Evidence of active herpesvirus 6 (variant-A) infection in patients with lymphadenopathy in Belém, Pará, Brazil

A total of 323 patients with lymphadenopathy were selected in Belém, Brazil, between January 1996 and December 2001, and screened for the presence of human herpesvirus-6 (HHV-6) IgM- and- IgG antibodies by enzyme-linked immunosorbent assay (ELISA). When seroprevalence is analyzed by gender, similar rates are found for female (60.6%) and male (55.7%) individuals. Seventy-seven (23.8%) patients were HHV-6-IgM-and- IgG-positive (IgM+ subgroup), with positivity rates of 29.7% and 17.7% (p = 0.0007) for female- and male individuals, respectively. Sera from a subgroup (n = 120) of these subjects, with high HHV-6 antibody levels (either IgM+ or IgG+ reactivities), were subsequently processed for the presence of HHV-6 DNA by polymerase chain reaction (PCR)/nested PCR. Active infections (IgM+ and/or IgG+ high levels specific antibodies plus detection of viral DNA) were diagnosed in 20/77 (20.0%) and 8/43 (18.6%); subgroup of the 120 individuals suspected of having HHV-6 suggestive recent infection. All (n = 28) cases of active infection were found to be associated with HHV-6 variant-A (HHV-6A), as detectable by PCR/nested PCR, using variant-specific primer that amplify regions of 195 base pairs (bp) (HHV-6A) and 423 bp (HHV-6B). Rates of HHV-6 DNA detection between female and male patients were similar (p > 0.05) in the IgM+ and IgG+ groups: 20.4% versus 35.7% and 25.0% versus 13.0%, respectively. HHV-6 DNA was detected across < 5 through 41-50-year age-groups for patients whose serum samples were IgM+, with rates ranging from 7.7% (female subjects aged < 5 years) to 80.0% (male, 11-20 years). Among patients whose serological status was IgG+, HHV-6 DNA was detected in < 5, 6-10, 21-30 and > 50 age-groups at rates that ranged from 15.4% (male, < 5 years of age) to 100.0% (female aged 11-20 years). Swelling cervical lymph nodes were the most common sign, accounting for 9 (32.0%) cases in each gender group. Among patients (n = 28) with active infection by HHV-6A variant, duration of symptoms lasted 1-5 days in 35.7% of subjects, whereas in 64.3% of them the disease lasted 6-20 days. Our data suggest that it is worth seeking for HHV-6 infection whenever a patient (infant or adult) presents with lymphadenopathy as a prominent symptom in the course of an acute febrile illness.

Determination of human cytomegalovirus genetic diversity in different patient populations in Costa Rica

Seroprevalence of HCMV in Costa Rica is greater than 95% in adults; primary infections occur early in life and is the most frequent congenital infection in newborns. The objectives of this study were to determine the genetic variability and genotypes of HCMV gB gene in Costa Rica. Samples were collected from alcoholics, pregnant women, blood donors, AIDS patients, hematology-oncology (HO) children and HCMV isolates from neonates with cytomegalic inclusion disease. A semi-nested PCR system was used to obtain a product of 293-296 bp of the gB gene to be analyzed by Single Stranded Conformational Polymorphism (SSCP) and sequencing to determine the genetic polymorphic pattern and genotypes, respectively. AIDS patients showed the highest polymorphic diversity with 14 different patterns while fifty-six percent of HO children samples showed the same polymorphic pattern, suggesting in this group a possible nosocomial infection. In neonates three genotypes (gB1, gB2 and gB3), were determined while AIDS patients and blood donors only showed one (gB2). Of all samples analyzed only genotypes gB1, 2 and 3 were determined, genotype gB2 was the most frequent (73%) and mixed infections were not detected. The results of the study indicate that SSCP could be an important tool to detect HCMV intra-hospital infections and suggests a need to include additional study populations to better determine the genotype diversity and prevalence.

Human herpesvirus-7 as a cause of exanthematous ilnesses in Belém, Pará, Brazil

We screened sera from 370 patients suffering from exanthematous illnesses in Belém, North Brazil, for the presence of human herpesvirus-7 (HHV-7) IgM and IgG antibodies. Samples were obtained from January 1996 to December 2002 and were processed by a HHV-7-specific indirect immunofluorescence assay (IFA). HHV-7-specific IgM and/or IgG antibodies were found in 190 (51.4%) of these patients, with similar prevalence rates (IgM+ and IgG+ subgroups taken together) for female and male subjects: 52.5% and 50.3%, respectively. Serological status as defined by IgG was identified in 135 (36.5%) patients. In 55 (14.9%) of the patients HHV-7 IgM antibodies were detected. HHV-7 IgM- and- IgG antibody rates were similar (p > 0.05) when male and female subjects are compared: 14.4% versus 15.3% and 38.1% versus 35.0%, respectively. Statistically significant difference (p = 0.003) was noted when HHV-7-IgM-positive female and male patients aged 5-8 months are compared. Prevalence rates ranging from 4.6% (female, 5-8 months of age) to 93.3% (female, > 10 years of age) and 12.2% (male, 5-8 months) to 80.0% (male, 8-10 years of age) were noted in the IgG- positive subgroups. A subgroup (n = 131) of patients with IgM or IgG HHV-7 antibodies were examined for the presence of DNA using a polymerase chain reaction/nested PCR. Recent/active HHV-7 infection occurred at a rate of 11.0% (6/55) among patients whose samples presented IgM+ specific antibodies. In a subgroup (n = 76) of patients with high HHV-7-IgG antibody levels (titre > 1:160) DNA could not be detected in sera examined by PCR/nested PCR. Of the six recent/active infections, four subjects with less than 1 year and two with 3 and 6 years of age, presented typical exanthem subitum (E.S), as defined by higher fever (> 38.0 &ordm;C) with duration of 24 to 72 hours, followed by a maculopapular skin rash. Our results underscore the need for searching HHV-7 infection in patients with exanthematous diseases, particularly those presenting with typical E.S. HHV-7 appears therefore to emerge as a newly recognized pathogen of exanthem in our region.

Analysis of HIV- type 1 protease and reverse transcriptase in Brazilian children failing highly active antiretroviral therapy (HAART)

The aim of this study was to evaluate the genotypic resistance profiles of HIV-1 in children failing highly active antiretroviral therapy (HAART). Forty-one children (median age = 67 months) receiving HAART were submitted to genotypic testing when virological failure was detected. cDNA was extracted from PBMCs and amplified by nested PCR for the reverse transcriptase and protease regions of the pol gene. Drug resistance genotypes were determined from DNA sequencing. According to the genotypic analysis, 12/36 (33.3%) and 6/36 (16.6%) children showed resistance and possible resistance, respectively, to ZDV; 5/36 (14%) and 4/36 (11.1%), respectively, showed resistance and possible resistance to ddI; 4/36 (11.1%) showed resistance to 3TC and D4T; and 3/36 (8.3%) showed resistance to Abacavir. A high percentage (54%) of children exhibited mutations conferring resistance to NNRTI class drugs. Respective rates of resistance and possible resistance to PIs were: RTV (12.2%, 7.3%); APV (2.4%, 12.1%); SQV(0%, 12.1%); IDV (14.6%, 4.9%), NFV (22%, 4.9%), LPV/RTV (2.4%, 12.1%). Overall, 37/41 (90%) children exhibited virus with mutations related to drug resistance, while 9% exhibited resistance to all three antiretroviral drug classes.

PCR-based identification of Burkholderia pseudomallei

DNA amplification techniques are being used increasingly in clinical laboratories to confirm the identity of medically important bacteria. A PCR-based identification method has been in use in our centre for 10 years for Burkholderia pseudomallei and was used to confirm the identity of bacteria isolated from cases of melioidosis in Ceará since 2003. This particular method has been used as a reference standard for less discriminatory methods. In this study we evaluated three PCR-based methods of B. pseudomallei identification and used DNA sequencing to resolve discrepancies between PCR-based results and phenotypic identification methods. The established semi-nested PCR protocol for B. pseudomallei 16-23s spacer region produced a consistent negative result for one of our 100 test isolates (BCC #99), but correctly identified all 71 other B. pseudomallei isolates tested. Anomalous sequence variation was detected at the inner, reverse primer binding site for this method. PCR methods were developed for detection of two other B. pseudomallei bacterial metabolic genes. The conventional lpxO PCR protocol had a sensitivity of 0.89 and a specificity of 1.00, while a real-time lpxO protocol performed even better with sensitivity and specificity of 1.00, and 1.00. This method identified all B. pseudomallei isolates including the PCR-negative discrepant isolate. The phaC PCR protocol detected the gene in all B. pseudomallei and all but three B. cepacia isolates, making this method unsuitable for PCR-based identification of B. pseudomallei. This experience with PCR-based B. pseudomallei identification methods indicates that single PCR targets should be used with caution for identification of these bacteria, and need to be interpreted alongside phenotypic and alternative molecular methods such as gene sequencing.

Comparison of serology, antigenemia assay and the polymerase chain reaction for monitoring active cytomegalovirus infections in hematopoietic stem cell transplantation patients

Forty-six allogeneic hematopoietic stem cell transplantation (HSCT) patients were monitored for the presence of CMV antibodies, CMV-DNA and CMV antigens after transplantation. Immunoenzymatic serological tests were used to detect IgM and the increase in CMV IgG antibodies (increase IgG), a nested polymerase chain reaction (N-PCR) was used to detect CMV-DNA, and an antigenemia assay (AGM) was used to detect CMV antigens. The presence of CMV-IgM and/or CMV-increase IgG antibodies was detected in 12/46 (26.1%) patients, with a median time between HSCT and the detection of positive serology of 81.5 days. A positive AGM was detected in 24/46 (52.2%) patients, with a median time between HSCT and antigen detection of 62 days. Two or more consecutive positive N-PCR results were detected in 32/46 (69.5%) patients, with a median time between HSCT and the first positive PCR of 50.5 days. These results confirmed that AGM and mainly PCR are superior to serology for the early diagnosis of CMV infection. Six patients had CMV-IgM and/or CMV-increase IgG with a negative AGM (five cases) or N-PCR assay (one case). In five of these cases the serological markers were detected during the first 100 days after HSCT, the period of highest risk. These findings support the idea that serology may be useful for monitoring CMV infections in HSCT patients, especially when PCR is unavailable.

Molecular screening of Plasmodium sp. asymptomatic carriers among transfusion centers from Brazilian Amazon region

The transmission of malaria in Brazil is heterogeneous throughout endemic areas and the presence of asymptomatic Plasmodium sp. carriers (APCs) in the Brazilian Amazon has already been demonstrated. Malaria screening in blood banks is based on the selection of donors in respect to possible risks associated with travel or residence, clinical evidence and/or inaccurate diagnostic methods thereby increasing the probability of transfusion-transmitted infection. We evaluated the frequency of APCs in four blood services in distinct areas of the Brazilian Amazon region. DNA was obtained from 400 human blood samples for testing using the phenol-chloroform method followed by a nested-PCR protocol with species-specific primers. The positivity rate varied from 1 to 3% of blood donors from the four areas with an average of 2.3%. All positive individuals had mixed infections for Plasmodium vivax and Plasmodium falciparum. No significant differences in the results were detected among these areas; the majority of cases originated from the transfusion centres of Porto Velho, Rondônia State and Macapá, Amapá State. Although it is still unclear whether APC individuals may act as reservoirs of the parasite, efficient screening of APCs and malaria patients in Brazilian blood services from endemic areas needs to be improved.

Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6)

HHV-6 is the etiological agent of Exanthem subitum which is considered the sixth most frequent disease in infancy. In immuno-compromised hosts, reactivation of latent HHV-6 infection may cause severe acute disease. We developed a Sybr Green Real Time PCR for HHV-6 and compared the results with nested conventional PCR. A 214 pb PCR derived fragment was cloned using pGEM-T easy from Promega system. Subsequently, serial dilutions were made in a pool of negative leucocytes from 10-6 ng/µL (equivalent to 2465.8 molecules/µL) to 10-9 (equivalent to 2.46 molecules/µL). Dilutions of the plasmid were amplified by Sybr Green Real Time PCR, using primers HHV3 (5' TTG TGC GGG TCC GTT CCC ATC ATA 3)'and HHV4 (5' TCG GGA TAG AAA AAC CTA ATC CCT 3') and by conventional nested PCR using primers HHV1 (outer): 5'CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (outer): 5' ACA TCT ATA ATT TTA GAC GAT CCC 3'; HHV3 (inner) and HHV4 (inner) 3'. The detection threshold was determined by plasmid serial dilutions. Threshold for Sybr Green real time PCR was 24.6 molecules/µL and for the nested PCR was 2.46 molecules/µL. We chose the Real Time PCR for diagnosing and quantifying HHV-6 DNA from samples using the new Sybr Green chemistry due to its sensitivity and lower risk of contamination.

Cytomegalovirus in colorectal cancer and idiopathic ulcerative colitis

Cytomegalovirus (CMV) is a genus in the family Herpesviridae that has been associated with gastrointestinal syndromes. In this work we looked for a possible association of CMV infection with colorectal cancer and ulcerative colitis (UC). Blood and enteric tissue samples of 14 patients with colorectal cancer and of 21 with UC were subjected to a nested-PCR that amplifies part of the gB gene of CMV and also to immunohistochemistry using a specific monoclonal antibody to IE 76kDa protein of CMV. CMV was detected by nested-PCR in the blood and/or the enteric tissue of nine (64.3%) colorectal cancer and 16 (76.2%) ulcerative colitis patients. In the immunohistochemistry it was observed that 12 (12/21, 57.1%) positive enteric tissue samples of patients with UC and none from patients with colorectal cancer (0/14) were positive to CMV. The positivity of CMV infections in the UC patient group (12/21, 57.1%) showed by both techniques, was significantly higher (p = 0.015) than that observed for colorectal cancer patients (2/14, 14.3%). These results suggest an association of ulcerative colitis with CMV infection of the enteric tissue.

Genotypic identification of Cryptosporidium spp. isolated from hiv-infected patients and immunocompetent children of São Paulo, Brazil

Cryptosporidium isolates identified in fourteen stool samples, collected from five HIV-infected patients and nine immunocompetent children, living in the Sate of São Paulo, Brazil, were submitted to a molecular analysis using a nested PCR followed of restriction fragment length polymorphism (RFLP), for genetic characterization. The analysis was based on digestion with RsaI restriction enzyme of a DNA fragment amplified from the Cryptosporidium oocyst wall protein (COWP) gene. Based on this analysis, four samples were identified as Cryptosporidium parvum, eight as Cryptosporidium hominis and two presented a profile that correspondedto Cryptosporidium meleagridis when compared to the standards used in the analysis. The use of molecular methods can be helpful to identify source of infections and risk factors related to Cryptosporidium infection in our communities.

Pesquisa de Sequências do Cromossoma Y em Indivíduos com Síndroma de Turner

O síndroma de Turner (TS) tem sido descrito em associação com diversas anomalias dos cromossomas sexuais. Embora a maioria dos individuos com TS não apresentem evidência citogenética de sequências do cromossoma Y, diferentes autores consideram que algumas doentes com TS podem possuir uma linha celular minoritária contendo material do cromossoma Y, que não é detectada pela análise citogenética convencional. A identificação de moisacismos minoritários ou subrepresentados contendo o cromossoma Y é de importância fundamental em termos clínicos devido ao risco aumentado que estas doentes possuem para desenvolvimento de gonadoblastoma. No presente estudo procedeu-se à análise citogenética convencional de linfócitos de sangue periférico obtidos de 22 doentes com TS. Destas doentes, doze possuíam cariotipo 45,X, em sete foram detectados mosaicos com ou sem anomalias estruturais do cromossoma X e nas restantes três foram identificados os seguintes cariotipos: 46,X,i (X)(q10); 46,X,+mar/47,X,idic(Y),+mar e 45,X/46,X,+r. Os estudos moleculares foram realizados em DNA genómico obtido a partir de linfócitos de sangue periférico e de células de mucosa bucal, dois tecidos que derivam de folhetos embrionários diferentes, respectivamente, mesoderme e ectoderme. A pesquisa de moisacismos minoritários envolvendo o cromossoma Y foi efectuada por PCR simples e PCR nested para os seguintes loci específicos do cromossoma Y: SRY (sex determining region Y), TSPY (testis specific protein Y encoded), DYZ3 (locus centromérico) e DAZ1 (deleted in azoospermia). O uso de STSs localizados nos braços curto e longo do cromossoma Y permitiu a caracterização de um idic (Y)e de um cromossoma em anel, detectados em duas das doentes estudadas. A eleveda sensibilidade da PCR nested (1 célila masculina/125 000 células femininas) permitiu excluir a presença de moisacismos minoritários do Y em 20 das 22 doentes com TS. Na doente com um idic(Y) a análise por PCR simples foi posistiva para todos os loci estudados com excepção da região heterocromática. Este resultado permitiu identificar o ponto de quebra no braço longo entre sY158 e sY159, tendo-se confirmado por hibridação in situ de fluorescência (FISH), a duplicação da eurocromatina do braço longo, centrómero e braço curto do cromossoma Y. A caracterização do cromossoma em anel, detectado num das doentes com TS só foi possível por FISH e por PCR. Neste cromossoma, derivado de Y, foi detectada, no braço curto, a delecção da região pseudoautossómica 1 (PARY1)e, no braço longo, a delecção dos intervalos 6 e 7. Contudo, o referido cromossoma foi positivo para os loci SRY, RPS4Y, AMGY, TSPY localizados no braço curto, DYZ3 (centrómero) e, sY85, DFFRY, GY6, sY87, sY113, sY119, sY122, sY126 e RBMY1 localizados no braço longo do cromossoma Y. Este estudo permitiu, assim, excluir a presença de moisacismos minoritários do cromossoma Y em dois tecidos obtidos de 20 doentes com TS, e caracterizar por FISH e análise molecular, um idic(Y) e um cromossoma em anel, em que a natureza deste último não tinha sido identificada por análise citogenética convencional. O risco elevado de desenvolvimento de gonadoblastoma nos indivíduos com TS que possuem sequências do cromossoma Y justifica a aplicação de FISH e PCR para a caracterização de cromossomas marcadores e a utilização de PCR nested para a detecção de moisacismos minoritários do Y sempre que o material deste cromossoma não seja detectado pela análise citogenética convencional em doentes com cariotipo 45,X e/ou virilização.

Molecular characterization of Cryptosporidium spp. from HIV infected patients from an urban area of Brazil

Cryptosporidium spp. are important cause of enteric disease in humans, but may also infect animals. This study describes the relative frequency of several Cryptosporidium species found in human specimens from HIV infected patients in the São Paulo municipality obtained from January to July 2007. Sequence analysis of the products of nested-PCR based on small subunit rRNA and Cryptosporidium oocyst wall protein coding genes revealed 17 (63.0%) isolates of C. hominis, four (14.8%) C. parvum, five (18.5%) C. felis and one (3.7%) C. canis. These findings suggest that, in urban environments of Brazil, the cat adapted C. felis may play a potential role in the zoonotic transmission of cryptosporidiosis whereas the anthroponotic transmission of cryptosporidiosis caused by C. hominis seems to predominate.

Preliminary investigation of Culicidae species in South Pantanal, Brazil and their potential importance in arbovirus transmission

In view of the high circulation of migratory birds and the environmental and climatic conditions which favor the proliferation of arthropods, the Brazilian Pantanal is susceptible to circulation of arboviruses. However, the amount of data concerning arbovirus vectors in this area is scarce; therefore the aim of this study was to conduct a preliminary investigation of Culicidae species in the Nhecolândia Sub-region of South Pantanal, Brazil and their potential importance in the arbovirus transmission. A total of 3684 specimens of mosquitoes were captured, 1689 of which caught in the rainy season of 2007, were divided into 78 pools and submitted to viral isolation, Semi-Nested RT-PCR and Nested RT-PCR, with a view to identifying the most important arboviruses in Brazil. Simultaneously, 70 specimens of ticks found blood-feeding on horses were also submitted to the same virological assays. No virus was isolated and viral nucleic-acid detection by RT-PCR was also negative. Nevertheless, a total of 22 Culicidae species were identified, ten of which had previously been reported as vectors of important arboviruses. The diversity of species found blood-feeding on human and horse hosts together with the arboviruses circulation previously reported suggest that the Nhecolândia Sub-region of South Pantanal is an important area for arbovirus surveillance in Brazil.

Transmissão do vírus citomegálico através do aleitamento materno em prematurosÂ

RESUMO: Nas últimas quatro décadas, desenvolveram-se vários estudos, dispersos por todo o mundo, visando analisar a importância da transmissão perinatal do CMV pelo leite materno, nos recém-nascidos prematuros e de baixo peso à nascença. Comparando estes estudos, as taxas de incidência de infecção e doença são extremamente variáveis, não havendo consenso entre os autores. Surgiu, assim, a necessidade de realizar a presente dissertação, pioneira ao nível nacional, com o objectivo de determinar a incidência da infecção perinatal citomegálica, transmitida através do aleitamento materno a recémnascidos prematuros, e identificar as suas consequências clínicas. Para a elaboração deste trabalho foram seleccionados todos os recém-nascidos com idade gestacional inferior a 35 semanas e respectivas mães seropositivas para CMV, internados na Unidade de Cuidados Intensivos de Neonatalogia do Hospital de São Francisco Xavier, entre o período de 1 de Outubro de 2010 e 31 Julho de 2011. Foram analisadas as urinas dos recém-nascidos e o leite materno das mães na primeira, sexta e décima segunda semana pós-parto, utilizando a técnica de Nested-PCR e, posteriormente, a PCR em Tempo Real, para determinar a carga viral do leite materno. Constatou-se que a aquisição da infecção perinatal de CMV no recém-nascido prematuro ocorre com alguma frequência (40,5%), sendo muito provável que a via de transmissão em 38,1% destes casos seja o leite materno infectado, não devendo esta ser desvalorizada. Relativamente às consequências clínicas, não foram observadas alterações clínico-laboratoriais associadas à infecção citomegálica. Analisando os factores que condicionam a transmissão da infecção citomegálica perinatal, estabeleceu-se uma correlação entre a carga viral do leite materno e a transmissão do vírus, permitindo deduzir que quanto maior a carga viral, maior o risco de transmissão. Finalmente, os resultados obtidos sugerem que o risco de transmissão da infecção e suas consequências clínicas não justificam a contra-indicação da amamentação, pois esta comporta, também, elevados benefícios. ----------------ABSTRACT: In the last four decades, several studies were developed all over the world to analyze the importance of CMV perinatal transmission through mother´s milk in the preterm and low birthweight newborns. Comparing these studies, the infection and disease incidence rates are extremely variable, without agreement between the authors. The aim of this study, the first of the kind made in Portugal, is to define the rate of perinatal cytomegalovirus infection in preterm newborns via breastfeeding and its clinical consequences. All the newborns with gestational age below 35 weeks and their CMV seropositive mothers, admitted between the October 1, 2010 and July 31, 2011 in the Neonatology Intensive Care Unit of the São Francisco Xavier Hospital, were included in this study. Human breast milk and urine specimens were collected from mothers and their preterms infants around the 1st, 6th and 12th week after delivery and analyzed by Nested-PCR. The PCR in Real Time was used to determine the breast milk viral load. It was found that the CMV perinatal infection in preterm infants occurs in 40,5% of the cases and most likely 38,1% of these were infected via breastmilk and therefore this should not be overlooked. Considering the clinical consequences, no clinical and laboratory changes associated with cytomegalovirus infection were observed. Analyzing the factors that determine the perinatal transmission of the cytomegalovirus, it was established a correlation between breast milk viral load and viral transmission, allowing to conclude that the higher the viral load the greater the risk of transmission. Finally, the obtained results suggest that the risk of CMV infection and its clinical consequences don´t justify the breastfeeding contraindication because it also provides high benefits.

Efeito dos factores do hospedeiro e parasitários na susceptibilidade à Malária e gravidade da Doença

A malária é considerada como a doença parasitária mais séria em humanos, infectando cerca de 5 a 10% da população mundial, estimando-se entre 300 a 600 milhões de casos e mais de dois milhões de mortes, anualmente. Apesar da severidade da doença, a compreensão da variabilidade da resposta do hospedeiro à infecção (traduzida desde a infecção silenciosa até formas crónicas da doença, passando por quadros clínicos potencialmente fatais), continua a ser um dos grandes desafios da investigação médica. Vários factores genéticos parasitários ou do hospedeiro, estado imune e níveis de exposição, contribuem para esta variabilidade, mas a sua importância relativa para a carga total da doença tem sido pouco estudada. Entre outros, é possível salientar como fontes importantes de variabilidade na susceptibilidade à malária e gravidade da doença, factores intrínsecos ao hospedeiro tais como polimorfismos genéticos relacionados com as células sanguíneas e factores parasitários como a composição da(s) população(ões) parasitária(s) presentes na infecção. Factores do hospedeiro humano relacionados com as células sanguíneas (drepanocitose e deficiência na glucose-6-fosfato desidrogenase - G6PD) têm sido tradicionalmente estudados e relacionados com a gravidade da malária causada por Plasmodium falciparum. Relativamente às populações parasitárias, das cinco espécies que infectam humanos, P. falciparum continua a ser responsável pela malária grave, apesar de muito recentemente alguma gravidade ser atribuída a Plasmodium vivax. Sabe-se que nas mesmas áreas outras espécies coexistem em muito maior prevalência, contrariando o que se pensava há algum tempo. Apesar de poucos estudos terem focado o tema das infecções mistas, existem alguns relatos de que eventuais interacções entre as diferentes espécies presentes simultaneamente no mesmo hospedeiro poderem afectar a susceptibilidade à doença. Com o objectivo geral de avaliar o efeito e a contribuição destes factores na susceptibilidade e gravidade da malária, analisando e comparando três grupos (estudo de caso-controlo) doentes com malária grave (MG), doentes com malária não-complicada (Mnc) e indivíduos infectados assintomáticos (IA), realizámos em Angola de 2007 a 2010, um estudo em sete das 18 províncias com distintos níveis de endemicidade. Foram obtidas 1.416 amostras de sangue periférico de 1.198 indivíduos assintomáticos e 218 doentes. O DNA obtido a partir destes isolados foi utilizado para detecção da presença de variantes genéticos relacionados com os eritrócitos (drepanocitose – análise do gene HBB, deficiência G6PD – análise do gene G6PD, antigénio Duffy-análise do gene DARC) e identificação das espécies de Plasmodium presentes, através de nested-PCR mediante a amplificação dos genes que codificam a subunidade menor do RNA ribossomal. Os resultados demonstraram prevalências superiores às anteriormente descritas em relação às seguintes espécies parasitárias: P. falciparum 98,2% vs 92,0% e Plasmodium malariae 10,7% vs 1,0% e inferior em relação a P. vivax 2,5% vs 7,0%. Foi reconfirmada a presença de Plasmodium ovale (descrita anteriormente), mas não publicada em documentos oficiais e relatada pela primeira vez em Angola a presença de infecções mistas (duplas e triplas) (15,7%) e de infecção por P. vivax em indivíduos Duffy-negativos (dados publicados). Relativamente aos polimorfismos relacionados com os genes HBB e G6PD e provável associação com a protecção à malária, os nossos resultados confirmam a associação do traço falciforme (heterozigotia HbS), com a protecção à doença (OR = 0,30; IC 95% 0,18-0,49; p<0,001). Contudo, não foi encontrada, quer nos indivíduos hemizigóticos, quer nos heterozigóticos para o alelo G6PD (A-) nenhuma evidência para a protecção a malária (MG e Mnc) (OR = 1,69; IC 95% 0,91-3,13; p=0,096). Os resultados desta investigação requerem um estudo mais aprofundado, com uma dimensão amostral maior, necessário à confirmação da nossa observação.