Low rate of oropharyngeal human papillomavirus infection of women with cervical lesions and their partners: new data from Brazilian population.

BACKGROUND: Although the natural history of cervical and oral infection by human papillomavirus (HPV) has been intensely investigated, the ability of this virus to infect oral and genital mucosae in the same individual and its potential of communicability are still unclear. OBJECTIVES: This study aimed at assessing the presence of oropharyngeal HPV infection in women with cervical lesions and in their current sexual partners in a Brazilian population. METHODS: It included a total of 65 patients, 43 women and 22 male partners. Medical history and the sociobehavioral profile were assessed through interviews that included the association of oropharyngeal HPV and the sexual behavior of patients, and also extra and intra-oral examinations were performed. Brushing was used to collect cells from the oropharyngeal mucosa. HPV DNA was checked through nested PCR with primers PGMY09/11 and GP5+/6+, and Pappilocheck to genotyping. RESULTS: Oropharyngeal HPV infection was detected in four of 65 (6.15%) cases, with one of 43 (2.3%) women, and three of 22 (13.6%) male partners. Clinically no patient showed HPV-related oral lesions. Pappilocheck assay showed the absence of HPV genotype commonly found in cervical mucosa. Moreover, there was no correlation between the presence of oropharyngeal HPV and sexual behavior risk factors. CONCLUSIONS: The results suggest that the presence of cervical lesions does not lead to HPV oropharyngeal infection. It also highlights the low rate of HPV infection in the oropharyngeal mucosa of women with cervical lesions and their partners in a researched sample.

Allele frequencies for 15 autosomal STRs in a population sample from the Federal District (Brazil)-a territory that arose from nothing.

The Federal District (Brazil) was created in 1960 in the Central-West Region of Brazil in a previously unpopulated area. In 2010, this artificially founded district was populated by 2,562,963 inhabitants. In this study, the genetic variations of the 15 Next Generation Multiplex (NGM(TM)) short tandem repeat loci were analyzed. The results indicate that the NGM(TM) is a highly informative genetic system in this population, which is more similar to the southeastern, northeastern, and overall Brazil populations. This conclusion agrees with the population composition reported in the 2010 National Survey Inquiries, in which most of the immigrants were from the northeast and the southeast.

Haplotype diversity of 23 Y-chromosomal STRs in a population sample from the Federal District (Brazil)--a territory that arose from nothing.

In this study, the genetic variations of 23 short tandem repeats on the Y-chromosome were analyzed in a sample of 201 males from the Federal District (Brazil). The Federal District (Brazil) was built in 1960 in Brazil's Central West region, where there was no previous population. In 2010, the population of this artificially founded district consisted of 2,500,000 inhabitants. We observed 200 different haplotypes, 199 of which were unique and one of which occurred two times. The haplotype diversity was 0.9999, and the discrimination capacity was 0.995. The data are available in the Y chromosome haplotype reference database under accession number YA003843. The results were compared to the haplotypes from other Brazilian macroregions.

Clinical and molecular features of four Brazilian families with multiple endocrine neoplasia type 1.

Objective: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant syndrome characterized by its clinical variability and complexity in diagnosis and treatment. We performed both clinical and molecular descriptions of four families with MEN1 in a follow-up at a tertiary center in Brasília. Methods: From a preliminary review of approximately 500 medical records of patients with pituitary neuroendocrine tumor (PitNET) from the database of the Neuroendocrinology Outpatient Clinic of the University Hospital of Brasília, a total of 135 patients met the criteria of at least two affected family members. From this cohort, we have identified 34 families: only four with a phenotype of MEN1 and the other 30 families with the phenotype of familial isolated pituitary adenoma (FIPA). Eleven patients with a clinical diagnosis of MEN1 from these four families were selected. Results: Variants in MEN1 gene were identified in all families. One individual from each family underwent genetic testing using targeted high-throughput sequencing (HTS). All patients had primary hyperparathyroidism (PHPT), and the second most common manifestation was PitNET. One individual had well-differentiated liposarcoma, which has been previously reported in a single case of MEN1. Three variants previously described in the database and a novel variant in exon 2 have been found. Conclusions: The study allowed the genotypic and phenotypic characterization of families with MEN1 in a follow-up at a tertiary center in Brasília.

Detection of anti-SARS-CoV-2 salivary antibodies in vaccinated adults.

Since the introduction of efficient anti-SARS-CoV-2 vaccines, the detection of antibodies becomes useful for immunological monitoring and COVID-19 control. Therefore, this longitudinal study aimed to evaluate the detection of SARS-CoV-2 antibodies in the serum and saliva of COVID-19-vaccinated adults. The study included 13 not vaccinated and 35 vaccinated participants with two doses of CoronaVac (Sinovac/Butantan) vaccine who subsequently received BNT162b2 (Pfizer-BioNTech) vaccine as a booster dose. Vaccinated participants donated saliva and serum in three different time points. Enzyme-linked immunosorbent assay was used for antibody detection. In our results, the serum neutralizing antibodies (NAb) were detected in 34/35 samples after second dose and in 35/35 samples one and five months after the booster dose. In saliva, NAb were detected in 30/35 samples after second dose and in 35/35 of samples one and five months after the booster dose. IgA was detected in 19/34 saliva samples after second dose, in 18/35 one month after the booster and in 30/35 five months after. IgG in saliva was detected in 1/34 samples after second dose, 33/35 samples one month after the booster dose and in 20/35 five months after. A strong correlation was found between IgG and neutralizing activity in saliva, and salivary IgA would be a sign of recent exposure to the virus. In conclusion, saliva can be suitable for monitoring antibodies anti-SARS-CoV-2 after vaccination. Heterologous vaccination contributed to increase anti-SARS-CoV-2 antibodies in the Brazilian health context. Complementary studies with large groups are mandatory to conclude the interest in following mucosal immunity.

Overcoming Supply Shortage for SARS-CoV-2 Detection by RT-qPCR.

In February 2020, our laboratory started to offer a RT-qPCR assay for the qualitative detection of severe acute respiratory syndrome coronavirus 2. A few months after the assay was released to our patients, some materials, reagents, and equipment became in short supply. Alternative protocols were necessary in order to avoid stopping testing to the population. However, the suitability of these alternatives needs to be validated before their use. Here, we investigated if saliva is a reliable alternative specimen to nasopharyngeal swabs; if 0.45% saline is a reliable alternative to guanidine hydrochloride as a collection viral transport media; the stability of SARS-COV-2 in guanidine hydrochloride and in 0.45% saline for 10 and 50 days at room temperature; and if the primers/probe concentration and thermocycling times could be reduced so as to overcome the short supply of these reagents and equipment, without a significant loss of the assay performance. We found that saliva is not an appropriated specimen for our method-nasopharyngeal swabs perform better. Saline (0.45%) and guanidine hydrochloride have a similar SARS-CoV-2 diagnostic capability as tube additives. Reliable SARS-CoV-2 RNA detection can be performed after sample storage for 10 days at room temperature (18-23 °C) in both 0.45% saline and guanidine hydrochloride. Using synthetic RNA, and decreasing the concentration of primers by five-fold and probes by 2.5-fold, changed the assay limit of detection (LOD) from 7.2 copies/reaction to 23.7 copies/reaction and the subsequent reducing of thermocycling times changed the assay LOD from 23.7 copies/reaction to 44.2 copies/reaction. However, using real clinical samples with Cq values ranging from ~12.15 to ~36.46, the results of the three tested conditions were almost identical. These alterations will not affect the vast majority of diagnostics and increase the daily testing capability in 30% and increase primers and probe stocks in 500% and 250%, respectively. Taken together, the alternative protocols described here overcome the short supply of tubes, reagents and equipment during the SARS-CoV-2 pandemic, avoiding the collapse of test offering for the population: 105,757 samples were processed, and 25,156 SARS-CoV-2 diagnostics were performed from 9 May 2020 to 30 June 2020.

Worldwide prevalence of PI3K-AKT-mTOR pathway mutations in head and neck cancer: A systematic review and meta-analysis.

A systematic review (SR) and meta-analysis were conducted to determine the prevalence of PI3K-AKT-mTOR signaling pathway mutations in patients with head and neck cancer (HNC). Overall, 105 studies comprising 8630 patients and 1306 mutations were selected. The estimated mutations prevalence was 13 % for PIK3CA (95 % confidence interval [CI] = 11-14; I2 = 82 %; p < 0.0001), 4% for PTEN (95 % CI = 3-5; I2 = 55 %; p < 0.0001), 3% for MTOR (95 % CI = 2-4; I2 = 5%; p = 0.40), and 2% for AKT (95 % CI = 1-2; I2 = 50 %; p = 0.0001). We further stratified the available data of the participants according to risk factors and tumor characteristics, including HPV infection, tobacco use, alcohol exposure, TNM stage, and histological tumor differentiation, and performed subgroup analysis. We identified significant associations between PI3K-AKT-mTOR pathway-associated mutations and advanced TNM stage (odds ratio [OR] = 0.20; 95 % CI = 0.09-0.44; I² = 71 %; p = 0.0001) and oropharyngeal HPV-positive tumors and PIK3CA mutations (OR = 17.48; 95 % CI = 4.20-72.76; I² = 69 %; p < 0.0002). No associations were found between alcohol and tobacco exposure, and tumor differentiation grade. This SR demonstrated that the PI3K-AKT-mTOR pathway emerges as a potential prognostic factor and could offer a molecular basis for future studies on therapeutic targeting in HNC patients.

Luteinizing hormone beta mutation and hypogonadism in men and women.

Selective luteinizing hormone deficiency due to mutations in the luteinizing hormone beta-subunit gene (LHB) is a rare cause of hypogonadism. We describe the clinical features of a consanguineous family in which three siblings, two men and one woman, had hypogonadism related to isolated luteinizing hormone deficiency. These subjects have a newly discovered homozygous mutation of a 5' splice site in LHB: IVS2+1G-->C. This mutation disrupts the splicing of messenger RNA (mRNA), generating a gross abnormality in the processing of the luteinizing hormone beta-subunit mRNA, which abrogates the secretion of luteinizing hormone. We also determined that the female phenotype of this LHB mutation is characterized by normal pubertal development, secondary amenorrhea, and infertility.

Co-infection of SARS-CoV-2 and dengue virus: a clinical challenge.

Many regions of the world where dengue epidemics are seasonal are also facing the COVID-19 pandemic. This is a medical concern because both diseases are difficult to distinguish since they have similar clinical symptoms and laboratory findings, and because they have different clinical management. So far, co-infection of SARS-CoV-2 and dengue virus (DENV) has not been studied. Herein we report the first case of a patient with co-infection of COVID-19 and dengue. Both infections were simultaneously laboratory confirmed by positive RT-qPCR for SARS-CoV-2 and RT-qPCR for DENV, NS1, IgM and IgG antibody tests for dengue. The patient had a favorable clinical improvement, without severe symptoms. This case emphasize that, in pandemic era, having a diagnostic of one infection does not rule out the possibility of having another infection concomitantly. In addition, underscores the importance of an accurate and timely diagnosis to prevent the spread of COVID-19.

Analytical Sensitivity and Specificity of Two RT-qPCR Protocols for SARS-CoV-2 Detection Performed in an Automated Workflow.

WHO declared the novel coronavirus (COVID-19) outbreak a global pandemic on 11 March 2020. The establishment of standardized RT-qPCR protocols for respiratory secretions testing, as well as sharing of specimens, data, and information became critical. Here, we investigate the analytical performance of two interim RT-qPCR protocols (Charité and Centers for Disease Control (CDC)) for the qualitative detection of SARS-CoV-2 executed in a fully automated platform. Analytical specificity, PCR amplification efficiency, analytical sensitivity (limit of detection), and cross-reactivity were evaluated using contrived samples. The on-going accuracy was evaluated by retrospective analysis of our test results database (real clinical samples). N1, E, and a modified version of RdRP assays presented adequate analytical specificity, amplification efficiency, and analytical sensitivity using contrived samples. The three assays were applied to all individuals who requested the SARS-CoV-2 molecular test assay in our laboratory and it was observed that N1 gave more positive results than E, and E gave more positive results than RdRP (modified). The RdRP and E were removed from the test and its final version, based on N1 assay only, was applied to 30,699 Brazilian individuals (from 19 February 2020 to 8 May 2020). The aggregated test results available in the database were also presented.

Serum Has Higher Proportion of Janus Kinase 2 V617F Mutation Compared to Paired EDTA-Whole Blood Sample: A Model for Somatic Mutation Quantification Using qPCR and the 2-∆∆Cq Method.

Detection of the Janus Kinase-2 (JAK2) V617F mutation is a diagnostic criterion for myeloproliferative neoplasms, and high levels of mutant alleles are associated with worse outcomes. This mutation is usually tested on blood DNA by allele-specific qPCR (AS-qPCR) and measured using absolute quantification. However, some automated DNA extractions co-extracts of PCR inhibitors from blood and qPCR absolute quantification need increased efforts in order to maintain standard curves. JAK2 V617F can also be detected in serum using droplet digital PCR (ddPCR), a specimen with less inhibitors and favorable to automated extractions, but ddPCR instruments are not wide available as qPCR thermocyclers. Here, we evaluate whether JAK2 V617F could be accurately quantified by AS-qPCR using the 2-∆∆Cq method on blood DNA and validate the assay using gold-standard molecular diagnostic protocols. Next, we apply the validated method to assess if the mutation could be reliably detected/quantified in serum. JAK2 V617F could be quantified by AS-qPCR using the 2-∆∆Cq method-the assay was highly accurate (bias of 1.91%) compared to a commercial kit, highly precise (total CV% of 0.40%, 1.92%, 11.12% for samples with 93%, 54%, and 2.5% of mutant allele), highly sensitive (limit of detection of 0.15%), and demonstrated a linear detection response from 1.1% to 99.9%. Serum presented a higher mutant allele burden compared to the paired whole blood (mean of 4%), which allows for an increased JAK2 mutant detection rate and favors increased JAK2 V617F high-throughput analysis.

Correlation between salivary and serum CA15-3 concentrations in patients with breast cancer.

The early detection of breast cancer enables the use of less aggressive treatment and increases patient survival. The transmembrane glycoprotein mucin 1, which is also known as cancer antigen 15-3 (CA15-3), is aberrantly glycosylated and overexpressed in a variety of epithelial cancers, and serves a crucial role in the progression of the disease. CA15-3 is currently used as a marker of breast cancer. In the present study, CA15-3 concentrations in saliva and blood of patients with breast cancer were evaluated to test new assays to detect salivary CA15-3 in addition to ELISA and its diagnostic value. To the best of our knowledge, there are no previous reports of the use of chemiluminescence assay (CLIA) and electrochemiluminescence assay (ECLIA) in saliva. Saliva and blood were collected on the same day from patients with breast cancer (n=26) and healthy controls (n=28). For each subject, the level of serum CA15-3 was measured using ECLIA, and the level of salivary CA15-3 was measured using ECLIA, CLIA and enzyme-linked immunosorbent assay (ELISA). ELISA and CLIA were able to detect CA15-3 in saliva; however, ECLIA could not detect salivary CA15-3. There was no significant difference between the mean serum and salivary CA15-3 levels in patients with breast cancer or healthy controls. The levels of CA15-3 were highest for luminal breast cancer subtypes and stage IV cases. A moderate correlation was observed between salivary and serum CA15-3 levels as measured by ELISA in breast cancer patients (r=0.56; P=0.0047). The results demonstrated that ECLIA was not a good method to detect salivary CA15-3, although it is the gold standard for detecting serum CA15-3. The presence of CA15-3 in saliva was confirmed, and this will be useful in future research. Further investigations are necessary to confirm the ability to detect salivary CA15-3 and its correlation with serum CA15-3.

Genotyping of Single Nucleotide Polymorphisms Using Allele-Specific qPCR Producing Amplicons of Small Sizes Directly from Crude Serum Isolated from Capillary Blood by a Hand-Powered Paper Centrifuge.

The cell-free genomic DNA (gDNA) concentration in serum ranges from 1500 to 7500 copies/mL within 2 h after phlebotomy (6⁻24 times the concentration observed in plasma). Here, we aimed to evaluate the gDNA size distribution in serum with time after coagulation and to test if crude serum can be directly used as a source of gDNA for qPCR. Next, we investigated if single nucleotide polymorphisms (SNPs) could be genotyped directly from the crude serum isolated from capillary blood using a hand-powered paper centrifuge. All tested PCR targets (65, 100, 202 and 688 base pairs) could be successfully amplified from DNA extracted from serum, irrespective of their amplicon size. The observed qPCR quantitation cycles suggested that the genomic DNA yield increased in serum with incubation at room temperature. Additionally, only 65 and 101 base pair qPCR targets could be amplified from crude serum soon after the coagulation. Incubation for 4 days at room temperature was necessary for the amplification of PCR targets of 202 base pairs. The 688 base pair qPCR target could not be amplified from serum directly. Lastly, serum was successfully separated from capillary blood using the proposed paper centrifuge and the genotypes were assigned by testing the crude serum using allele-specific qPCR, producing small amplicon sizes in complete agreement with the genotypes assigned by testing the DNA extracted from whole blood. The serum can be used directly as the template in qPCR for SNP genotyping, especially if small amplicon sizes are applied. This shortcut in the SNP genotyping process could further molecular point-of-care diagnostics due to elimination of the DNA extraction step.

Flow Cytometric Analysis of Erythrocytes Osmotic Fragility in Hereditary Spherocytosis: A Case-Controlled Study Evaluating the Best Anticoagulant, Sample Pre-Treatment and NaCl Concentration for Reliable Screening of this Red Blood Cell Membrane Disorder.

BACKGROUND: The cytometric flow osmotic fragility test (FC-OFT) was recently introduced. However, the test is still under development and some variables have not yet been fully tested. METHODS: The osmotic fragility of hereditary spherocytosis (HS) cases and healthy controls were evaluated by FC-OFT using a series of tubes containing decreasing concentrations of NaCl. The analyses were executed in fresh and incubated (37°C for 24 h) blood samples anticoagulated with EDTA and heparin. The percentages of residual red blood cells were used to plot the osmotic fragility curves. The OF curves of each tested condition were compared using the median corpuscular fragility (MCF). ROC curve analyses identified the most accurate NaCl concentrations for differentiation between HS cases and healthy controls. RESULTS: FC-OFT curves assumed a sigmoidal dose-response shape and the MCF of cases and controls were different in all instances. MCF comparisons revealed that incubation and anticoagulant have major and minor effects on the FC-OFT, respectively. One hundred percent of sensitivity and specificity was obtained from 5.5 to 6.0 g/L of NaCl in EDTA-treated fresh blood, from 6.0 to 8.0 g/L of NaCl in EDTA-treated incubated blood, and in none of the tested NaCl concentration in heparinized blood. CONCLUSIONS: EDTA is the anticoagulant of choice for the assay. Incubation at 37°C for 24 h increased its diagnostic capability. The most reliable NaCl concentration for the discrimination of HS case from controls was 6.0 g/L of NaCL in fresh EDTA-treated blood, and was 7.5 g/L of NaCl in incubated EDTA-treated blood. © 2018 International Clinical Cytometry Society.

Association between suspected Zika virus disease during pregnancy and giving birth to a newborn with congenital microcephaly: a matched case-control study.

OBJECTIVE: In early 2015, an outbreak of an acute exanthematous illness with dengue-like symptoms occurred in northeastern Brazil. By the end of the same year, an unexpected increase in the number of cases of microcephaly was observed in the region. The microcephaly outbreak cause was unknown and rumors pointing to various potential causes arose. Since we were unaware at the time if this scenario would attract the interest of the broader scientific community, due to the neglected regions associated and as often happens with many others health conditions related to infectious diseases in Latin America. This coupled with the fact that diagnostic testing for Zika virus was not available, prompted us to design a study that could demonstrate the correlation between the development of an exanthematous illness with Zika-like symptoms during pregnancy and the delivery of a newborn with congenital microcephaly. RESULTS: Mothers who experienced symptoms associated with the Zika virus during pregnancy had 10 times higher odds of delivering newborns with congenital microcephaly when compared with mothers who did not exhibit Zika-like symptoms. Thus, the acute exanthematous illness outbreak could be associated with the congenital microcephaly outbreak. We could not distinguish which virus caused the acute exanthematous illness in the study subjects (Zika, dengue or chikungunya), but these results could help to reduce the misquided speculation in regards to the cause of the microcephaly and could have expedited public health policies intended for controlling the mosquito vector. In addition to the lower head circumference, microcephalic neonates also had lower thoracic circumference, lower height and lower weight compared to non-microcephalic babies suggesting intrauterine growth restriction. Additionally, we found borderline association between mothers classified as homemakers and, who had past dengue infections with microcephaly. Prior contraction of dengue virus seems to play a role in the risk for the condition reflecting the domestication of the Aedes Aegypti and the enhancement of the Zika virus infection by dengue antibodies, respectively. The limitations of this study are: (a) participants recall bias, (b) absence of laboratory test results for Zika virus and other arboviruses and (c) incomplete test results for other pathogens that could lead to microcephaly. The study protocol was registered at ClinicalTrial.gov under the identifier NCT02741882. Registered on April 13th, 2016.

Co-infection of SARS-CoV-2 and dengue virus: a clinical challenge

Abstract Many regions of the world where dengue epidemics are seasonal are also facing the COVID-19 pandemic. This is a medical concern because both diseases are difficult to distinguish since they have similar clinical symptoms and laboratory findings, and because they have different clinical management. So far, co-infection of SARS-CoV-2 and dengue virus (DENV) has not been studied. Herein we report the first case of a patient with co-infection of COVID-19 and dengue. Both infections were simultaneously laboratory confirmed by positive RT-qPCR for SARS-CoV-2 and RT-qPCR for DENV, NS1, IgM and IgG antibody tests for dengue. The patient had a favorable clinical improvement, without severe symptoms. This case emphasize that, in pandemic era, having a diagnostic of one infection does not rule out the possibility of having another infection concomitantly. In addition, underscores the importance of an accurate and timely diagnosis to prevent the spread of COVID-19.

Prevalence of the rs1801282 single nucleotide polymorphism of the PPARG gene in patients with metabolic syndrome.

OBJECTIVE: This study aimed to get the genotypic and allelic frequencies of rs1801282 in 179 volunteer donors and 154 patients with Metabolic syndrome (MetS) in Brasilia, Brazil and also examine the association with anthropometric, biochemical and hemodynamic variables in the latter group. MetS comprises a group of diseases resulting from insulin resistance, in-creased risk of type 2 diabetes and atherosclerotic cardiovascular disease. MetS is defined by the presence of increased visceral fat, atherogenic dyslipidemia (elevated triglycerides (TGL)), with decreased high density lipoprotein (HDL) and increased low density lipoprotein (LDL) levels, hypertension (BPH) and disturbances in glucose homeostasis representing a significant burden across the world due to the alarming increase in the incidence over the last decades besides their significant morbidity and mortality. Peroxisome proliferator activated receptor-gamma (PPARg) has been mentioned as a candidate gene for determining the risk of MetS. It is a member of the nuclear receptors superfamily and a ligand-activated transcription factor, which regulates the expression of genes involved in the network lipogenesis and adipogenesis, insulin sensitivity, energy balance, inflammation, angiogenesis and atherosclerosis. Among the PPARG genetic variants, single nucleotide polymorphism rs1801282 has been the most extensively studied one since it was first described by Yen and cols. in 1997. This polymorphism is characterized by the replacement of a proline (CCC) to an alanine (GCA) at codon 12 of exon B, due to the exchange of a cytosine with a guanine. The Ala allele frequency varies in different ethnic groups. MATERIALS AND METHODS: DNA was extracted using Chelex-100 method and determinations of genotypes were performed by allele-specific chain reaction. RESULTS: The distribution of genotype frequency of the MetS group was not statistically different from the frequency in the donor population at large. In the first group, genotype frequency was CC to 0.869 and 0.103 for CG, while allelic frequencies were 0.948 for C and 0.052 for G allele. In the group of donors, the genotype and allele frequencies were 0.882 for CC, 0.117 to CG; and 0.941 to 0.059 for G and C, respectively. GG genotype was not found in any of the two groups. The genotype distribution and allele frequencies were in Hardy-Weinberg equilibrium. No marker could be detected from the analysis of anthropometric, biochemical and hemodynamic variables in the MetS group. CONCLUSION: Our data suggest that this polymorphism is not correlated with predisposition to MetS. The results obtained on a small sample of the population of Brasilia, corroborate the data reported in the literature on the prevalence of this polymorphism in PPAR in populations of different ethnic origins.

Fetal male lineage determination by analysis of Y-chromosome STR haplotype in maternal plasma.

The aim of this study is to determine the fetus Y-STR haplotype in maternal plasma during pregnancy and estimate, non-invasively, if the alleged father and fetus belong to the same male lineage. The study enrolled couples with singleton pregnancies and known paternity. All participants signed informed consent and the local ethics committee approved the study. Peripheral blood was collected in EDTA tubes (mother) and in FTA paper (father). Maternal plasma DNA was extracted by using NucliSens EasyMAG. Fetal gender was determined by qPCR targeting DYS-14 in maternal plasma and it was also confirmed after the delivery. From all included volunteers, the first consecutive 20 mothers bearing male fetuses and 10 mothers bearing female fetuses were selected for the Y-STR analysis. The median gestational age was 12 weeks (range 12-36). All DNA samples were subjected to PCR amplification by PowerPlex Y23, ampFLSTR Yfiler, and two in-house multiplexes, which together accounts for 27 different Y-STR. The PCR products were detected with 3500 Genetic Analyzer and they were analyzed using GeneMapper-IDX. Fetuses' haplotypes (Yfiler format) were compared to other 5328 Brazilian haplotypes available on Y-chromosome haplotypes reference database (YHRD). As a result, between 22 and 27 loci were successfully amplified from maternal plasma in all 20 cases of male fetuses. None of the women bearing female fetuses had a falsely amplified Y-STR haplotype. The haplotype detected in maternal plasma completely matched the alleged father haplotype in 16 out of the 20 cases. Four cases showed single mismatches and they did not configure exclusions; 1 case showed a mutation in the DYS 458 locus due to the loss of one repeat unit and 3 cases showed one DYS 385I/II locus dropout. All mismatches were confirmed after the delivery. Seventeen fetuses' haplotypes were not found in YHRD and one of them had a mutation, which corresponded to the paternity probability of 99.9812% and 95.7028%, respectively. Three fetuses' haplotypes occurred twice in YHRD, which corresponded to paternity probability of 99.9437%. In conclusion, high discriminatory fetal Y-STR haplotype could be determined from maternal plasma during pregnancy starting at 12 weeks of gestation. All male fetuses could be attributed to the alleged father male lineage early in pregnancy. The high probability of paternity associated with each case suggests that the relationship is not random and this strategy can be use as an alternative for male fetal kinship analysis.

Cerebrospinal Fluid CRH Levels in Late Pregnancy Are Not Associated With New-Onset Postpartum Depressive Symptoms.

CONTEXT: CRH participates in the hypothalamic-pituitary-adrenal axis and in neural circuits involved in the pathophysiology of depression. During pregnancy, the placenta produces large amounts of CRH, and production ceases abruptly after delivery. The relationship between CRH in the cerebrospinal fluid (CSF) during pregnancy and peripartum mood disorders has not been investigated. OBJECTIVES: The objectives were to determine whether there are differences in CSF CRH concentrations of pregnant and nonpregnant women and whether CSF CRH concentrations in late pregnancy are associated with the presence of depressive symptoms during pregnancy and in the early postpartum period. DESIGN: This was a prospective cohort study conducted from January to April, 2011. SETTING: The study was conducted in one public and two private hospitals in Brasilia, Brazil. PATIENTS: Patients included 107 healthy pregnant women who underwent elective cesarean delivery and 22 nonpregnant healthy women who underwent spinal anesthesia for elective surgical sterilization. INTERVENTION: CRH in CSF was measured in pregnant and nonpregnant women by ELISA. MAIN OUTCOME MEASURE: The association between CSF CRH concentration at delivery and maternal depression assessed before cesarean section and postpartum (4 to 8 wk) with the Edinburgh Postnatal Depression Scale (EPDS), with a cutoff of ≥ 13. RESULTS: CRH concentration in the CSF was significantly higher in pregnant (4.1 ± 0.51 log CRH) than in nonpregnant women (3.6 ± 0.26 log CRH) (P < .001). Depressive symptoms starting after delivery occurred in 5.6% of women. CRH concentration in CSF was not different between women without depressive symptoms and women showing such symptoms during pregnancy or in the postpartum period. CONCLUSION: CRH concentration in the CSF was higher in pregnant women than in nonpregnant women. However, in this sample, CSF CRH in late pregnancy was not associated with new-onset depressive symptoms in the early postpartum period.