Frequency of allogenic blood transfusion in patients with gastrointestinal cancer: a cross-sectional study in Peru.

BACKGROUND: Gastrointestinal cancer demands a high frequency of transfusions, and the high availability of blood products. We aimed to determine the frequency of blood transfusions and the most used blood products according to the type of gastrointestinal cancer. METHODS: A cross-sectional study was conducted in a Peruvian Type I Hemotherapy and Blood Bank Service of a Private Oncological Clinic during 2016-2018. We included patients with gastrointestinal cancer using the International Code of Diseases. The donations were made in compliance with the requirements of the Programa Nacional de Hemoterapía y Banco de Sangre and in accordance with the Standardised Operational Procedure of the clinic. RESULTS: We analysed 3,022 patients, of which 163 (5.4%) had gastrointestinal cancer (67.1 ± 12 years). The 80 (49.1%) men did not show significant differences with the 83 (50.9%) women (p = 0.178). The most frequent neoplasia was the colon (41.7%) and pancreas (37.4%). Three hundred and four blood products were transfused (average 1.8 ± 2.5 units (range: 1-30 units/patient)), of which 81.3% (247 units) were red blood cells concentrated, 8.6% (26 units) were fresh-frozen-plasma (FFP) and 6.6% (20 units) were cryoprecipitate. The type of cancer that most blood products demanded was colon neoplasia (41.8%), followed by pancreatic cancer (26.3%) and liver cancer (10.9%). We determined that ~55% of patients were O Rh(D)+ and in five patients we were poly-transfused. CONCLUSION: Our findings suggested that patients with gastrointestinal cancer require large numbers of transfusions of blood cell concentrate and FFP. Also, we showed that cancer of the colon, pancreas and liver demanded more than 75% of blood products.

Seroprevalence of hepatitis E virus in Argentinean blood donors.

BACKGROUND: Hepatitis E virus (HEV) is the main cause of enteric acute viral hepatitis worldwide. In this epidemiological framework, it has become a threat to blood safety and a relevant issue for blood transfusions. However, there is a paucity of data regarding prevalence of HEV infection. The aim of this study was to determine HEV seroprevalence in blood donors from different regions from Argentina. MATERIAL AND METHODS: Serum samples from 391 individuals attending five blood donor centers located in different regions from Argentina were analyzed for anti-HEV IgG and anti-HEV IgM. RESULTS: Overall, anti-HEV IgG was detected in 44 out of 391 (11.3%) samples. HEV prevalence ranged from 5.1 to 20.0% among different country regions. A significant difference in blood donors' age was observed between anti-HEV IgG positive and negative individuals [44 (37-51) vs. 35 (27-43), P < 0.001, respectively]. Anti-HEV IgM was detected in 8 out of 44 (18.2%) anti-HEV IgG positive cases. CONCLUSION: Anti-HEV IgG was detected in blood donor samples from five analyzed Argentinean regions, highlighting the wide distribution of the virus in the country. HEV prevalence was variable among different regions and significantly higher in older donors. Given the evidence of anti-HEV IgM presence in blood donors, suggesting a potential risk of transfusion-transmitted HEV, screening for HEV in blood units to be used in vulnerable population would be desirable. Molecular studies for detection of viremic donors and donor-recipients follow-up are necessary to certainly determine the risk of transfusion-transmitted HEV in Argentina.

Genetic diversity and phylogeographic analysis of human herpesvirus type 8 (HHV-8) in two distant regions of Argentina: Association with the genetic ancestry of the population.

BACKGROUND: The genetic diversity of persistent infectious agents, such as HHV-8, correlates closely with the migration of modern humans out of East Africa which makes them useful to trace human migrations. However, there is scarce data about the evolutionary history of HHV-8 particularly in multiethnic Latin American populations. OBJECTIVES: The aims of this study were to characterize the genetic diversity and the phylogeography of HHV-8 in two distant geographic regions of Argentina, and to establish potential associations with pathogenic conditions and the genetic ancestry of the population. STUDY DESIGN: A total of 101 HIV-1 infected subjects, 93 Kaposi's Sarcoma (KS) patients and 411 blood donors were recruited in the metropolitan (MET) and north-western regions of Argentina (NWA). HHV-8 DNA was detected by ORF-26 PCR in whole blood, saliva and FFPE tissues. Then, ORF-26 and ORF-K1 were analyzed for subtype assignment. Mitochondrial DNA and Y chromosome haplogroups, as well as autosomal ancestry markers were evaluated in samples in which subtypes could be assigned. Phylogeographic analysis was performed in the ORF-K1 sequences from this study combined with 388 GenBank sequences. RESULTS: HHV-8 was detected in 50.7%, 59.2% and 8% of samples from HIV-1 infected subjects, KS patients and blood donors, respectively. ORF-K1 phylogenetic analyses showed that subtypes A (A1-A5), B1, C (C1-C3) and F were present in 46.9%, 6.25%, 43.75% and 3.1% of cases, respectively. Analyses of ORF-26 fragment revealed that 81.95% of strains were subtypes A/C followed by J, B2, R, and K. The prevalence of subtype J was more commonly observed among KS patients when compared to the other groups. Among KS patients, subtype A/C was more commonly detected in MET whereas subtype J was the most frequent in NWA. Subtypes A/C was significantly associated with Native American maternal haplogroups (p = 0.004), whereas subtype J was related to non-Native American haplogroups (p < 0.0001). Sub-Saharan Africa, Europe and Latin America were the most probable locations from where HHV-8 was introduced to Argentina. CONCLUSIONS: These results give evidence of the geographic circulation of HHV-8 in Argentina, suggest the association of ORF-26 subtype J with KS development and provide new insights about its relationship with ancient and modern human migrations and identify the possible origins of this virus in Argentina.

Hepatitis B surface antibodies seroprevalence among people born before and after implementation of universal HBV vaccination.

Universal vaccination is the most effective strategy to control hepatitis B virus (HBV) infection. In Argentina, vaccination against HBV was incorporated in year 2000 for newborns and in 2003 for 11 years old children. However, there is a paucity of data about protection levels against HBV infection. The aim of this work was to determine the prevalence of seroprotective anti-HBs antibodies (aHBs) in Argentina. Serum samples negative for HBsAg and anti-HBc from 132 children born after year 2000 and 762 blood donors, older than 18 years, from five centers across the country, were analyzed for aHBs. Titers ≥10 mIU/mL were observed in 74/132 children (56.1%) and 336/762 (44.1%) in blood donors. The median age for blood donors was 33.9 (23-43); from them, 210 (27.6%) were born after 1992 and, therefore, were catch-up by vaccine implementation at 11 years old age. Donors born in 1992 or before showed a significantly lower frequency of protection (32.2%) compared to donors born after 1992 (75.2%), p < 0.0001. In addition, significant differences were observed in the status of seroprotection between different participating centers (p = 0.024). Implementation of HBV vaccine in 2000 and 2003 implied an overall increase of the aHBs seroprotective rates, with a particularly adequate response in children vaccinated at 11 years old age. The observed results suggest that population born in 1992 or before is currently the most susceptible. Consequently, it would be advisable to become aware of the risk of transmission in this age group and to stress this population vaccination campaigns.

Seroprevalence, cost per donation and reduction in blood supply due to positive and indeterminate results for infectious markers in a blood bank in Lima, Peru.

INTRODUCTION: Safety in Transfusion Medicine is subject to regulations and government legislation within a total quality framework. The aim of this study was to evaluate the impact of seroprevalence and indeterminate results on lost units and cost per donation. METHODS: A prospective cross-sectional study was performed in the Blood Bank and Transfusion Therapy Department of the Hospital Central de la Policia Nacional del Perú in Lima, Peru. All completed donations (replacement/voluntary) without complications were included in this study. Every donation met the institutional requirements and quality criteria of Programa Nacional de Hemoterapia y Bancos de Sangre (PRONAHEBAS). Data analysis was achieved using the Statistical Package for the Social Sciences. RESULTS: A total of 7723 donations were evaluated during 2014 and 2015 with 493 being seropositive (overall prevalence 5.25%) and 502 having indeterminate results (overall prevalence 5.35%). Thus total loss was 995units, 437.8L of blood and 49,750 US dollars. The most common seropositive infectious markers were the core antibody of hepatitis B virus (2.82%) and syphilis (1.02%), and the most common indeterminate results were Chagas disease (1.27%) and the core antibody of hepatitis B virus (1.26%). There was no significant change in the prevalence of seropositivity (p-value=0.243) or indeterminate results (p-value=0.227) over the two-year period of the study. A statistical correlation was found between the cost per lost donation and the most prevalent markers (rho=0.848; p-value=<0.001). CONCLUSION: Seroprevalence was lower than the regional mean, but the prevalence of indeterminate results was elevated causing a great impact on blood supply and economic losses to this institution.

Seroprevalence, cost per donation and reduction in blood supply due to positive and indeterminate results for infectious markers in a blood bank in Lima, Peru

ABSTRACT Introduction: Safety in Transfusion Medicine is subject to regulations and government legislation within a total quality framework. The aim of this study was to evaluate the impact of seroprevalence and indeterminate results on lost units and cost per donation. Methods: A prospective cross-sectional study was performed in the Blood Bank and Transfusion Therapy Department of the Hospital Central de la Policia Nacional del Perú in Lima, Peru. All completed donations (replacement/voluntary) without complications were included in this study. Every donation met the institutional requirements and quality criteria of Programa Nacional de Hemoterapia y Bancos de Sangre (PRONAHEBAS). Data analysis was achieved using the Statistical Package for the Social Sciences. Results: A total of 7723 donations were evaluated during 2014 and 2015 with 493 being seropositive (overall prevalence 5.25%) and 502 having indeterminate results (overall prevalence 5.35%). Thus total loss was 995 units, 437.8 L of blood and 49,750 US dollars. The most common seropositive infectious markers were the core antibody of hepatitis B virus (2.82%) and syphilis (1.02%), and the most common indeterminate results were Chagas disease (1.27%) and the core antibody of hepatitis B virus (1.26%). There was no significant change in the prevalence of seropositivity (p-value = 0.243) or indeterminate results (p-value = 0.227) over the two-year period of the study. A statistical correlation was found between the cost per lost donation and the most prevalent markers (rho = 0.848; p-value = <0.001). Conclusion: Seroprevalence was lower than the regional mean, but the prevalence of indeterminate results was elevated causing a great impact on blood supply and economic losses to this institution.

Role of HLA-DP and HLA-DQ on the clearance of hepatitis B virus and the risk of chronic infection in a multiethnic population.

BACKGROUND & AIMS: HBV infection exhibits geographical variation in its distribution in South America. While HBV rates are low in central Argentina, the north-western region exhibits intermediate HBV rates. Unfortunately, the reasons that could explain this difference are still unknown. METHODS: A total of 1440 Argentines were recruited and grouped into HBV patients, HBV-resolved individuals and healthy controls. Genetic ancestry was assessed by analysis of biparental lineages and ancestry autosomal typing. SNPs of HLA-DPA1 (rs3077), HLA-DPB1 (rs9277542), HLA-DQB1 (rs2856718) and HLA-DQB2 (rs7453920) were determined, and HBV genotyping was performed by phylogenetic analysis in HBV patients. RESULTS: Native American ancestry prevailed in the north-western region when compared with central Argentina (P<.0001). However, no differences were observed among the three groups of each region. The distribution of HBV genotypes revealed significant differences (P<.0001). Three SNPs (rs3077, rs9277542 and rs7453920) showed a significant association with protection against chronic HBV and viral clearance in both regions. The remaining SNP showed a significant association with susceptibility to chronic HBV. The frequency rates of rs3077-T, related to protection against chronic HBV and viral clearance, were lower in north-western Argentina when compared with central Argentina. The same uneven frequency rates were observed for SNP rs9277542. CONCLUSIONS: This is the first study addressing the associations between the HLA-DP and HLA-DQ loci and the protection against chronic HBV and viral clearance in a multiethnic South American population. The uneven distribution of HLA-DP and HLA-DQ supports the HBV epidemiological differences observed in these two regions of Argentina with dissimilar ancestry genetic background.

Prevalence and trends of markers of hepatitis B virus, hepatitis C virus and human Immunodeficiency virus in Argentine blood donors.

BACKGROUND: Transfusion-transmitted infections are a major problem associated with blood transfusion. The aim of this study was to determine prevalence and trends of HBV, HCV and HIV in blood donors in Argentina. METHODS: A retrospective study was carried out in blood donors of 27 transfusion centers covering the whole country over a period of eight years (2004-2011). Serologic screening assays for HBsAg, anti-HBc, anti-HCV, and anti-HIV were performed in all centers and nucleic acid amplification testing (NAT) was performed in 2 out of the 27 centers. RESULTS: The 2,595,852 samples tested nationwide from 2004 to 2011 showed that the prevalence of HBsAg decreased from 0.336% to 0.198% (p < 0.0001), that of anti-HBc from 2.391% to 2.007% (p < 0.0001), that of anti-HCV from 0.721% to 0.460%, (p < 0.0001) and that of anti-HIV from 0.208% to 0.200 (p = 0.075). The prevalence of HBV, HCV and HIV was unevenly distributed among the different regions of the country. Two out of 74,838 screening- negative samples were positive in NAT assays (1 HIV-RNA and 1 HCV-RNA); moreover, HBV-DNA, HCV-RNA and HIV-RNA were detected in 60.29, 24.54 and 66.67% of screening-positive samples of the corresponding assays. As regards donors age, positive HBV-DNA and HCV-RNA donors were significantly older than healthy donors (46.6, 50.5 and 39.5 y respectively, p < 0.001). CONCLUSIONS: Argentina has a low prevalence of HBsAg, anti-HCV and anti-HIV in blood donors, with a decreasing trend for HBsAg, anti-HBc and anti-HCV but not for anti-HIV over the last 8 years. The uneven distribution of transfusion-transmitted infections prevalence among the different regions of the country highlights the need to implement regional awareness campaigns and prevention. The discrepancy between samples testing positive for screening assays and negative for NAT assays highlights the problem of blood donors who test repeatedly reactive in screening assays but are not confirmed as positive upon further testing. The uneven distribution of age between healthy donors and NAT-positive donors could be related to changes in risks of these pathogens in the general population and might be attributed to a longer exposure to transmission risk factors in elderly people.

Hepatitis B virus and hepatitis D virus in blood donors from Argentina: circulation of HBsAg and reverse transcriptase mutants.

In Argentina, current procedures to ensure the safety of the blood supply for transfusion include the serologic detection of specific blood-borne infections. The aim of this study was to evaluate the prevalence and the genetic diversity of hepatitis B virus (HBV) and hepatitis D virus (HDV) in blood donor populations from two distantly located Argentine regions. Data from 56,983 blood donations from the Favaloro Foundation, in the city of Buenos Aires (Central Region), and the Central Blood Bank of Misiones Province (Northeast Region) were analyzed. Samples that were reactive for HBsAg were analyzed for HBV-DNA characterization and HDV serological and molecular analysis. The HBV prevalence was 0.12 % for HBsAg and 1.68 % for anti-HBc antibodies in Buenos Aires, and 0.73 % and 8.55 %, respectively, in Misiones. Seventy-seven HBsAg-reactive samples were analyzed by polymerase chain reaction for HBV-DNA. Subgenotypes A2, B2, C2, F1b and F4 (Buenos Aires) and F1b and D3 (Misiones) were detected. Several mutations within the major hydrophilic region of HBsAg, the reverse transcriptase, the basal core promoter, and the precore/core were detected. HDV genotype 1 was identified in Buenos Aires. This study confirms the circulation of several HBV subgenotypes, as well as known and newly identified variants, and the presence of HDV1 in this population. A thorough investigation has to be carried out to evaluate the clinical importance of some of the documented mutations as well as those detected in the HDV1 case.

Virus emergentes de importancia en medicina transfusional: paradigmas, hitos, aprendizajes / Emergent virus with importance in transfusional medicine: paradigms, milestones, learnings

Las infecciones emergentes juegan un papel importantísimo en medicina transfusional. La experiencia con HIV puso en evidencia la necesidad de actuar rápidamente. La lentitud en la respuesta de los Bancos de Sangre y la falta de un liderazgo en la adopción de medidas preventivas dieron lugar a una transmisión importante por vía transfusional. En cuanto a las hepatitis postransfusionales NANB, aprendimos las lecciones acerca de las pruebas subrogantes. Sin embargo, la respuesta para prevenir la transmisión de HCV fue lenta porque la comunidad científica estaba focalizada en la transmisión de HIV. En el caso del XMRV, la presión ejercida por la comunidad fue muy importante. Se formaron grupos multidisciplinarios de expertos que realizaron gran cantidad de estudios y la respuesta ocurrió rápidamente, aunque al poco tiempo se demostró que este patógeno no era relevante para la Medicina Transfusional. Con respecto al WNV, la familiaridad con los modelos desarrollados por el CDC para estimar los riesgos y las lecciones aprendidas por las experiencias con HIV y HCV facilitaron una respuesta rápida y se implementaron medidas rápidamente para minimizar el riesgo de transmisión por vía transfusional. Se abrió un nuevo paradigma: la importancia de considerar los riesgos de transfusión que pueden derivar de agentes que causan viremias breves, usualmente asintomáticas, pero con el potencial de generar brotes estacionales de alta incidencia. La respuesta a la amenaza con WNV fue rápida, apropiada y exitosa. Las nuevas herramientas de biología molecular han permitido el aislamiento de numerosos gérmenes emergentes y lo seguirán haciendo en el futuro. Estar alertas ante nuevos patógenos de potencial importancia es nuestra responsabilidad. (AU)

Emerging infections play an extremely important role in transfusion medicine. Experience with HIV highlighted the necessity to act quickly. The slow Blood Banks response and the lack of leadership in the adoption of preventive measures resulted in a significant transfusional transmission. Regarding the post-transfusion NANB hepatitis, we have learned the lessons about the surrogate tests. However, the response to prevent HCV transmission was slow given that the scientific community was focused on HIV transmission. In the case of XMRV, pressure from the community was extremely important. Multidisciplinary groups of experts who conducted many studies were formed and the answer came quickly, but soon it was proved that this pathogen was not relevant to Transfusion Medicine. With respect to WNV, familiarity with the models developed by the CDC to estimate the risks and lessons learned from experiences with HIV and HCV facilitated a quick response, and measures were quickly implemented to minimize the risk of transmission by transfusion. A new paradigm came up: the importance of con¡sidering the risks of transfusion that may result from agents that cause brief, usually asymptomatic viremia, but with the potential to generate high incidence seasonal outbreaks of viralloads. The response to the threat with WNV was rapid, appropriate and successful. The new tools of molecular biology have allowed the isolation of many emerging germs and will continue to do so in the future. Being alert to new pathogens of potential importance is our responsibility. (AU)

Virus emergentes de importancia en medicina transfusional: paradigmas, hitos, aprendizajes / Emergent virus with importance in transfusional medicine: paradigms, milestones, learnings

Las infecciones emergentes juegan un papel importantísimo en medicina transfusional. La experiencia con HIV puso en evidencia la necesidad de actuar rápidamente. La lentitud en la respuesta de los Bancos de Sangre y la falta de un liderazgo en la adopción de medidas preventivas dieron lugar a una transmisión importante por vía transfusional. En cuanto a las hepatitis postransfusionales NANB, aprendimos las lecciones acerca de las pruebas subrogantes. Sin embargo, la respuesta para prevenir la transmisión de HCV fue lenta porque la comunidad científica estaba focalizada en la transmisión de HIV. En el caso del XMRV, la presión ejercida por la comunidad fue muy importante. Se formaron grupos multidisciplinarios de expertos que realizaron gran cantidad de estudios y la respuesta ocurrió rápidamente, aunque al poco tiempo se demostró que este patógeno no era relevante para la Medicina Transfusional. Con respecto al WNV, la familiaridad con los modelos desarrollados por el CDC para estimar los riesgos y las lecciones aprendidas por las experiencias con HIV y HCV facilitaron una respuesta rápida y se implementaron medidas rápidamente para minimizar el riesgo de transmisión por vía transfusional. Se abrió un nuevo paradigma: la importancia de considerar los riesgos de transfusión que pueden derivar de agentes que causan viremias breves, usualmente asintomáticas, pero con el potencial de generar brotes estacionales de alta incidencia. La respuesta a la amenaza con WNV fue rápida, apropiada y exitosa. Las nuevas herramientas de biología molecular han permitido el aislamiento de numerosos gérmenes emergentes y lo seguirán haciendo en el futuro. Estar alertas ante nuevos patógenos de potencial importancia es nuestra responsabilidad.

Emerging infections play an extremely important role in transfusion medicine. Experience with HIV highlighted the necessity to act quickly. The slow Blood Banks response and the lack of leadership in the adoption of preventive measures resulted in a significant transfusional transmission. Regarding the post-transfusion NANB hepatitis, we have learned the lessons about the surrogate tests. However, the response to prevent HCV transmission was slow given that the scientific community was focused on HIV transmission. In the case of XMRV, pressure from the community was extremely important. Multidisciplinary groups of experts who conducted many studies were formed and the answer came quickly, but soon it was proved that this pathogen was not relevant to Transfusion Medicine. With respect to WNV, familiarity with the models developed by the CDC to estimate the risks and lessons learned from experiences with HIV and HCV facilitated a quick response, and measures were quickly implemented to minimize the risk of transmission by transfusion. A new paradigm came up: the importance of con­sidering the risks of transfusion that may result from agents that cause brief, usually asymptomatic viremia, but with the potential to generate high incidence seasonal outbreaks of viralloads. The response to the threat with WNV was rapid, appropriate and successful. The new tools of molecular biology have allowed the isolation of many emerging germs and will continue to do so in the future. Being alert to new pathogens of potential importance is our responsibility.

HDAg-L variants in covert hepatitis D and HBV occult infection among Amerindians of Argentina: new insights.

BACKGROUND: Guidelines suggest that all HBsAg-positive patients should be tested for anti-HDV IgG antibodies and to confirm active hepatitis D virus (HDV) infection by detection of HDV RNA by reverse transcriptase (RT) polymerase chain reaction (PCR). OBJECTIVES: The aim of this study was to determine the serological prevalence and molecular features of HDV within an Amerindian community from Argentina exhibiting positivity for HBsAg and/or anti-HBc total Ig. STUDY DESIGN: Forty-six plasma samples were tested for the detection of total anti-HDV antibodies by ELISA. Concomitantly, a partial RNA region coding for the delta antigen (HDAg) was amplified by RT-nested PCR (RT-nPCR). In silica translation of DNA sequences into the amino acid (aa) sequence of HDAg-S (aa110-195) and HDAg-L (aa110-214) was performed. RESULTS: Out of 46 HDV non-reactive samples by ELISA, 3 were HDV RNA positive by RT-nPCR. These samples were anti-HBc-only positive, 2 of them identified as cases of occult hepatitis B infection (OBI). The 3 cases were HBeAg-negative and showed normal ALT/AST levels. All sequences were ascribed to HDV genotype 1, but exhibited nucleotide differences in HDAg-L coding region, among which, mutations at codons 197 and 201 - reportedly known to promote in vitro an unsuitable interaction with HBsAg - were observed. CONCLUSIONS: These results provide evidence of covert HDV infection even among OBI, highlighting the need to reevaluate the currently applied guidelines for HDV diagnostic algorithms, as well as to explore if the observed mutations promote any effect on HDV pathogenesis.

New natural variants of hepatitis B virus among Amerindians from Argentina with mainly occult infections.

BACKGROUND: Hepatitis B virus infection is frequent among Amerindians. In Argentina HBV genotypes A, B, C, D, E, F and H were described in different populations, while some cases of occult hepatitis B infection (OBI) were reported in human immunodeficiency virus (HIV) coinfected patients. OBJECTIVE: To determine the prevalence, genetic diversity of HBV and to analyze the deduced amino acid sequence of both S and viral polymerase (P) genes among Amerindians of Argentina. STUDY DESIGN: A cross-sectional study including 561 individuals belonging to distinct ethnic groups, the Mbyá-guaraní (MG), the Kolla (K), the Sagua-Huarpe (SH) and the Wichi (W) was performed. RESULTS: The prevalence of HBsAg was 1.7% and 1.4% for the MG and SH, respectively, while anti-HBc was detected in all communities. HBV DNA of S/P and preCore-Core genomic regions were amplified by nested polymerase chain reaction in 59 reactive samples for anti-HBc total Ig and/or HBsAg. Of them, thirteen exhibited detectable HBV DNA, eleven of which were identified as OBI. Genotype F was predominant in the MG community with co-circulation of subgenotypes F4, F1b, A2 and D3, while subgenotype C2 prevailed within the SH community. All cases exhibited the polymorphism rtL217R within the RT domain associated to resistance to adefovir. Mutations rtD206E and rtV207I associated with lamivudine resistance were found in two MG and three SH respectively. Other new substitutions were described within the P sequence. CONCLUSIONS: This study shows for the first time the predominance of OBI, HBV subgenotypes and natural variants in Amerindians from Argentina.