Identification of body fluids—menstrual blood, saliva, and nasal secretions—over different periods of time, using mRNA

Background Forensic examination of biological samples started at the beginning of the twentieth century by applying the ABO blood group system in evidence related to crimes or human identification. In the present study, real-time PCR multiplex was used to identify dried and stored swabs (saliva, nasal secretions, and menstrual blood) through the target genes of saliva (histatin 3 and statherin), nasal secretions (statherin and BPIFA1), and menstrual blood (metalloproteinases 10 and 7). Results The expressions of histatin 3 and statherin in the dried saliva decreased over days of storage with a significant p value of <0.001. BPIFA1 was highly expressed in nasal secretions, and the expression level significantly decreased throughout the study with a significant p value of <0.001. The MMP7 and MMP10 genes were highly expressed in the menstrual blood, and the expression level decreased over days of storage with a significant p value of p<0.001. Conclusions Dried swabs of the saliva, Nasal secretions, Menstrual blood can be identified over the storage duration of the study using mRNA profiling of specific markers.

Avanços em testes genéticos pré-implantacionais: revisão de literatura

O desenvolvimento das tecnologias genéticas pré-implantacionais nos permite, hoje, identificar anomalias genéticas hereditárias, realizar triagem em busca de mutações e analisar previamente a viabilidade de um embrião, inclusive conhecer a compatibilidade genética embrionária com a de uma pessoa já nascida e doente, que necessite de um doador compatível. Os testes genéticos pré-implantacionais consistem em um conjunto de técnicas, realizadas após o processo de Fertilização in vitro (FIV) e antes da implantação do embrião. Devido à escassez de artigos na literatura brasileira sobre o assunto, foi desenvolvida uma revisão de literatura, que tem como objetivo explanar sobre os avanços que ocorreram nos últimos 30 anos nos testes utilizados para o diagnóstico genético pré-implantacional (DGPI), esclarecendo e apresentando seus protocolos. Para isso, foram selecionadas as principais técnicas adotadas, sendo elas Nested PCR, Multiplex PCR, qPCR, FISH, aCGH, SNP’s, NGS Ion Torrent e Illumina, além de contextualizar a cronologia dos testes e apresentar todo o contexto ético que os envolve. Para tanto, foram utilizadas 48 referências de língua inglesa, portuguesa e espanhola, em sua maioria datadas entre 2017 a 2021. É fato que o DGPI confere um grande avanço no campo da reprodução assistida e possibilita mulheres com idade avançada ou histórico recorrente de aborto, além de pessoas com anomalias genéticas, terem o DNA de seus embriões analisados antes de sua implantação, permitindo identificar mutações que poderão afetar a saúde do bebê e garantindo a sua compatibilidade com a vida.

Determining common variants in patients with haemophilia A in South Vietnam and screening female carriers in their family members

AimsThe aim of this study was to determine common variants in F8, including intron 22 inversion (Inv22), intron 1 inversion (Inv1) and point mutations, the transmission of these variants between patients with haemophilia A (HA) and their family members.MethodsGenetic analysis was conducted in 71 patients who were clinically diagnosed with HA and 152 related female members in South Vietnam by a combination of inversion PCR (I-PCR), multiplex PCR and direct sequencing.ResultsVariants in F8, including Inv22, point mutations (with 37 genotypes) and two novel variants, occupied 60 patients with HA. Among severe patients, the rate of Inv22 was 44%. Missense was the common point mutation of over 50% in patients with moderate HA and mild HA. Inv1 was absent in all patients. F8 variants were also found in 119 female carriers (FCs) (78.3%) from families related to patients with HA. There were 56 mothers (93.3%) carrying F8 variants and passing the same variants to their sons.ConclusionsThese findings were the first to provide important information about the presence of Inv22 and point mutation in Vietnamese patients with HA, the mothers and their female family members. It demonstrated that genetic diagnosis and counselling for HA carriers were essential factors for future improvements in comprehensive and equitable healthcare polices for patients with HA and FCs in Vietnam.

Viral and Immunologic Factors Associated with Fatal Outcome of Patients with Severe Fever with Thrombocytopenia Syndrome in Korea

Significant progress has been made on the molecular biology of the severe fever with thrombopenia virus (SFTSV); however, many parts of the pathophysiological mechanisms of mortality in SFTS remain unclear. In this study, we investigated virologic and immunologic factors for fatal outcomes of patients with SFTS. We prospectively enrolled SFTS patients admitted from July 2015 to October 2020. Plasma samples were subjected to SFTSV RNA RT-PCR, multiplex microbead immunoassay for 17 cytokines, and IFA assay. A total of 44 SFTS patients were enrolled, including 37 (84.1%) survivors and 7 (15.9%) non-survivors. Non-survivors had a 2.5 times higher plasma SFTSV load than survivors at admission (p < 0.001), and the viral load in non-survivors increased progressively during hospitalization. In addition, non-survivors did not develop adequate anti-SFTSV IgG, whereas survivors exhibited anti-SFTSV IgG during hospitalization. IFN-α, IL-10, IP-10, IFN-γ, IL-6, IL-8, MCP-1, MIP-1α, and G-CSF were significantly elevated in non-survivors compared to survivors and did not revert to normal ranges during hospitalization (p < 0.05). Severe signs of inflammation such as a high plasma concentration of IFN-α, IL-10, IP-10, IFN-γ, IL-6, IL-8, MCP-1, MIP-1α, and G-CSF, poor viral control, and inadequate antibody response during the disease course were associated with mortality in SFTS patients.

Characterization of Faecal Enterococci from Wild Birds in Turkey and Its Importance in Antimicrobial Resistance

This research aimed to investigate the diversity of faecal enterococci isolated from wild birds, to detecttheir antibiotic resistance patterns and to determine their distribution of genes related to vancomycin resistance. Additionally, to investigate their virulence factors that are important in the development of the disease. One hundred seven cloacal/rectal samples were inoculated onto Enterococcus Agar, and presumptive colonies were identified and confirmed by PCR. Multiplex PCR assays were used to screen vanA, vanB, vanC1 and vanC2/3. The virulence-related genes; ace, gelE, efa and agg were determined by PCR. Among the 103 enterococci, 62 E.faecalis, 23 E.faecium 3 E.gallinarum, 2 E.durans, 1 E.casseliflavus and 12 Enterococcus spp. were identified. Of the 103 enterococci, 26 were found to be resistant against to three or more antibiotics. The highest percentages were detected for chloramphenicol (52%), tetracycline (33%) and erythromycin (30%). Two E.gallinarum isolates were harboring three virulence factors, and one isolate was carrying a single virulence factor. There is no virulence factor in the E.casseliflavus isolate. Also, vanA and vanB genes were not found. Forty-two of 103 enterococci were harboring virulence factors, more frequently in E.faecalis. Forty-two enterococci carried efa A, 31 isolates carried gel E, and ace was found in 18 isolates. Virulence gene agg was not detected. When the results of the study were evaluated in general, multiple drug resistance was described as 25%. Considering the risk of polluting the water resources of wild animals, it is suggested that the continuity of this type of epidemiological study in wildlife animals is necessary. In conclusion, the wild birds may act as substantial reservoirs carrying antimicrobial resistance among enterococci and estimate the potential risk for man, pets and farm animals.

Molecular Detection and Characterization of Pasteurella multocida Infecting Camels in Marsabit and Turkana Counties, Kenya

BackgroundInfection with Pasteurella multocida is abundant in Kenya yet there is scarce information on their genetic diversity. Pasteurella multocida is considered to be one of the normal flora in the respiratory tract of camels and other animals but it becomes pathogenic and causes pasteurellosis when the resistance of the camel body is diminished by harmful environmental influences. Close herding, overwork, limited food supply, and wet climatic conditions are stresses that seem to speed the spread of the infection. Conventional PCR, Multiplex PCR and sequencing were applied to enhance identification of Pasteurella multocida at any level of specificity viz; strain, species, and genus. These molecular tools were applied to confirm the presence and genetic diversity of Pasteurella multocida in 102 blood and 30 nasal swab samples collected from Marsabit and Turkana counties in Kenya. Kmt1 gene was used as the marker gene for Pasteurella multocida and hyaD-hyaC, bcbD, dcbF, ecbJ, and fcbD as marker genes for capsular groups. A study done in northern Kenya noted that in Africa pasteurellosis infections causing death in camels (Camelus dromedarius) have been existing since 1890 though the real cause of this disease remains elusive and needs further study. The study was done to detect Pasteurella multocida and characterize its capsular types by application of molecular biology toolsResultsTwenty one Kenyan isolates were confirmed to be Pasteurella multocida and only capsular group E was detected in both counties. Pasteurella multocida sequences were found to be highly conserved, however isolates detected in Kenya were found to be genetically related to other isolates from African and other parts of the world. ConclusionsThe study confirm that the camels were infected by Pasteurella multocida of capsular type E in Marsabit and Turkana Counties of Kenya. DNA sequences were found to be homologous to Pasteurella multocida thereby confirming that the camels were infected by Pasteurella multocida.

Human Polyomaviruses (HPyV) in Wastewater and Environmental Samples from the Lisbon Metropolitan Area: Detection and Genetic Characterization of Viral Structural Protein-Coding Sequences

Due to the lack of reliable epidemiological information regarding the geographic distribution and genetic diversity of human polyomaviruses (HPyV) in Portugal, we addressed these issues in this initial study by focusing on the Lisbon Metropolitan area, the most populated and culturally diverse hub in the country. The HPyV structural protein-coding sequence was partially amplified using two touch-down PCR multiplex protocols, starting from water samples, collected between 2018 and 2020, where viral genomes were detected.. The obtained results disclosed the frequent detection of HPyV1, HPyV2, HPyV5, and HPyV6 in 35.3% (n = 6), 29.4% (n = 5), 47.1% (n = 8) and 29.4% (n = 5), respectively, of the water samples analyzed. The sequences assigned to a given viral species did not segregate to a single genotype, this being especially true for HPyV2 for which five genotypes (including a putative new genotype 9) could be identified. The phylogenetic trees obtained for HPyV5 and HPyV6 had less resolving power than those obtained for HPyV1/HPyV2, but both viruses were shown to be genetically diverse. This analysis emphasizes the epidemiological helpfulness of these detection/genetic characterization studies in addition to being relevant tools for assessment of human waste contamination.

The role of Escherichia coli in the etiology of piglet diarrhea in selected pig producing districts of central Uganda

Background: Pig production in Uganda is highly constrained by rampant piglet mortalities with diarrhea being a key feature. The present study was conducted to determine possible involvement of Escherichia coli (E. coli) as agents of diarrhea in piglets and elucidate the factors for their spread and virulence, towards development of mitigation strategies in the smallholder pig value chains in Uganda. Methodology: This was a cross-sectional study carried out from January to August 2020 on pre- and post-weaned piglets from households in Kayunga and Mityana districts of Central Uganda, selected by snowballing method to redundancy. Data about herd management and risk factors for colibacillosis were collected from selected farmers in the two districts. A total of 179 faecal samples were collected from randomly selected neonatal and pre-weaning piglets for bacteriological isolation of Escherichia coli. Virulence (enterotoxin and fimbrial) genes from the isolates were detected by multiplex polymerase chain reaction (PCR) assay. Results: From the 179 faecal samples, a total of 158 (88.3%) E. coli isolates were obtained. Virulence gene markers were detected in 18.4% (29/158) of the isolates. Among the investigated genes encoding for enterotoxin production, STb was the most prevalent (16/158, 10.13%), followed by STa (12/158, 7.59%), while gene for LT was not detected. The gene coding for F4 adhesin was the only one detected while F18 adhesin was not detected from the isolates. On multiple logistic regression analysis, only tertiary educational level (OR=0.141; 95% CI=0.30-0.666; p=0.013) and infrequent use of antibiotics (OR=0.231, 95% CI=0.062-0.859; p=0.029) among the farmers, were the two factors significantly protective of the piglets from diarrhoea. Conclusion: This study reports a high prevalence of enterotoxin gene markers among E. coli isolates in piglets and revealed the potential role of these bacteria in the aetiology of piglet diarrhoea and mortalities in Uganda. Additionally, this study identified risk factors that can be useful in formulating treatment and control strategies of infection caused by these bacteria. Further studies are needed to identify more adhesins these E. coli isolates employ for intestinal colonization, a step that will help inform vaccine development. French title: Le rôle d'Escherichia coli dans l'étiologie de la diarrhée des porcelets dans certains districts producteurs de porcs du centre de l'Ouganda Contexte: La production porcine en Ouganda est fortement limitée par la mortalité généralisée des porcelets, la diarrhée étant une caractéristique clé. La présente étude a été menée pour déterminer l'implication possible Escherichia coli piglet diarrhea in Uganda d'Escherichia coli (E. coli) en tant qu'agents de diarrhée chez les porcelets et élucider les facteurs de leur propagation et de leur virulence, vers le développement de stratégies d'atténuation dans les chaînes de valeur des petits producteurs de porcs en Ouganda. Méthodologie: Il s'agit d'une étude transversale réalisée de janvier à août 2020 sur des porcelets pré- et post-sevrés issus de ménages des districts de Kayunga et Mityana du centre de l'Ouganda, sélectionnés par la méthode boule de neige jusqu'à la redondance. Les données sur la gestion du troupeau et les facteurs de risque de colibacillose ont été recueillies auprès d'éleveurs sélectionnés dans les deux districts. Au total, 179 échantillons de matières fécales ont été prélevés sur des porcelets néonatals et en pré-sevrage sélectionnés au hasard pour l'isolement bactériologique d'Escherichia coli. Les gènes de virulence (entérotoxine et fimbrial) des isolats ont été détectés par une amplification en chaîne par polymérase (PCR) multiplex. Résultats: À partir des 179 échantillons de matières fécales, un total de 158 (88,3%) isolats d'E. coli ont été obtenus. Des marqueurs du gène de virulence ont été détectés dans 18,4% (29/158) des isolats. Parmi les gènes étudiés codant pour la production d'entérotoxines, STb était le plus répandu (16/158, 10,13%), suivi de STa (12/158, 7,59%), tandis que le gène de la LT n'a pas été détecté. Le gène codant pour l'adhésine F4 était le seul détecté alors que l'adhésine F18 n'a pas été détectée dans les isolats. Sur l'analyse de régression logistique multiple, seul le niveau d'enseignement supérieur (OR=0,141; IC à 95%=0,30-0,666; p=0,013) et l'utilisation peu fréquente d'antibiotiques (OR=0,231, IC à 95 %=0,062-0,859; p=0,029) parmi les éleveurs, étaient les deux facteurs de protection significative des porcelets contre la diarrhée. Conclusion: Cette étude rapporte une prévalence élevée de marqueurs génétiques d'entérotoxines parmi les isolats d'E. coli chez les porcelets et a révélé le rôle potentiel de ces bactéries dans l'étiologie de la diarrhée et de la mortalité des porcelets en Ouganda. De plus, cette étude a identifié des facteurs de risque qui peuvent être utiles dans la formulation de stratégies de traitement et de contrôle de l'infection causée par ces bactéries. D'autres études sont nécessaires pour identifier plus d'adhésines que ces isolats d'E. coli utilisent pour la colonisation intestinale, une étape qui aidera à éclairer le développement de vaccins.