Ethics statement

The study was approved by the Institutional Research Board of the Escola Bahiana de Medicina e Saúde Pública (CAAE: 70078517.6.0000.5544), and informed written consent was obtained from participants when possible. In accordance with the ethical principles outlined in Brazilian regulations, the need for consent was waived only in cases of death or loss to follow-up.

The participants in this study were People living with HTLV-1 treated at the Integrative and Multidisciplinary Center for HTLV (CHTLV) between 2016 and 2018. The CHTLV outpatient clinic provides comprehensive biopsychosocial care to the public and is supported by public health services of the Brazilian National Health System (Sistema Único de Saúde [SUS]) [19]. HTLV-1 infection was confirmed by Elisa and Western blotting. For the determination of CPK levels, the Siemens Atellica CH Creatine Kinase (CK_L) assay was employed, utilizing upper reference limits of 192.0 U/L for females and 308.0 U/L for males.

The inclusion criteria were elevated creatine phosphokinase (CPK) levels above the upper limit on two separate tests at different time points. Participants who had taken myotoxic medications or were co-infected with HIV, hepatitis B, hepatitis C, syphilis, or toxoplasmosis were excluded from this study. A neurological examination was performed by a single neurologist (ARFV) for all included patients, and the diagnosis of HAM/TSP was based on Castro Costa’s criteria [20]. A comprehensive series of laboratory tests was performed as part of the study. These tests included the measurement of several parameters, including CPK, which is important in the assessment of muscle damage; lactate dehydrogenase (LDH), an enzyme that is elevated in several diseases, including muscle disease; alanine aminotransferase (ALT) and aspartate aminotransferase (AST), enzymes that indicate liver cell damage; and serologies for various infectious diseases, such as anti-HIV, anti-hepatitis B, anti-hepatitis C, syphilis, cytomegalovirus, and toxoplasmosis; antinuclear factor (FAN), an antibody that may be present in autoimmune diseases; antihistidy l antibody (anti-Jo1), an antibody associated with certain autoimmune muscle diseases and vitamin B12 levels: to exclude myelopathy due to B12 deficiency. Measurement of HTLV-1 proviral load (PVL), which determines the amount of HTLV-1 genetic material integrated into the genome of peripheral blood mononuclear cells (PBMCs). DNA was extracted using a Spin Column DNA Extraction System (Qiagen, Hilden, Germany). PVL was quantified in real time using the TaqMan polymerase chain reaction (PCR), as previously described [21]. Muscle biopsies of the left deltoid muscle were performed using an open technique. The samples were analyzed using the following techniques: 1) conventional histology of sections stained with Hematoxylin & Eosin, periodic acid-Schiff for glycogen, and modified Gomori trichrome for connective tissue matrix; 2) histochemistry with oil red 0 for fat and techniques for evaluating the enzymatic activity of succinate dehydrogenase, nicotinamide adenine dinucleotide, ATPase at pH 9.4, 4.3, and 4.6, alkaline phosphatase, acid phosphatase, and cytochrome C oxidase (COX); 3) immunohistochemistry using the indirect peroxidase method with primary antibodies against dystrophins (D1, D2, D3), merosin, dysferlin, CD4/CD8 lymphocytes, CD68 macrophages, and major histocompatibility complex class I (MHCI). The following parameters were used to standardize the final muscle biopsy report: variation in muscle fiber size, perimysial and endomysial fibrosis, number of nuclei and their position in the muscle cells (peripheral or central), presence of necrosis, presence of inflammatory cell infiltration, assessment of the internal architecture of the muscle cells, preservation of the mosaic pattern related to the distribution of the different types of muscle fibers, accumulation of fat and glycogen, acid phosphatase activity, accumulation of fat and glycogen, preservation of mosaic pattern by distribution of fiber types, and presence of COX-negative fibers. When indicated, a molecular panel for neuromuscular diseases [22] was used to examine the muscular dystrophies. Polymyositis was diagnosed according to the diagnostic criteria of the European Alliance of Associations for Rheumatology (EULAR) and the American College of Rheumatology (ACR) [23]; These criteria include various clinical, laboratory, and histopathological aspects for the evaluation and diagnosis of muscle diseases. Clinical aspects include the pattern of muscle weakness, age at symptom onset, and involvement of the cervical flexors. Laboratory aspects included evaluation of hyperCKemia, elevated transaminase levels, and the presence of positive anti-Jo1 antibodies. Histopathological findings included inflammatory infiltrates of mononuclear cells in the endomysium.

Description of the cases

Case 1 concerns a 54-year-old Black woman who tested positive for HTLV-1 during a blood donation at the age of 40. At the age of 43 years, she began to experience fatigue and proximal muscle weakness, initially in the lower limbs, which then gradually progressed to the upper limbs. At this point, the patient was diagnosed with neurogenic bladder. Laboratory examination revealed a CPK level of 655 U/L. At the age of 45, she underwent a muscle biopsy, which revealed inflammatory myopathy. Thereafter, the patient received immunosuppressive treatment with methotrexate. At 52 years of age, the patient presented with tetraparesis, mainly affecting the proximal and upper limbs, along with weakness of the neck muscles. The symptoms worsened over time. In 2018, at 54 years, a new muscle biopsy was performed, which showed muscle degeneration, variations in fiber size and centralization of the nuclei, endomysial and perimysial fibrosis, and foci of inflammatory infiltrate in the endomysium (Fig 2A). Necrosis was not seen. Immunohistochemical analysis of the muscle tissue showed normal expression of proteins, such as dysferlin, dystrophin, merosin, sarcoglycans, collagen 6, and caveolin-3. CPK measured in the same month as the second muscle biopsy was 255 U/L.. HTLV PVL was 16,178 x 10⁶ PBMC, approximately two years after the second muscle biopsy Based on the clinical presentation and findings from muscle biopsies, the patient fulfilled the EULAR/ACR criteria for the diagnosis of polymyositis.

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Fig 2. A) Case 1: The arrows delimit an area in which muscle fibers (M) are separated by fibrosis.

The muscle fibers have varying sizes and shapes. There is a focus of inflammatory infiltration of mononuclear cells (arrowhead) and there are adipocytes (A) permeating the muscle (Hematoxylin-eosin staining); B) Case 2: A focus of inflammatory infiltration of mononuclear cells can be observed in the endomysium (arrow) and adipocytes (A) are seen permeating the muscle. C) Case 2: Immunostaining shows CD8-positive lymphocytes permeating the endomysium.

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Case 2 concerns a 56-year-old Brown woman who, at the age of 41, began to experience pain, fatigue and weakness in her lower limbs. At the age of 43, she was diagnosed with HTLV-1 infection. At the age of 46, her CPK level was elevated (610 U/L), and polymyositis was suspected. As a result, immunosuppressive treatment with methotrexate was prescribed. At the age of 55 years, the patient presented with grade III muscle strength in the proximal muscles of the lower and upper limbs, moderate weakness of the neck muscles and abolished deep reflexes. At this point, the HTLV-1 PVL was 5,957 x 10⁶ PBMC, and CPK was 122 U/L. A muscle biopsy showed a myopathic pattern with moderate variability in fiber size, an increase in endomysial and perimysial connective tissue, heightened nuclear centralization (Fig 2B), and increased labeling for CD8 + cells (Fig 2C) and MHC I in the lymphocytic perimysial inflammatory infiltrate. Based on clinical presentation and muscle biopsy findings, this patient also met the EULAR/ACR criteria for the diagnosis of polymyositis.

Case 3 concerns a Brown male patient born in 1954 who was diagnosed with HTLV-1 infection at the age of 50 when he presented with a neurogenic bladder. At the age of 59, he began to experience proximal lower limb muscle weakness and recurrent falls. Due to elevated CPK levels (1,125 U/L), a diagnosis of myopathy was made, and the patient was subsequently treated with corticosteroids. At the age of 62 years, the patient presented with tetraparesis mainly affecting the proximal muscles of the lower limbs, axial weakness centered on the neck flexors, increased deep reflexes in all limbs, atrophy of the deltoid and supraspinatus muscles, and moderate spasticity in the lower limbs. The patient’s clinical condition deteriorated considerably and also affected the respiratory muscles. Non-invasive ventilatory support was initiated, but the patient eventually developed respiratory failure and died four months after the onset of respiratory symptoms. Although a muscle biopsy could not be performed, this patient met the EULAR/ACR criteria for the diagnosis of polymyositis due to the clinical presentation, elevated CPK levels and progressive deterioration of muscle strength. The development of respiratory muscle involvement emphasizes the severity of the disease. Despite treatment with corticosteroids, the patient died, likely due to complications related to respiratory failure. No HTLV-1 PVL testing was performed for this patient.

Case 4 involves a White woman born in 1978 who developed proximal weakness, mainly in the lower limbs, at age 30. At age 34, she was serologically diagnosed with HTLV-1 infection associated with the appearance of a neurogenic bladder. In September 2016, at the age of 38, the patient was wheelchair-dependent, exhibiting Grade 3 muscle strength in the thighs, areflexia of the lower extremities, and an elevated CPK level of 828 U/L. At age 39, she presented with tetraparesis, marked weakness of the pelvic girdle and lower limbs, and normal deep reflexes in all limbs. As a result, she was confined to a wheelchair. Laboratory testing revealed a high HTLV-1 PVL of 31,888 x 10⁶ PBMC. A muscle biopsy showed a dystrophic appearance with significant variability in fiber size, increased connective tissue in the endomysium and perimysium, heightened nuclear centralization, rare necrotic fibers, cytoplasmic vacuolization, a moderate endomysial inflammatory infiltrate containing CD8⁺ and MHC I+ lymphocytes, as well CD68⁺ macrophages, and intermyofibrillar disorganization. Based on these findings, the diagnosis in this case was muscular dystrophy. The presence of dystrophic changes in the muscle cell along with the clinical presentation of progressive proximal weakness and the histopathologic features support the conclusion of muscular dystrophy as the underlying disease in this patient.

Case 5 concerns a Brown woman born in 1983 who was diagnosed with HTLV-1 infection during prenatal screening at the age of 27. At the age of 29, she began to present with diffuse muscle weakness accompanied by elevated CPK levels (1,255 U/L). At the age of 30, she was diagnosed with a neurogenic bladder. At the age of 35, she developed tetraparesis with more pronounced proximal weakness, spasticity in the lower limbs, markedly increased deep tendon reflexes in all four limbs, and bilateral positive Hoffman signs. The HTLV-1 PVL was 10,959 x 10⁶ PBMC. A muscle biopsy showed nonspecific myopathic changes characterized by variations in muscle fiber size and a tendency towards central core degeneration (Fig 3). Immunohistochemical labeling for dysferlin, dystrophins (D1, D2, D3), merosin, alpha-sarcoglycan, gamma-sarcoglycan, beta-sarcoglycan, collagen VI, caveolin-3 and desmin showed normal expression. Despite thorough examination, no specific myopathy was detected. At the time of the biopsy (October 2018), the patient was under methotrexate and prednisone therapy (40 mg/day). Electromyography was performed in February 2023 with normal results. CPK levels at age 40 were 1,059 U/L (March 2023) and 1,372 U/L (July 2023), and 862 U/L in April 2024 at age 41. No additional viral load testing was performed.

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Fig 3. Case 5 - Histological section stained with Hematoxylin & Eosin shows muscle fiber with size variation.

https://doi.org/10.1371/journal.pntd.0013409.g003

Case 6 concerns a Black man born in 1945 who developed weakness in his lower limbs at the age of 49, which gradually spread to his upper limbs. At the age of 59, he tested positive for HTLV-1 during an examination for neurogenic bladder symptoms. An elevated CPK level (1337 U/L) was detected, leading to the suspicion of myopathy, and immunosuppressive therapy was initiated. Over time, his clinical condition deteriorated, accompanied by weight loss. At the age of 73, he was tetraparetic and confined to a wheelchair. The proximal lower limbs were primarily affected, and spasticity and increased reflexes were observed in all four limbs. He also had bilateral ptosis with restricted eye movement. The HTLV-1 PVL was elevated (111,312x10⁶ PBMC), however no prior PVL values were available, and CPK level was 137 U/L. A muscle biopsy revealed abnormalities in muscle fiber size, increased nuclear centralization with changes in the internal architecture of the fibers, and the absence of COX-positive fibers. Based on these findings, a diagnosis of mitochondrial disease was made. Nonetheless, the clinical features of spasticity and hyperreflexia, along with a high HTLV-1 PVL, raise the possibility that HTLV-1 infection contributed to progressive neuromuscular impairment, potentially exacerbating an underlying genetic myopathy.

Case 7 concerns a Brown woman born in 1994 who was diagnosed with HTLV-1 infection at the age of 24 during a routine laboratory examination. Further investigations revealed hyperCKemia (CPK values of 9,648 U/I) and slightly elevated ALT and AST levels. No CPK isoforms were available. The HTLV-1 PVL was 80,308 x 10⁶ PBMC. Neurological examination was normal, with preserved muscle strength, reflexes, and sensitivity. There were no sphincter complaints. The muscle biopsy showed no abnormalities in the staining of dysferlin, dystrophins (D1, D2, D3), merosin, alpha-sarcoglycans, gamma-sarcoglycans, beta-sarcoglycans, collagen 6, caveolin-3 or desmin. No specific cause for her hyperCKemia was identified throughout the study.

In summary, histological examination of muscle tissue from the two cases of polymyositis (cases 1 and 2) revealed inflammatory myopathies. In case 1, the muscles exhibited advanced stages of degeneration, characterized by abnormal fiber size, increased endomysial and perimysial connective tissue, and foci of endomysial inflammatory infiltrates. Case 2 displayed variability in fiber size, increased endomysial and perimisial connective tissue, nuclear centralization, infiltration of CD68 + macrophages and MHC I expression by lymphocytes in the perimysial inflammatory infiltrate. One patient (case 4) was diagnosed with myofibrillar dystrophy. Muscle biopsy revealed variable fiber size, a tendency toward nuclear centralization, endomysial inflammatory infiltrate, and changes in the internal architecture of muscle fibers due to intermyofibrillar disorganization. Another patient (case 5) had nonspecific myopathy with mild variability in fiber size and an initial “moth-eaten” pattern in muscle tissue. Case 6 was diagnosed with mitochondrial myopathy characterized by variability in muscle fiber size, centralization of the nucleus, and mitochondrial alterations and the absence of COX-positive fibers. Cases 1, 2, and 3 were diagnosed as polymyositis, and cases 1, 3, 5, and 7 showed signs of myelopathy (Table 1).