values among the 117 HTLV positive samples, suggesting they had relatively low anti-HTLV antibody titers. Given that the EIA platforms utilize the chemiluminescence-based signal amplification techniques and they are favourable of the detection of extremely low levels of analytes, it was possible that the antibody titers of these two positive samples characterized by CLIA platforms exceeded the detection limit of ASSURE HTLV-I/II Rapid Test. Regarding the other positive sample missed by ASSURE HTLV-I/II Rapid Test, it was only reactive for GD21, p19 and rgp46-I bands in its HTLV Blot 2.4 result. Notably, the antibody profile of this sample appeared to be distinct from other positive samples, in which a majority of native HTLV antigen bands would typically test positive in HTLV Blot 2.4. Such variations in antibody profiles were expected, varying from individual to individual and even between different viral strains. This variability could contribute to this negative result observed by the ASSURE HTLV-I/II Rapid Test. Another limitation of the study is that more potential interferences remain to be tested to better demonstrate the accuracy of ASSURE HTLV-I/II Rapid Test, such as common over-the-counter anti-inflammatory medications (aspirin, paracetamol, ibuprofen).

Even though the ASSURE HTLV-I/II Rapid Test may not detect HTLV antibodies as effectively as the EIA platform, it is still a useful diagnostic tool in areas where the EIA platform is not available. For example, the rapid screening capability of this test could be especially beneficial in countries with lower HTLV prevalence, where cost-effectiveness is prioritized when deciding on testing algorithms for screening large populations. Another example would be the organ donation scenario. World Health Organization (WHO) has emphasized the importance of having a lateral flow assay format on HTLV infection for pre-surgery organ donation and blood bank screening. The use of rapid assays can significantly decrease costs without resulting in the loss of donor tissue and organs.

Conclusions

ASSURE HTLV-I/-II Rapid Test has been successfully developed and validated using a wide range of well-characterized clinical samples. The assay is easy to perform, and the results can be interpreted at 15 min of test run. In addition, the possibility to use whole blood specimens further facilitate the readiness and rapidness of the assay at all levels of the healthcare system. As the assay showed a satisfactory level of performance against the HTLV-I/-II positive specimens, the ASSURE HTLV-I/-II Rapid Test could be equally employed as a screening tool in the HTLV endemic countries, or as one of the preventive measures in non-endemic countries.