1. The study developed a new PCR/RFLP technique to identify the 3 genotypes of Plasmodium vivax circumsporozoite protein (VK210, VK247, and P. vivax-like) using DNA extracted from blood samples.
2. The technique uses PCR amplification of the central immunodominant region of the CSP gene followed by restriction enzyme digestion and fragment analysis to distinguish the genotypes.
3. Testing demonstrated the technique could accurately identify the genotypes using plasmid controls for each variant, and that it had high sensitivity detecting parasitemia levels as low as 0.0069 parasites per microliter.
This study sequenced the genomes of 11 clinical Mycobacterium abscessus isolates from 8 US patients with pulmonary infections. Core genome analysis compared these isolates to 30 globally diverse strains to investigate population structure. Longitudinally sampled isolates showed very few genetic differences, suggesting homogenous infection populations. Genome content variation between isolates was 0.3-8.3% compared to the reference strain, indicating plasticity.
Nuhu et al_Poster NAPA2016 correction and observationNuhu Tanko
The study determined the prevalence and genetic profiles of ESBL-producing uropathogens among members of the Enterobacteriaceae family at Specialist Hospital Sokoto, Nigeria. A total of 64 Gram-negative uropathogens were isolated from 365 urine samples, with E. coli and Salmonella arizonae being most prevalent. The isolates showed high resistance to cotrimoxazole, nalidixic acid, ciprofloxacin and norfloxacin. 64.1% of isolates were multidrug resistant. ESBL production was detected in 23.4% of isolates. PCR analysis showed 73.3% of ESBL producers contained the blaCTX-M gene and 26.7
Candidemia in HIV-positive patients in Dschang District Hospital (West Region...Claude Nangwat
Candidemia has been identified as a public health problem in HIV-infected patients. The evaluation of CD4 count, transaminases and blood glucose, are being used as a means to monitor the health of HIV-infected patients, without excluding the diagnosis of candidemia and other opportunistic infections. In order to contribute in improving the care of HIV-infected patients attending Dschang District Hospital and later on, in other hospitals in Cameroon, we conducted from June to September 2014 a cross-sectional study, with general objective; to determine the association between candidemia and selected biochemical and haematological parameter changes in HIV-infected patients, as a possible indicator in monitoring HIV disease progression.
To do this, blood samples were collected from HIV-infected patients assigned to the UPEC of Dschang District Hospital for follow up, and haemogram report, CD4 counts, ALAT level, ASAT level, and glucose level in blood were evaluated by cytometric and spectrophotometric assays. Candida species were isolated from some blood samples, and then identified using CHROMagar Candida culture medium. The broth microdilution method was afterwards used to test the susceptibility of the fungal isolates vis-a-vis three conventional antifungal agents.
Mycological analysis of blood samples showed that eight (08) patients had candidemia, a prevalence of 6.11%. Eight (08) isolates were obtained from these eight (08) candidemic HIV-infected patients; this consisted of 4(50%) Candida albicans, 3(37.5%) Candida parapsilosis and 1(12.5%) Candida glabrata. All these isolates were resistant (MICs ranged from 2 to >256 µg/mL) to the antifungals used, that is, ketoconazole, amphotericin B and nystatin.
A significant correlation was found between candidemia and white blood cell count, with a correlation coefficient of r = 0.240 (p < 0.05). Based on the results obtained, the systematic diagnosis of candidemia should be performed in patients infected with HIV in Cameroon in order to improve on their care.
Key words: Candidemia, HIV, biochemical parameters, hematological parameters, Antifungals activities.
1. The document provides information about the educational and professional background of an individual, including degrees obtained from various institutions between 1973-2013 related to prenatal diagnosis, genetics, and rare syndromes.
2. It discusses various levels of genetics from DNA to populations and highlights key concepts like meiosis, mitosis, chromosomes, and mitochondrial DNA.
3. Genetics and genomics provides information at the cellular level about structure, expression, regulation and pathways/networks, but understanding the whole organism is still incomplete and requires integration of concepts from other fields like physics, chemistry and mathematics.
This study investigated the relationship between variants of the glycophorin B (GPB) gene and susceptibility to Plasmodium falciparum infection in the Brazilian Amazon. The researchers found that individuals carrying the GYPB*S allele were more likely to be infected with P. falciparum than those without this allele. Sequence analysis of GYPB also suggested it has been under natural selection due to malaria. This study provides evidence that genetic variation in GPB receptor influences the ability of P. falciparum to invade red blood cells in this population.
This document contains a summary of Sandra G. Nishikawa's skills and experience. She has over 30 years of experience in molecular biology techniques including DNA/RNA isolation, PCR, cloning, sequencing, cell culture and more. She has worked as a research technician for various professors at the University of Calgary studying topics like hepatitis B virus, prion diseases, and cancer.
This document discusses using molecular genotyping methods to investigate the genetic diversity of Mycobacterium tuberculosis strains in Taiwan. It provides background on M. tuberculosis and describes several genotyping methods including restriction fragment length polymorphism (RFLP), spoligotyping, variable number tandem repeats (VNTR), and mycobacterial interspersed repetitive units (MIRU). The study aims to evaluate the genotyping efficiency of these methods, select appropriate genetic markers, and establish high-throughput protocols. Preliminary results on 479 samples from Taiwan show discrimination of strains by region, age, gender and genotype. Locus discrimination power and Hunter-Gaston discrimination index are also reported for different genotyping methods.
Detection of Wuchereria bancrofti DNA in paired serum and urine samples using...dewisetiyana52
This study aimed to standardize PCR-based systems for the diagnosis of lymphatic filariasis using serum and urine samples. Paired biological samples were collected from 20 individuals known to be infected with Wuchereria bancrofti. Conventional and semi-nested PCR assays were optimized to detect W. bancrofti DNA. The internal PCR system was able to detect as little as 10 fg of W. bancrofti DNA and detected infection in all 20 patients using both serum and urine samples. In contrast, the semi-nested PCR only detected infection in 2 of the 20 patients. This study demonstrates the potential of using a simple internal PCR system and urine samples for the diagnosis of W. b
This study sequenced the genomes of 11 clinical Mycobacterium abscessus isolates from 8 US patients with pulmonary infections. Core genome analysis compared these isolates to 30 globally diverse strains to investigate population structure. Longitudinally sampled isolates showed very few genetic differences, suggesting homogenous infection populations. Genome content variation between isolates was 0.3-8.3% compared to the reference strain, indicating plasticity.
Nuhu et al_Poster NAPA2016 correction and observationNuhu Tanko
The study determined the prevalence and genetic profiles of ESBL-producing uropathogens among members of the Enterobacteriaceae family at Specialist Hospital Sokoto, Nigeria. A total of 64 Gram-negative uropathogens were isolated from 365 urine samples, with E. coli and Salmonella arizonae being most prevalent. The isolates showed high resistance to cotrimoxazole, nalidixic acid, ciprofloxacin and norfloxacin. 64.1% of isolates were multidrug resistant. ESBL production was detected in 23.4% of isolates. PCR analysis showed 73.3% of ESBL producers contained the blaCTX-M gene and 26.7
Candidemia in HIV-positive patients in Dschang District Hospital (West Region...Claude Nangwat
Candidemia has been identified as a public health problem in HIV-infected patients. The evaluation of CD4 count, transaminases and blood glucose, are being used as a means to monitor the health of HIV-infected patients, without excluding the diagnosis of candidemia and other opportunistic infections. In order to contribute in improving the care of HIV-infected patients attending Dschang District Hospital and later on, in other hospitals in Cameroon, we conducted from June to September 2014 a cross-sectional study, with general objective; to determine the association between candidemia and selected biochemical and haematological parameter changes in HIV-infected patients, as a possible indicator in monitoring HIV disease progression.
To do this, blood samples were collected from HIV-infected patients assigned to the UPEC of Dschang District Hospital for follow up, and haemogram report, CD4 counts, ALAT level, ASAT level, and glucose level in blood were evaluated by cytometric and spectrophotometric assays. Candida species were isolated from some blood samples, and then identified using CHROMagar Candida culture medium. The broth microdilution method was afterwards used to test the susceptibility of the fungal isolates vis-a-vis three conventional antifungal agents.
Mycological analysis of blood samples showed that eight (08) patients had candidemia, a prevalence of 6.11%. Eight (08) isolates were obtained from these eight (08) candidemic HIV-infected patients; this consisted of 4(50%) Candida albicans, 3(37.5%) Candida parapsilosis and 1(12.5%) Candida glabrata. All these isolates were resistant (MICs ranged from 2 to >256 µg/mL) to the antifungals used, that is, ketoconazole, amphotericin B and nystatin.
A significant correlation was found between candidemia and white blood cell count, with a correlation coefficient of r = 0.240 (p < 0.05). Based on the results obtained, the systematic diagnosis of candidemia should be performed in patients infected with HIV in Cameroon in order to improve on their care.
Key words: Candidemia, HIV, biochemical parameters, hematological parameters, Antifungals activities.
1. The document provides information about the educational and professional background of an individual, including degrees obtained from various institutions between 1973-2013 related to prenatal diagnosis, genetics, and rare syndromes.
2. It discusses various levels of genetics from DNA to populations and highlights key concepts like meiosis, mitosis, chromosomes, and mitochondrial DNA.
3. Genetics and genomics provides information at the cellular level about structure, expression, regulation and pathways/networks, but understanding the whole organism is still incomplete and requires integration of concepts from other fields like physics, chemistry and mathematics.
This study investigated the relationship between variants of the glycophorin B (GPB) gene and susceptibility to Plasmodium falciparum infection in the Brazilian Amazon. The researchers found that individuals carrying the GYPB*S allele were more likely to be infected with P. falciparum than those without this allele. Sequence analysis of GYPB also suggested it has been under natural selection due to malaria. This study provides evidence that genetic variation in GPB receptor influences the ability of P. falciparum to invade red blood cells in this population.
This document contains a summary of Sandra G. Nishikawa's skills and experience. She has over 30 years of experience in molecular biology techniques including DNA/RNA isolation, PCR, cloning, sequencing, cell culture and more. She has worked as a research technician for various professors at the University of Calgary studying topics like hepatitis B virus, prion diseases, and cancer.
This document discusses using molecular genotyping methods to investigate the genetic diversity of Mycobacterium tuberculosis strains in Taiwan. It provides background on M. tuberculosis and describes several genotyping methods including restriction fragment length polymorphism (RFLP), spoligotyping, variable number tandem repeats (VNTR), and mycobacterial interspersed repetitive units (MIRU). The study aims to evaluate the genotyping efficiency of these methods, select appropriate genetic markers, and establish high-throughput protocols. Preliminary results on 479 samples from Taiwan show discrimination of strains by region, age, gender and genotype. Locus discrimination power and Hunter-Gaston discrimination index are also reported for different genotyping methods.
Detection of Wuchereria bancrofti DNA in paired serum and urine samples using...dewisetiyana52
This study aimed to standardize PCR-based systems for the diagnosis of lymphatic filariasis using serum and urine samples. Paired biological samples were collected from 20 individuals known to be infected with Wuchereria bancrofti. Conventional and semi-nested PCR assays were optimized to detect W. bancrofti DNA. The internal PCR system was able to detect as little as 10 fg of W. bancrofti DNA and detected infection in all 20 patients using both serum and urine samples. In contrast, the semi-nested PCR only detected infection in 2 of the 20 patients. This study demonstrates the potential of using a simple internal PCR system and urine samples for the diagnosis of W. b
The study characterized the Campylobacter jejuni IAL 2383 strain isolated from humans in Brazil. They found that the strain harbored important virulence genes and expressed major virulence factor transcripts. It grew better at 41°C than 37°C, indicating ability to colonize avian hosts. The strain was sensitive to most antibiotics tested and could serve as an experimental model for interactions with host cells and acquisition of antibiotic resistance.
Elevated mitochondrial DNA copy number is observed in aneuploid embryos, indicating this parameter could become an additional tool for prioritizing embryo transfer. The study analyzed mitochondrial DNA copy number in 606 embryos using next generation sequencing and found higher numbers in aneuploid versus normal embryos. While most factors like embryo quality, sex, and patient age showed no correlation, implanted 5-day embryos had significantly higher mitochondrial DNA amounts than non-implanted embryos. This suggests mitochondrial DNA copy number may predict embryo viability and improve selection.
This document summarizes a study that analyzed 267 fecal samples from cattle, goats, and poultry in Botswana for the presence of Cryptococcus neoformans. A total of 72 samples (26.9%) tested positive for C. neoformans, mostly from cattle. The isolates were further analyzed to determine their mating type (MATα, MATa, or hybrids). Mating type analysis revealed the presence of all three types in the isolates from cattle, poultry, and goats, with MATα being most common. The results suggest that veterinary animals can act as reservoirs for C. neoformans and highlight the need to prevent transmission to at-risk human populations.
This patent document describes the isolation and characterization of a novel human coronavirus (SARS-CoV) that is the causative agent of severe acute respiratory syndrome (SARS). It provides the nucleic acid sequence of the SARS-CoV genome and the amino acid sequences of its open reading frames. Methods are described for using these molecules to detect SARS-CoV and detect infections. Immune stimulatory compositions are also provided, along with methods for their use.
This study evaluated the use of targeted next-generation sequencing (NGS) for preimplantation genetic diagnosis (PGD) of single-gene disorders. The study compared NGS results from embryo biopsies to results from two established PGD methods. NGS provided 100% consistency with the established methods in diagnosing point mutations and small insertions/deletions in six couples at risk of transmitting single-gene disorders. Additionally, NGS allowed for parallel single-gene disorder screening and comprehensive chromosome screening from the same biopsy sample. The study demonstrates NGS can provide accurate and consistent PGD results and could serve as a model for further development of this emerging technology in PGD.
The document is a United States patent describing methods for producing recombinant coronavirus particles. Specifically, it describes developing a helper cell that contains: 1) a coronavirus permissive cell; 2) a coronavirus replicon RNA containing a heterologous sequence and packaging signal but lacking a structural protein gene; and 3) a separate helper RNA encoding the missing structural protein. When these components are expressed in the cell, coronavirus particles are assembled that contain the heterologous RNA sequence but cannot replicate without the helper RNA. The patent claims compositions and methods for making and using these viral particles to deliver heterologous genes.
Diversity of O Antigens within the Genus Cronobacter - MartinaPauline Ogrodzki
This study analyzed the diversity of O antigens in the bacterial genus Cronobacter by testing 82 strains representing all Cronobacter species. Restriction fragment length polymorphism analysis of the O-antigen gene cluster identified 11 previously reported and 6 new serotypes. Whole genome sequencing of reference strains confirmed the new serotypes and showed some existing PCR probes did not correctly identify genomic variations. Analysis of lipopolysaccharide phenotypes also differentiated 24 total serotypes among Cronobacter strains. Certain serotypes including C. sakazakii O2, O1, and O4 and C. turicensis O1 were found to predominately cause clinical infections. This work provides an updated systematic classification of Cronobacter serotypes.
Join Fight CRC in a webinar about biomarkers. In this session, Dr. Chris Lieu will focus the discussion on the NTRK biomarker, in addition to ctDNA, and Next-Generation Sequencing.
Antibiotic resistance and virulence genes in enterococcusMohamed Hassan
This document summarizes a study on antibiotic resistance and virulence genes in Enterococcus strains isolated from hospitals in Saudi Arabia. 89 bacterial isolates were obtained from hospital patients, of which 12 were Enterococcus species. These Enterococcus isolates were tested for antibiotic resistance, identified using specific gene primers, and characterized using repetitive sequence-based PCR (Rep-PCR). The results showed that 58.3% of isolates were Enterococcus faecium, 16.6% were Enterococcus durans, and 25.1% were other Enterococcus species. 67% of isolates showed multi-drug resistance. Virulence genes were also detected in some isolates. 16S rDNA sequencing was used to further identify the isolates. The isolates exhibited genetic diversity
Development and validation of an accurate quantitative real time polymerase c...t7260678
This document describes the development and validation of a quantitative real-time polymerase chain reaction (qPCR) method for comprehensive chromosomal aneuploidy screening of human blastocysts. The method was found to be highly accurate, correctly diagnosing aneuploidies in 97.6% of cell line samples and 98.6% of human blastocyst samples compared to conventional methods. The qPCR method can provide a diagnosis for all 24 chromosomes in only 4 hours, making it suitable for screening of blastocyst biopsies without the need for cryopreservation. This rapid method could allow for fresh euploid embryo transfers and improve outcomes for couples undergoing in vitro fertilization.
This document summarizes a study on the isolation and molecular characterization of human adenovirus. The study found that out of 83 samples collected from eye secretions, 69 (83.13%) tested positive for human adenovirus using rapid tests and PCR. The highest rate of infection was found in individuals aged 16-30 years old (55.04%) and males had a higher rate of infection than females. Human adenovirus was successfully isolated by inoculating samples on chicken embryo fibroblast cell cultures and embryonated eggs, where cytopathic effects were observed. Molecular characterization was also conducted to identify the adenovirus strains present.
This document provides a review of Capnocytophaga canimorsus, a commensal bacterium in the oral flora of dogs and cats that can cause zoonotic infections in humans. C. canimorsus infections in humans range from mild flu-like symptoms to fatal sepsis. The review describes the infectious agent, its pathogenicity and virulence factors, infections in animals and humans, diagnosis, prevalence, therapy and prevention. It notes that C. canimorsus is a fastidious, facultative anaerobic, Gram-negative rod that requires CO2 for growth and is transmitted through dog and cat bites, scratches or close contact. Human infections following contact with dogs or cats can
This study evaluated the use of quantitative PCR (qPCR) to genotype single nucleotide polymorphisms (SNPs) near a mutation in the RTEL1 gene for preimplantation genetic diagnosis of Dyskeratosis Congenita, compared to the standard method using short tandem repeats (STRs). The standard STR method misdiagnosed 3 of 14 embryos due to recombination between the distant STR marker and mutation. In contrast, qPCR of closely linked SNPs identified recombination in 9 of 17 embryos and correctly diagnosed all embryos. This case demonstrates that qPCR of SNPs provides improved sensitivity over STRs for detecting recombination near telomeric mutations.
This document summarizes information presented about Mycobacterium tuberculosis and tuberculosis. It discusses that M. tuberculosis grows slowly, doubling every 24 hours, and takes 3-4 weeks to culture. It also notes that tuberculosis infects around 2 billion people globally and causes 1.6 million deaths per year. The document also mentions that multidrug resistant tuberculosis is emerging worldwide and there are an estimated 50 million people infected with multidrug resistant strains.
This document describes the development and application of a reverse transcription PCR (RT-PCR) assay for genotyping noroviruses (NoV) based on the major capsid protein VP1. The authors analyzed 100 NoV VP1 sequences to identify a conserved region, called region D, that differentiates between genotypes. They designed two primer sets targeting region D that are broadly reactive for genogroups I and II. Testing on a panel of 81 NoV strains showed the region D primers detected 95% of samples. Phylogenetic analysis of region D and full VP1 sequences grouped strains identically, confirming three newly identified clusters. The region D RT-PCR provides a reliable method for NoV genotyping.
Next Generation Sequencing application in virologyEben Titus
Next Generation Sequencing (NGS) is a promising technique for virus diagnosis that provides several advantages over traditional Sanger sequencing. NGS workflows involve sample preparation, sequencing, and data analysis. NGS has various applications in virology including identifying viral quasispecies, detecting antiviral drug resistance mutations, discovering novel virus genotypes, and performing quality control of live vaccines. While NGS reduces costs and improves throughput over Sanger sequencing, analyzing large NGS datasets requires strong bioinformatics skills. Overall, NGS represents a significant improvement for virus research and diagnosis.
This document provides an overview of Capnocytophaga, including its classification, characteristics, species, culture methods, incidence, detection, identification, interactions, virulence factors, antibiotic therapy and resistance. Capnocytophaga is an anaerobic, gram-negative bacterium that is facultative and exhibits gliding movement. It includes species such as C. ochracea, C. gingivalis, C. sputigena, and C. canimorsus. The document discusses methods for culturing and identifying Capnocytophaga, its role in periodontal diseases, potential virulence factors, and antibiotic susceptibility.
This document summarizes Kgothatso Meno's BSc honors research project on norovirus genotype I (NoV GI). Some key points:
- NoV is a major cause of gastroenteritis worldwide and is highly contagious. It predominantly affects children, elderly, and immunocompromised individuals.
- The project aims to optimize genotyping primers for NoV GI and apply them to study GI diversity in clinical and environmental samples in South Africa.
- Preliminary results found that 35% of sewage samples from April 2015-March 2016 were positive for NoV GI, and all sequenced samples were genotype GI.4.
- The document outlines challenges in genotyping GI due
Actuación monitores ante hemorragia en campamentoMaikisaurio
Este documento proporciona instrucciones sobre cómo los monitores deben responder a una hemorragia en un campamento. Explica cómo preparar materiales como una garrafa con agua y una botella para simular la pérdida de sangre. Luego detalla los tipos de hemorragias y las consideraciones a tener en cuenta sobre la velocidad del sangrado. Finalmente, recomienda aplicar presión directa a la herida durante 5 minutos y luego un vendaje compresivo, y especifica cómo tratar hemorragias externas, internas y exteriorizadas.
Paragon Honda is offering a 2009 red Honda Accord with 31,839 miles for $19,500. The vehicle has a 5-speed automatic transmission and is located in Woodside, NY. Interested buyers can contact Paragon Honda at 888-703-4259 or view the vehicle on their website for more details and pricing.
The document discusses the steps to design and implement a Configuration Manager 2012 environment for a company called XYZ. It recommends placing primary site servers in San Francisco and Paris, and secondary servers in London and Tokyo based on office sizes. It also outlines prerequisites like extending the Active Directory schema, configuring the System Management container, and adding Windows roles and features to site servers.
The study characterized the Campylobacter jejuni IAL 2383 strain isolated from humans in Brazil. They found that the strain harbored important virulence genes and expressed major virulence factor transcripts. It grew better at 41°C than 37°C, indicating ability to colonize avian hosts. The strain was sensitive to most antibiotics tested and could serve as an experimental model for interactions with host cells and acquisition of antibiotic resistance.
Elevated mitochondrial DNA copy number is observed in aneuploid embryos, indicating this parameter could become an additional tool for prioritizing embryo transfer. The study analyzed mitochondrial DNA copy number in 606 embryos using next generation sequencing and found higher numbers in aneuploid versus normal embryos. While most factors like embryo quality, sex, and patient age showed no correlation, implanted 5-day embryos had significantly higher mitochondrial DNA amounts than non-implanted embryos. This suggests mitochondrial DNA copy number may predict embryo viability and improve selection.
This document summarizes a study that analyzed 267 fecal samples from cattle, goats, and poultry in Botswana for the presence of Cryptococcus neoformans. A total of 72 samples (26.9%) tested positive for C. neoformans, mostly from cattle. The isolates were further analyzed to determine their mating type (MATα, MATa, or hybrids). Mating type analysis revealed the presence of all three types in the isolates from cattle, poultry, and goats, with MATα being most common. The results suggest that veterinary animals can act as reservoirs for C. neoformans and highlight the need to prevent transmission to at-risk human populations.
This patent document describes the isolation and characterization of a novel human coronavirus (SARS-CoV) that is the causative agent of severe acute respiratory syndrome (SARS). It provides the nucleic acid sequence of the SARS-CoV genome and the amino acid sequences of its open reading frames. Methods are described for using these molecules to detect SARS-CoV and detect infections. Immune stimulatory compositions are also provided, along with methods for their use.
This study evaluated the use of targeted next-generation sequencing (NGS) for preimplantation genetic diagnosis (PGD) of single-gene disorders. The study compared NGS results from embryo biopsies to results from two established PGD methods. NGS provided 100% consistency with the established methods in diagnosing point mutations and small insertions/deletions in six couples at risk of transmitting single-gene disorders. Additionally, NGS allowed for parallel single-gene disorder screening and comprehensive chromosome screening from the same biopsy sample. The study demonstrates NGS can provide accurate and consistent PGD results and could serve as a model for further development of this emerging technology in PGD.
The document is a United States patent describing methods for producing recombinant coronavirus particles. Specifically, it describes developing a helper cell that contains: 1) a coronavirus permissive cell; 2) a coronavirus replicon RNA containing a heterologous sequence and packaging signal but lacking a structural protein gene; and 3) a separate helper RNA encoding the missing structural protein. When these components are expressed in the cell, coronavirus particles are assembled that contain the heterologous RNA sequence but cannot replicate without the helper RNA. The patent claims compositions and methods for making and using these viral particles to deliver heterologous genes.
Diversity of O Antigens within the Genus Cronobacter - MartinaPauline Ogrodzki
This study analyzed the diversity of O antigens in the bacterial genus Cronobacter by testing 82 strains representing all Cronobacter species. Restriction fragment length polymorphism analysis of the O-antigen gene cluster identified 11 previously reported and 6 new serotypes. Whole genome sequencing of reference strains confirmed the new serotypes and showed some existing PCR probes did not correctly identify genomic variations. Analysis of lipopolysaccharide phenotypes also differentiated 24 total serotypes among Cronobacter strains. Certain serotypes including C. sakazakii O2, O1, and O4 and C. turicensis O1 were found to predominately cause clinical infections. This work provides an updated systematic classification of Cronobacter serotypes.
Join Fight CRC in a webinar about biomarkers. In this session, Dr. Chris Lieu will focus the discussion on the NTRK biomarker, in addition to ctDNA, and Next-Generation Sequencing.
Antibiotic resistance and virulence genes in enterococcusMohamed Hassan
This document summarizes a study on antibiotic resistance and virulence genes in Enterococcus strains isolated from hospitals in Saudi Arabia. 89 bacterial isolates were obtained from hospital patients, of which 12 were Enterococcus species. These Enterococcus isolates were tested for antibiotic resistance, identified using specific gene primers, and characterized using repetitive sequence-based PCR (Rep-PCR). The results showed that 58.3% of isolates were Enterococcus faecium, 16.6% were Enterococcus durans, and 25.1% were other Enterococcus species. 67% of isolates showed multi-drug resistance. Virulence genes were also detected in some isolates. 16S rDNA sequencing was used to further identify the isolates. The isolates exhibited genetic diversity
Development and validation of an accurate quantitative real time polymerase c...t7260678
This document describes the development and validation of a quantitative real-time polymerase chain reaction (qPCR) method for comprehensive chromosomal aneuploidy screening of human blastocysts. The method was found to be highly accurate, correctly diagnosing aneuploidies in 97.6% of cell line samples and 98.6% of human blastocyst samples compared to conventional methods. The qPCR method can provide a diagnosis for all 24 chromosomes in only 4 hours, making it suitable for screening of blastocyst biopsies without the need for cryopreservation. This rapid method could allow for fresh euploid embryo transfers and improve outcomes for couples undergoing in vitro fertilization.
This document summarizes a study on the isolation and molecular characterization of human adenovirus. The study found that out of 83 samples collected from eye secretions, 69 (83.13%) tested positive for human adenovirus using rapid tests and PCR. The highest rate of infection was found in individuals aged 16-30 years old (55.04%) and males had a higher rate of infection than females. Human adenovirus was successfully isolated by inoculating samples on chicken embryo fibroblast cell cultures and embryonated eggs, where cytopathic effects were observed. Molecular characterization was also conducted to identify the adenovirus strains present.
This document provides a review of Capnocytophaga canimorsus, a commensal bacterium in the oral flora of dogs and cats that can cause zoonotic infections in humans. C. canimorsus infections in humans range from mild flu-like symptoms to fatal sepsis. The review describes the infectious agent, its pathogenicity and virulence factors, infections in animals and humans, diagnosis, prevalence, therapy and prevention. It notes that C. canimorsus is a fastidious, facultative anaerobic, Gram-negative rod that requires CO2 for growth and is transmitted through dog and cat bites, scratches or close contact. Human infections following contact with dogs or cats can
This study evaluated the use of quantitative PCR (qPCR) to genotype single nucleotide polymorphisms (SNPs) near a mutation in the RTEL1 gene for preimplantation genetic diagnosis of Dyskeratosis Congenita, compared to the standard method using short tandem repeats (STRs). The standard STR method misdiagnosed 3 of 14 embryos due to recombination between the distant STR marker and mutation. In contrast, qPCR of closely linked SNPs identified recombination in 9 of 17 embryos and correctly diagnosed all embryos. This case demonstrates that qPCR of SNPs provides improved sensitivity over STRs for detecting recombination near telomeric mutations.
This document summarizes information presented about Mycobacterium tuberculosis and tuberculosis. It discusses that M. tuberculosis grows slowly, doubling every 24 hours, and takes 3-4 weeks to culture. It also notes that tuberculosis infects around 2 billion people globally and causes 1.6 million deaths per year. The document also mentions that multidrug resistant tuberculosis is emerging worldwide and there are an estimated 50 million people infected with multidrug resistant strains.
This document describes the development and application of a reverse transcription PCR (RT-PCR) assay for genotyping noroviruses (NoV) based on the major capsid protein VP1. The authors analyzed 100 NoV VP1 sequences to identify a conserved region, called region D, that differentiates between genotypes. They designed two primer sets targeting region D that are broadly reactive for genogroups I and II. Testing on a panel of 81 NoV strains showed the region D primers detected 95% of samples. Phylogenetic analysis of region D and full VP1 sequences grouped strains identically, confirming three newly identified clusters. The region D RT-PCR provides a reliable method for NoV genotyping.
Next Generation Sequencing application in virologyEben Titus
Next Generation Sequencing (NGS) is a promising technique for virus diagnosis that provides several advantages over traditional Sanger sequencing. NGS workflows involve sample preparation, sequencing, and data analysis. NGS has various applications in virology including identifying viral quasispecies, detecting antiviral drug resistance mutations, discovering novel virus genotypes, and performing quality control of live vaccines. While NGS reduces costs and improves throughput over Sanger sequencing, analyzing large NGS datasets requires strong bioinformatics skills. Overall, NGS represents a significant improvement for virus research and diagnosis.
This document provides an overview of Capnocytophaga, including its classification, characteristics, species, culture methods, incidence, detection, identification, interactions, virulence factors, antibiotic therapy and resistance. Capnocytophaga is an anaerobic, gram-negative bacterium that is facultative and exhibits gliding movement. It includes species such as C. ochracea, C. gingivalis, C. sputigena, and C. canimorsus. The document discusses methods for culturing and identifying Capnocytophaga, its role in periodontal diseases, potential virulence factors, and antibiotic susceptibility.
This document summarizes Kgothatso Meno's BSc honors research project on norovirus genotype I (NoV GI). Some key points:
- NoV is a major cause of gastroenteritis worldwide and is highly contagious. It predominantly affects children, elderly, and immunocompromised individuals.
- The project aims to optimize genotyping primers for NoV GI and apply them to study GI diversity in clinical and environmental samples in South Africa.
- Preliminary results found that 35% of sewage samples from April 2015-March 2016 were positive for NoV GI, and all sequenced samples were genotype GI.4.
- The document outlines challenges in genotyping GI due
Actuación monitores ante hemorragia en campamentoMaikisaurio
Este documento proporciona instrucciones sobre cómo los monitores deben responder a una hemorragia en un campamento. Explica cómo preparar materiales como una garrafa con agua y una botella para simular la pérdida de sangre. Luego detalla los tipos de hemorragias y las consideraciones a tener en cuenta sobre la velocidad del sangrado. Finalmente, recomienda aplicar presión directa a la herida durante 5 minutos y luego un vendaje compresivo, y especifica cómo tratar hemorragias externas, internas y exteriorizadas.
Paragon Honda is offering a 2009 red Honda Accord with 31,839 miles for $19,500. The vehicle has a 5-speed automatic transmission and is located in Woodside, NY. Interested buyers can contact Paragon Honda at 888-703-4259 or view the vehicle on their website for more details and pricing.
The document discusses the steps to design and implement a Configuration Manager 2012 environment for a company called XYZ. It recommends placing primary site servers in San Francisco and Paris, and secondary servers in London and Tokyo based on office sizes. It also outlines prerequisites like extending the Active Directory schema, configuring the System Management container, and adding Windows roles and features to site servers.
El documento describe el desarrollo de Facebook Places, una aplicación que permite a los usuarios registrar su ubicación y compartirla con sus amigos. Explica que más del 30% de las visitas a Facebook y el 40% de las visitas a Twitter ahora son a través de dispositivos móviles. También analiza las ventajas e inconvenientes que Places ofrece a los usuarios y las empresas, como la publicidad personalizada, información de perfiles valiosa y mayores oportunidades de construir comunidades y lealtad a la marca.
Este documento proporciona una introducción a las fórmulas en Excel. Explica la definición y sintaxis de una fórmula, los niveles de operadores, y proporciona ejemplos de fórmulas comunes y ejercicios para practicar. También incluye una tabla de ejemplos para realizar fórmulas adicionales.
1. A study analyzed genetic and immune response differences between P. vivax circumsporozoite (CS) genotypes VK210 and P. vivax-like.
2. Phylogenetic analysis of 18S rRNA and CytB genes found high similarity between the genotypes, with zero nucleotide diversity, placing them in the same clade.
3. Individuals infected with P. vivax-like had a lower antibody response against CS repetitive region peptides than those with VK210, suggesting variation is limited to the CS repetitive region.
This document describes the development of a PCR-RFLP assay to identify Plasmodium species and variants of P. vivax infecting Anopheles mosquitoes. Specific primers were designed that target regions of the circumsporozoite gene to distinguish P. falciparum, P. malariae, and P. vivax variants VK210, VK247, and P. vivax-like. The assay was tested on artificially infected mosquitoes and showed good agreement with nested PCR. The PCR-RFLP method provides a sensitive way to detect Plasmodium species and variants, which can help understand malaria transmission dynamics.
Prevalence of Rota Virus Detection by Reverse TranscriptasePolymerase Chain R...IOSRJPBS
The present study was conducted for the period from 1/6/2016 to 20/1/2017 in Baquba city. The study aimed to detection of rotavirus in stool specimens of children fewer than five age and also explore the effects of certain demographic factors on the detection rates by revers transcriptase- polymerase chain reaction. The study included 49 patients with acute diarrhea, 32 were male and 17 were female. The age range was two months to 5 years. Demographic information on the patients regarding age, sex, residence, type of feeding and source of drinking water were collected from their parents. Stool specimens were collected from each patients and. Detection of rotavirus in stool specimens was done by conventional reverse transcriptase polymerase chain reaction (RT-PCR). The results of present study showed that the overall infection rate by rotavirus among patients with acute diarrhea by RT-PCR tests was 93.88%. The highest infection rate was recorded among those >10-≤15 months of age. None of the results showed significantly difference between female and male, PCR (88% vs 96.87%). Likewise, there was insignificantly difference between urban and rural residence, PCR (95.65% vs 92.30%). The results revealed insignificantly higher infection rate among patients (those below 2 years) feed mixing (91.66%) and bottled (100%) compared to that breast feeding (77.77%) by RT-PCR. The rotavirus infection rate was insignificantly higher among patients consuming municipal water for drinking (97.22%) compared to those consuming bottled water (84.61%) by the RT-PCR. The study concluded that rotavirus was detected in high rates among children less than 5 years old with acute diarrhea in Baquba city, particularly those less than 2 year old.
This study evaluated the distribution of Plasmodium vivax variants (VK210, VK247, and P. vivax-like) in three areas of Brazil using a new GFM-PCR-ELISA technique. All variants were found in all three areas. VK210 was most commonly found as a single infection while the others occurred in mixed infections. VK210 was associated with the highest parasitemia levels while P. vivax-like had the lowest. Parasitemia clearance times did not differ based on variant or treatment schedule. The new technique was accurate for epidemiological surveys of the vivax complex.
This study analyzed faecal specimens from 2,495 diarrhoea cases in Kolkata, India between 2007-2009 to determine the seasonal distribution and characteristics of norovirus (NoV) infections. NoV was detected in 78 cases, mostly children under 2 years old, sometimes as the sole pathogen but often along with other enteric pathogens. Sequencing of the NVGII strains showed clustering with GII.4, GII.13 and GII.6 NoV types. NoV infections occurred year-round and were associated with mild dehydration in children and adults in Kolkata.
The 'omics' revolution: How will it improve our understanding of infections a...WAidid
This slideset explains the ‘Omics’ technology and its role in the study of infections and vaccination. It is a revolution as it offers powerful tools to interrogate the animal / human immune response to vaccines and infections.
1) The study examined the effects of infliximab treatment on Crohn's disease patients infected with Mycobacterium avium ssp. paratuberculosis (MAP), which has been associated with Crohn's disease.
2) Patients treated with infliximab showed significantly decreased levels of antibodies against two MAP proteins compared to untreated patients, suggesting infliximab decreases secretion of these proteins.
3) The study also found infliximab treatment was detrimental to the survival of MAP within infected macrophages, with significantly decreased survival of MAP in macrophages exposed to infliximab.
The document summarizes preliminary research toward developing a method for stably transfecting the malaria parasite Plasmodium vivax. The researchers tested various transfection conditions and found the highest parasite survival rates using the Amaxa Nucleofector program U-033 and pre-loading erythrocytes with DNA. However, no fluorescent parasites were observed, likely due to the low efficiency of Plasmodium transfection. Future work aims to increase parasite numbers for transfection and further optimize methods.
Amany m. elshamy why the negative covid19 pcr test is a misguide resultsAmany Elshamy
The document discusses limitations of PCR tests for COVID-19 diagnosis. PCR tests have sensitivity of 70-90% and false negatives are common within the first week of infection or after 15 days. Commercial PCR kits target genes like ORF1ab, E, N and RdRP which have high genetic diversity and mutations, reducing accuracy. Serology tests have increasing sensitivity after the first week but depend on viral activity and cannot determine active infection. Chest CT is more reliable than PCR for diagnosis, showing characteristic lung opacities. Improved COVID-19 diagnostics are needed to enhance early reporting and accurately diagnose asymptomatic cases.
This document provides an introduction and table of contents for a book on advanced laboratory techniques in avian medicine. The introduction gives an overview of traditional diagnostic methods and emerging molecular biological techniques. The table of contents outlines two main sections - traditional diagnostic methods, and molecular biological techniques - covering topics like isolation and identification of microorganisms, serological procedures, and nucleic acid and protein methods.
I reviewed several manuscripts, books, grants and project proposals. This is one of the paper I reviewed recently published in Plant Biotechnology Journal
Primordial Germ Cells- A tool for avian genome manipulationDr. MAYUR VISPUTE
This presentation deals with the scope and technique of avian genome manipulation by using avian primordial germ cells to obtain the pharmaceuticals using chicken egg as a bioreactor system and also to enhance the overall poultry production, and disease resistance, etc.
The document describes a study that used MALDI-TOF MS to identify mycobacterial isolates. It compared two protein extraction protocols (A and B) on reference strains and clinical isolates, finding protocol A identified 92.1% of isolates to the species level compared to 50% for protocol B. Protocol A was then used to identify 27 environmental mycobacterial isolates, with two isolates misidentified by PRA-hsp65 but correctly identified by MALDI-TOF MS. Sequencing of the hsp65 and 16S rRNA genes confirmed the MALDI-TOF MS identifications. The results support the use of MALDI-TOF MS as a rapid and valuable tool for identifying
This study investigated an outbreak of neonatal sepsis in a neonatal intensive care unit attributed to coagulase-negative staphylococci (CoNS). The aims were to assess the association between intravenous catheters and CoNS sepsis, identify persistent CoNS strains, and determine antibiotic resistance patterns. Random amplified polymorphic DNA (RAPD) analysis identified two main clusters of CoNS isolates, with some strains showing similarity over 88%, suggesting persistence. Staphylococcus capitis was the most prevalent pathogen, infecting 80% of patients and showing multi-drug resistance. The results support that CoNS are a significant cause of neonatal intensive care unit sepsis, can infect via intravenous catheters, and persistent strains may cir
We present evidence of Plasmodium vivax infection in two homozygous FY*B-33 (Duffy-negative) individuals from the Brazilian Amazon region. Molecular analysis confirmed P. vivax infection through detection of P. vivax small subunit rRNA and circumsporozoite protein genes. One individual had a mixed infection of VK210 and P. vivax-like genotypes, while the other was infected with VK210. Both individuals also had antibodies to the P. vivax merozoite surface protein 1. This provides rare evidence that P. vivax may be capable of invading Duffy-negative red blood cells in some regions, though additional studies are needed to better understand this finding.
This document describes a study that analyzed the transcriptome of the gut epithelium in Helicoverpa zea (corn earworm) larvae 24 hours after being infected with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV) or a mock treatment. The study found 1,139 genes were differentially expressed between the two groups, with 63% downregulated and 37% upregulated in infected larvae. Genes related to digestion, detoxification, and some immune responses like viral recognition were generally downregulated upon infection, while antimicrobial peptides and prophenoloxidase were upregulated. This provided insight into how baculovirus infection alters gene expression in the gut, likely the most heavily
Cloning and sequence analysis of banana streak virus dna. harper 1998Paloma Susan
Banana streak virus (BSV) causes severe problems for banana cultivation. The researchers cloned and sequenced the genome of a Nigerian isolate of BSV. The genome was found to be 7,389 base pairs and organized similarly to other badnaviruses, with three open reading frames. Comparison showed BSV is distinct from but closely related to sugarcane bacilliform virus. PCR primers designed from the sequence data detected BSV sequences in banana plants, indicating portions of the BSV genome may integrate into the banana genome. The BSV sequence provides a basis for more sensitive PCR-based detection methods.
Simultaneous, specific and real time detection of biothreat and frequently en...Tiensae Teshome
This document describes the development of a platform for the simultaneous detection of six important foodborne pathogens (Escherichia, Salmonella, Shigella, Vibrio, Yersinia, and Francisella) using real-time PCR. The researchers mined genomic databases to identify unique genomic targets for primer design. They designed new primer sets and validated them in silico and in vitro, demonstrating specific detection of each pathogen at the genus and species levels. A sensitivity assay showed the platform could detect as few as 640 fg of Francisella tularensis DNA. Preliminary testing also showed it could detect Shigella in milk down to 6-60 CFU/ml within an hour, demonstrating
GCMA-Tackling the menace of MDR gram negative pathogens with a novel BL-BLI-...Jigar Mehta
The patient presented with worsening respiratory status and fever. Klebsiella pneumoniae was isolated from BAL culture and rapid molecular testing detected OXA-48. OXA-48 is commonly reported in India and often co-produced with ESBLs. Given the detection of OXA-48 and the likelihood of ESBL co-production based on local epidemiology, ceftazidime-avibactam was initiated due to its activity against OXA-48 producers and many ESBLs.
1. The study characterized antibody responses to Plasmodium falciparum invasion ligands EBA-140 and EBA-181 in individuals from malaria-endemic areas of Brazil and Cameroon.
2. Responses differed between populations, with African individuals strongly reacting to most EBA fragments while Brazilian individuals from Mato Grosso reacted weakly and those from the Amazon had elevated but lower responses than Africans.
3. When compared to responses against other malaria proteins, the Brazilian population appeared to have more variable ability to recognize P. falciparum invasion ligands, distinct from the African population.
This document analyzes the frequency of two polymorphisms (C282Y and H63D) in the HFE gene in malaria patients and blood donors from four states in the Brazilian Amazon region. The study found:
1) No individuals were homozygous for the C282Y polymorphism, and only 5 heterozygous individuals were detected, all from Pará State.
2) The most common genotype for the H63D polymorphism was heterozygous in both patient groups.
3) Allele frequencies for the H63D polymorphism were higher than for the C282Y polymorphism, consistent with other studies on Brazilian populations.
This study investigated the relationship between variants of the glycophorin B (GPB) gene and susceptibility to Plasmodium falciparum infection in the Brazilian Amazon. The researchers found that individuals carrying the GYPB*S allele were more likely to be infected with P. falciparum than those without this allele. Additionally, population genetics analysis suggested that natural selection has shaped patterns of genetic diversity at the GYPB locus. This study provides evidence that genetic variation in GPB influences susceptibility to P. falciparum infection in this population.
This document summarizes a study that investigated the influence of HLA-DRB1 alleles on the production of antibodies against malaria antigens in individuals naturally infected with Plasmodium vivax in Brazil. The study found an association between HLA-DR3 and HLA-DR5 alleles and lack of antibody response to the amino terminal region of the circumsporozoite protein (CSP). It also found an association between HLA-DR3 and higher antibody response to merozoite surface protein 1 (MSP1). No significant associations were found between HLA-DRB1 alleles and antibody responses to other malaria antigens tested.
1. Researchers studied Plasmodium falciparum field isolates from Colombia, Peru, and Brazil to determine the invasion pathways and ligand polymorphisms used.
2. They found that most isolates from Colombia and Peru invade red blood cells through an atypical pathway that is resistant to all enzyme treatments, unlike known pathways.
3. This unusual pathway was associated with specific variants of PfRh2a, PfRh5, and EBA-181 ligands, suggesting these proteins play a major role in this pathway. The study demonstrates diversity in invasion mechanisms between regions.
This study examined 51 Brazilian Plasmodium falciparum isolates for polymorphisms in the Pfmdr1 gene thought to be associated with chloroquine resistance. 49 of the isolates were found to be resistant to chloroquine in vitro, while all were sensitive to mefloquine, amodiaquine, and quinine. The isolates were analyzed for three Pfmdr1 polymorphisms: Asn86Tyr, Asn1042Asp, and Asp1246Tyr. Asn86Tyr was not detected in any isolates, while Asn1042Asp was found in 50 isolates and Asp1246Tyr was found in all 51 isolates. This provides support that As
This document analyzes genetic variation at the Pfs48/45 gene and microsatellite loci in 255 Plasmodium falciparum isolates from 5 populations. It finds:
1) Alleles and haplotypes of 5 SNPs in the Pfs48/45 gene varied extremely between populations, much more so than alleles at 11 neutral microsatellite loci.
2) Measurements of between-population allele frequency variation (FST) were 4-7 times higher for Pfs48/45 than microsatellites, both within and between continents.
3) The highly skewed Pfs48/45 patterns suggest divergent selection on the protein's amino acid sequence between populations, indicating it may determine game
This study evaluated four recombinant proteins representing the 19 kDa C-terminal region of the Plasmodium vivax merozoite surface protein-1 (MSP119) for detecting P. vivax antibodies in human serum samples. The sensitivity of an ELISA using the recombinant proteins to detect antibodies in 200 samples from individuals with P. vivax infection ranged from 90-93.5%. Specificity using control samples without P. vivax exposure was 98.3-100%. The study demonstrates the potential of an ELISA using recombinant MSP119 proteins for serological detection of P. vivax infection.
This study compared ELISA and PCR-ELISA techniques for detecting human Plasmodium parasites in Anopheles mosquitoes from the Amazon region of Brazil. The PCR-ELISA technique confirmed all positive and negative ELISA results but detected additional Plasmodium species in 5 of the 32 positive mosquitoes that were not detected by ELISA alone. The PCR-ELISA is more sensitive than ELISA for detecting human malaria parasites in mosquitoes.
1) O documento analisa os casos de malária no estado de Santa Catarina entre 1996-2001, com 5,5% das 4.707 amostras sendo positivas.
2) Plasmodium vivax causou 69% dos casos, Plasmodium falciparum 25,6%, infecções mistas 5% e Plasmodium malariae 0,4%.
3) 67,4% dos casos foram importados e 32,6% autóctones, com aumento de casos importados nos anos subsequentes.
This study analyzed genetic diversity and population structure of Plasmodium falciparum in 5 populations in the Brazilian Amazon region. Microsatellite markers were analyzed in 196 parasite isolates. There was significant multilocus linkage disequificance within populations, particularly those with fewer mixed infections. However, most isolates had unique multilocus genotypes, indicating genetic diversity. Genetic divergence between populations was substantial but did not correlate simply with geographical distance. The findings suggest distinct population structures and minimal gene flow between foci in the region.
1) The study evaluated the performance of the OptiMal malaria rapid diagnostic test under different storage conditions of 25°C, 30°C, and 39°C for 24, 48, and 72 hours.
2) The test detected all 111 positive blood samples except for 2 low parasitemia Plasmodium malariae samples.
3) The study suggests that the OptiMal test can be used for malaria diagnosis in Brazilian regions, though further research is needed to evaluate its performance under different environmental conditions like humidity.
This study evaluated the frequency of asymptomatic Plasmodium carriers (APCs) among blood donors in four blood banks in the Brazilian Amazon region. Blood samples from 400 donors who passed screening were tested using PCR to detect Plasmodium DNA. The positivity rate varied from 1-3% between blood banks, with an overall rate of 2.3%. All positive samples contained mixed infections of P. vivax and P. falciparum. While screening methods used by the blood banks did not detect the infections, PCR revealed its superiority for detecting low levels of parasites. The results emphasize the need to improve screening for APCs in blood banks in malaria endemic areas to control transfusion-transmitted malaria.
This study investigated genetic mutations associated with chloroquine resistance in Plasmodium falciparum samples from the Brazilian Amazon region. The study analyzed 40 samples from 4 localities for mutations in the pfmdr1, cg2, and pfcrt genes. It found 100% of samples contained mutations in pfmdr1 codons 184, 1042, and the cg2 gamma region associated with chloroquine resistance. Most samples also contained the pfcrt K76T mutation, except some from Porto Velho which matched a Thai resistant genotype. This research contributes to understanding the molecular basis of widespread chloroquine resistance in this region.
This document summarizes a study that analyzed Duffy blood group genotypes in Plasmodium vivax malaria patients and blood donors in four areas of the Brazilian Amazon region. The study found:
1) A high frequency of the FYA/FYB genotype in both patients and donors, followed by FYB/FYB.
2) Some Duffy genotypes showed significant differences in frequency between patients and donors.
3) Individuals with the FYA/FYB genotype may have higher susceptibility to malaria, while the presence of the FYB-33 allele could provide resistance.
Mixed Plasmodium falciparum infections were common in four areas of the Brazilian Amazon region. Molecular diagnosis found 73% of infections were single P. falciparum infections, while 27% were mixed infections, mostly double infections. Mixed infections were associated with weaker clinical malaria symptoms like lower rates of fever and headache compared to single P. falciparum infections. The study highlights the need for improved malaria diagnosis to better understand mixed infections and their impact on disease severity.
This study aimed to investigate the presence of arboviruses like dengue virus in clinical samples from 111 malaria patients in the Amazon region of Brazil.
Dengue virus serotype 2 was detected in two patients from Novo Repartimento, Pará who also had active Plasmodium vivax malaria infections. Despite limited data, concurrent dengue and malaria infections are likely more common in the Amazon region than detected, as both diseases co-circulate and have similar transmission vectors and clinical presentations, making dual diagnosis possible. Further research is needed to better understand the frequency of co-infection in this region.
This study investigated the frequencies of ABO blood group genotypes and alleles in Plasmodium falciparum malaria patients and blood donors from the Brazilian Amazon region. The researchers found that over half of individuals had the ABO*O01O01 genotype. The ABO*AO01 genotype was the second most common. No significant differences were detected in genotype or allele frequencies between the malaria patients and blood donors. Analysis of O alleles found the O1 variant allele to be most frequent in both groups, with no evidence of the homozygous O2 allele.
This study evaluated antibody responses to the Pv200L fragment of Plasmodium vivax merozoite surface protein-1 (PvMSP-1) in individuals from 4 malaria-endemic regions in Brazil. Plasma samples from 261 P. vivax infected individuals were tested for antibodies to Pv200L by ELISA. The frequency of antibody responders ranged from 71.9-98.7% between regions and correlated with malaria transmission intensity. Higher antibody levels were also associated with greater past exposure to malaria parasites. Results provide evidence that Pv200L elicits naturally acquired antibodies and could be a potential vaccine candidate.
This document summarizes a study that investigated the influence of HLA-DRB1 alleles on the production of antibodies against malaria antigens in individuals naturally infected with Plasmodium vivax in Brazil. The study found an association between HLA-DR3 and HLA-DR5 alleles and lack of antibody response to the amino terminal region of the circumsporozoite protein (CSP). It also found an association between HLA-DR3 and higher antibody response to merozoite surface protein 1 (MSP1). No significant associations were found between HLA-DRB1 alleles and antibody responses to other malaria antigens tested.
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2. 416 R.T. Alves et al. / Diagnostic Microbiology and Infectious Disease 59 (2007) 415–419
The proportion of positive sera, specific for the VK210 and fresh blood samples collected in different endemic areas of
VK247 variants, ranged from 28% to 66% in Thailand the Brazilian Amazon region, all with positive results for
(Wirtz et al., 1990). Nevertheless, VK247 genotype was P. vivax thick blood films (TBFs). TBFs were examined by
identified in 58% of all patients infected with both genotypes independent experienced microscopists who were unaware of
(Kain et al., 1992, 1993). In Brazil, all variants were each result as recommended by the World Health Organiza-
genotyped, but only VK210 was found as a single agent of tion. Furthermore, molecular confirmation of P. vivax was
infection, whereas the other 2 occurred as mixed infections performed for all samples according to the method described
(Machado and Póvoa, 2000; Silva et al., 2006). On the other by Kimura et al. (1997). The protocol for this study was
hand, serological approaches had shown higher levels of reviewed and approved by the Ethics Research Board of the
positivity for antibodies against the 3 variants in Brazilian Medicine School in São José do Rio Preto, Brazil.
endemic and nonendemic areas (Arruda et al., 1996;
Oliveira-Ferreira et al., 2004). VK247 variant was mainly 2.2. Target DNA sequences and design of synthetic
found as a single infection in West Africa and the Indian oligonucleotides
subcontinent. In addition, the majority of the studied DNA was extracted from blood samples by the phenol–
individuals had mixed infections with both variants, the chloroform method (Pena et al., 1991). To amplify the CSP
predominant and VK210 (Kain et al., 1991; Gonzales et al., gene, 2 sets of forward and reverse primers were designed
2001). In Southern Mexico, it was observed that all patients based on the conserved central portion of the CSP gene.
were infected with VK210 and most of them also had VK247 The CSP sequences are available in the GenBank database
(Rodriguez et al., 2000). All variants were detected in field (VK210, accession number 11926; VK247, accession
isolates from malarious regions of Papua New Guinea, number M69061; P. vivax-like, accession number
Indonesia, and Madagascar, although no pure P. vivax-like L13724). The sequences were amplified using the follow-
isolate was verified (Qari et al., 1991, 1993). ing set of primers: PR1 (5′-ACT TTT ATT CGA CTT TGT
Kain et al. (1992) developed a genotype-specific poly- TGG TC-3′) and PR2 (5′-ATG GAC TCC ATG CAG TGT
merase chain reaction (PCR) technique by 32P-end–labeled AAC C-3′). The optimal specificity was achieved using the
oligoprobes to detect the VK210 and VK247 variants, BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/).
whereas Kho et al. in 1999 investigated the polymorphisms A conformational analysis was made to investigate the
of the CSP gene in isolates from Korea by the PCR/ possibility of secondary structure formations (primer
restriction fragment length polymorphism (RFLP) technique. dimer). All oligonucleotide primers were synthesized by
The first methodology developed to identify P. vivax the Integrated DNA Technologies (Coralville, IA).
genotypes was PCR/hybridization, which also uses radi-
olabeled oligoprobes, but the technique is expensive and 2.3. PCR standardization
time-consuming, and also requires an adequate laboratorial
structure for elimination of the oligoprobes proper disposal Different PCR conditions were tested, varying PCR mixer
(Qari et al., 1993). Six years ago, Machado and Póvoa (2000) concentrations, primer annealing, and number of cycles.
optimized the Glass Fiber Membrane (GFM)/PCR/enzyme- After optimization, DNA (1.5 μL) was amplified in a total
linked immunosorbent assay (ELISA) method; however, it reaction volume of 25 μL consisting of 1× PCR buffer
needs much time, as well as uses initiating biotinylated (10 mmol/L Tris–HCl, pH 8.3, 50 mmol/L KCl), 1.5 mmol/L
primers and digoxigenin-labeled probes, raising the cost of of MgCl2, 1.0 μmol/L of each primer, 200 μmol/L
the procedure. In 2006, a protocol of nested-PCR/RFLP was deoxyribonucleotide triphospate (dNTPs), 2.5 U ampli-Taq
standardized for the diagnosis of 2 of the 3 genotypes: DNA polymerase, 1% betaine, and water (25 μL). Twenty-
VK210 and VK247 (Zakeri et al., 2006). five cycles of amplification were performed in a thermo-
The analysis of RFLPs of PCR products is a fast and cycler (DNA MasterCycler, Eppendorf, Hamburg, Germany)
simple technique (Trost et al., 2004) normally used in after initial denaturation of DNA at 94 °C for 5 min. Each
molecular biology laboratories in malaria endemic countries, cycle consisted of a denaturation step at 93 °C for 60 s, an
requiring only basic equipment (Tahar et al., 1998). Here, we annealing step at 41 °C for 90 s, and an extension step at
report on the standardization of a new PCR/RFLP for the 72 °C for 2 min, with a final extension at 72 °C for 10 min
identification of the 3 described P. vivax CSP gene variants. after the last cycle. The PCR products were analyzed by
electrophoresis using 1.5% agarose gels and stained with
ethidium bromide.
2. Materials and methods
2.4. Restriction digests of PCR products
2.1. Samples
The selected enzymes were required to have at least 1
For PCR standardization, we used 3 different plasmids cleavage site in the amplification of each variant, resulting in
(BlueScript, Stratagene, La Jolla, CA), one for the DNA fragments that are easily visible in polyacrylamide gel.
characteristic CSP repetitive region of each variant Restriction digests were set up with 10 μL of PCR product
(VK210, VK247, and P. vivax-like), and 45 frozen plus 10 and 1 U of the respective enzyme (AluI and DpnI, Promega,
3. R.T. Alves et al. / Diagnostic Microbiology and Infectious Disease 59 (2007) 415–419 417
San Diego, CA), incubated for 1 h at 37 °C. Restriction
fragments were separated by electrophoresis in 12.5%
polyacrylamide gels. The gels were stained with ethidium
bromide and analyzed with a Gel Doc 2000 illuminator (Bio-
Rad, Hercules, CA).
2.5. PCR sensitivity threshold
Three P. vivax blood samples from patients with
parasitemia ranging from 300 to 12,500 parasites per
microliter were used. These samples were serially diluted
in blood from an uninfected donor to a final level of
parasitemia corresponding to 10−6 and further processed
for PCR amplification. After that, a parasitologic evalua-
tion was performed to compare the sensitivity among the Fig. 2. P. vivax CSP gene RFLP patterns after enzymatic digestion with AluI
(from lanes 2 to 4) and DpnI (from lanes 5 to 7). Lane 1, 50-bp DNA ladder
PCR products. (Invitrogen); lanes 2 and 5, VK210 plasmid; lanes 3 and 6, VK247 plasmid;
lanes 4 and 7, P. vivax-like plasmid; lane 8, 100-bp DNA ladder.
2.6. PCR specificity
As a negative control, blood samples obtained from 20
molecularly diagnosed Plasmodium falciparum-infected with human DNA alone or with samples containing only
patients and from 10 blood donors living in the same areas P. falciparum parasites. The sensitivity of PCR was determined
with negative molecular results for Plasmodium were used. by serial dilutions of P. vivax blood samples with known
parasitemia. PCR of the P. vivax CSP gene detected levels of
2.7. P. vivax CSP gene amplification control parasitemia corresponding to 0.0069 parasites per microliter.
A single amplification of a CSP gene fragment using a set 3.2. RFLP analysis
of previously described oligonucleotide primers (AL60 5′-
GTC GGA ATT CAT GAA GAA CTT CAT TCT C-3′and To distinguish among the 3 P. vivax genotypes, RFLP
AL61 5′-CAG CGG ATC CTT AAT TGA ATA ATG CTA using AluI identified fragments of 10, 27, 38, 54, 106, and 135
GG-3′) was performed for all DNA samples (Machado and bp for VK210 and 10, 38, and 673 bp for VK247, whereas
Póvoa, 2000). P. vivax-like showed an unique fragment of 10 and 726 bp. In
respect to RFLP, using DpnI, we observed fragments with
sizes of 27, 42, 54, 81, 108, and 301 bp (VK247), whereas
3. Results P. vivax-like presented as 2 fragments (39 and 697 bp). The
3.1. Amplification of the P. vivax CSP gene fragment second enzyme has no restriction site for VK210 (Fig. 2).
Other fragments below 38 bp, not considered for variant
As shown in Fig. 1, DNA from all samples of P. vivax determination, were also formed after the RFLP procedure.
included in this study were amplified with the PR1 and PR2
primers. PCR products had lengths of 694 bp (VK210),
4. Discussion
721 bp (VK247), to 736 bp (P. vivax-like), as expected from
the BLAST program analysis. No amplifications were observed
Malaria is one of the most prevalent severe infectious
diseases in tropical and subtropical regions worldwide.
Because P. vivax malaria has been endemic in many
countries and its CSP genotypes are found worldwide, its
effective diagnosis is very important. Indeed, P. vivax
malaria variants may have different characteristics with
respect to the intensity of symptoms, the response to drugs,
and vector preference, which could cause drug resistance and
failure of control measures (Gopinath et al., 1994).
A new PCR/RFLP system was developed to identify
P. vivax genotypes. PCR primers were designed to amplify
the central immunodominant region of the CSP gene of this
protozoan. In our method, PCR primers were optimized to
Fig. 1. P. vivax CSP gene PCR products. Lane 1, 100-bp DNA ladder achieve easily distinguishable restriction fragments. The
(Invitrogen, Carlsbad, CA); lane 2, VK210 plasmid; lane 3, VK247 plasmid;
lane 4, P. vivax-like plasmid: lanes 5–6, P. vivax DNA from blood samples;
choice of restriction enzymes was also influenced by our
lanes 2–6, P. vivax CSP gene amplified with PR1 and PR2 primers; lanes objective of creating an efficient test with optimal resolution
7–8, P. vivax CSP gene amplified with AL60 and AL61. of restriction profiles. Based on the sequence analysis of
4. 418 R.T. Alves et al. / Diagnostic Microbiology and Infectious Disease 59 (2007) 415–419
P. vivax variants available in GenBank, the AluI and DpnI parasite in globally collected blood samples. J Infect Dis 170:
endonucleases were found to be the most suitable enzymes 1630–1633.
Herrera S, Corradim G, Arévalo-Herrera M (2007) An update on the search
for this purpose. AluI showed optimal discriminatory power for a Plasmodium vivax vaccine. Trends Parasitol :122–127.
to distinguish VK210 and P. vivax-like, but it was not adequate Imwong M, Pukrittayakamee S, Gruner AC, Renia L, Letourneur F,
to identify VK247 in mixed infections with the P. vivax-like Looareesuwan S, White NJ, Snounou G (2005) Practical PCR
variant. We solved this problem by adding a second genotyping protocols for Plasmodium vivax using Pvcs and Pvmsp1.
restriction step of the PCR product using DpnI, thereby Malar J 27:20.
Kain KC, Keystone J, Franke ED, Lanar DE (1991) Global distribution of a
unequivocally separating the VK247 and P. vivax-like variant of the circumsporozoite gene of Plasmodium vivax. J Infect Dis
variants, which allowed us the detection of mixed infections. 164:208–210.
The high cost and the need for adequate laboratory Kain KC, Brown AE, Webster HK, Wirtz RA, Keystone JS, Rodriguez MH,
conditions are the most frequently used arguments against Kinahan J, Rowland M, Lanar DE (1992) Circumsporozoite genotyping
using PCR in developing countries (Torres et al., 2006). of global isolates of Plasmodium vivax from dried blood specimens.
J Clin Microbiol 30:1863–1866.
However, PCR-based assays have advantages over micro- Kain KC, Brown AE, Lanar DE, Ballou WR, Webster HK (1993) Response
scopic tests because of their great capacity to distinguish P. of Plasmodium vivax variants to chloroquine as determined by
vivax genotypes, as all 3 variants are morphologically similar microscopy and quantitative polymerase chain reaction. Am J Trop
(Qari et al., 1993). Our method is sensitive and specific to Med Hyg 49:478–484.
Kho WG, Park YH, Chung JY, Kim JP, Hong ST, Lee WJ, Kim TS, Lee
detect P. vivax variants in both fresh and frozen samples. A
JS (1999) Two new genotypes Plasmodium vivax circumsporozoite
previous PCR/RFLP assay described by Zakeri et al. (2006) protein found in the Republic of Korea. Korean J Parasitol 37:
can only detect VK210 and VK247 and is inadequate for 265–270.
complete large-scale studies. In addition, our methodology Kimura M, Kaneko O, Liu Q, Zhou M, Kawamoto F, Wataya Y,
saves time and reduces the cost by more than one-half of the Otani S, Yamaguchi Y, Tanabe K (1997) Identification of the four
price of the GFM/PCR/ELISA method developed by species of human malaria parasites by nested PCR that targets
variant sequences in the small subunit rRNA gene. Parasitol Int
Machado and Póvoa (2000). Moreover, it does not use 46:91–95.
oligonucleotide primers labeled with radioisotopes as Machado RLD, Póvoa MM (2000) Distribution of Plasmodium vivax
described by Qari et al. (1993). variants (VK210, VK247 and P. vivax-like) in three endemic areas of
RFLP is more useful in distinguishing P. vivax genotypes Amazonian Brazil and their correlation with chloroquine-treatment.
than classifying them by using the CSP gene sequencing Trans R Soc Trop Med Hyg 94:377–381.
Machado RLD, Figueriredo-Filho AF, Calvosa VSP, Figueredo MC,
technique (Kho et al., 1999). Finally, the simplicity, Nascimento JM, Póvoa MM (2003) Correlation between Plasmodium
specificity, and sensitivity of the PCR/RFLP system vivax variants in Belém, Pará State, Brazil and symptoms and clearance
described here should be sufficient to determine the of parasitemia. Braz J Infect Dis 7:175–177.
prevalence and the distribution of infections of these Oliveira-Ferreira J, Pratt-Riccio LR, Santos M, Ribeiro F, Goldberg CT,
Banic AC (2004) HLA class II and antibody responses to
P. vivax genotypes in endemic and nonendemic malaria
circumsporozoite protein repeats of P. vivax (VK210, VK247 and P.
areas, enabling a better understanding their phylogeny. vivax-like) in individuals naturally exposed to malaria. Acta Trop 92:
63–69.
Acknowledgments Pena SDJ, Macedo AM, Gontijo NF (1991) DNA bioprints: simple non-
isotopic DNA fingerprints with biotinylated probes. Electrophoresis 12:
The authors thank Ana Carolina Silva, Gustavo Capatti, 14–52.
Qari SH, Goldman IF, Povoa MM, Oliveira S, Alpers MP, Lal AA (1991)
Valéria Fraga, and Luciana Moran for help in laboratory Wide distribution of the variant form of the human malaria parasite
work. Financial support was provided by FAPESP (Funda- Plasmodium vivax. J Biol Chem 266:16297–16300.
ção de Amparo à Pesquisa do Estado de São Paulo, São Qari SH, Shi YP, Goldman IF, Udhayakumar V, Alpers MP, Collins WE, Lal
Paulo, Brazil, process number 04/15486-7) and CNPq AA (1993) Identification of Plasmodium vivax-like human malaria
(Conselho Nacional de Desenvolvimento, Científico e parasite. Lancet 341:780–783.
Rieckmann KH, Davis DR, Hutton DC (1989) Plasmodium vivax resistance
Tecnológico, Brasília, DE, Brazil, process number 475524/ to chloroquine? Lancet 18:1183–1184.
2004-7). R.T.A. is a Masters student from the Genetic Rodriguez MH, Gonzalez-Ceron L, Hernandez JE, Nettel JA, Villarreal C,
Program Pos Graduation of the IBILCE/UNESP and has Kain KC, Wirtz RA (2000) Different prevalence of Plasmodium vivax
received research studentship from CNPq. phenotypes VK210 e VK247 associated with the distribution of Ano-
pheles albimanus and Anopheles pseudopunctipenis in Mexico. Am J
Trop Med Hyg 62:122–127.
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