Orlando C. Ferreira

Congenital Zika Virus Infection: Beyond Neonatal Microcephaly

Importance Recent studies have reported an increase in the number of fetuses and neonates with microcephaly whose mothers were infected with the Zika virus (ZIKV) during pregnancy. To our knowledge, most reports to date have focused on select aspects of the maternal or fetal infection and fetal effects. Objective To describe the prenatal evolution and perinatal outcomes of 11 neonates who had developmental abnormalities and neurological damage associated with ZIKV infection in Brazil. Design, Setting, and Participants We observed 11 infants with congenital ZIKV infection from gestation to 6 months in the state of Paraíba, Brazil. Ten of 11 women included in this study presented with symptoms of ZIKV infection during the first half of pregnancy, and all 11 had laboratory evidence of the infection in several tissues by serology or polymerase chain reaction. Brain damage was confirmed through intrauterine ultrasonography and was complemented by magnetic resonance imaging. Histopathological analysis was performed on the placenta and brain tissue from infants who died. The ZIKV genome was investigated in several tissues and sequenced for further phylogenetic analysis. Main Outcomes and Measures Description of the major lesions caused by ZIKV congenital infection. Results Of the 11 infants, 7 (63.6%) were female, and the median (SD) maternal age at delivery was 25 (6) years. Three of 11 neonates died, giving a perinatal mortality rate of 27.3%. The median (SD) cephalic perimeter at birth was 31 (3) cm, a value lower than the limit to consider a microcephaly case. In all patients, neurological impairments were identified, including microcephaly, a reduction in cerebral volume, ventriculomegaly, cerebellar hypoplasia, lissencephaly with hydrocephalus, and fetal akinesia deformation sequence (ie, arthrogryposis). Results of limited testing for other causes of microcephaly, such as genetic disorders and viral and bacterial infections, were negative, and the ZIKV genome was found in both maternal and neonatal tissues (eg, amniotic fluid, cord blood, placenta, and brain). Phylogenetic analyses showed an intrahost virus variation with some polymorphisms in envelope genes associated with different tissues. Conclusions and Relevance Combined findings from clinical, laboratory, imaging, and pathological examinations provided a more complete picture of the severe damage and developmental abnormalities caused by ZIKV infection than has been previously reported. The term congenital Zika syndrome is preferable to refer to these cases, as microcephaly is just one of the clinical signs of this congenital malformation disorder.

Evaluation of rapid tests for anti-HIV detection in Brazil

Objectives:This assessment in Brazil was to evaluate the performance of commercially available HIV rapid test (RT) against the gold standard testing and to establish a highly sensitive and specific RT algorithm for HIV diagnosis. Design:A prospective, anonymous and unlinked study. Methods:An evaluation of seven commercially available RT to compare their performance against the gold standard tests for Brazil. This includes two competing enzyme immunoassays plus a Western blot for confirmation. After informed consent, whole blood samples were collected from volunteers in voluntary counselling and testing sites (n = 400), antenatal clinics (n = 500) and from HIV-positive controls in AIDS treatment centres (n = 200). Two seroconversion panels, one HIV-1 subtype (B, B′, C and F) panel and an operational assay performance evaluation were also part of the study parameters. Results:For the seven RT the clinical sensitivity ranged from 97.74 to 100% and clinical specificity from 99.43 to 100%. However, only four RT were considered acceptable after full evaluation. The two EIA had a clinical sensitivity of 100% and clinical specificity of 99.32 and 99.66%. Two RT had the same performance on the seroconversions panels as the EIA. The operational assay performance evaluation for the RT indicated that Hexagon and Capillus could not be classified as simple assays. Conclusion:We have provided evidence that RT assays can perform equally or better than EIA for the detection of HIV antibodies. The simplicity and rapidity of the RT warrants its utilization in an algorithm for a rapid diagnosis of HIV infection.

Two lineages of dengue virus type 2, Brazil.

To the Editor: Dengue viruses (DENVs) belong to the genus Flavivirus (family Flaviviridae) and exist as 4 antigenic types, serotypes 1–4, each with well-defined genotypes. Dengue virus is associated with clinical manifestations that range from asymptomatic infections and relatively mild disease (classic dengue fever) to more severe forms of dengue hemorrhagic fever and dengue shock syndrome. Dengue has become one of the most serious vector-borne diseases in humans. The World Health Organization estimates that 2.5 billion persons live in dengue-endemic areas and >50 million are infected annually (1). In 1986, dengue virus type 1 (DENV-1) caused an outbreak in the state of Rio de Janeiro and has since become a public health concern and threat in Brazil. (2). In 1990, DENV-2 was reported in the state of Rio de Janeiro, where the first severe forms of dengue hemorrhagic fever and fatal cases of dengue shock syndrome were documented. The disease gradually spread to other regions of the country (3). In 2002, DENV-3 caused the most severe dengue outbreak in the country and sporadic outbreaks continued to be documented through 2005 (4). Since 1990, two additional epidemics caused by DENV-2 have occurred (1998 and 2007–2008) in Brazil. A severe DENV-2 epidemic in the state of Rio de Janeiro began in 2007 and continued in 2008; a total of 255,818 cases and 252 deaths were reported (5). This epidemic prompted us to investigate the genetic relatedness of DENV-2 for all of these epidemics. DENV-2 isolates from these epidemic periods were subjected to sequencing and comparison. Gross sequences of DENV-2 isolates from all epidemic periods grouped with sequences from DENV-2 American/Asian genotype; this finding was expected because this genotype is circulating in the Americas (6,7). Sequences of DENV-2 isolates from the 1998 epidemic grouped with sequences of DENV-2 isolates from the 1990 epidemic (data not shown) suggesting that viruses circulating during these 2 epidemic periods belong to the same lineage of the DENV-2 strain originally found in the state of Rio de Janeiro. However, sequences of DENV-2 isolates from 2007/2008 epidemics grouped separately and distinctly from the 1990 and 1998 DENV-2 isolates and represented a monophyletic group in the phylogenetic tree with bootstraps of 98% (Figure). This result shows a temporal circulation of genetically different viruses in Rio de Janeiro that could be a result of local evolution of DENV-2 since its introduction in 1990, or even an introduction of a new lineage of DENV-2 in the region. Figure Neighbor-joining phylogenetic tree of 68 complete envelope (E) gene sequences of dengue virus type 2 (DENV-2). Only bootstrap values >80% are shown. DENV-2 sequences obtained from 21 patients infected during the 1990, 1998, and 2007–2008 ... A study conducted by Aquino et al. (7) showed that Paraguayan DENV-2 strains could be grouped as 2 distinct variants within the American/Asian genotype, thus further supporting that the introduction of new DENV-2 variants may likely associate with the shift of dominant serotypes from DENV-3 to DENV-2 in 2005 and might have caused an outbreak of DENV-2. Our results are consistent with this scenario because was a shift of a dominant serotype from DENV-3 to DENV-2 that was observed in 2008 in Rio de Janeiro. However, other factors, such as immunity level to DENV-3 and DENV-2, could explain the shift of dominant serotype besides the circulation of a new viral variant. Because the dengue outbreaks of 2007 and 2008 were the most severe of the dengue infections in Brazil in terms of number of cases and deaths, this genetically distinct DENV-2 could have contributed to this pathogenic profile. Additionally, these samples came from disperse locations in Rio de Janeiro and we do not believe that there is a clustering issue in our sampling. However, again, other factors must be considered as contributors to this scenario because of the intrinsic properties of this distinct virus, host susceptibility, and secondary cases of infection. In addition, detailed examination of amino acid sequences of Brazilian DENV-2 strains isolated in 1998 and 2008 showed 6 aa substitutions in the envelope gene: V129I, L131Q, I170T, E203D, M340T, and I380V. Our results support the notion that aa positions at 129 and 131 in the envelope gene are critical genetic markers for phylogenetic classification of DENV-2 (7–9). Notably, residue 131 in the envelope gene is located within a pH-dependent hinge region at the interface between domains I and II of the envelope protein. Mutations at this region may affect the pH threshold of fusion and the process of conformational changes (10). Our results suggest the circulation of genetically different DENV-2 in Brazil and that these viruses may have a role in severity of dengue diseases. These findings can help to further understand the complex dynamic pathogenic profile of dengue viruses and their circulation in dengue-endemic regions.

Human T-lymphotropic virus type I and type II infections and correlation with risk factors in blood donors from São Paulo, Brazil

BACKGROUND: Little is known about the prevalence of and risk factors for human T‐lymphotropic virus type I and type II (HTLV‐I, HTLV‐II) infections in Brazil. STUDY DESIGN AND METHODS: Sera from 17,063 healthy Brazilian donors were screened by enzyme‐linked immunosorbent assay for antibody to HTLV‐I/II between August 1991 and July 1993. Repeatedly reactive samples were confirmed by Western blot, and discrimination between HTLV‐I and HTLV‐II was made by polymerase chain reaction or synthetic peptide enzyme‐linked immunosorbent assay. A univariate analysis was performed on demographic and serologic data. RESULTS: HTLV‐I infection was demonstrated in 83 percent of the 30 donors with reactive serologic tests (0.15% of the total tested [17,063]; 95% CI, 0.09‐0.20) and HTLV‐II infection in 17 percent (0.03% of the total tested [17,063]; 95% CI, 0.01‐0.05). HTLV‐I‐positive donors were more likely than reference groups to be of Asian ethnicity (odds ratio [OR] 15.1; reference group: whites), more than 50 years old (OR 4.2; reference group: 20–29 years old), and positive for antibody to hepatitis C virus (anti‐HCV) (OR 21.8) or to hepatitis B core (antigen) (anti‐HBc) (OR 5.7). HTLV‐II showed a significant association with anti‐HCV (OR 75.2) and anti‐HBc (OR 21.8). Eleven of the 25 HTLV‐I‐ positive donors were counseled. Family origin in endemic areas of Japan (n = 4), prior blood transfusion (n = 3), or sexual contact with prostitutes (n = 1) were the risk factors reported by 8 donors. In 3 white men, no risk factors could be identified. CONCLUSION: Both HTLV‐I and HTLV‐II occur among Brazilian blood donors. HTLV‐I is associated with Asian ethnicity, greater age, and the presence of anti‐HCV and anti‐HBc. Three HTLV‐I‐positive donors had a history of blood transfusion, which emphasizes the need for HTLV‐I/II screening in Brazil.

Evaluation of three methods for hemoglobin measurement in a blood donor setting

CONTEXT The hemoglobin (Hb) level is the most-used parameter for screening blood donors for the presence of anemia, one of the most-used methods for measuring Hb levels is based on photometric detection of cyanmetahemoglobin, as an alternative to this technology, HemoCue has developed a photometric method based on the determination of azide metahemoglobin. OBJECTIVE To evaluate the performance of three methods for hemoglobin (Hb) determination in a blood bank setting. DESIGN Prospective study utilizing blood samples to compare methods for Hb determination. SETTING Hemotherapy Service of the Hospital Israelita Albert Einstein, a private institution in the tertiary health care system. SAMPLE Serial blood samples were collected from 259 individuals during the period from March to June 1996. MAIN MEASUREMENTS Test performances and their comparisons were assessed by the analysis of coefficients of variation (CV), linear regression and mean differences. RESULTS The CV for the three methods were: Coulter 0.68%, Cobas 0.82% and HemoCue 0.69%. There was no difference between the mean Hb determination for the three methods (p>0.05). The Coulter and Cobas methods showed the best agreement and the HemoCue method gave a lower Hb determination when compared to both the Coulter and Cobas methods. However, pairs of methods involving the HemoCue seem to have narrower limits of agreement (+/- 0.78 and +/- 1.02) than the Coulter and Cobas combination (+/- 1.13). CONCLUSION The three methods provide good agreement for hemoglobin determination.

High HIV incidence in the postpartum period sustains vertical transmission in settings with generalized epidemics: a cohort study in Southern Mozambique

Acute infection with HIV in the postpartum period results in a high risk of vertical transmission through breastfeeding. A study was done to determine the HIV incidence rate and associated risk factors among postpartum women in Southern Mozambique, where HIV prevalence among pregnant women is 21%.

Serologic and molecular typing of human T-lymphotropic virus among blood donors in Maputo City, Mozambique

BACKGROUND: Screening for human T‐lymphotropic virus‐1/2 (HTLV‐1/2) infection is not performed in blood banks in Mozambique. The aim was to determine the prevalence of HTLV‐1/2 among blood donors of the Maputo Central Hospital Blood Bank and measure the coinfection rate of HTLV‐1/2 with human immunodeficiency virus (HIV), hepatitis B virus (HBV), and syphilis.

Frequency of human immunodeficiency virus type-2 in hiv infected patients in Maputo City, Mozambique

The HIV/AIDS pandemic is primarily caused by HIV-1. Another virus type, HIV-2, is found mainly in West African countries. We hypothesized that population migration and mobility in Africa may have facilitated the introduction and spreading of HIV-2 in Mozambique. The presence of HIV-2 has important implications for diagnosis and choice of treatment of HIV infection. Hence, the aim of this study was to estimate the prevalence of HIV-2 infection and its genotype in Maputo, Mozambique.HIV-infected individuals (N = 1,200) were consecutively enrolled and screened for IgG antibodies against HIV-1 gp41 and HIV-2 gp36 using peptide-based enzyme immunoassays (pepEIA). Specimens showing reactivity on the HIV-2 pepEIA were further tested using the INNO-LIA immunoblot assay and HIV-2 PCR targeting RT and PR genes. Subtype analysis of HIV-2 was based on the protease gene.After screening with HIV-2 pepEIA 1,168 were non-reactive and 32 were reactive to HIV-2 gp36 peptide. Of this total, 30 specimens were simultaneously reactive to gp41 and gp36 pepEIA while two samples reacted solely to gp36 peptide. Only three specimens containing antibodies against gp36 and gp105 on the INNO-LIA immunoblot assay were found to be positive by PCR to HIV-2 subtype A.The proportion of HIV-2 in Maputo City was 0.25% (90%CI 0.01-0.49). The HIV epidemic in Southern Mozambique is driven by HIV-1, with HIV-2 also circulating at a marginal rate. Surveillance program need to improve HIV-2 diagnosis and consider periodical survey aiming to monitor HIV-2 prevalence in the country.

KIR and a specific HLA-C gene are associated with susceptibility and resistance to hepatitis B virus infection in a Brazilian population

KIR and a specific HLA-C gene are associated with susceptibility and resistance to hepatitis B virus infection in a Brazilian population

Genetic Characterization of Human T-Cell Lymphotropic Virus Type 1 in Mozambique: Transcontinental Lineages Drive the HTLV-1 Endemic

Background Human T-Cell Lymphotropic Virus Type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). It has been estimated that 10–20 million people are infected worldwide, but no successful treatment is available. Recently, the epidemiology of this virus was addressed in blood donors from Maputo, showing rates from 0.9 to 1.2%. However, the origin and impact of HTLV endemic in this population is unknown. Objective To assess the HTLV-1 molecular epidemiology in Mozambique and to investigate their relationship with HTLV-1 lineages circulating worldwide. Methods Blood donors and HIV patients were screened for HTLV antibodies by using enzyme immunoassay, followed by Western Blot. PCR and sequencing of HTLV-1 LTR region were applied and genetic HTLV-1 subtypes were assigned by the neighbor-joining method. The mean genetic distance of Mozambican HTLV-1 lineages among the genetic clusters were determined. Human mitochondrial (mt) DNA analysis was performed and individuals classified in mtDNA haplogroups. Results LTR HTLV-1 analysis demonstrated that all isolates belong to the Transcontinental subgroup of the Cosmopolitan subtype. Mozambican HTLV-1 sequences had a high inter-strain genetic distance, reflecting in three major clusters. One cluster is associated with the South Africa sequences, one is related with Middle East and India strains and the third is a specific Mozambican cluster. Interestingly, 83.3% of HIV/HTLV-1 co-infection was observed in the Mozambican cluster. The human mtDNA haplotypes revealed that all belong to the African macrohaplogroup L with frequencies representatives of the country. Conclusions The Mozambican HTLV-1 genetic diversity detected in this study reveals that although the strains belong to the most prevalent and worldwide distributed Transcontinental subgroup of the Cosmopolitan subtype, there is a high HTLV diversity that could be correlated with at least 3 different HTLV-1 introductions in the country. The significant rate of HTLV-1a/HIV-1C co-infection, particularly in the Mozambican cluster, has important implications for the controls programs of both viruses.