Human T-cell leukemia virus type 1 infection among Japanese immigrants and their descendants living in Southeast Brazil: A call for preventive and control responses.

Human T-cell leukemia virus type 1 (HTLV-1) has worldwide distribution and is considered endemic in southwestern Japan. HTLV-1 infection has been associated with adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) besides other diseases. This cross-sectional study aimed to investigate the prevalence, risk factors and molecular characterization of HTLV-1, among the world's largest population of Japanese immigrants and their descendants outside of Japan, in São Paulo, Southeast Brazil, as well as to analyze the phylogenetic relationship among isolates of HTLV-1. From July to December 2017, 2,139 individuals from five Japanese associations were interviewed and submitted to blood collection. All serum samples were first tested for the presence of anti-HTLV-1/2 antibodies by ELISA and then peripheral blood from individuals with positive serological results were analyzed for the presence of HTLV-1 5'LTR proviral DNA. Partial sequencing of the 5'LTR region of HTLV-1 proviral DNA was performed by Sanger. The prevalence of HTLV-1 infection was 5.1% (CI 95%: 4.2-6.0). In the multiple logistic regression model, HTLV-1 infection was associated with age ≥ 45 years, female sex, being first and second-generation Japanese immigrants, and having sexual partners with history of blood transfusion. The phylogenetic analysis revealed that all HTLV-1 were classified as Cosmopolitan (1a) subtype. Of them, 47.8% were classified as Transcontinental (A) subgroup and 52.2% as belonging to the Japanese (B) subgroup. Although most HTLV-1-infected patients were asymptomatic (97.3%), blurred vision was associated with HTLV-1 infection. The high prevalence of HTLV-1 infection found in this studied population and especially the intra- and interfamily HTLV-1 transmission presents an urgent call for preventive and control responses of this infection in Brazil.

Yellow fever epizootics in non-human primates, Southeast and Northeast Brazil (2017 and 2018).

BACKGROUND: Yellow fever (YF) is a severe, infectious, but non-communicable arboviral hemorrhagic disease. In the last decades, yellow fever virus (YFV) infections have been prevalent in endemic areas in Brazil, affecting human and non-human primate (NHP) populations. Monitoring of NHP infection started in 1999, and reports of epizootic diseases are considered important indicators of viral transmission, particularly in relation to the sylvatic cycle. This study presents the monitoring of YFV by real-time RT-PCR and the epidemiological findings related to the deaths of NHPs in the south-eastern states and in the north-eastern state of Bahia, during the outbreak of YF in Brazil during 2017 and 2018. METHODS: A total of 4198 samples from 2099 NHPs from south-eastern and north-eastern Brazilian states were analyzed by real-time reverse transcription polymerase chain reaction (rtRT-PCR). RESULTS: A total of 4198 samples from 2099 NHPs from south-eastern and north-eastern Brazilian states were collected between 2017 and 2018. The samples were subjected to molecular diagnostics for YFV detection using real-time reverse transcription polymerase chain reaction (rtRT-PCR) techniques. Epizootics were coincident with human YF cases. Furthermore, our results showed that the YF frequency was higher among marmosets (Callithrix sp.) than in previous reports. Viremia in species of the genus Alouatta and Callithrix differed greatly. DISCUSSION: Our results indicate a need for further investigation of the role of Callithrix spp. in the transmission cycles of YFV in Brazil. In particular, YFV transmission was observed in a region where viral circulation has not been recorded for decades and thus vaccination has not been previously recommended. CONCLUSIONS: This highlights the need to straighten epizootic surveillance and evaluate the extent of vaccination programmes in Brazil in previously considered "YFV-free" areas of the country.

Chikungunya virus: clinical aspects and treatment - A Review.

Chikungunya is a severe and debilitating disease. Currently, Brazil is experiencing an epidemic caused by three arboviruses, which has changed the way health professionals have diagnosed and treated infected patients. The difficulty of diagnosis and the lack of a protocol for patient treatment, which fits Brazilian health system models, have made it difficult to manage this disease. It is necessary to implement a multidisciplinary network of patient care, in which primary care units play the main role. This review aims to present current information regarding the clinical aspects and treatment of Chikungunya virus infection.

Chikungunya virus: clinical aspects and treatment: a review

Chikungunya is a severe and debilitating disease. Currently, Brazil is experiencing an epidemic caused by three arboviruses, which has changed the way health professionals have diagnosed and treated infected patients. The difficulty of diagnosis and the lack of a protocol for patient treatment, which fits Brazilian health system models, have made it difficult to manage this disease. It is necessary to implement a multidisciplinary network of patient care, in which primary care units play the main role. This review aims to present current information regarding the clinical aspects and treatment of Chikungunya virus infection.

Seroprevalence of Chikungunya Virus in a Rural Community in Brazil.

BACKGROUND: The emergence of the Chikungunya virus (CHIKV) is currently expanding. In 2015, 38,332 cases of Chikungunya were reported to the Brazilian epidemiological surveillance system. Eighteen months after notification of the first case in the city of Feira de Santana, we conducted the first serosurvey to define the magnitude of transmission in a rural community in Brazil. METHODOLOGY/MAIN FINDINGS: The serosurvey was conducted in a random sample of 450 residences in the Chapada district, located 100 kilometers from Feira de Santana. We administered questionnaires and tested 120 sera from Chapada district residents for CHIKV IgM- and IgG-specific antibodies. An individual with CHIKV infection was defined as any person with CHIKV IgM or IgG antibodies detected in the serum. One Hundred cases of Chikungunya were reported after prolonged rainfall, which reinforced the relationship between the rainfall index and CHIKV transmission. Eighteen months after the start of the outbreak, we identified a seroprevalence of 20% (95% CI, 15.4-35%). CHIKV IgG- and IgM-specific antibodies were detected in 22/120 (18.3%) and 6/120 (5.0%) individuals, respectively. Among seropositive patients, 13/24 (54.2%) reported fever and joint pain over the previous two years (p<0.01). The rate of symptomatic CHIKV infection was 40.7%. CONCLUSIONS/SIGNIFICANCE: We identified a moderate seroprevalence of Chikungunya in the Chapada district, and in half of the confirmed CHIKV infections, patients reported arthralgia and fever over the previous two years.

Arterial hypertension and skin allergy are risk factors for progression from dengue to dengue hemorrhagic fever: a case control study.

BACKGROUND: Currently, knowledge does not allow early prediction of which cases of dengue fever (DF) will progress to dengue hemorrhagic fever (DHF), to allow early intervention to prevent progression or to limit severity. The objective of this study is to investigate the hypothesis that some specific comorbidities increase the likelihood of a DF case progressing to DHF. METHODS: A concurrent case-control study, conducted during dengue epidemics, from 2009 to 2012. Cases were patients with dengue fever that progressed to DHF, and controls were patients of dengue fever who did not progress to DHF. Logistic regression was used to estimate the association between DHF and comorbidities. RESULTS: There were 490 cases of DHF and 1,316 controls. Among adults, progression to DHF was associated with self-reported hypertension (OR = 1.6; 95% CI 1.1-2.1) and skin allergy (OR = 1.8; 95% CI 1.1-3.2) with DHF after adjusting for ethnicity and socio-economic variables. There was no statistically significant association between any chronic disease and progression to DHF in those younger than 15 years. CONCLUSIONS: Physicians attending patients with dengue fever should keep those with hypertension or skin allergies in health units to monitor progression for early intervention. This would reduce mortality by dengue.

Prospective cohort study with active surveillance for fever in four dengue endemic countries in Latin America.

To prepare for a Phase III dengue vaccine efficacy trial, 20 investigational sites were selected for this observational study to identify dengue infections in a closed cohort (N = 3,000 children 9-16 years of age). Of 255 acute febrile episodes experienced by 235 children, 50 (21.3%) were considered serologically probable dengue, and 18 (7.7%) were considered virologically confirmed (i.e., dengue NS1 antigen positive) dengue cases. Considering the disease-free and at-risk period from study start to onset of symptoms, the overall incidence density of acute febrile episodes was 17.7 per 100 person-years of follow-up, ranging from 15.3 in Colombia to 22.0 in Puerto Rico. This study showed that all sites were capable of capturing and following up acute febrile episodes within a specific timeframe among the established cohort and to detect dengue cases.

Evidence that lipopolisaccharide may contribute to the cytokine storm and cellular activation in patients with visceral leishmaniasis.

BACKGROUND: Visceral leishmaniasis (VL) is characterized by parasite-specific immunosuppression besides an intense pro-inflammatory response. Lipopolisaccharide (LPS) has been implicated in the immune activation of T-cell deficient diseases such as HIV/AIDS and idiopathic lymphocytopenia. The source of LPS is gram-negative bacteria that enter the circulation because of immunological mucosal barrier breakdown. As gut parasitization also occurs in VL, it was hypothesized that LPS may be elevated in leishmaniasis, contributing to cell activation. METHODOLOGY/PRINCIPAL FINDINGS: Flow cytometry analysis and immunoassays (ELISA and luminex micro-beads system) were used to quantify T-cells and soluble factors. Higher LPS and soluble CD14 levels were observed in active VL in comparison to healthy subjects, indicating that LPS was bioactive; there was a positive correlation between these molecules (r = 0.61;p<0.05). Interestingly, LPS was negatively correlated with CD4(+) (r = -0.71;p<0.01) and CD8(+) T-cells (r = -0.65;p<0.05). Moreover, higher levels of activation-associated molecules (HLA-DR, CD38, CD25) were seen on T lymphocytes, which were positively associated with LPS levels. Pro-inflammatory cytokines and macrophage migration inhibitory factor (MIF) were also augmented in VL patients. Consistent with the higher immune activation status, LPS levels were positively correlated with the inflammatory cytokines IL-6 (r = 0.63;p<0.05), IL-8 (r = 0.89;p<0.05), and MIF (r = 0.64;p<0.05). Also, higher plasma intestinal fatty acid binding protein (IFABP) levels were observed in VL patients, which correlated with LPS levels (r = 0.57;p<0.05). CONCLUSIONS/SIGNIFICANCE: Elevated levels of LPS in VL, in correlation with T-cell activation and elevated pro-inflammatory cytokines and MIF indicate that this bacterial product may contribute to the impairment in immune effector function. The cytokine storm and chronic immune hyperactivation status may contribute to the observed T-cell depletion. LPS probably originates from microbial translocation as suggested by IFABP levels and, along with Leishmania antigen-mediated immune suppression, may play a role in the immunopathogenesis of VL. These findings point to possible benefits of antimicrobial prophylaxis in conjunction with anti-Leishmania therapy.

Usefulness and applicability of the revised dengue case classification by disease: multi-centre study in 18 countries.

BACKGROUND: In view of the long term discussion on the appropriateness of the dengue classification into dengue fever (DF), dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS), the World Health Organization (WHO) has outlined in its new global dengue guidelines a revised classification into levels of severity: dengue fever with an intermediary group of "dengue fever with warning sings", and severe dengue. The objective of this paper was to compare the two classification systems regarding applicability in clinical practice and surveillance, as well as user-friendliness and acceptance by health staff. METHODS: A mix of quantitative (prospective and retrospective review of medical charts by expert reviewers, formal staff interviews), semi-quantitative (open questions in staff interviews) and qualitative methods (focus group discussions) were used in 18 countries. Quality control of data collected was undertaken by external monitors. RESULTS: The applicability of the DF/DHF/DSS classification was limited, even when strict DHF criteria were not applied (13.7% of dengue cases could not be classified using the DF/DHF/DSS classification by experienced reviewers, compared to only 1.6% with the revised classification). The fact that some severe dengue cases could not be classified in the DF/DHF/DSS system was of particular concern. Both acceptance and perceived user-friendliness of the revised system were high, particularly in relation to triage and case management. The applicability of the revised classification to retrospective data sets (of importance for dengue surveillance) was also favourable. However, the need for training, dissemination and further research on the warning signs was highlighted. CONCLUSIONS: The revised dengue classification has a high potential for facilitating dengue case management and surveillance.

Prevalence, genotypes and risk factors associated with hepatitis C virus infection in hemodialysis patients in Campo Grande, MS, Brazil.

A survey was conducted among the hemodialysis units of the city of Campo Grande, located in the state of Mato Grosso do Sul in the Mid-west region of Brazil, with the aim of investigating the prevalence, risk factors, and genotypes of hepatitis C virus (HCV) infection. A total of 163 patients were interviewed in five dialysis units. Serum samples were screened for anti-HCV. Positive samples were tested for HCV RNA and genotyped. The prevalence of anti-HCV was 11% (95% CI: 6.8-17.1). A history of transfusion with blood that was not screened for anti-HCV and length of time on hemodialysis were associated with HCV infection. HCV RNA was detected in 12 samples: ten were of genotype 1, subtypes 1a (75%) and 1b (8.3%), and two were of genotype 3, subtype 3a (16.7%).

Prevalence, genotypes and risk factors associated with hepatitis C virus infection in hemodialysis patients in Campo Grande, MS, Brazil

A survey was conducted among the hemodialysis units of the city of Campo Grande, located in the state of Mato Grosso do Sul in the Mid-west region of Brazil, with the aim of investigating the prevalence, risk factors, and genotypes of hepatitis C virus (HCV) infection. A total of 163 patients were interviewed in five dialysis units. Serum samples were screened for anti-HCV. Positive samples were tested for HCV RNA and genotyped. The prevalence of anti-HCV was 11 percent (95 percent CI: 6.8-17.1). A history of transfusion with blood that was not screened for anti-HCV and length of time on hemodialysis were associated with HCV infection. HCV RNA was detected in 12 samples: ten were of genotype 1, subtypes 1a (75 percent) and 1b (8.3 percent), and two were of genotype 3, subtype 3a (16.7 percent).

Comparison of conventional methods for diagnosis of visceral leishmaniasis in children of the Center-West Region of Brazil.

In Brazil, sophisticated techniques currently employed for diagnosis of visceral leishmaniasis, such as polymerase chain reaction-based assays, are only available in major research centers, whereas conventional methods are still used in many areas where the disease occurs. In the state of Mato Grosso do Sul, in the country's Center-West Region, visceral leishmaniasis has recently emerged in many cities, and duration of the disease, from the onset of symptoms to diagnosis, has been short. Considering that results of diagnostic tests may depend on the phase of the disease, we compared direct examination of bone marrow aspirates (BMAs), BMA culture, and serology by Indirect Immunofluorescence Antibody Test (IFAT) for diagnosis in children, according to time of evolution (< or = 30 days or > 30 days) and to spleen size (< or = 5 cm or > 5 cm) at admission. Duration of the illness did not interfere with test positivity: direct smear examination and IFAT were positive in more than 80% of patients, as was culture in around 60%. Results of positive microscopy, however, where predominant in patients with larger spleens. Thanks to the association of traditional techniques, only a few patients had to begin a treatment trial without confirming the diagnosis. Conventional methods for diagnosis of visceral leishmaniasis are still indispensable in our region, and training professionals in basic techniques should be incremented. The highest sensitivity in laboratory diagnosis among the cases investigated was that obtained with a combination of BMA direct examination and IFAT, nearing 100%.

Comparison of conventional methods for diagnosis of visceral leishmaniasis in children of the Center-West Region of Brazil

In Brazil, sophisticated techniques currently employed for diagnosis of visceral leishmaniasis, such as polymerase chain reaction-based assays, are only available in major research centers, whereas conventional methods are still used in many areas where the disease occurs. In the state of Mato Grosso do Sul, in the country's Center-West Region, visceral leishmaniasis has recently emerged in many cities, and duration of the disease, from the onset of symptoms to diagnosis, has been short. Considering that results of diagnostic tests may depend on the phase of the disease, we compared direct examination of bone marrow aspirates (BMAs), BMA culture, and serology by Indirect Immunofluorescence Antibody Test (IFAT) for diagnosis in children, according to time of evolution (< 30 days or >30 days) and to spleen size (< 5 cm or > 5 cm) at admission. Duration of the illness did not interfere with test positivity: direct smear examination and IFAT were positive in more than 80 percent of patients, as was culture in around 60 percent. Results of positive microscopy, however, where predominant in patients with larger spleens. Thanks to the association of traditional techniques, only a few patients had to begin a treatment trial without confirming the diagnosis. Conventional methods for diagnosis of visceral leishmaniasis are still indispensable in our region, and training professionals in basic techniques should be incremented. The highest sensitivity in laboratory diagnosis among the cases investigated was that obtained with a combination of BMA direct examination and IFAT, nearing 100 percent.

Etiologia de exantema em crianças em uma área endêmica de dengue / Etiology of exanthema in children in a dengue endemic area

OBJETIVO: Estudar a etiologia dos casos de exantema com ou sem febre em crianças atendidas no pronto-socorro de um hospital de uma zona endêmica para dengue. MÉTODOS: No período de 21/09/2001 a 20/09/2002, foram inscritas no estudo 95,9 por cento (71/74) das crianças atendidas no pronto-socorro do Hospital Universitário de Campo Grande (MS) que apresentassem exantema (percentual de recusa de 4,1 por cento). Após preenchimento do protocolo com os dados das crianças, as mesmas foram submetidas a exame físico seguido da coleta de amostras de sangue para realizar hemograma com contagem de plaquetas e sorologias (IgM e IgG); inicialmente para dengue, rubéola e toxoplasmose e, posteriormente, naqueles casos com resultado negativo, realizou-se sorologia para parvovirose, herpes vírus tipo 6 e sarampo. RESULTADOS: O diagnóstico laboratorial foi confirmado através da pesquisa de anticorpo IgM em 88,7 por cento dos casos investigados: dengue (77,5 por cento), herpes vírus tipo 6 (8,4 por cento), parvovirose (2,8 por cento) e diagnóstico inconclusivo em oito pacientes (11,3 por cento). Não foi evidenciada sorologia positiva (IgM) para sarampo, rubéola ou toxoplasmose naquela ocasião. As manifestações clínicas mais freqüentes nos pacientes com dengue foram: febre, prurido, prostração, mialgia e prova do laço positiva. Nos pacientes cujo diagnóstico foi dengue, a prova do laço foi positiva em 58,4 por cento (32/55) dos casos, demonstrando diferença estatisticamente significativa quando comparada com o grupo cujo diagnóstico não foi dengue. CONCLUSÕES: Nas crianças com exantema, dengue pode ser a principal enfermidade causal, atentando-se para a epidemiologia do local. É necessário um controle constante da vigilância epidemiológica e sorológica das doenças exantemáticas.

OBJECTIVE: To study the etiology of exanthema cases, with or without fever, in children seen in the emergency room of a hospital located in a region where dengue is endemic. METHODS: Enrollment took place between 21/09/2001 and 20/09/2002 and included 95.9 percent (71/74) of children presenting with exanthema at the emergency room of the Hospital Universitário de Campo Grande, MS (4.1 percent refusals). After the children had had their details taken and entered on the study protocol, they were subjected to physical examination followed by collection of blood samples for blood testing with platelet counts and serology (IgM and IgG); initially for dengue, rubella and toxoplasmosis and then, in negative cases, serology was also run for parvovirus, herpes virus type 6 and measles. RESULTS: Laboratory diagnoses were confirmed by means of IgM antibody assay in 88.7 percent of the cases investigated: dengue (77.5 percent), herpes virus type 6 (8.4 percent), parvovirus (2.8 percent) and in eight patients diagnosis was inconclusive (11.3 percent). On this occasion no positive serology (IgM) was observed for measles, rubella or toxoplasmosis. The most common clinical manifestations among the dengue patients were: fever, itching, prostration, myalgia and positive tourniquet test results. In 58.4 percent (32/55) of those cases diagnosed with dengue, the tourniquet test was positive, which was a statistically significant difference when compared with the remainder of the sample. CONCLUSIONS: When children present with exanthema, it is possible that dengue is the primary causative disease, depending on the epidemiology of the location. Constant control of epidemiological and serological surveillance of exanthematous diseases is necessary.

Etiology of exanthema in children in a dengue endemic area.

OBJECTIVE: To study the etiology of exanthema cases, with or without fever, in children seen in the emergency room of a hospital located in a region where dengue is endemic. METHODS: Enrollment took place between 21/09/2001 and 20/09/2002 and included 95.9% (71/74) of children presenting with exanthema at the emergency room of the Hospital Universitário de Campo Grande, MS (4.1% refusals). After the children had had their details taken and entered on the study protocol, they were subjected to physical examination followed by collection of blood samples for blood testing with platelet counts and serology (IgM and IgG); initially for dengue, rubella and toxoplasmosis and then, in negative cases, serology was also run for parvovirus, herpes virus type 6 and measles. RESULTS: Laboratory diagnoses were confirmed by means of IgM antibody assay in 88.7% of the cases investigated: dengue (77.5%), herpes virus type 6 (8.4%), parvovirus (2.8%) and in eight patients diagnosis was inconclusive (11.3%). On this occasion no positive serology (IgM) was observed for measles, rubella or toxoplasmosis. The most common clinical manifestations among the dengue patients were: fever, itching, prostration, myalgia and positive tourniquet test results. In 58.4% (32/55) of those cases diagnosed with dengue, the tourniquet test was positive, which was a statistically significant difference when compared with the remainder of the sample. CONCLUSIONS: When children present with exanthema, it is possible that dengue is the primary causative disease, depending on the epidemiology of the location. Constant control of epidemiological and serological surveillance of exanthematous diseases is necessary.

Seroprevalence of Hepatitis B virus infection among an Afro-descendant community in Brazil.

Furnas dos Dionísios is an Afro-Brazilian black community whose descendants were mainly fugitive slaves that established themselves in the State of Mato Grosso do Sul (MS), Brazil. The population is comprised mainly of low socioeconomic individuals who are engaged in agricultural activities. The objective of this study was to investigate the prevalence of hepatitis B (HB) and its correlation with epidemiological data obtained from the community. The studied population totaled 260 individuals with ages varying from 1 to 79 years (median 20). One hundred thirty-three (51.2%) were females and 127 (48.8%) were males. A high prevalence for anti-HBc was observed (42.7%), with present infection detected in 9.2% of the subjects who were also HB surface antigens (HBs Ag) positive; 27.3% were anti-HBc and anti-HBs reactive, and 6.2% had anti-HBc as only marker. The prevalence for anti-HBc was proportional to age, reaching its highest peak in age categories greater than 50. No serological marker was detected in children under the age of 2 years, however anti-HBc was present in 12 subjects with ages between 2 and 14 years, of these 8 (7.4%) were HBsAg positive. Among individuals over the age of 15 years, 99 were anti-HBc reactive, of these 16 (10.5%) were also HBsAg positive, thus suggesting an increased prevalence of HBV carriers among children and adolescents. The risk factors observed in this community that were significantly associated with anti-HBc positivity were age (over 20 years) and having an anti-HBc positive mother. Both HBeAg and anti-HBe were detected in 44.4% of the samples tested. HBsAg subtypes found in the studied population were adw2 (77.7%) and ayw2 (23.3%). While intrafamilial transmission was most likely responsible for HBV infection among children, other routes such as sexual contact might be considered for individuals with ages over 15 years.

Seroprevalence of Hepatitis B virus infection among an afro-descendant community in Brazil

Furnas dos Dionísios is an Afro-Brazilian black community whose descendants were mainly fugitive slaves that established themselves in the State of Mato Grosso do Sul (MS), Brazil. The population is comprised mainly of low socioeconomic individuals who are engaged in agricultural activities. The objective of this study was to investigate the prevalence of hepatitis B (HB) and its correlation with epidemiological data obtained from the community. The studied population totaled 260 individuals with ages varying from 1 to 79 years (median 20). One hundred thirty-three (51.2 percent) were females and 127 (48.8 percent) were males. A high prevalence for anti-HBc was observed (42.7 percent), with present infection detected in 9.2 percent of the subjects who were also HB surface antigens (HBs Ag) positive; 27.3 percent were anti-HBc and anti-HBs reactive, and 6.2 percent had anti-HBc as only marker. The prevalence for anti-HBc was proportional to age, reaching its highest peak in age categories greater than 50. No serological marker was detected in children under the age of 2 years, however anti-HBc was present in 12 subjects with ages between 2 and 14 years, of these 8 (7.4 percent) were HBsAg positive. Among individuals over the age of 15 years, 99 were anti-HBc reactive, of these 16 (10.5 percent) were also HBsAg positive, thus suggesting an increased prevalence of HBV carriers among children and adolescents. The risk factors observed in this community that were significantly associated with anti-HBc positivity were age (over 20 years) and having an anti-HBc positive mother. Both HBeAg and anti-HBe were detected in 44.4 percent of the samples tested. HBsAg subtypes found in the studied population were adw2 (77.7 percent) and ayw2 (23.3 percent). While intrafamilial transmission was most likely responsible for HBV infection among children, other routes such as sexual contact might be considered for individuals with ages over 15 years