A novel high performing multiplex immunoassay Multi-HTLV for serological confirmation and typing of HTLV infections.

BACKGROUND: Human T-Cell Lymphotropic Viruses (HTLV) type 1 and type 2 account for an estimated 5 to 10 million infections worldwide and are transmitted through breast feeding, sexual contacts and contaminated cellular blood components. HTLV-associated syndromes are considered as neglected diseases for which there are no vaccines or therapies available, making it particularly important to ensure the best possible diagnosis to enable proper counselling of infected persons and avoid secondary transmission. Although high quality antibody screening assays are available, currently available confirmatory tests are costly and have variable performance, with high rates of indeterminate and non-typable results reported in many regions of the world. The objective of this project was to develop and validate a new high-performance multiplex immunoassay for confirmation and discrimination of HTLV-1 and HTLV-2 strains. METHODOLOGY/PRINCIPAL FINDINGS: The multiplex platform was used first as a tool to identify suitable antigens and in a second step for assay development. With data generated on over 400 HTLV-positive blood donors sourced from USA and French blood banks, we developed and validated a high-precision interpretation algorithm. The Multi-HTLV assay demonstrated very high performance for confirmation and strain discrimination with 100% sensitivity, 98.1% specificity and 100% of typing accuracy in validation samples. The assay can be interpreted either visually or automatically with a colorimetric image reader and custom algorithm, providing highly reliable results. CONCLUSIONS/SIGNIFICANCE: The newly developed Multi-HTLV is very competitive with currently used confirmatory assays and reduces considerably the number of indeterminate results. The multiparametric nature of the assay opens new avenues to study specific serological signatures of each patient, follow the evolution of infection, and explore utility for HTLV disease prognosis. Improving HTLV diagnostic testing will be critical to reduce transmission and to improve monitoring of seropositive patients.

Estimation of the window period of human T-cell leukemia virus type 1 and 2 tests by a lookback study of seroconverters among Japanese voluntary blood donors.

BACKGROUND: Japan is endemic for human T-cell leukemia virus type 1 (HTLV-1), and the horizontal transmission of HTLV-1 is often reported. However, the window period (WP) for serologic or molecular screening is unclear. STUDY DESIGN AND METHODS: Results for anti-HTLV-1 screening and confirmatory tests obtained from 648 591 repeated blood donors in the Kyushu district, one of the most endemic areas of HTLV-1 in the world, were evaluated. A lookback study was conducted for seroconverters. RESULTS: During 2012 to 2019, 436 seroconverters (155 men, 281women) were identified with use of a screening chemiluminescence enzyme-immunoassay (CLEIA) and multiple confirmatory tests. Because the period between the latest seronegative donation and seroconversion was highly variable (2.1-276.7 months), 19 cases that seroconverted within 6 months were subjected to the analysis. The WP of the particle agglutination assay and CLEIA was estimated to be 2.2 ± 0.6 and 2.6 ± 1.7 months, respectively. The WP of the indirect immunofluorescence assay was 4.8 ± 6.5 months. Although the WP of western blotting was estimated to be 6.3 ± 8.7 months, four cases were still indeterminate through the study period. Chemiluminescence and line immunoassays, the current screening and confirmatory tests used in the Japanese blood program, showed the shortest WP of 2.2 ± 0.6 months. The WP of real-time polymerase chain reaction for HTLV-1 was estimated to be 4.1 ± 7.8 months. CONCLUSIONS: The WP in commercially available testing systems for HTLV-1/2 was determined for natural infection among repeated blood donors. Considering the HTLV-1 WP will help increase transfusion safety and facilitate the accurate diagnosis of HTLV-1 infection.

Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid and Accurate Confirmatory Diagnosis of HTLV-1/2 Infection.

Laboratory diagnosis of human T-lymphotropic viruses (HTLV) 1 and 2 infection is performed by serological screening and further confirmation with serological or molecular assays. Thus, we developed a loop-mediated isothermal nucleic acid amplification (LAMP) assay for the detection of HTLV-1/2 in blood samples. The sensitivity and accuracy of HTLV-1/2 LAMP were defined with DNA samples from individuals infected with HTLV-1 (n = 125), HTLV-2 (n = 19), and coinfected with HIV (n = 82), and compared with real-time polymerase chain reaction (qPCR) and PCR-restriction fragment length polymorphism (RFLP). The overall accuracy of HTLV-1/2 LAMP (95% CI 74.8-85.5%) was slightly superior to qPCR (95% CI 69.5-81.1%) and similar to PCR-RFLP (95% CI 79.5-89.3%). The sensitivity of LAMP was greater for HTLV-1 (95% CI 83.2-93.4%) than for HTLV-2 (95% CI 43.2-70.8%). This was also observed in qPCR and PCR-RFLP, which was associated with the commonly lower HTLV-2 proviral load. All molecular assays tested showed better results with samples from HTLV-1/2 mono-infected individuals compared with HIV-coinfected patients, who present lower CD4 T-cell counts. In conclusion, HTLV-1/2 LAMP had similar to superior performance than PCR-based assays, and therefore may represent an attractive alternative for HTLV-1/2 diagnosis due to reduced working time and costs, and the simple infrastructure needed.

Myelopathy secondary to human T-lymphotropic virus and Treponema pallidum infection: case report.

Introduction: The human T-lymphotropic virus has been associated with human disease, affecting CD4+ T, CD8+ T, and B lymphocytes. It can cause T-cell leukemia/lymphoma and HTLV-associated myelopathy. Case presentation: A 31-year-old woman was admitted after 2 months of cramps, paraparesis, and fecal/urinary incontinence. She was diagnosed with neurosyphilis according to the cerebrospinal fluid analysis. Despite treatment with crystalline penicillin there was no recovery, and anti-HTLV-1/2 tests were positive; therefore, the diagnosis of HTLV-associated myelopathy was made. The patient rejected glucocorticoid treatment; baclofen and carbamazepine were used to treat spasticity and cramps, respectively. The patient has not had progression. Discussion: HTLV-associated myelopathy is generated by an exaggerated inflammatory response in the central nervous system with clonal expansion of CD4+ T and CD8+ T lymphocytes. There is not a specific and useful treatment; glucocorticoids can reduce inflammation, but do not improve clinical functional outcomes. There is a high prevalence of syphilis and human T-lymphotropic virus co-infection in tropical countries; however, myelopathy as the first clinical manifestation is unusual. The treatment of neurosyphilis could reduce the inflammation into the central nervous system and could decrease the progression of sequelae. This is the first case of myelopathy secondary to viral and treponemal co-infection confirmed in Colombia.

Isolation of the Arawete and Asurini Indians keeps the tribes free from HTLV infection during 36 years of follow-up.

Arawete and Asurini Indian tribes were revisited after a 36-year follow-up in search of HTLV infections. 46 persons (23 from each tribe) were tested for HTLV-1/2 antibodies and viral DNA. None were positive; this was probably because of their social/cultural isolation from neighboring tribes where HTLV-2c is hyperendemic.

Human T-lymphotropic virus 1 and 2 among people who used illicit drugs in the state of Pará, northern Brazil.

People who used illicit drugs (PWUDs) represent an important population group for acquisition and viral dispersion. In Brazil, high rates of the human T lymphotropic virus 1 (HTLV-1) and 2 (HTLV-2) have been reported in epidemiological studies. However, the epidemiological scenario of HTLV-1/2 infections in PWUDs is still poorly understood. Thus, this cross-sectional study determined the prevalence, frequency of subtypes and factors associated with HTLV-1/2 infections among PWUDs in the Brazilian state of Pará, an area considered endemic for this virus and with poor health services. Among 826 PWUDs, 53 (6.4%) presented anti-HTLV-1/2 antibodies by EIA and 44 (5.3%) presented proviral DNA by PCR. HTLV-1 and HTLV-2 were detected in 25 (3.0%) and 19 (2.3%) PWUDs, respectively. Subtypes 1a (25/44), transcontinental (23/44) and Japanese subgroups (2/44), 2b (6/44) and 2c (13/44) were identified. Involvement in illicit/criminal activity, daily use of illicit drugs, illicit drug use over 12 years, unprotected sex with other PWUDs, changes in genitalia (including ulcers and wounds), and more than 12 sexual partners were associated with HTLV-1/2 in PWUDs. This high prevalence and intense circulation of subtypes and subgroups of HTLV-1/2 is very worrying, and indicate the need for urgent actions for its control, prevention and treatment.

Pregnancy does not adversely impact diagnostic tests for HTLV-1/2 infection.

Mother-to-child-transmission (MTCT) of human T-cell lymphotropic virus type-1(HTLV-1) contributes disproportionately to the burden of HTLV-1 associated diseases. All preventive measures to avoid MTCT rely on the identification of infected mothers. However, the impact of pregnancy on HTLV-1 diagnosis has not been clearly assessed. Paired samples from 21 HTLV-1 infected women taken during pregnancy and while not pregnant were analysed by CMIA and PCR. The signal-to-cut-off values (S/CO) were higher during pregnancy than in the paired non-pregnant samples. HTLV-1 proviral load did not alter significantly by pregnant state. S/CO positively correlated with HTLV proviral load. Pregnancy does not impair the diagnosis of HTLV-1/2, by either immunological (CMIA) or molecular (qPCR/nPCR) tests.

Development and evaluation of human T-cell leukemia virus-1 and -2 multiplex quantitative PCR.

The diagnosis of human T -cell leukemia virus type 1 (HTLV-1) infection in Japan is usually performed by serological testing, but the high rate of indeterminate results from western blotting makes it difficult to assess the infection accurately. Nucleic acid tests for HTLV-1 and/or HTLV-2 are used to confirm infection with HTLV-1 and/or HTLV-2 and are also used for the follow-up of HTLV-1 related diseases. To prepare a highly sensitive method that can discern infection with HTLV-1 and HTLV-2, a multiplex quantitative polymerase chain reaction (qPCR) by large-scale primer screening was developed. Sensitivity and specificity were evaluated by serial dilution of cell lines and by testing with known clinical samples. The resulting multiplex qPCR can detect about four copies of HTLV-1 provirus per 105 cells. Moreover, HTLV-1 provirus could be detected in 97.2% (205 of 211) of HTLV-1 seropositive clinical samples. These sensitivities were sufficiently high compared with the methods reported previously. Also, all the HTLV-2 seropositive clinical samples tested were found to be positive by this method (three of three). In conclusion, this method can successfully and simultaneously detect both types of HTLV-1 and HTLV-2 provirus with extremely high sensitivity.

[Detection of Human T lymphotropic virus 1 (HTLV-1) Cosmopolitan subtype Transcontinental subgroup (Aa) and HTLV-2 subtype b in blood donors of Corrientes]. / Hallazgo del virus linfotrópico T humano 1 (HTLV-1) subtipo Cosmopolita subgrupo Transcontinental (Aa) y del HTLV-2 subtipo b en donantes de sangre de Corrientes.

A molecular epidemiological study was conducted in a population of 9422 blood donors in the province of Corrientes, Northeastern Argentina, to determine the prevalence of Human T-cell lymphotropic virus types 1 and 2 (HTLV-1/2), the phylogenetic identification of HTLV-1 and 2 subtypes/subgroups and perform a mutation analysis. Based on the results obtained, it was shown that both HTLV-1 and HTLV-2 are circulating in a low-risk population of Corrientes, although with a similar prevalence to that of non-endemic areas. Phylogenetic studies identified the HTLV-1 Cosmopolitan subtype Transcontinental subgroup (Aa), and the HTLV-2 subtype b. Infected donors reported neither a history of risk factors such as transfusions, intravenous drug use, nor risky or HTLV-1/2 seropositive sexual partners. These results suggest that these viruses were transmitted from mother to child, possibly from generation to generation, and that these strains were introduced into the Caucasian population of this region from ancestors originating from endemic areas of the country either from or through contact with individuals from other countries years ago. Our results demonstrate for the first time the presence of HTLV-1 and HTLV-2 in the province of Corrientes. Moreover, although the province can be considered a non-endemic area, the need to include these retroviruses in a national Public Health program is highlighted, in order to have qualified professionals duly trained to make their diagnosis and provide the necessary information in relation to primary care and patient follow-up.

Dermatological manifestations in patients with human T-cell lymphotropic virus at a reference service in Amazon.

INTRODUCTION: Individuals infected with the human T-cell lymphotropic virus type 1 (HTLV-1) commonly present skin lesions, which may be a warning sign for the diagnosis of infection. This study describes the most prevalent skin manifestations in HTLV carriers attended at the clinic of Núcleo de Medicina Tropical (NMT) of the Universidade Federal do Pará (UFPA) in Belém, Pará, Brazil. METHODS: This is a study of a series of cases of patients infected with human T-cell lymphotropic virus types 1 and 2 (HTLV-1/2) treated at NMT UFPA between 1999 and 2016. A descriptive analysis of data was applied. RESULTS: Among 788 surveyed medical records in the service, 15.10% (n = 119) were referred to the dermatology clinic. From the series of cases that presented with skin lesions, 66.39% were female and 33.61% were male, and the average age of this group was 48 years. There was a predominance of patients with noninfectious inflammatory manifestations (64.2%), followed by infectious ones (24.6%), and 1.58% with lymphoproliferative diseases. As for the group of lesions, 45.26% of the erythematous-squamous type were observed, followed by dyschromia (24.21%), and eczematous (14.74%). One patient with a diagnosis of adult T-cell leukemia/lymphoma, another with parapsoriasis, and four with infective dermatitis are highlighted. CONCLUSION: Skin disorders in the HLTV positive patient are important causes of referral to the dermatologist with etiological and skin lesions groups diversity. In the series of cases studied, lymphoproliferatives diseases and infective dermatitis associated with HTLV-1 were presented as a challenge for the diagnosis and clinical management of these patients.

Hairpin probes based click polymerization for label-free electrochemical detection of human T-lymphotropic virus types II.

A novel, simple, and label-free amplification electrochemical impedance method for quantitative detection of Human T-lymphotropic virus types II(HTLV-II) via click chemistry-mediated of hairpin DNA probes (hairpins) with polymers was developed. The hairpins were firstly attached to the gold electrode surface by an S-Au bond, the azido terminals of hairpins were close to the electrode surface, which make it difficult to be approached. After hybridizing with HTLV-II, the hairpins were unfolded and experienced a big configuration change, which made the azido terminals of the hairpins available to conjugate with alkynyl-containing polymer, called P(DEB-DSDA), formed by 1,4-diacetylenebenzene (DEB) and 4,4'-Diazido-2,2'-stilbenedisulfonic acid disodium salt tetrahydrate (DSDA) via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). With amount of P(DEB-DSDA) conjugated with the hairpin probes via click polymerization, its electrochemical signal can have a great amplification. Under optimized experimental conditions, this new probe showed a low detection limit of 0.171 pM with a good liner in the range of 1 pM-1 nM. Meanwhile, the biosensor also exhibited good selectivity and reliability in detection of real serum samples, indicating that it has great application potential in clinical DNA diagnosis and detection.

Seroprevalence of HTLV-1/2 Among Voluntary Blood Donors in Vietnam.

Infection with human T lymphotropic virus (HTLV), although asymptomatic in most cases, can lead to severe illnesses, such as adult T cell leukemia/lymphoma or myelopathy/tropical spastic paraparesis. HTLV can be transmitted by whole-blood (WB) transfusion. The prevalence of HTLV among blood donor populations has not been characterized in Vietnam, although the screening has been partially implemented on voluntary basis since 2016. To determine the seroprevalence of HTLV-1/2 among blood donors, a total of 14,819 healthy blood donors in northern, central, and southern Vietnam and 1,003 samples from hepatitis B surface antigen (HbsAg), anti-hepatitis C (anti-HCV), or HIV Ag/Ab reactive blood donors were screened for anti-HTLV-1/2 antibodies by a chemiluminescence immunoassay using the Abbott ARCHITECT rHTLV-I/II assay. The anti-HTLV-1/2 repeat reactive (RR) samples were further tested by immunoblot (IB) method using MP Biomedicals HTLV Blot 2.4 for confirmation and differentiation of HTLV-1/2 infection. Proviral HTLV subgenomic amplification of the gag and tax regions was performed on the available WB RR samples (N = 11) by polymerase chain reaction (PCR). Among 14,819 blood donors, 34 samples (0.23%) were RR for anti-HTLV-1/2 antibodies, but only 1 case was confirmed HTLV-2 positive (0.0067%) and 5 cases were classified as indeterminate (0.034%) by IB. The RR rate was 0.39% among HBsAg/anti-HCV/HIV reactive sample groups, but none of them was confirmed by IB. Subgenomic PCR failed to amplify proviral DNA from WB samples of 11 RR samples. HTLV-1/2 prevalence was found to be low among blood donors in the study. Continued vigilance remains essential to maintain a low transfusion-transmitted risk in Vietnam.

Performance of Commercially Available Serological Screening Tests for Human T-Cell Lymphotropic Virus Infection in Brazil.

Serological screening for human T-cell lymphotropic virus type 1 (HTLV-1) is usually performed using enzyme-linked immunosorbent assay (ELISA), particle agglutination, or chemiluminescence assay kits. Due to an antigen matrix improvement entailing the use of new HTLV antigens and changes in the format of HTLV screening tests, as well as newly introduced chemiluminescence assays (CLIAs), a systematic evaluation of the accuracy of currently available commercial tests is warranted. We aimed to assess the performance of commercially available screening tests for HTLV infection diagnosis. A diagnostic accuracy study was conducted on a panel of 397 plasma samples: 200 HTLV-negative plasma samples, 170 HTLV-positive plasma samples, and 27 plasma samples indeterminate by Western blotting (WB). WB-indeterminate samples (i.e., those yielding no specific bands for HTLV-1 and/or HTLV-2) were assessed by PCR, and the results were used to compare agreement among the commercially available ELISA screening tests. For performance analysis, WB-indeterminate samples were excluded, resulting in a final study panel of 370 samples. Three ELISA kits (Murex HTLV-1/2 [Murex], anti-HTLV-1/2 SYM Solution [SYM Solution], and Gold ELISA HTLV-1/2 [Gold ELISA]) and one CLIA kit (Architect rHTLV-1/2) were evaluated. All screening tests demonstrated 100% sensitivity. Concerning the HTLV-negative samples, the SYM Solution and Gold ELISA kits had specificity values of >99.5%, while the Architect rHTLV-1/2 test presented 98.1% specificity, followed by Murex, which had a specificity of 92.0%. Regarding the 27 samples with WB-indeterminate results, after PCR confirmation, all ELISA kits showed 100% sensitivity but low specificity. Accuracy findings were corroborated by the use of Cohen's kappa value, which evidenced slight and fair agreement between PCR analysis and ELISAs for HTLV infection diagnosis. Based on the data, we believe that all evaluated tests can be safely used for HTLV infection screening.

Serological detection of human T-cell lymphotropic virus types I and II among pregnant women in Ilorin, Nigeria.

Human T-cell lymphotropic virus types I/II (HTLV-I/II) is endemic in some parts of the world including Nigeria. Reported prevalence rates in Nigeria have largely focused on blood donors. This study aims at determining the prevalence of HTLV infection among pregnant women in Ilorin North-central Nigeria. Serum samples from 276 pregnant women who were antenatal clinic attendees at General and Civil Service Hospitals in Ilorin were tested for the presence of HTLV-I/II antibodies using Enzyme Linked Immunosorbent Assay test kits from Diagnostic Automation INC., USA. Out of the 276 women tested, 3 tested positive giving a prevalence rate of 1.1%. The result was analyzed on the basis of age, marital status, nature of family, educational status, occupation, religion, parity, and gestational stage of the women. There was no statistical association of HTLV positivity with any of the variables. Although relatively lower than prevalence rate recorded among similar study groups in other parts of the country, the 1.1% prevalence in this study underscores the need for proper education and creation of awareness among antenatal clinic attendees, so as to reduce viral transmission and incidence of HTLV-related diseases.

Droplet digital PCR for absolute quantification of proviral load of human T-cell lymphotropic virus (HTLV) types 1 and 2.

BACKGROUND: Human T-lymphotrophic virus (HTLV) types 1 and 2 cause lifelong infection whereby most infected individuals are asymptomatic whilst a minority develop infection-related disease. These latter patients invariably have been found to have high proviral load (PVL). Therefore, infected patients are monitored by determining the proportion of lymphocytes that are infected with HTLV-1/2. An increase in PVL has been shown to represent an increasing risk of developing HTLV-associated diseases. Monitoring of PVL requires a reliable and sensitive method. In this study assays based on droplet digital PCR (ddPCR) were established and evaluated for detection and quantification of HTLV-1/2. OBJECTIVES: To develop two parallel assays to detect the tax genes and determine the PVL of HTLV-1 and -2. STUDY DESIGN: Sixty-seven clinical samples from patients infected with HTLV-1 or HTLV-2 were analysed. The samples had previously been analysed with a qPCR and a comparison between ddPCR and qPCR was performed. The specificity of the assays were determined by analyzing samples from 20 healthy blood donors. RESULTS: The ddPCR was a stable and sensitive method for detection and quantification of HTLV-1 and -2. When comparing the qPCR and ddPCR the correlation was high (Pearsons correlation coefficient 0.96). The variability of the ddPCR was very low with intra-assay coefficient of variation (CV) of 0.97-3.3% (HTLV-1) and 1.7-8.2% (HTLV-2) and inter-assay CV of 1.8-6.1% (HTLV-1) and 1.2-12.9% (HTLV-2). CONCLUSIONS: The ddPCR reliably quantified HTLV DNA in clinical samples and could be a useful tool for monitoring of PVLs in HTLV-infected individuals.

Low prevalence of HTLV1/2 infection in a population of immigrants living in southern Italy.

AIMS: To assess the prevalence of HTLV-1 and HTLV-2 infections in a cohort of immigrants living in southern Italy. FINDINGS: We screened for antibody to HTLV-1/2 infection 1,498 consecutive immigrants born in endemic areas (sub-Saharan Africa or southern-Asia) by a commercial chemiluminescent microparticle immunoassay. If confirmed in a Western blot assay, which differentiates anti-HTLV-1 from anti-HTLV-2, the positive sera were tested for specific HTLV RNA by a home-made PCR. The immigrants investigated were more frequently males (89.05%), young (median age 26 years), with a low level of education (median schooling 6 years), born in sub-Saharan Africa (79.70%). They had been living in Italy for a median period of 5 months. Only one (0.07%) subject was anti-HTLV-1 -positive/HTLV-1 RNA-negative; he was an asymptomatic 27-year-old male from Nigeria with 6 years' schooling who stated unsafe sexual habits and unsafe injection therapy. CONCLUSIONS: The data suggest screening for HTLV1 and HTLV-2 infections all blood donors to Italy from endemic countries at least on their first donation; however, a cost-effectiveness study is needed to clarify this topic.