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Japan is one of the world's highly endemic areas for human T cell leukemia virus type 1 (HTLV-1), and it is known that the infection rate of HTLV-1 increases with age. The infection rate among the elderly has been estimated based on data from blood donors under the age of 65, and the actual number and rate of infection among the elderly are unknown. Data of 26,090 preoperative HTLV-1 screening tests conducted at Kagoshima University Hospital from 2001 to 2020, including 2726 HTLV-1-positive patients, were used for calculating the decadal infection rates for the year of birth. Estimated infection rates by birth year and demographic tables were used to estimate the current number of infected people in Kagoshima. The estimated total numbers of people infected with HTLV-1 in Kagoshima prefecture were 139,436 in 2005 and 80,975 in 2019. The infection rate increased with age for both men and women, reaching 17.3% for women born before the 1920s. Next, we tried to clarify whether the increase in infection rates with age was due to post-school age infections. The age of birth with the greatest increase in infection rate after 10 years was women born in the 1970s, and the increase in infection rate was only 0.98%, which is not a statistically significant increase. The number of infected people in Kagoshima was80,000 in 2019. No data were available in this study to point to the involvement of horizontal transmission after school age in the high infection rate among the elderly. The high infection rate among the elderly is thought to have been high even when they were infants.

Human T-cell leukemia virus type 1 (HTLV-1) is a causative agent of the life-threatening diseases, adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy. Following implementation of antenatal screening in Japan, novel transmission of HTLV-1 in adolescent and adult generations is expected to replace vertical transmission as the main route for transmission.To obtain the current status of HTLV-1 horizontal infection and to assess the fluctuation of transmission occurring among adolescents and adults in Japan.We followed-up 5,017,916 eligible repeat blood donors for 8 years from 2013 to 2021. We evaluated HTLV-1 transmission rate by age group (16-69 years-old), and calculated the total number of novel transmissions in Japan using demographic statistics published by the government of Japan.We identified 457 seroconverters (men, 203; women, 254) in a total of 19,244,604 person-years during the study period. The number of seroconversions per 100,000 person-years was 1.54 for men and 4.21 for women. An increase in the number of novel infections was observed in both sexes in adolescent and young adult generations despite the health bias of blood donors.We estimate that more than 2,800 new HTLV-1 infections occur annually in Japan. It is a serious concern that without immediate measures against new HTLV-1 infections, such as guideline formulation, an inclusion of HTLV as routine screening in sexual health services, an information campaign, and surveillance of the general population, novel HTLV-1 infection could continue to increase in Japan and be a source of global transmission.

Background The reliable diagnosis of human T-cell leukemia virus type 1 (HTLV-1) infection is important, particularly as it can be vertically transmitted by breast feeding mothers to their infants. However, current diagnosis in Japan requires a confirmatory western blot (WB) test after screening/primary testing for HTLV-1 antibodies, but this test often gives indeterminate results. Thus, this collaborative study evaluated the reliability of diagnostic assays for HTLV-1 infection, including a WB-based one, along with line immunoassay (LIA) as an alternative to WB for confirmatory testing. Results Using peripheral blood samples from blood donors and pregnant women previously serologically screened and subjected to WB analysis, we analyzed the performances of 10 HTLV-1 antibody assay kits commercially available in Japan. No marked differences in the performances of eight of the screening kits were apparent. However, LIA determined most of the WB-indeterminate samples to be conclusively positive or negative (an 88.0% detection rate). When we also compared the sensitivity to HTLV-1 envelope gp21 with that of other antigens by LIA, the sensitivity to gp21 was the strongest. When we also compared the sensitivity to envelope gp46 by LIA with that of WB, LIA showed stronger sensitivity to gp46 than WB did. These findings indicate that LIA is an alternative confirmatory test to WB analysis without gp21. Therefore, we established a novel diagnostic test algorithm for HTLV-1 infection in Japan, including both the performance of a confirmatory test where LIA replaced WB on primary test-reactive samples and an additional decision based on a standardized nucleic acid detection step (polymerase chain reaction, PCR) on the confirmatory test-indeterminate samples. The final assessment of the clinical usefulness of this algorithm involved performing WB analysis, LIA, and/or PCR in parallel for confirmatory testing of known reactive samples serologically screened at clinical laboratories. Consequently, LIA followed by PCR (LIA/PCR), but neither WB/PCR nor PCR/LIA, was found to be the most reliable diagnostic algorithm. Conclusions Because the above results show that our novel algorithm is clinically useful, we propose that it is recommended for solving the aforementioned WB-associated reliability issues and for providing a more rapid and precise diagnosis of HTLV-1 infection.

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.

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.

Background An increased risk of total death owing to human T-lymphotropic virus type-I (HTLV-I) infection has been reported. However, its etiology and protective factors are unclear. Various studies reported fluctuations in immune-inflammatory status among HTLV-I carriers. We conducted a matched cohort study among the general population in an HTLV-I-endemic region of Japan to investigate the interaction between inflammatory gene polymorphisms and HTLV-I infection for total death, incidence of cancer, and atherosclerosis-related diseases. Method We selected 2180 sub-cohort subjects aged 35–69 years from the cohort population, after matching for age, sex, and region with HTLV-I seropositives. They were followed up for a maximum of 10 years. Inflammatory gene polymorphisms were selected from TNF-α, IL-10, and NF-κB1. A Cox proportional hazard model was used to estimate the hazard ratio (HR) and the interaction between gene polymorphisms and HTLV-I for risk of total death and incidence of cancer and atherosclerosis-related diseases. Results HTLV-I seropositivity rate was 6.4% in the cohort population. The interaction between TNF-α 1031T/C and HTLV-I for atherosclerosis-related disease incidence was statistically significant (p = 0.020). No significant interaction was observed between IL-10 819T/C or NF-κB1 94ATTG ins/del and HTLV-I. An increased HR for total death was observed in the Amami island region, after adjustment of various factors with gene polymorphisms (HR 3.03; 95% confidence interval, 1.18–7.77). Conclusion The present study found the interaction between TNF-α 1031T/C and HTLV-I to be a risk factor for atherosclerosis-related disease. Further follow-up is warranted to investigate protective factors against developing diseases among susceptible HTLV-I carriers., Highlights • We observed death and incidence risk with HTLV-I and SNPs in a matched cohort study. • An increased risk for total death with HTLV-I was observed in the island region. • Inflammatory SNP interacted with HTLV-I for atherosclerosis-related disease risk.

Western blotting (WB) for human T cell leukemia virus type 1 (HTLV-1) is performed to confirm anti-HTLV-1 antibodies detected at the initial screening of blood donors and in pregnant women. However, the frequent occurrence of indeterminate results is a problem with this test. We therefore assessed the cause of indeterminate WB results by analyzing HTLV-1 provirus genomic sequences. A quantitative PCR assay measuring HTLV-1 provirus in WB-indeterminate samples revealed that the median proviral load was approximately 100-fold lower than that of WB-positive samples (0.01 versus 0.71 copy/100 cells). Phylogenic analysis of the complete HTLV-1 genomes of WB-indeterminate samples did not identify any specific phylogenetic groups. When we analyzed the nucleotide changes in 19 HTLV-1 isolates from WB-indeterminate samples, we identified 135 single nucleotide substitutions, composed of four types, G to A (29%), C to T (19%), T to C (19%), and A to G (16%). In the most frequent G-to-A substitution, 64% occurred at GG dinucleotides, indicating that APOBEC3G is responsible for mutagenesis in WB-indeterminate samples. Moreover, interestingly, five WB-indeterminate isolates had nonsense mutations in Pol and/or Tax, Env, p12, and p30. These findings suggest that WB-indeterminate carriers have low production of viral antigens because of a combination of a low proviral load and mutations in the provirus, which may interfere with host recognition of HTLV-1 antigens.

Adult T-cell leukemia/lymphoma (ATL) occurs in approximately 5% of individuals infected with human T-cell leukemia virus type 1 (HTLV-1). A high proviral load (PVL; more than four copies per 100 peripheral blood mononuclear cells (PBMCs) or 1.6 copies per 100 blood leukocytes) and being male are risk factors for ATL development. Whether anti-HTLV-1 antibody level is related to such risk is unknown. Here, PVL and antibody levels were examined using real-time PCR and other tests in 600 HTLV-1 positive screened Japanese blood donors to understand the relationship between PVL and antibody level in asymptomatic carriers and to gain insights toward better antibody testing for HTLV-1 infection. The 430 donors in whom proviral DNA was detected were considered as true positives for HTLV-1 infection. Among donors aged 40 years or older, more males than females had a PVL corresponding to more than 1.6% infected leukocytes, and an antibody titer below the median (P = 0.0018). In antibody tests using an HTLV-1 positive cell line or Env antigens there was a large discrepancy in antibody titer among 13 provirus-positive samples, probably suggesting that antibody-based screening tests should incorporate multiple HTLV-1 antigens, such as Gag and Env antigens.

Human T-lymphotropic virus 1 (HTLV-1) infection has an especially high prevalence in Japan. Transmission has been confirmed in infancy through breastfeeding; however, little is known about the epidemiological aspects of new HTLV-1 infections later in life. We aimed to estimate the nationwide annual number of new HTLV-1 infections among adolescents and adults in Japan.In this retrospective cohort analysis, we assessed new HTLV-1 infections of repeat blood donors aged 16-69 years between Jan 1, 2005, and Dec 31, 2006, in the Japanese Red Cross Blood Centres database. We used results of antibody tests done in repeat blood samples collected until Dec 31, 2011, to assess the number who seroconverted to HTLV-1. We calculated the incidence density by dividing the number of seroconverters by the number of person-years of follow-up, and then extrapolated densities to regional populations to estimate the annual number of new HTLV-1 infections.We included 3 375 821 HTLV-1-seronegative blood donors (2 100 915 men and 1 274 906 women). Within a median follow-up of 4·5 years (IQR 2·3-5·8), 532 people (204 men and 328 women) had seroconverted. The incidence density was significantly higher in women (6·88 per 100 000 person-years; 95% CI 6·17-7·66) than in men (2·29 per 100 000 person-years; 95% CI 1·99-2·62; p0·0001). The estimated annual number of new HTLV-1 infections was 4190 (95% CI 4064-4318) with 975 (914-1038) infections in men and 3215 (3104-3328) in women.New HTLV-1 infections in adolescents and adults are an important public health concern in Japan and preventive strategies are needed to reduce new transmission.Ministry of Health, Labour, and Welfare of Japan; Japan Agency for Medical Research and Development.

Elevated levels of soluble CD30 (sCD30) are linked with various T-cell neoplasms. However, the relationship between sCD30 levels and the development of adult T-cell leukemia (ATL) in human T-cell leukemia virus type 1 (HTLV-1) carriers remains to be clarified. We here investigated whether plasma sCD30 is associated with risk of ATL in a nested case-control study within a cohort of HTLV-1 carriers. We compared sCD30 levels between 11 cases (i.e., HTLV-1 carriers who later progressed to ATL) and 22 age-, sex- and institution-matched control HTLV-1 carriers (i.e., those with no progression). The sCD30 concentration at baseline was significantly higher in cases than in controls (median 65.8, range 27.2-134.5 U/mL vs. median 22.2, range 8.4-63.1 U/mL, P=0.001). In the univariate logistic regression analysis, a higher sCD30 (≥30.2 U/mL) was significantly associated with ATL development (odds ratio 7.88 and the 95% confidence intervals 1.35-45.8, P = 0.02). Among cases, sCD30 concentration tended to increase at the time of diagnosis of aggressive-type ATL, but the concentration was stable in those developing the smoldering-type. This suggests that sCD30 may serve as a predictive marker for the onset of aggressive-type ATL in HTLV-1 carriers.

Quantitative PCR (qPCR) of human T-cell leukemia virus type 1 (HTLV-1) provirus is used for HTLV-1 testing and for assessment of risk of HTLV-1-related diseases. In this study, a reference material was developed for standardizing HTLV-1 qPCR. Freeze-dried TL-Om1 cells diluted with Jurkat cells were prepared and an assigned value for proviral load (PVL) of 2.71 copies/100 cells was determined by digital PCR. Nine Japanese laboratories using their own methods evaluated the PVLs of this reference material as 1.08-3.49 copies/100 cells. The maximum difference between laboratories was 3.2-fold. Correcting measured PVLs by using a formula incorporating the assigned value of this reference material should minimize such discrepancies.

We investigated the incidence of human T-cell leukemia virus type I (HTLV-1) infection in a total of 17 regions in four countries, including 13 regions in Japan, as well as Korea (Seoul and Busan), China, and Vietnam, by testing breast milk using a particle agglutination assay (PA) and line immunoassay (LIA). Among 266 samples from Japan, 24 (9.0%) were positive on PA and 3 (1.1%) were positive on LIA. Among 50 samples from Seoul, 2 were positive on PA and 1 was positive on LIA. In contrast, all 50 samples from Busan were negative on both tests, suggesting the maldistribution of HTLV-1 infectants in South Korea. The numbers of positive samples were 2/91 on PA and 1/91 on LIA for China and 1/88 on both PA and LIA for Vietnam. In China, one sample with a high probability of HTLV-2 infection was identified by LIA and synthetic peptide enzyme-linked immunosorbent assay (ELISA). We examined HTLV-1 antibody in breast milk samples using commercially available test kits, suggesting the existence of HTLV-1 carriers in endemic areas in Southeast Asia and an HTLV-2 infectant in China. As a part of human ethno-epidemiological research, these results constitute valuable epidemiological data. Further studies on the sensitivity, specificity, and reliability of assays using antibodies to HTLV-1 and 2 in breast milk will be necessary for large-scale epidemiological surveys of HTLV infection.

Definitive risk factors for the development of adult T-cell leukemia (ATL) among asymptomatic human T-cell leukemia virus type I (HTLV-1) carriers remain unclear. Recently, HTLV-1 proviral loads have been evaluated as important predictors of ATL, but a few small prospective studies have been conducted. We prospectively evaluated 1218 asymptomatic HTLV-1 carriers (426 males and 792 females) who were enrolled during 2002 to 2008. The proviral load at enrollment was significantly higher in males than females (median, 2.10 vs 1.39 copies/100 peripheral blood mononuclear cells [PBMCs]; P < .001), in those 40 to 49 and 50 to 59 years of age than that of those 40 years of age and younger (P = .02 and .007, respectively), and in those with a family history of ATL than those without the history (median, 2.32 vs 1.33 copies/100 PBMCs; P = .005). During follow-up, 14 participants progressed to overt ATL. Their baseline proviral load was high (range, 4.17-28.58 copies/100 PBMCs). None developed ATL among those with a baseline proviral load lower than approximately 4 copies. Multivariate Cox analyses indicated that not only a higher proviral load, advanced age, family history of ATL, and first opportunity for HTLV-1 testing during treatment for other diseases were independent risk factors for progression of ATL.

Human T-lymphotropic virus type 1 (HTLV-1) infection is known to affect hepatitis C virus (HCV) clearance and to accelerate the development of hepatocellular carcinoma in HCV-infected patients. In this study, we found the prevalence and titer of an antibody recognizing the central region of the HTLV-1 Gp46 protein to be associated with the severity of chronic liver disease. The antibody prevalence was significantly correlated with the stage of chronic liver disease (P < 0.0001): 3 (14.3%) of 21 patients with minimal-mild chronic hepatitis, 12 (24%) of 50 with moderate-severe chronic hepatitis, 7 (87.5%) of 8 with liver cirrhosis, and 13 (100%) of 13 with hepatocellular carcinoma. These results indicate that the antibody may be a useful marker of the deterioration of liver disease in patients co-infected with HCV and HTLV-1. This antibody may be useful for the diagnosis of liver diseases and the development of more effective treatments.

Quantitative PCR (qPCR) analysis of human T-cell leukemia virus type 1 (HTLV-1) was used to assess the amount of HTLV-1 provirus DNA integrated into the genomic DNA of host blood cells. Accumulating evidence indicates that a high proviral load is one of the risk factors for the development of adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis. However, interlaboratory variability in qPCR results makes it difficult to assess the differences in reported proviral loads between laboratories. To remedy this situation, we attempted to minimize discrepancies between laboratories through standardization of HTLV-1 qPCR in a collaborative study. TL-Om1 cells that harbor the HTLV-1 provirus were serially diluted with peripheral blood mononuclear cells to prepare a candidate standard. By statistically evaluating the proviral loads of the standard and those determined using in-house qPCR methods at each laboratory, we determined the relative ratios of the measured values in the laboratories to the theoretical values of the TL-Om1 standard. The relative ratios of the laboratories ranged from 0.84 to 4.45. Next, we corrected the proviral loads of the clinical samples from HTLV-1 carriers using the relative ratio. As expected, the overall differences between the laboratories were reduced by half, from 7.4-fold to 3.8-fold on average, after applying the correction. HTLV-1 qPCR can be standardized using TL-Om1 cells as a standard and by determining the relative ratio of the measured to the theoretical standard values in each laboratory.

We previously showed that 71-kDa heat shock cognate protein (HSC70) functions as a cellular receptor for gp46 protein via the gp46-197 region, corresponding to Asp197 to Leu216 of human T-cell lymphotropic virus type 1 (HTLV-1), leading to cell-to-cell transmission of HTLV-1. We found that HSC70 protein was contained in goat serum and casein used as blocking agents in the usual ELISA method. Here, it was demonstrated that HSC70 contamination in the blocking agents causes a false-negative result in the detection of anti-gp46-197 antibody in serum samples from HTLV-1-infected individuals. By using ELISA without the blocking agents, we detected antibodies recognizing the HSC70-binding site of gp46, and the anti-gp46-197 antibody specifically appeared in sera from patients with HTLV-1-associated diseases. The frequency of serum anti-gp46-197 antibody-positive individuals was 98% and 100% among ATLL and HAM/TSP patients, respectively, but only 6% among asymptomatic HTLV-1-infected carriers (ACs). The antibody titer in ATLL and HAM/TSP patients was higher than that in ACs (P < 0.002 for ATLL; P < 0.0001 for HAM/TSP). These findings suggest that appearance of the anti-gp46-197 antibody is a predictive marker for the onset of HTLV-1-associated disease.

We previously reported that the region corresponding to amino acids 197 to 216 of the gp46 surface glycoprotein (gp46-197) served as a binding domain for the interaction between gp46 and trypsin-sensitive membrane components of the target cell, leading to syncytium formation induced by human T-cell lymphotropic virus type 1 (HTLV-1)-bearing cells. Our new evidence shows that the 71-kDa heat shock cognate protein (HSC70) acts as a cellular receptor for syncytium formation. Using affinity chromatography with the peptide gp46-197, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we isolated three components (bands A, B, and C) from MOLT-4 cell lysate which exhibited specific interactions with gp46 and inhibitory activities for syncytium formation induced by HTLV-1-bearing cells. Band A and B components were identified as HSC70 and β-actin, respectively, through amino acid sequencing by tandem mass spectrometry and immunostaining with specific monoclonal antibodies. Band C is likely to be a nonprotein component, because full activity for syncytium formation was seen after extensive trypsin digestion. Anti-HSC70 monoclonal antibody clearly blocked syncytium formation in a coculture of HTLV-1-bearing cells and indicator cells, whereas no inhibition was seen with anti-β-actin monoclonal antibody. Furthermore, flow cytometric analysis indicated that anti-HSC70 antibody reacted with MOLT-4 cells. Thus, we propose that HSC70 expressed on the target cell surface acts as a cellular acceptor to gp46 exposed on the HTLV-1-infected cell for syncytium formation, thereby leading to cell-to-cell transmission of HTLV-1.