Seronegative human T-cell lymphotropic virus 1 carriers in blood banks: A potential viral source for silent transmission?

BACKGROUND AND OBJECTIVES: Transfusion-transmitted viruses count among the greatest threats to blood safety. In Argentina, current laws oblige testing all donated blood for the presence of antibodies against human T-cell lymphotropic viruses 1 and 2 (HTLV-1/2). In endemic zones of the country, a high rate of seronegative HTLV-1 individuals with clear evidence of infection because of symptoms and/or presence of tax sequences of HTLV-1 and/or IgG anti-Tax antibodies has been recently described. Migration from endemic to nonendemic zones of Argentina is very frequent. MATERIALS AND METHODS: During a 1-year period, in the blood bank of Córdoba city, we performed molecular screening of all donors who were born in or arose from endemic zones for HTLV-1/2 in Argentina and neighbouring countries. RESULTS: By screening 219 bp of HTLV-1/2 tax gene, 0.6% (2/317) of the blood donors proved to be positive for HTLV-1 tax sequence. One of the donors presented anti-Tax antibodies, demonstrating the transcriptional activity of the tax gene, and the other donor was also positive for LTR and pol gene sequences. The HTLV-1 genetic analysis of the LTR sequence determined that it belonged to the Cosmopolitan subtype HTLV-1aA. CONCLUSION: These findings suggest potential limitations of some currently approved screening assays for HTLV-1 detection applied in some donor populations and the possibility of an HTLV-1 seronegative carrier state with the potential for silent transmission by blood.

[A population-based study on seroprevalence and factors associated with SARS-CoV-2 infection in Córdoba, Argentina]. / Estudio de base poblacional de seroprevalencia y factores asociados a la infección por SARS-CoV-2 en Córdoba, Argentina.

Seroepidemiological studies help identify the indirect distribution of diseases, detecting serological markers of immunity and demonstrating undiagnosed infections in the general population. The objectives were to estimate the seroprevalence of SARS-CoV-2 antibodies in Córdoba, Argentina, from December 2020 to January 2021 and to identify factors associated with the virus' contagiousness. A population-based cross-sectional observational study was performed in 3,225 individuals over two years of age living in Córdoba city, selected by multiple-stage random sampling proportional to distribution by gender, age group, and socioeconomic status in the city's population. Clinical characteristics, anthropometry, and comorbidities were collected by interview. Qualitative serological testing was performed for detection of SARS-CoV-2 nucleocapsid IgG antibodies (ARCHITECT, Abbott). SARS-CoV-2 seroprevalence was estimated for the total population and by age group, sex, socioeconomic status, and presence of target diseases. Prevalence ratios (PR) were estimated using a log-binomial regression model. SARS-CoV-2 seropositivity was 16.68% (95%CI: 15.41-18.01). Age 2 to 18 years, living in neighborhoods with low socioeconomic status, and obesity increased the odds of seropositivity (PR = 1.50; 95%CI: 1.10-2.04, PR = 1.91; 95%CI: 1.34-2.67 and PR = 1.39; 95%CI: 1.04-1.85). The results indicate that the city of Córdoba displays differential attributes that increase the likelihood of a positive SARS-CoV-2 antibody test. This allows targeting epidemiological surveillance strategies to reduce the spread of the virus.

Los estudios seroepidemiológicos permiten conocer la distribución indirecta de las enfermedades, detectando marcadores séricos de inmunidad y demostrando infecciones no diagnosticadas en la población general. El objetivo fue estimar la seroprevalencia de anticuerpos contra el SARS-CoV-2, en Córdoba, Argentina, entre diciembre de 2020 y enero de 2021, e identificar factores asociados a la contagiosidad del virus. Se realizó un estudio observacional transversal, de base poblacional, con 3.225 individuos mayores de 2 años, residentes en Córdoba Capital, que fueron seleccionados mediante un diseño de muestreo aleatorio en múltiples etapas, proporcional a la distribución por género, franja etaria y nivel socioeconómico de la población de Córdoba. Las características clínicas, antropometría y comorbilidades se recogieron mediante entrevistas. Se realizó un test serológico cualitativo para la detección de anticuerpos IgG antinucleocápside para SARS-CoV-2 (ARCHITECT, Abbott). La seroprevalencia del SARS-CoV-2 se estimó en la población y por franja de edad, sexo, nivel socioeconómico y presencia de las patologías estudiadas. Las razones de prevalencia (RP) se estimaron usando un modelo de regresión log-binomial. La seropositividad para SARS-CoV-2 fue de 16,68% (IC95%: 15,41-18,01). Tener entre 2 y 18 años, residir en barrios con nivel socioeconómico bajo y la presencia de obesidad, aumentaron la oportunidad de seropositividad (RP = 1,50; IC95%: 1,10-2,04, RP = 1,91; IC95%: 1,34-2,67 y RP = 1,39; IC95%: 1,04-1,85). Los resultados indican que en Córdoba Capital existen atributos diferenciales que aumentan la posibilidad de ser seropositivo para SARS-CoV-2. Esto permite dirigir estrategias de vigilancia epidemiológica para reducir la propagación del virus.

Os estudos soroepidemiológicos permitem conhecer a distribuição indireta das doenças, detectando marcadores séricos de imunidade e demonstrando infecções não diagnosticadas na população geral. O objetivo foi estimar a soroprevalência de anticorpos contra o SARS-CoV-2, em Córdoba, Argentina, entre dezembro de 2020 e janeiro de 2021, e identificar fatores associados à contagiosidade do vírus. Um estudo observacional transversal foi realizado, de base populacional, com 3.225 indivíduos maiores de 2 anos, residentes em Córdoba Capital, que foram selecionados por meio de um delineamento de amostragem aleatória em múltiplos estágios, proporcional à distribuição de gênero, a faixa etária e o nível socioeconômico da população de Córdoba. As características clínicas, antropometria e comorbidades foram coletadas por meio de entrevistas. Um teste sorológico qualitativo foi realizado para a detecção de anticorpos IgG anti-nucleocapsídeo para SARS-CoV-2 (ARCHITECT, Abbott). A soroprevalência do SARS-CoV-2 foi estimada na população e por faixa etária, sexo, nível socioeconômico e presença das patologias estudadas. Razões de prevalência (RP) foram estimadas usando um modelo de regressão log-binomial. A soropositividade para SARS-CoV-2 foi de 16,68% (IC95%: 15,41-18,01). Ter entre 2 e 18 años, residir em bairros com nível socioeconômico baixo e a presença de obesidade aumentaram a chance de soropositividade (RP = 1,50; IC95%: 1,10-2,04, RP = 1,91; IC95%: 1,34-2,67 e RP = 1,39; IC95%: 1,04-1,85). Os resultados indicam que em Córdoba Capital existem atributos diferenciais que aumentam a possibilidade de ser soropositivo para SARS-CoV-2. Isso permite direcionar estratégias de vigilância epidemiológica para reduzir a propagação do vírus.

Estudio de base poblacional de seroprevalencia y factores asociados a la infección por SARS-CoV-2 en Córdoba, Argentina / A population-based study on seroprevalence and factors associated with SARS-CoV-2 infection in Córdoba, Argentina / Estudo populacional de soroprevalência e fatores associados à infecção por SARS-CoV-2 em Córdoba, Argentina

Los estudios seroepidemiológicos permiten conocer la distribución indirecta de las enfermedades, detectando marcadores séricos de inmunidad y demostrando infecciones no diagnosticadas en la población general. El objetivo fue estimar la seroprevalencia de anticuerpos contra el SARS-CoV-2, en Córdoba, Argentina, entre diciembre de 2020 y enero de 2021, e identificar factores asociados a la contagiosidad del virus. Se realizó un estudio observacional transversal, de base poblacional, con 3.225 individuos mayores de 2 años, residentes en Córdoba Capital, que fueron seleccionados mediante un diseño de muestreo aleatorio en múltiples etapas, proporcional a la distribución por género, franja etaria y nivel socioeconómico de la población de Córdoba. Las características clínicas, antropometría y comorbilidades se recogieron mediante entrevistas. Se realizó un test serológico cualitativo para la detección de anticuerpos IgG antinucleocápside para SARS-CoV-2 (ARCHITECT, Abbott). La seroprevalencia del SARS-CoV-2 se estimó en la población y por franja de edad, sexo, nivel socioeconómico y presencia de las patologías estudiadas. Las razones de prevalencia (RP) se estimaron usando un modelo de regresión log-binomial. La seropositividad para SARS-CoV-2 fue de 16,68% (IC95%: 15,41-18,01). Tener entre 2 y 18 años, residir en barrios con nivel socioeconómico bajo y la presencia de obesidad, aumentaron la oportunidad de seropositividad (RP = 1,50; IC95%: 1,10-2,04, RP = 1,91; IC95%: 1,34-2,67 y RP = 1,39; IC95%: 1,04-1,85). Los resultados indican que en Córdoba Capital existen atributos diferenciales que aumentan la posibilidad de ser seropositivo para SARS-CoV-2. Esto permite dirigir estrategias de vigilancia epidemiológica para reducir la propagación del virus.

Seroepidemiological studies help identify the indirect distribution of diseases, detecting serological markers of immunity and demonstrating undiagnosed infections in the general population. The objectives were to estimate the seroprevalence of SARS-CoV-2 antibodies in Córdoba, Argentina, from December 2020 to January 2021 and to identify factors associated with the virus' contagiousness. A population-based cross-sectional observational study was performed in 3,225 individuals over two years of age living in Córdoba city, selected by multiple-stage random sampling proportional to distribution by gender, age group, and socioeconomic status in the city's population. Clinical characteristics, anthropometry, and comorbidities were collected by interview. Qualitative serological testing was performed for detection of SARS-CoV-2 nucleocapsid IgG antibodies (ARCHITECT, Abbott). SARS-CoV-2 seroprevalence was estimated for the total population and by age group, sex, socioeconomic status, and presence of target diseases. Prevalence ratios (PR) were estimated using a log-binomial regression model. SARS-CoV-2 seropositivity was 16.68% (95%CI: 15.41-18.01). Age 2 to 18 years, living in neighborhoods with low socioeconomic status, and obesity increased the odds of seropositivity (PR = 1.50; 95%CI: 1.10-2.04, PR = 1.91; 95%CI: 1.34-2.67 and PR = 1.39; 95%CI: 1.04-1.85). The results indicate that the city of Córdoba displays differential attributes that increase the likelihood of a positive SARS-CoV-2 antibody test. This allows targeting epidemiological surveillance strategies to reduce the spread of the virus.

Os estudos soroepidemiológicos permitem conhecer a distribuição indireta das doenças, detectando marcadores séricos de imunidade e demonstrando infecções não diagnosticadas na população geral. O objetivo foi estimar a soroprevalência de anticorpos contra o SARS-CoV-2, em Córdoba, Argentina, entre dezembro de 2020 e janeiro de 2021, e identificar fatores associados à contagiosidade do vírus. Um estudo observacional transversal foi realizado, de base populacional, com 3.225 indivíduos maiores de 2 anos, residentes em Córdoba Capital, que foram selecionados por meio de um delineamento de amostragem aleatória em múltiplos estágios, proporcional à distribuição de gênero, a faixa etária e o nível socioeconômico da população de Córdoba. As características clínicas, antropometria e comorbidades foram coletadas por meio de entrevistas. Um teste sorológico qualitativo foi realizado para a detecção de anticorpos IgG anti-nucleocapsídeo para SARS-CoV-2 (ARCHITECT, Abbott). A soroprevalência do SARS-CoV-2 foi estimada na população e por faixa etária, sexo, nível socioeconômico e presença das patologias estudadas. Razões de prevalência (RP) foram estimadas usando um modelo de regressão log-binomial. A soropositividade para SARS-CoV-2 foi de 16,68% (IC95%: 15,41-18,01). Ter entre 2 e 18 años, residir em bairros com nível socioeconômico baixo e a presença de obesidade aumentaram a chance de soropositividade (RP = 1,50; IC95%: 1,10-2,04, RP = 1,91; IC95%: 1,34-2,67 e RP = 1,39; IC95%: 1,04-1,85). Os resultados indicam que em Córdoba Capital existem atributos diferenciais que aumentam a possibilidade de ser soropositivo para SARS-CoV-2. Isso permite direcionar estratégias de vigilância epidemiológica para reduzir a propagação do vírus.

Human T-Lymphotropic virus type 1 infection in absence of tax gene: A challenge for molecular diagnosis.

This is the first report of HTLV-1 infection without detectable tax gene. Even though the tax gene of HTLV-1 presents high genetic stability, in the case presented here no sequence of tax was detected by three different and widely used molecular assays targeting several sequences of the gene. Nevertheless, HTLV-1 pol and env genes and LTR region were properly detectable. Several PCRs targeting tax sequences have been developed and largely used for molecular diagnosis of HTLV infection since the tax gene of HTLV-1 is known to be well preserved and intolerant to changes or mutations. In the case reported here, molecular detection of the virus was challenging. HTLV prevalence is complex and in many regions remains unknown. The identification of HTLV-infected individuals is important to determine its actual prevalence and design strategies to reduce viral spread. The finding and communication of HTLV-1 defective-provirus strains is important and necessary to guide the selection of representative target sequences on HTLV genome to design molecular assays, highlighting that different sequences should be combined to ensure adequate diagnosis. The latter is especially relevant in cases when discordant results between serological and molecular assays. This report contributes to the knowledge of the overall molecular epidemiology of HTLV-1 and encourages the need of surveillance of HTLV-1 "missed tax gene profiles" and the evaluation of the impact of these defective viral variants on molecular diagnosis and human health.

First Description of Seronegative HTLV-1 Carriers in Argentina.

In some areas of Argentina endemic for human T-lymphotropic virus type 1 (HTLV-1), tropical spastic paraparesis is frequent in subjects who lack antibodies against the virus; however, the relevance of this seronegative status in the country has not been investigated. In neighboring countries, HTLV-1 seronegative status has been described in patients with different diseases; however, data regarding features of seronegative HTLV-1 carriers are scarce. We investigated the seronegative status in 124 relatives of 28 HTLV-1 infected subjects from an endemic area in Northwest Argentina. Blood samples and clinical/epidemiological data were collected. Human T-lymphotropic virus type 1 infection was diagnosed by serology and long terminal repeat (LTR) sequence, env and tax gene detection. IgG anti-Tax HTLV-1 antibody, tax gene sequence, and DNA proviral load were also evaluated. Seventy-five percent of the 124 relatives were negative for HTLV-1/2 antibodies; 35.5% were also negative by molecular assays and 64.5% were negative for HTLV-1 LTR and env sequences, but positive for two sequences of HTLV-1 tax gene. Also, 35.7% of these subjects had IgG anti-Tax antibodies. The seronegative HTLV-1 status was significantly associated with male gender, youth, and sensory symptoms/autonomic nervous system dysfunction. High rates of seronegative symptomatic and asymptomatic HTLV-1 carriers in Argentina are described. The evidence highlights that HTLV-1 prevalence may be underestimated worldwide. Larger cohort studies are required to assess disease outcome in these seronegative subjects. Also, the findings emphasize the limitations of ongoing screening assays for diagnosis and blood safety. Therefore, algorithms for HTLV-1 diagnosis should include not only serological but also molecular assays.

Silent dissemination of HTLV-1 in an endemic area of Argentina. Epidemiological and molecular evidence of intrafamilial transmission.

BACKGROUND: Molecular and epidemiological studies of transmission routes and risk factors for infection by HTLV-1 are extremely important in order to implement control measures, especially because of the high prevalence of HTLV-1 in several regions of the world. San Salvador de Jujuy, Northwest Argentina, is a highly endemic area for HTLV-1 and foci of tropical spastic paraparesis/HTLV-1-associated myelopathy. OBJECTIVE: To gain further insight into the role of intrafamilial transmission of HTLV-1 in a highly endemic region in Argentina. METHOD: Cross-sectional study in Northwest Argentina. Epidemiological data and blood samples were collected from 28 HTLV-1 infected subjects (index cases) and 92 close relatives/cohabitants. HTLV-1 infection was diagnosed by detection of antibodies and proviral DNA. The LTR region was sequenced and analyzed for genetic distances (VESPA software), in addition to determination and identification of polymorphisms to define HTLV-1 family signatures. RESULTS: Fifty seven of the 120 subjects enrolled had antibodies against HTLV-1 and were typified as HTLV-1 by PCR. The prevalence rate of HTLV-1 infection in family members of infected index cases was 31.52% (29/92). The infection was significantly associated with gender, age and prolonged lactation. Identity of LTR sequences and presence of polymorphisms revealed high prevalence of mother-to-child and interspousal transmission of HTLV-1 among these families. CONCLUSION: There is an ongoing and silent transmission of HTLV-1 through vertical and sexual routes within family clusters in Northwest Argentina. This evidence highlights that HTLV-1 infection should be considered as a matter of public health in Argentina, in order to introduce preventive measures as prenatal screening and breastfeeding control.

Usefulness of nucleic acid testing to reduce risk of hepatitis B virus transfusion-transmitted infection in Argentina: high rate of recent infections.

BACKGROUND: Results from 10-year experience using nucleic acid test (NAT) screening in a blood bank of Córdoba are presented, showing the first data on prevalence of recent hepatitis B virus (HBV) infections and occult HBV infections (OBIs) in Argentina. STUDY DESIGN AND METHODS: Molecular screening was performed by COBAS AmpliScreen human immunodeficiency virus Type 1 (HIV-1) test Version 1.5 and COBAS AmpliScreen hepatitis C virus (HCV) test Version 2.0 and COBAS TaqScreen MPX and MPX Version 2.0 test (Roche Molecular Systems). To characterize OBI, additional molecular and serologic assays were performed. RESULTS: As results of NAT, 0.075% of the donors (155/205,388) tested positive for HIV, 0.05% (106/205,388) for HCV, and 0.045% (76/168,215) for HBV. Donors who tested positive for HIV or HCV by NAT were also positive by serology. There was one of 33,643 donors recently infected with HBV. At time of donation, six of 76 (7.9%) donors with confirmed HBV infection presented virologic and serologic profiles consistent with OBI. By additional studies three were OBI, two were window period infections, and one remained unclassified. CONCLUSION: NAT contributed significantly to the reduction of the potential risk of HBV transmission with a frequency of one in 56,072, detecting three in 168,215 donors without serologic evidence of infection. NAT also detected three in 168,215 OBIs. The finding of high frequency of recent infections (1/33,643), unexpected for this country, highlights the need of promoting unified effective regulations that enforce the use of NAT in all blood banks in Argentina and points out the importance of assessing the risk of HBV transmission in blood banks of other countries considered to be low-endemic.

[Sensitivity of the COBAS AmpliScreen™ HIV-1 test v1.5 for HIV-1 detection]. / Sensibilidad del equipo Cobas AmpliScreen™ HIV-1 Test, v1.5, para la detección de HIV-1.

The introduction of nucleic acid amplification techniques (NAT) in blood banks was intended to reduce the residual risk of transfusion-transmitted infections. Co-circulation of a great diversity of HIV-1 variants in Argentina portrays the need to assess the sensitivity of serological and molecular assays available for their detection. In this study, we evaluated the sensitivity of the COBAS AmpliScreen™ HIV-1 Test, version 1.5 (Roche) for the detection of HIV-1 RNA in plasma samples of infected individuals from Argentina. The results of this study reveal that this technique has high sensitivity for the detection of HIV-1 RNA under assay conditions: using mini-pool testing, pools ≥ 50 RNA copies per ml achieved ≥ 92 % sensitivity, whereas in the standard procedure, samples ≥ 207 RNA copies/ml achieved 100 % sensitivity. Moreover, the COBAS AmpliScreen™ HIV-1 Test, version 1.5 (Roche) is suitable for detecting prevailing HIV-1 variants.

Sensibilidad del equipo Cobas AmpliScreenTM HIV-1 Test, v1. 5, para la detección de HIV-1 / Sensitivity of the COBAS AmpliScreenTM HIV-1 Test v1.5 for HIV-1 detection

Las técnicas de amplificación de ácidos nucleicos (NAT) se incorporaron en los bancos de sangre para reducir el riesgo residual de transmisión de infecciones por vía transfusional. La cocirculación de distintas variantes del HIV-1 en Argentina indica la necesidad de evaluar la sensibilidad de los ensayos serológicos y moleculares disponibles para su detección. En este trabajo se evaluó la sensibilidad del equipo COBAS AmpliScreenTM HIV-1 Test, versión 1.5 (Roche), para detectar ARN viral en plasmas de individuos infectados con HIV-1 de Argentina. Los resultados demuestran que esta técnica tiene una alta sensibilidad para detectar ARN de HIV-1 en las condiciones ensayadas: para ensayo de mini-pooles (pooles ≥ 50 copias de ARN/ml), la sensibilidad fue ≥ 92 %, y para procedimiento estándar (plasmas ≥ 207 copias de ARN/ml), la sensibilidad fue 100 %. Además, la técnica COBAS AmpliScreenTM HIV-1 Test, versión 1.5 (Roche), es adecuada para la detección de las variantes de HIV-1 prevalentes

The introduction of nucleic acid amplification techniques (NAT) in blood banks was intended to reduce the residual risk of transfusion-transmitted infections. Co-circulation of a great diversity of HIV-1 variants in Argentina portrays the need to assess the sensitivity of serological and molecular assays available for their detection. In this study, we evaluated the sensitivity of the COBAS AmpliScreenTM HIV-1 Test, version 1.5 (Roche) for the detection of HIV-1 RNA in plasma samples of infected individuals from Argentina. The results of this study reveal that this technique has high sensitivity for the detection of HIV-1 RNA under assay conditions: using mini-pool testing, pools ≥ 50 RNA copies per ml achieved ≥ 92 % sensitivity, whereas in the standard procedure, samples ≥ 207 RNA copies/ ml achieved 100 % sensitivity. Moreover, the COBAS AmpliScreenTM HIV-1 Test, version 1.5 (Roche) is suitable for detecting prevailing HIV-1 variants

Molecular epidemiology of hepatitis B virus in Córdoba, Argentina.

BACKGROUND: The analysis of the genomes of hepatitis B virus (HBV) identifies phylogenetic variants called genotypes, which may lead to distinct biological and clinical behaviors. OBJECTIVES: The aim of this study was to describe the current molecular epidemiology and genetic diversity of HBV in Córdoba, Argentina. STUDY DESIGN: A total of 52 HBV samples, 40 from HBV mono-infected and 12 from human immunodeficiency virus (HIV)-HBV co-infected patients, were sequenced in the S gene and in the basal core promoter-precore (BCP-pC) region. RESULTS: Presence of subgenotypes F1b (35%) and F4 (17.5%), subgenotype A2 (37.5%), C (5.0%) (subgenotype could not be defined) and D (5.0%) (subgenotype D2, and the other could not be defined) were observed among mono-infected patients. The co-infected individuals displayed a different genotype distribution: sub-genotype A2 was the most common (75.0%), followed by subgenotype F1b (25.0%). CONCLUSIONS: These results showed two epidemiologic scenarios: the mono-infected population may represent the ethnic composition of the current human population of Córdoba, where the Amerindian (genotype F) and European origins (subgenotype A2) account for the 90% of the samples; for the co-infected patients, the high prevalence of subgenotype A2 resemble previous analyses from Buenos Aires. In addition, mutations in hepatitis B surface antigen (HBsAg), polymerase and BCP-pC regions were identified, mainly in chronic or co-infected patients.

Sensibilidad del equipo Cobas AmpliScreenÔäó HIV-1 Test, v1.5, para la detección de HIV-1. / [Sensitivity of the COBAS AmpliScreenÔäó HIV-1 test v1.5 for HIV-1 detection].

The introduction of nucleic acid amplification techniques (NAT) in blood banks was intended to reduce the residual risk of transfusion-transmitted infections. Co-circulation of a great diversity of HIV-1 variants in Argentina portrays the need to assess the sensitivity of serological and molecular assays available for their detection. In this study, we evaluated the sensitivity of the COBAS AmpliScreen HIV-1 Test, version 1.5 (Roche) for the detection of HIV-1 RNA in plasma samples of infected individuals from Argentina. The results of this study reveal that this technique has high sensitivity for the detection of HIV-1 RNA under assay conditions: using mini-pool testing, pools  50 RNA copies per ml achieved  92

sensitivity, whereas in the standard procedure, samples  207 RNA copies/ml achieved 100

sensitivity. Moreover, the COBAS AmpliScreenÔäó HIV-1 Test, version 1.5 (Roche) is suitable for detecting prevailing HIV-1 variants.

[Serological diagnosis of HTLV-1/2: combination of screening assays to define the serological status in blood donors]. / Diagnóstico serológico de HTLV-1/2: combinación de técnicas de tamizaje para definir el estatus serológico en donantes de sangre.

Alternative algorithms were evaluated in order to reduce the number of false reactive results for antibodies against HTLV-1/2. From 20,210 samples tested, 0.37% (74/20,210) was reactive by ELISA Murex. Of these, 23 were confirmed as positive by the indirect immunofluorescence assay whereas 51 were negative, being the positive predictive value (PPV) 31.08%. From a combination of the ELISA Murex assay with the particle agglutination assay (PA) and ELISA MP, the following results were obtained: 26/74 were reactive by ELISA Murex and PA, PPV 88.5% and 32/74 were reactive by ELISA Murex and ELISA MP, PPV 71.8 %. The ROC curve analysis determined that for an RP 4.74, the values for sensitivity, specificity, PPV and NPV by ELISA Murex were 100%, 98.04%, 95.8% and 100%, respectively. We propose that reactive samples by ELISA Murex with an RP d 4.74 should be retested in duplicate by PA, and the resulting concordantly nonreactive samples should be defined as negative for HTLV-1/2.

Diagnóstico serológico de HTLV-1/2: combinación de técnicas de tamizaje para definir el estatus serológico en donantes de sangre / [Serological diagnosis of HTLV-1/2: combination of screening assays to define the serological status in blood donors].

Alternative algorithms were evaluated in order to reduce the number of false reactive results for antibodies against HTLV-1/2. From 20,210 samples tested, 0.37

(74/20,210) was reactive by ELISA Murex. Of these, 23 were confirmed as positive by the indirect immunofluorescence assay whereas 51 were negative, being the positive predictive value (PPV) 31.08

. From a combination of the ELISA Murex assay with the particle agglutination assay (PA) and ELISA MP, the following results were obtained: 26/74 were reactive by ELISA Murex and PA, PPV 88.5

and 32/74 were reactive by ELISA Murex and ELISA MP, PPV 71.8

. The ROC curve analysis determined that for an RP 4.74, the values for sensitivity, specificity, PPV and NPV by ELISA Murex were 100

, respectively. We propose that reactive samples by ELISA Murex with an RP d 4.74 should be retested in duplicate by PA, and the resulting concordantly nonreactive samples should be defined as negative for HTLV-1/2.

Development and validation of a real-time PCR assay for a novel HTLV-1 tax sequence detection and proviral load quantitation.

A quantitative real-time PCR (qPCR) assay using SYBR Green dye was established in order to detect and quantify the proviral DNA of HTLV-1 in peripheral blood mononuclear cells (PBMCs). Primers were designed, and the assay was standardized to amplify a novel, conserved HTLV-1 tax region. Proviral load was normalized to the amount of cellular DNA by quantitation of the human albumin gene. Firstly, the qPCR was assessed determining the specificity, sensitivity, dynamic range and intra- and inter-assay reproducibility of the technique. The limit of detection as determined by PROBIT analysis using dilutions of the standard was 2.97 copies. The assay had an excellent dynamic range from 105 to 10¹ copies per reaction and good intra- and inter-assay reproducibility, CVs less than 2%. Secondly, the performance of the qPCR was tested on 40 HTLV-1 seropositive individuals. Proviral load for HTLV-1 carriers ranged from 2.2×10² to more than 8.3×104 copies/106 PBMCs. The high sensitivity and wide dynamic range allowed the determination of a broad range of HTLV-1 proviral loads in infected individuals. This assay is a valuable alternative diagnostic tool when current available serological assays are insufficient. In addition, it will facilitate the study of the relationship between proviral load and pathogenesis.

Diagnóstico serológico de HTLV-1/2: combinación de técnicas de tamizaje para definir el estatus serológico en donantes de sangre. / [Serological diagnosis of HTLV-1/2: combination of screening assays to define the serological status in blood donors].

Alternative algorithms were evaluated in order to reduce the number of false reactive results for antibodies against HTLV-1/2. From 20,210 samples tested, 0.37

(74/20,210) was reactive by ELISA Murex. Of these, 23 were confirmed as positive by the indirect immunofluorescence assay whereas 51 were negative, being the positive predictive value (PPV) 31.08

. From a combination of the ELISA Murex assay with the particle agglutination assay (PA) and ELISA MP, the following results were obtained: 26/74 were reactive by ELISA Murex and PA, PPV 88.5

and 32/74 were reactive by ELISA Murex and ELISA MP, PPV 71.8

. The ROC curve analysis determined that for an RP 4.74, the values for sensitivity, specificity, PPV and NPV by ELISA Murex were 100

, 98.04

, 95.8

and 100

, respectively. We propose that reactive samples by ELISA Murex with an RP d 4.74 should be retested in duplicate by PA, and the resulting concordantly nonreactive samples should be defined as negative for HTLV-1/2.

Correlation of HTLV-1 Tax genetic diversity with HTLV-1 associated myelopathy/tropical spastic paraparesis progression and HTLV-1a genotypes in an HTLV-1 endemic region in Argentina.

The oncoprotein Tax was characterized genetically from a large cohort of human T-cell lymphotropic virus type 1 (HTLV-1) seropositive individuals from the most endemic region of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and HTLV-1 infection in Argentina, the province of San Salvador de Jujuy. Sixteen HAM/TSP patients and 47 HTLV-1 healthy carriers were evaluated. Six Tax genetic polymorphisms were identified and observed in 70.8% of healthy carriers and 62.5% of HAM/TSP patients. Tax genetic polymorphisms were not associated with clinical status but A8344C polymorphism statistically provide a borderline protective effect of HAM/TSP outcome. Nucleotide diversity in healthy carriers was 0.00549, whereas HAM/TSP virus population revealed a low diversity of 0.00379, suggests a positive selection for Tax protein conservation in this group. It is concluded that tax genetic polymorphisms do not increase the risk of developing HAM/TSP in this endemic region. However, in spite of the low prevalence of HTLV-1aB genotype, statistical analysis revealed an important correlation of tax genetic signatures with HTLV-1aA trans-continental subgroup.

HTLV type 1 genetic types among native descendants in Argentina.

The province of San Salvador de Jujuy, located in the northwest of Argentina, is a highly endemic area for HTLV-1 infection and a foci of tropical spastic paraparesis/HTLV-1-associated myelopathy (HAM/TSP). Therefore, to better understand this, we carried out a genetic characterization of a large set of HTLV-1 strains (n = 65) of descendants of Amerindians from this region. The LTR and env regions were analyzed. The genetic analysis showed that all of these new HTLV-1 isolates from Argentina belong to the Transcontinental subgroup A of the HTLV-1a Cosmopolitan subtype, with the exception of three isolates that cluster within the Japanese subgroup B. Interestingly, the majority of the sequences from Jujuy province belonged to a distinct cluster within the Latin America Transcontinental subgroup, referred to here as the Jujuy subcluster, and were characterized by specific signatures in the LTR. Given that the samples analyzed in this study belong to the Amerindian population and the high prevalence of HTLV-1 in Jujuy in contrast to the low prevalence of this virus in the country, it could be that HTLV-1aA was spread in Argentina from the Amerindians to the cosmopolitan population. Moreover, this is the first report of an HTLV-1aB or Japanese subgroup in descendants of non-Japanese people in South America.

Retrospective study of the prevalence of human T-cell lymphotropic virus-type 1/2, HIV, and HBV in pregnant women in Argentina.

This study shows first data on HTLV-1/2 seroprevalence among pregnant women in the non-endemic region of Argentina. In a retrospective study a representative sample (n = 3,143) of the pregnant women registered in the health public service in the province of Córdoba was evaluated. HTLV-1/2 seroprevalence was 0.191% +/- 0.0857 [IC 0.022-0.359]. This prevalence was 10 times higher in pregnant women than in blood donors [0.019 (4/21.183)]. The pregnant women would reflect the epidemiology of the general population more accurately since it constitutes a more heterogeneous group than that of blood donors. The prevalence of infection with HIV was 2.8 times higher than that of HTLV-1/2 (P < 0.05) and the presence of any of these two viruses was not a subrogating indicator of the presence of the other (Goodman and Kruskal's Tau coefficient = 0.0092). The prevalence of HBV was not significantly different from that of HTLV-1/2 (P > 0.05). We consider that it is necessary to carry out continuous studies in order to define the main risk factors for infection of these women. Thus, a decision could be made to apply the best policy in public health to prevent vertical transmission of the virus in Argentina.

Propuesta de un algoritmo alternativo con pruebas complementarias en donantes con resultados reactivos en pruebas de tamizaje para HIV / HIV screening test. An alternative algorithm of complementary tests in seroactive donors

En este trabajo se presentan los resultados de la experiencia del Banco de Sangre de la Universidad Nacional de Córdoba con el uso de equipos de 4¬ generación en el screening de HIV y la valoración de la eficiencia del mismo utilizando diferentes técnicas serológicas y PCR. Además se propone un algoritmo para ser implementado en bancos de sangre con el fin de minimizar el descarte de bolsas y aclarar el status para HIV en los donantes con screening inicial reactivo. De 3822 donantes de sangre procesados por HIV Ag/Ac Combination ABBOTT Murex se descartaron 30 unidades de sangre repetidamente reactivas y repetidamente en zona gris, de las cuales solamente en 2 se confirmó infección por HIV. Las 30 muestras fueron procesadas por las siguientes técnicas: Murex HIV-1.2.0 ABBOT Murex (EIE), Murex HIV Antigen Mab ABBOTT Murex (EIE), Microelisa system Vironostika HIV Uni- Form II plus O BIOMERIEUX, SFD HIV 1/2 PA BIORAD FUJIREBIO (aglutinación de partículas de gelatina o APG), NEW LAV BLOT I BIO-RAD (Western Blot o WB), IFI-HIV-1-BIO-MANGUINHOS y PCR "in house". La detección de anticuerpos (3¬ generación) y del antígeno p24 por separado con el reactivo ABBOTT Murex resultó en 10 muestras repetidamente reactivas y en zona gris para los anticuerpos y 1 repetidamente reactiva para el antígeno. Por Western blot, de las 30 muestras 2 fueron positivas, 6 negativas y 22 indeterminadas. Con el ensayo de 4¬ generación utilizado hubo 0,73 por ciento de falsos positivos lo que determinó un descarte innecesario de unidades de sangre y un estado de incertidumbre para el donante. La PCR fue de gran utilidad para resolver el diagnóstico en donantes con resultados discordantes por los inmunoensayos. Consideramos que la estrategia propuesta resulta útil hasta que sea posible la incorporación de NAT para el screening de HIV en nuestros bancos de sangre. (AU)

Propuesta de un algoritmo alternativo con pruebas complementarias en donantes con resultados reactivos en pruebas de tamizaje para HIV / HIV screening test. An alternative algorithm of complementary tests in seroactive donors

En este trabajo se presentan los resultados de la experiencia del Banco de Sangre de la Universidad Nacional de Córdoba con el uso de equipos de 4ª generación en el screening de HIV y la valoración de la eficiencia del mismo utilizando diferentes técnicas serológicas y PCR. Además se propone un algoritmo para ser implementado en bancos de sangre con el fin de minimizar el descarte de bolsas y aclarar el status para HIV en los donantes con screening inicial reactivo. De 3822 donantes de sangre procesados por HIV Ag/Ac Combination ABBOTT Murex se descartaron 30 unidades de sangre repetidamente reactivas y repetidamente en zona gris, de las cuales solamente en 2 se confirmó infección por HIV. Las 30 muestras fueron procesadas por las siguientes técnicas: Murex HIV-1.2.0 ABBOT Murex (EIE), Murex HIV Antigen Mab ABBOTT Murex (EIE), Microelisa system Vironostika HIV Uni- Form II plus O BIOMÉRIEUX, SFD HIV 1/2 PA BIORAD FUJIREBIO (aglutinación de partículas de gelatina o APG), NEW LAV BLOT I BIO-RAD (Western Blot o WB), IFI-HIV-1-BIO-MANGUINHOS y PCR "in house". La detección de anticuerpos (3ª generación) y del antígeno p24 por separado con el reactivo ABBOTT Murex resultó en 10 muestras repetidamente reactivas y en zona gris para los anticuerpos y 1 repetidamente reactiva para el antígeno. Por Western blot, de las 30 muestras 2 fueron positivas, 6 negativas y 22 indeterminadas. Con el ensayo de 4ª generación utilizado hubo 0,73 por ciento de falsos positivos lo que determinó un descarte innecesario de unidades de sangre y un estado de incertidumbre para el donante. La PCR fue de gran utilidad para resolver el diagnóstico en donantes con resultados discordantes por los inmunoensayos. Consideramos que la estrategia propuesta resulta útil hasta que sea posible la incorporación de NAT para el screening de HIV en nuestros bancos de sangre.