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.

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.

Infecciones asintomáticas por Chlamydia trachomatis: un problema controlable en la población adolescente / Asymptomatic Chlamydia trachomatis infection in adolescent population: a manageable problem

La detección precoz de la infección por Chlamydia trachomatis (CT) asintomática (70-75%) reduce la incidencia de complicaciones, sobre todo las relacionadas a la infertilidad y esterilidad en ambos sexos siendo relevante en las mujeres. Objetivos: Conocer la prevalencia de CT en la población asintomática de jóvenes y adolescente (JA) de la ciudad de Córdoba (Argentina); evaluar los factores de riesgo y proponer un programa de detección adecuado a nuestro medio. Métodos: Se estudiaron, entre enero de 2004 y enero de 2006, 427 JA de ambos sexos entre 18 y 24 años (221 estudiantes universitarios (GU) y 206 adolescentes no universitarios (GNU) con nivel socio económico bajo). Se realizó PCR en orina con dos técnicas: plásmido y proteína de la membrana externa de CT; cultivo en líneas celulares para las muestras positivas; y tratamiento de los individuos positivos y control posterior. Resultados: La prevalencia global fue 8,7% (37/427) siendo mayor en las mujeres (13,7% vs. 4,1%; p = 0.0004), en el GNU (13.1 vs. 4.5%; p = 0.001) y en aquellos con las necesidades básicas (NB) insatisfechas (14.8% vs. 6.1%; p = 0.0006). No hubo diferencias significativas (DS) con respecto al comportamiento sexual y al uso de diferentes métodos anticonceptivos. Los antecedentes previos de exudado vaginal o uretral y adenomegalias inguinales tuvieron un elevado valor predictivo negativo (93,01% y 94,2%, respectivamente). Conclusiones: Es recomendable efectuar tamizaje de jóvenes en diferentes escenarios con técnicas sensibles. Los programas basados en los factores de riesgo son inadecuados en nuestro medio. La información y la educación general y sexual deben ser consideradas herramientas imprescindibles para controlar esta infección.

HTLV-1a tax gene and long terminal repeat sequences from Argentinean strains reveal disagreement with tax restriction fragment length polymorphism subtyping.

Sequence and cluster analysis have shown two HTLV-1a tax gene subgroups, tax A and tax B, which are related to long terminal repeat (LTR) molecular subtypes. On the basis of subgroup-specific nucleotide substitutions, restriction fragment length polymorphism (RFLP) analysis of the tax gene for subtyping HTLV-1a isolates was proposed. In this study we genetically characterized the tax gene from 63 HTLV-1-positive Argentinean individuals, including 14 patients with HTLV-1-associated myelopathy/tropical spastic paraparesis and 49 healthy HTLV-1 carriers. RFLP analysis showed that 48 samples yielded the tax A profile (76.19%) and that 15 samples contained the uncut tax B profile (23.81%). However, the LTR and tax sequence analysis revealed that in fact only 2 from the 15 samples belonged to the HTLV-1aB subgroup, presenting four tax B subgroup-specific nucleotide substitutions. The tax gene cluster analysis also confirmed that the majority of Argentinean strains belonged to the Transcontinental HTLV-1aA subgroup. These results indicate that the tax gene RFLP assay which has been proposed and used by some authors to screen HTLV-1a subgroups, is not a suitable tool to perform molecular epidemiological characterization of HTLV-1a populations.

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.

HTLV-1 p12I protein sequences from South America: truncated proteins and common genetic signatures.

p12 pX ORF-I (p12I) of simian T cell lymphotropic virus 1 from Central and West Africa is a 91-amino acid (aa) protein, contrasting with the known p12 sequences from human T cell lymphotropic virus type 1, which are 99 aa in length. An in silico sequence analysis shows a premature termination codon in HTLV-1 p12I sequences from South American and Japanese strains. A translation analysis indicated the presence of 86-aa and 81-aa p12I proteins, shorter than the 99-aa protein found in the majority of HTLV-1 strains around the world. This report shows one more common feature between these two viruses. Additionally, the HTLV-1 p12 signature reported as a genetic marker of Brazilian sequences (p12I P63 and P91) is also highly prevalent in p12 Argentinean sequences. Because p12I may confer a proliferative advantage on HTLV-1-infected cells the existence of naturally truncated proteins might supply insights into its pathogenic mechanisms.

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.

Usefulness of a Nested-polymerase chain reaction for molecular diagnosis of human T-cell lymphotropic virus type I/II.

This study aimed at implementing a Nested-polymerase chain reaction (Nested-PCR) for the molecular diagnosis of human T-cell lymphotropic virus type I/II (HTLV-I and HTLV-II) infections in peripheral blood mononuclear cells of infected subjects in Argentina. The sensitivity and specificity of the assay for the detection of regional strains were assessed by comparing them with the molecular assay of reference PCR-hybridization. The Nested-PCR detected 1 MT-2 cell (> or = 8 proviral copies)/1x10(6) non-infected cells showing high sensitivity for provirus detection. While both molecular assays showed high specificity (100%) for HTLV-I and HTLV-II detection, the sensitivity values differed: 100% for Nested-PCR and 67% for PCR-hybridization assay. Moreover, this technique showed less sensitivity for the detection of DNA sequences of HTLV-II (33%) than for the detection of DNA sequences of HTLV-I (75%). The high sensitivity and specificity of the Nested-PCR for regional strains and its low costs indicate that this assay could replace the PCR-hybridization assay for the molecular diagnosis of HTLV-I/II infections. It will be interesting to assess the usefulness of this assay as a tool for the molecular diagnosis of HTLV-I/II infections in other developing countries. Other studies that include a greater number of samples should be conducted.

Usefulness of a Nested-polymerase chain reaction for molecular diagnosis of human T-cell lymphotropic virus type I/II

This study aimed at implementing a Nested-polymerase chain reaction (Nested-PCR) for the molecular diagnosis of human T-cell lymphotropic virus type I/II (HTLV-I and HTLV-II) infections in peripheral blood mononuclear cells of infected subjects in Argentina. The sensitivity and specificity of the assay for the detection of regional strains were assessed by comparing them with the molecular assay of reference PCR-hybridization. The Nested-PCR detected 1 MT-2 cell ( 8 proviral copies)/1x106 non-infected cells showing high sensitivity for provirus detection. While both molecular assays showed high specificity (100 percent) for HTLV-I and HTLV-II detection, the sensitivity values differed: 100 percent for Nested-PCR and 67 percent for PCR-hybridization assay. Moreover, this technique showed less sensitivity for the detection of DNA sequences of HTLV-II (33 percent) than for the detection of DNA sequences of HTLV-I (75 percent). The high sensitivity and specificity of the Nested-PCR for regional strains and its low costs indicate that this assay could replace the PCR-hybridization assay for the molecular diagnosis of HTLV-I/II infections. It will be interesting to assess the usefulness of this assay as a tool for the molecular diagnosis of HTLV-I/II infections in other developing countries. Other studies that include a greater number of samples should be conducted.

Técnicas moleculares como herramientas para el tamizaje viral. Estado actual del desarrollo de una técnica de RT-Multiplex-Nested PCR para el control de HIV en sangre / Molecular techniques for viral screening. RT-Multiplex-Nested PCR for HIV control in blood

Los índices de morbilidad y mortalidad en receptores de sangre aumentan drásticamente debido a la transmisión de infecciones virales por vía transfusional. Entre los virus transmitidos por sangre, el de mayor impacto sanitario y social es el virus de la inmunodeficiencia humana tipo 1 (HIV-1). A pesar de que la implementación del tamizaje de anticuerpos para HIV en los Bancos de Sangre de Argentina ha permitido reducir considerablemente la transmisión del virus por vía sanguínea, existe en la actualidad evidencia suficiente de la transmisión de HIV por unidades de sangre con serología negativa. En los Bancos de Sangre de la mayoría de los países europeos y en los Estados Unidos se utilizan, desde el año 1999, técnicas de amplificación de ácidos nucleicos (NAT) para detectar HIV y HCV, con el fin de reducir el período de ventana inmunológica. En este trabajo realizamos una actualización del uso de las técnicas moleculares como herramientas de tamizaje de HIV en la sangre destinada a transfusión y presentamos los resultados preliminares obtenidos a partir del desarrollo de una técnica molecular artesanal: transcripción reversa y reacción en cadena de la polimerasa anidada (RT-Multiplex-Nested PCR), de alta sensibilidad y de bajo costo. Los resultados obtenidos demuestran que la sensibilidad y la especificidad de esta técnica para detectar cepas de HIV regionales están dentro de los límites establecidos por las reglamentaciones internacionales. En esta etapa del trabajo se está realizando la validación de la técnica desarrollada utilizando estándares internacionales. Esta última técnica podrá ser utilizada para el control de la sangre en Córdoba, con el fin de reducir el riesgo de transmisión del virus HIV por esta vía.

Seroepidemiology of HTLV-I/II in Argentina: an overview.

In this report, the results of seroepidemiologic studies of human T-lymphotropic virus type I (HTLV-I) and type II (HTLV-II) infections in different population groups in Argentina have been compiled. The studies have shown a high prevalence of HTLV-I/II infection in blood donors in the provinces in the north of Argentina (1.0% in Jujuy, 0.7% in Salta, and 0.6% in Formosa) and a low prevalence in the provinces in the central region of the country (

[Screening of HIV in blood banks. Evaluation of fourth generation kits]. / Screening de HIV en bancos de sangre. Evaluación de los equipos de cuarta generación.

Use of detection tests for p24 HIV antigen (p24Ag) in blood banks in Argentina is recommended by the Argentinean Society of Hemotherapy and Immunohematology. In the blood bank of the National University of Cordoba (Argentina), the recent implementation of the p24Ag screening test has considerably increased the cost of the battery of screening tests and its use in all blood donations has not produced the benefits expected. A 4th generation EIA was evaluated for the screening of HIV in comparison with the currently used assays in the blood bank of National University of Cordoba (3rd generation EIA + p24Ag assay). For this comparison, 11 serum samples from subjects with early HIV infection (early seroconversion period) were tested, as well as 27 serum samples from asymptomatic HIV-infected subjects and other 39 from non-HIV infected subjects. The 3rd generation EIA and the 4th generation EIA showed the same sensitivity value (100%) but the specificity of the 3rd generation EIA was higher (97.5%) comparing with 4th generation (95.1%). Besides, the p24Ag test failed to detect 2 samples from subjects with early HIV infection. These results indicate a good performance of both 3rd and 4th generation assays for screening of HIV. However, due to the lowest cost of 4th generation EIA kit, it could replace the currently used assays for HIV screening in regional blood banks. This screening assay will lead to gain in effectiveness and reduced costs until the detection of HIV RNA can be implemented in blood banks.

Immunofluorescence assay reactivity patterns of serum samples presenting indeterminate Western blot results for antibodies to HIV-1 and HTLV-I/II in Cordoba, Argentina

Serum samples (n: 110) from blood donors and high risk individuals from Cordoba, Argentina with indeterminate HIV-1 and HTLV-I/II Wb profiles were studied for specific antibodies to HTLV-I/II and HIV-1 by indirect immunofluorescence assay (IFA) and for the presence or absence of HIV-1 and HTLV-I/II specific bands by Wb. This study was carried out in order to characterize their putative reactions with HIV-1 and HTLV-I/II proteins and to resolve the retrovirus infection status of these individuals. Results indicated that blood donors sera displaying indeterminate HIV-1 or HTLV-I/II Wb patterns were not immunoreactive to HTLV-I/II and HIV-1 on IFA. However, a high rate of indeterminate HIV-1 and HTLV-I/II Wb samples from high risk individuals had positive HTLV-I/II and HIV-1 IFA results respectively. Our study supports the growing evidence that HTLV-HIV indeterminate seroreactivity in low risk population is due to a cross reaction against nonviral antigens, and in high risk populations the indeterminate samples show serological cross-recognition between HIV-1 proteins and HTLV-I/II proteins on Wb. These results point out the necessity to investigate the HTLV-I/II reactivity in indeterminate HIV-1 samples and viceversa in order to confirm the diagnosis. Finally, this study shows the potential usefulness of IFA in elucidating the status of HIV-1 and HTLV-I/II infection of individuals with indeterminate Wb profiles, thus enabling resolution of retrovirus infection status

Efficiency of indirect immunofluorescence assay as a confirmatory test for the diagnosis of human retrovirus infection (HIV-1 and HTLV-I/II) in different at risk populations

Avaliou-se a eficiencia da tecnica de immunofluorescencia indireta (IFI) como metodo confirmatorio no diagnostico da infeccao por HIV-1 e HTLV-I/II. Para isto, processaram-se amostras com sorologia positiva ou negativa por Western blot (Wb) para ambos virus, pertencentes a populacoes em diferentes graus de risco de adquirir a infecao e determinaram-se os valores de sensibilidade, especificidade, valores preditivos positivo e negativo e o indice de concordancia Kappa da IFI para cada sistema viral em comparacao com o Wb. Como fontes de antigenos da IFI empregaram-se as linhas celulares H9 (HTLV-III b), MT-2 e MT-4 (persistentemente infectadas com HTLV-I), MO-T (persistentemente infectadas com HTLV-II). Os valores globais de sensibilidade e especificidade para o sistema HIV-1, foram 96,80 por cento e 98,60 por cento respectivamente, enquanto os valores preditivos positivo e negativo 99,50 por cento e 92,00 por cento respectivamente...