Daniel Clark

Proviral load and immune markers associated with human T‐lymphotropic virus type 1 (HTLV‐1)‐associated myelopathy/tropical spastic paraparesis (HAM/TSP) in Peru

Human T‐lymphotropic virus type 1 (HTLV‐1) is the aetiological agent of HTLV‐1‐associated myelopathy/tropical spastic paraparesis (HAM/TSP). The objective of this study is to identify which ex vivo and in vivo markers are associated independently with HAM/TSP in a Peruvian population. Eighty‐one subjects (33 men/48 women) were enrolled: 35 presented with HAM/TSP, 33 were asymptomatic HTLV‐1 carriers (ACs) and 13 were HTLV‐1‐seronegative controls (SCs). Ex vivo markers included T cell proliferation and Th1 [interferon (IFN)‐γ], Th2 [interleukin (IL)‐4, IL‐5], proinflammatory [tumour necrosis factor (TNF)‐α] and anti‐inflammatory (IL‐10) cytokine production in non‐stimulated peripheral blood mononuclear cell (PBMC) cultures. In vivo CD4+ T cell count, markers of Th1 [interferon‐inducible protein (IP)‐10] and Th2 (sCD30) activity in plasma and HTLV‐1 proviral load in PBMCs were also evaluated. In univariate analysis, several markers, including T cell proliferation, IFN‐γ, IP‐10, sCD30 and proviral load were associated with HAM/TSP, but in a multiple logistic regression analysis only the proviral load remained associated significantly with disease manifestation [adjusted OR 9·10 (1·24–66·91)]. Our findings suggest that HAM/TSP is associated primarily with proviral load, whereas the observed association with some immune markers seems secondary.

Strongyloidiasis and infective dermatitis alter human T lymphotropic virus-1 clonality in vivo.

Human T-lymphotropic Virus-1 (HTLV-1) is a retrovirus that persists lifelong by driving clonal proliferation of infected T-cells. HTLV-1 causes a neuroinflammatory disease and adult T-cell leukemia/lymphoma. Strongyloidiasis, a gastrointestinal infection by the helminth Strongyloides stercoralis, and Infective Dermatitis associated with HTLV-1 (IDH), appear to be risk factors for the development of HTLV-1 related diseases. We used high-throughput sequencing to map and quantify the insertion sites of the provirus in order to monitor the clonality of the HTLV-1-infected T-cell population (i.e. the number of distinct clones and abundance of each clone). A newly developed biodiversity estimator called “DivE” was used to estimate the total number of clones in the blood. We found that the major determinant of proviral load in all subjects without leukemia/lymphoma was the total number of HTLV-1-infected clones. Nevertheless, the significantly higher proviral load in patients with strongyloidiasis or IDH was due to an increase in the mean clone abundance, not to an increase in the number of infected clones. These patients appear to be less capable of restricting clone abundance than those with HTLV-1 alone. In patients co-infected with Strongyloides there was an increased degree of oligoclonal expansion and a higher rate of turnover (i.e. appearance and disappearance) of HTLV-1-infected clones. In Strongyloides co-infected patients and those with IDH, proliferation of the most abundant HTLV-1+ T-cell clones is independent of the genomic environment of the provirus, in sharp contrast to patients with HTLV-1 infection alone. This implies that new selection forces are driving oligoclonal proliferation in Strongyloides co-infection and IDH. We conclude that strongyloidiasis and IDH increase the risk of development of HTLV-1-associated diseases by increasing the rate of infection of new clones and the abundance of existing HTLV-1+ clones.

Development and Validation of a Multiplex Real-Time PCR Assay for Simultaneous Genotyping and Human T-Lymphotropic Virus Type 1, 2, and 3 Proviral Load Determination

ABSTRACT The human T-lymphotropic virus (HTLV) proviral load remains the best surrogate marker for disease progression. Real-time PCR techniques have been developed for detection and quantification of cosmopolitan HTLV type 1a (HTLV-1a) and HTLV-2. Since a growing level of diversity in subtypes and genotypes is observed, we developed a multiplex quantitative PCR for simultaneous detection, genotyping, and quantification of proviral loads of HTLV-1, 2, and 3. Our assay uses tax type-specific primers and dually labeled probes and has a dynamic range of 105 to 10 HTLV copies. One hundred sixty-three samples were analyzed, among which all of the different subtypes within each HTLV genotype could be detected. The performance of proviral load determination of our multiplex assay was compared with that of a previously published HTLV-1 singleplex quantitative PCR based on SYBR green detection, developed at a different institute. Linear regression analysis showed a statistically significant (P < 0.0001) and strong (r2 = 0.87) correlation between proviral load values measured with the two distinct real-time PCR assays. In conclusion, our novel assay offers an accurate molecular diagnosis and genotyping, together with the determination of the proviral load of HTLV-infected individuals, in a single amplification reaction. Moreover, our molecular assay could offer an alternative when current available serological assays are insufficient.

IFN‐γ production in response to Tax 161–233, and frequency of CD4+ Foxp3+ and Lin− HLA‐DRhigh CD123+ cells, discriminate HAM/TSP patients from asymptomatic HTLV‐1‐carriers in a Peruvian population

Human T‐lymphotropic virus 1 (HTLV‐1) can cause HTLV‐1‐associated myelopathy/tropical spastic paraparesis (HAM/TSP). The objective of this study was to gain insight into the pathogenesis of HAM/TSP by focusing on the CD8+ T‐cell response. Twenty‐three HTLV‐1‐seronegative controls (SC), 29 asymptomatic HTLV‐1 carriers (AC) and 48 patients with HAM/TSP were enrolled in the study. We evaluated the production of interferon‐γ (IFN‐γ) in peripheral blood mononuclear cells stimulated with Tax overlapping peptides, the expression of genes related to the CD8+ cytotoxic T‐cell response, the frequency of CD4+ Foxp3+ cells and of dendritic cells, and the HTLV‐1 provirus load (PVL). The frequency of cells producing IFN‐γ in response to Tax 161–233, but not to Tax 11–19, discriminated patients with HAM/TSP from AC. The increased pro‐inflammatory response observed in patients with HAM/TSP was shared by AC with a high PVL, who also exhibited lower levels of granzyme H mRNA in unstimulated CD8+ T cells than AC with a low PVL. Patients with HAM/TSP showed higher frequencies of CD4+ Foxp3+ cells and lower frequencies of plasmacytoid dendritic cells (pDC) than AC. Our findings are consistent with a model in which HTLV‐1, along with the host genetic background, drives quantitative and qualitative changes in pDC and CD4+ Foxp3+ cells that lead to a predominance of inflammatory responses over lytic responses in the CD8+ T‐cell response of individuals predisposed to develop HAM/TSP.

Evaluation of Host Genetic and Viral Factors as Surrogate Markers for HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis in Peruvian HTLV-1-Infected Patients

Human T‐lymphotropic virus 1 (HTLV‐1)‐associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a complication that affects up to 5% of HTLV‐1‐infected individuals. Several host genetic and viral factors have been associated with the risk of HAM/TSP. The aim of this study was to evaluate the performance of a prognostic model for HAM/TSP developed in Japan in a Peruvian population of 71 HAM/TSP patients and 94 asymptomatic carriers (ACs). This model included age, proviral load (PVL), the presence of HLA‐A*02 and HLA‐Cw*08 alleles, SDF‐1 +801, and TNF‐α −863 polymorphisms, and viral subgroup. We describe frequencies for the four host genetic markers and demonstrate the presence of the HTLV‐1 tax B subgroup in Peru. Using cross‐validation, we show that the predictive ability of the prognostic model, as characterized by the area under the receiver‐operating characteristic curve (AUC), does not differ from a model containing PVL only (both AUC = 0.74). We found some suggestive evidence of a protective effect of the HLA‐A*02 allele but failed to replicate the associations with the other three genetic markers and with viral subgroup. A logistic model containing PVL, age, gender, and HLA‐A*02 provided the best predictive ability in the Peruvian cohort (AUC = 0.79). J. Med. Virol. 82:460–466, 2010.

Comparison of three ELISAs for the routine diagnosis of human T-lymphotropic virus infection in a high-prevalence setting in Peru

To compare three human T-lymphotropic virus (HTLV) ELISAs in a high-prevalence setting, we recruited 300 adults: 125 relatives of HTLV-infected subjects and 175 patients with possible diagnoses of HTLV-associated diseases. Sera were tested with Platelia, Murex, and Ortho ELISA. Samples with positive or discordant ELISA underwent confirmatory Inno-Lia testing. Inno-Lia gave 85/300 HTLV-1-positive and 1/300 HTLV-2-positive results. The positive predictive value was 98% for Platelia, and 100% for Murex and Ortho. Six samples had discordant ELISA; Murex gave one false-negative result, Ortho two and Platelia one. In high-prevalence settings, it is recommended to test samples with two ELISAs before considering them HTLV-seronegative.

Human T-lymphotropic virus type 1 infection is frequent in rural communities of the southern Andes of Peru

OBJECTIVES To evaluate the presence of human T-lymphotropic virus type 1 (HTLV-1) infection in isolated rural communities in the southern Andes of Peru. METHODS We conducted a cross-sectional study in five communities located in three provinces in Ayacucho, Peru. The five communities are located at >3000 meters above sea level and are mainly rural, and more than 85% of the population speaks Quechua. Volunteers aged 12 years and older were included. Clinical and epidemiological data were collected, along with a blood sample for serological testing. RESULTS We included 397 participants; their median age was 41 years (interquartile range 31-57 years) and 69% were women. According to our definitions, 98% were of Quechua origin. HTLV-1 was diagnosed in 11 people: 0/164 in Cangallo, 3/154 (2%) in Vilcashuaman, and 8/79 (10%) in Parinacochas. There were no cases of HTLV-2. All the HTLV-1-positive participants were born in Ayacucho and were of Quechua origin; they ranged in age from 29 to 87 years (median 56 years) and 10/11 were women. Ten were apparently healthy, and one woman was diagnosed with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Three out of 11 had a family member with a lower limb impairment compatible with HAM/TSP. CONCLUSION The fact that HTLV-1 infection was present in two out of three provinces suggests that HTLV-1 could be highly endemic in the southern Andes in the Quechua population.

Possible implication of NFKB1A and NKG2D genes in susceptibility to HTLV-1-associated myelopathy/tropical spastic paraparesis in Peruvian patients infected with HTLV-1.

The human T‐cell lymphotropic virus type 1 (HTLV‐1) is the etiological agent of HTLV‐1‐associated myelopathy/tropical spastic paraparesis (HAM/TSP), a progressive disease causing paraparesis of the lower limbs. Only a minority of persons infected with HTLV‐1 develop HAM/TSP. Universal susceptibility factors for HAM/TSP are not known. The viral genotype is similar in asymptomatic carriers and HAM/TSP patients. High proviral load has been associated consistently with HAM/TSP, but this factor does not explain fully the presence of disease in HTLV‐1‐infected subjects. Most likely, host genetic factors will play an important role in HAM/TSP development. A two‐stage case‐control study was carried out to evaluate the association between HAM/TSP and candidate single nucleotide polymorphisms (SNPs) from 45 genes in addition to six human leukocyte antigen (HLA) alleles. Ancestry‐informative markers were used to correct for population stratification. Several SNPs belonging to NFKB1A and NKG2D showed a trend of association in both stages. The fact that the direction of the association observed in the first stage was the same in the second stage suggests that NFKB1A and NKG2D may be implicated in the development of HAM/TSP. Further replication studies in independent HTLV‐1 patient groups should validate further these associations. J. Med. Virol. 84:319–326, 2012.

Role of killer cell immunoglobulin-like receptor gene content and human leukocyte antigen-C group in susceptibility to human T-lymphotropic virus 1-associated myelopathy/tropical spastic paraparesis in Peru.

Human T-lymphotropic virus 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) affects approximately 5% of HTLV-1-infected individuals. It is poorly understood why only some infected subjects develop this disease, but host genetic factors may determine susceptibility. The innate immune system may influence disease outcome in HTLV-1-infected individuals because of its role in early immune responses to viral infections. Variation in genes encoding killer cell immunoglobulin-like receptors (KIR) and their human leukocyte antigen (HLA) molecule ligands may affect the risk of HAM/TSP. We performed a two-stage case-control study to examine the distribution of KIR genes and HLA-Cw groups in Peruvian HTLV-1-infected HAM/TSP individuals and asymptomatic carriers. We also tested for epistatic effects between specific KIR genes and HLA-Cw groups. In the first stage, we found several trends toward association with HAM/TSP or proviral load (PVL). However, these results were not replicated in the second stage. In conclusion, this is the first report on KIR gene frequencies in the Peruvian population and may be of significance in hematopoietic stem-cell transplants. Our study did not reveal significant associations between KIR genes and HLA-Cw groups and HAM/TSP or PVL. However, because our study was powered to detect only larger effects, additional studies using larger cohorts are needed.

HAM/TSP in relatives of HAM/TSP cases and in relatives of asymptomatic HTLV-1 carriers

To assess the hypothesis that HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) runs in families, we compared the frequency of HAM/ TSP among HTLV-1-positive relatives of HAM/TSP patients with the frequency of HAM/TSP among HTLV1-positive relatives of asymptomatic HTLV-1 carriers. We reviewed available information at the Instituto de Medicina Tropical Alexander von Humboldt in Lima (period 1990-2012). Index cases with HAM/TSP were defined as unrelated, HTLV-1-positive patients with a clinical diagnosis of HAM/TSP for whom HAM/TSP was the motive for HTLV-1 testing and who brought ≥1 relative (blood relatives and/or partners) for HTLV-1 testing. Asymptomatic index cases were defined as unrelated, asymptomatic HTLV-1 carriers who were tested for HTLV-1 as candidate blood donors and brought ≥1 relative for screening. In the family studies of 334 index cases with HAM/TSP, 1124 relatives were tested, 318/1124 (28%) were HTLV-1 positive, and 30/318 (9%) had HAM/TSP. In the family studies of 230 asymptomatic index cases, 544 relatives were tested, 204/544 (38%) were HTLV-1 positive, and 15/204 (7%) had HAM/TSP. We classified the relatives in groups based on sex and age. HAM/TSP frequency increased with age and was higher in women than in men. We found no significant differences in HAM/TSP frequency between relatives of HAM/TSP index cases and relatives of asymptomatic index cases. In total, there were 21 families with 2 HAM/TSP cases, 4 families with 3 HAM/ TSP cases, and 1 family with 5 HAM/TSP cases. This analysis approach suggests that HAM/TSP usually affects isolated people, but that in some, particular families, HAM/ TSP clusters can occur.