RESUMO
Kidney biopsy interpretation is the gold standard for the diagnosis and prognosis for kidney disease. Pathognomonic diagnosis hinges on the correct assessment of different structures within a biopsy that is manually visualized and interpreted by a renal pathologist. This laborious undertaking has spurred attempts to automate the process, offloading the consumption of temporal resources. Segmentation of kidney structures, specifically, the glomeruli, tubules, and interstitium, is a precursory step for disease classification problems. Translating renal disease decision making into a deep learning model for diagnostic and prognostic classification also relies on adequate segmentation of structures within the kidney biopsy. This study showcases a semi-automated segmentation technique where the user defines starting points for glomeruli in kidney biopsy images of both healthy normal and diabetic kidney disease stained with Nile Red that are subsequently partitioned into four areas: background, glomeruli, tubules and interstitium. Five of 30 biopsies that were segmented using the semi-automated method were randomly selected and the regions of interest were compared to the manual segmentation of the same images. Dice Similarity Coefficients (DSC) between the methods showed excellent agreement; Healthy (glomeruli: 0.92, tubules: 0.86, intersititium: 0.78) and diabetic nephropathy: (glomeruli: 0.94, tubules: 0.80, intersititium: 0.80). To our knowledge this is the first semi-automated segmentation algorithm performed with human renal biopsies stained with Nile Red. Utility of this methodology includes further image processing within structures across disease states based on biological morphological structures. It can also be used as input into a deep learning network to train semantic segmentation and input into a deep learning algorithm for classification of disease states.
Assuntos
Aprendizado Profundo , Algoritmos , Biópsia , Humanos , Processamento de Imagem Assistida por Computador , Rim/diagnóstico por imagemRESUMO
BACKGROUND: The aim of this study was to determine the prevalence of human papillomavirus (HPV) and 15 species that cause sexually transmitted infections (STIs) in negative cytology. In addition, we compared the diagnostic performance of multiplex polymerase chain reaction (PCR) with widely available techniques used to detect HPV. METHODS: We recruited 235 women of reproductive age who had negative cytology findings in a liquid-based cervical smear. STIs were identified by multiplex PCR, and HPV genotypes by multiplex PCR, hybrid capture 2, and DNA microaray; discordant results were analyzed by direct sequencing. RESULTS: Approximately 96.6% of patients with negative cytology results were positive for pathogens that cause STIs. The pathogens most frequently detected were Gardnerella vaginalis, Ureaplasma urealyticum. The incidence of HPV in negative cytology was 23.3%. Low-risk HPV infection was significantly correlated with Chalmaydia trachomatis, and high-risk HPV infection was significantly correlated with Group ß streptococcus. The analytical sensitivities of the multiplex PCR and DNA microarray were higher than 80%, and the analytical specificity was nearly 100% for all tests. CONCLUSIONS: Multiplex PCR yielded results that most of patients with negative cytology were positive for pathogens that cause STIs, and were more similar to that of DNA microarray, than that of hybrid capture 2 in terms of analytical sensitivity and prediction value of HPV infection.
Assuntos
Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Reação em Cadeia da Polimerase/métodos , Doenças Virais Sexualmente Transmissíveis/diagnóstico , Virologia/métodos , Adulto , Colo do Útero/citologia , Feminino , Gardnerella vaginalis/isolamento & purificação , Humanos , Análise em Microsséries/métodos , Pessoa de Meia-Idade , Hibridização de Ácido Nucleico/métodos , Papillomaviridae/genética , Prevalência , Sensibilidade e Especificidade , Ureaplasma urealyticum/isolamento & purificação , Esfregaço VaginalRESUMO
We describe a matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS)-based assay for human papillomavirus (HPV) genotyping--the restriction fragment mass polymorphism (RFMP) assay, which is based on mass measurement of genotype-specific oligonucleotide fragments generated by TypeIIS restriction endonuclease cleavage after recognition sites have been introduced by PCR amplification. The use of a TypeIIS restriction enzyme makes the RFMP assay independent of sequence and applicable to a wide variety of HPV genotypes, because these enzymes have cleavage sites at a fixed distance from their recognition sites. After PCR amplification, samples are subjected to restriction enzyme digestion with FokI and BtsCI and desalting using Oasis purification plates, followed by analysis by MALDI-TOF MS. Overall, the protocol is simple, takes approximately 4-4.5 h and can accurately detect and identify at least 74 different HPV genotypes.
Assuntos
Papillomavirus Humano 16/genética , Polimorfismo de Fragmento de Restrição/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Pareamento de Bases , Sequência de Bases , Genótipo , Dados de Sequência Molecular , Oligonucleotídeos/genéticaRESUMO
BACKGROUND/AIMS: The objective of this study was to document the prevalence rate of occult hepatitis B virus (HBV) in healthy pregnant woman and the possibility of transmission to the foetus. METHODS: This study was performed prospectively with 202 healthy pregnant women. HBV-DNA testing was performed using two specific quantitative tests with two independent sets of sera and cord blood. DNA sequencing analysis was carried out to confirm the specificity of polymerase chain reaction (PCR) product of HBV-DNA testing. RESULTS: Eight of 202 (4%) individuals with the TaqMan PCR assay and 23 of 202 (11.4%) with the COBAS Amplicor HBV Monitor test were HBV-DNA positive. Six (3%) individuals were positive with both methods. Sequencing and genotyping analysis of HBV polymerase gene with sera of the 75th subject resulted in genotype C. HBV-DNA testing with four cord blood samples showed that all were HBV-DNA negative. CONCLUSION: Occult HBV infection shows a difference in prevalence rate depending on the test method but the existence has been confirmed by sequencing analysis. Our results also suggest that vertical transmission through the cord blood is not so high as to be clinical problems and warrants further investigation.
Assuntos
DNA Viral/sangue , Hepatite B/epidemiologia , Complicações Infecciosas na Gravidez/epidemiologia , Adulto , Feminino , Produtos do Gene pol/genética , Hepatite B/transmissão , Hepatite B/virologia , Humanos , Recém-Nascido , Transmissão Vertical de Doenças Infecciosas , Coreia (Geográfico)/epidemiologia , Reação em Cadeia da Polimerase , Gravidez , Complicações Infecciosas na Gravidez/virologia , Prevalência , Estudos Prospectivos , Análise de Sequência de DNARESUMO
BACKGROUND: Infection with human papilloma virus (HPV) is the main cause of cervical cancer, and HPV genotyping is of increasing importance for determining clinical course and management of the disease based on the HPV genotypes. Here, we established a novel matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF MS) assay, termed restriction fragment mass polymorphism (RFMP) that is suitable for genotyping multiple HPV in an accurate and high-throughput manner. We evaluated the performance of the RFMP assay in HPV genotyping by comparing the results with those of direct or clonal sequencing and hybrid capture (HC) assays. METHODS: The study population consisted of 50 patients with histologically confirmed cervical lesions and a positive test for HPV DNA. HPV genotyping was performed with RFMP, sequencing, and HC assays. The assigned genotypes and risk groups were compared among the methods. RESULTS: Concordance rates in the genotype level between RFMP vs sequencing, sequencing vs HC, and HC vs RFMP were 98% (49/50), 88% (44/50), and 88% (44/50), respectivley. Especially, RFMP and sequencing were 100% concordant when assigned high-risk group was considered identical in 1 case of mixed genotypes identified only in RFMP. The observed discrepancy between HC and the other two methods is due to the assignment of six cases of low, intermediate, or unassigned risk genotypes as high-risk group in HC method. CONCLUSIONS: RFMP, sequencing, and HC assays were highly concordant with each other in HPV genotyping. Compared to sequencing assay, RFMP assay is found to be advantageous in detecting mixed genotype infections. The accuracy and amenability to high-throughput analysis should make the RFMP assay suitable for reliable screening of HPV genotypes in clinical laboratories.
Assuntos
Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Neoplasias do Colo do Útero/diagnóstico , Feminino , Genótipo , Humanos , Hibridização de Ácido Nucleico/métodos , Papillomaviridae/classificação , Papillomaviridae/genética , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA/métodosRESUMO
BACKGROUND/AIMS: Immunogenetic factors may play a role in determining the susceptibility of an individual to viral infection. CCR5 promoter polymorphisms are known to be associated with HIV infection. However, there has been no report on the association between CCR5 promoter polymorphism and HBV infection. Therefore, we investigated the relationship between the CCR5 promoter polymorphism and HBV infection. METHODS: A total of 377 patients were classified into two groups according to their HBV infection status: (1) the spontaneous clearance group (SC); HBsAg (-), anti-HBc (+), anti-HBs (+) (2) the chronic HBsAg (+) carrier group (CC); HBsAg (+), anti-HBc (+), anti-HBs (-). CCR5 polymorphisms were detected by employing matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS)- based SNP scoring assay, termed Restriction Fragment Mass Polymorphism (RFMP), which exploits the differences in molecular masses between the common allele and rare allele bases of interest. RESULTS: We found that the genotype frequencies of CCR5 A59029G significantly differed between the SC group (n=138) and CC group (n=239) (P<0.05). The CCR5 59029A allelic genotype was associated with an increased risks of chronic infection rather than spontaneous clearance (P=0.002), and the presence of the CCR5 59029G allele was significantly associated with the spontaneous clearance of HBV (P=0.001). Strong linkage disequilibrium between the CCR5-59029 and the CCR5-59353 polymorphic variants was identified. None of the 377 subjects had the CCR5-32 bp deletion mutation. CONCLUSIONS: The CCR5 promoter polymorphisms at position 59029 might play a role in the clearance of HBV infection. This primary experimental evidence needs further studies to clarify the clinical usefulness of CCR5 promoter polymorphisms as a target for the screening or treatment of HBV infection.
Assuntos
Hepatite B/genética , Polimorfismo Genético , Regiões Promotoras Genéticas/genética , Receptores CCR5/genética , Adulto , Feminino , Predisposição Genética para Doença , Genótipo , Vírus da Hepatite B/genética , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Identifying hepatitis C virus (HCV) genotypes has become increasingly important for determining clinical course and the outcome of antiviral therapy. Here we describe the development of restriction fragment mass polymorphism (RFMP) analysis, a novel matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assay suitable for high-throughput, sensitive, specific genotyping of multiple HCV species. METHODS: The assay is based on PCR amplification and mass measurement of oligonucleotides containing genotype-specific motifs in the 5' untranslated region, into which a type IIS restriction endonuclease recognition was introduced by PCR amplification. Enzymatic cleavage of the products led to excision of multiple oligonucleotide fragments representing variable regions whose masses were determined by MALDI-TOF MS. RESULTS: The RFMP assay identified viral genotypes present at concentrations as low as 0.5% and reliably determined their relative abundance. When sera from 318 patients were analyzed, the RFMP assay exhibited 100% concordance with results obtained by clonal sequencing and identified mixed-genotype infections in 22% of the samples, in addition to several subtype variants. CONCLUSIONS: The RFMP assay has practical advantages over existing methods, including better quantitative detection of mixed populations and detection of genotype variants without need for population-based cloning, enabling reliable viral genotyping in laboratories and efficient study of the relationship between viral genotypes and clinical outcome.
Assuntos
DNA Viral/genética , Hepacivirus/genética , Regiões 5' não Traduzidas , DNA Viral/sangue , Hepatite C Crônica/genética , Humanos , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based genotyping assay, termed restriction fragment mass polymorphism (RFMP) has been recently developed for detecting hepatitis B virus (HBV) mutants. The assay is based on PCR amplification and mass measurement of oligonucleotides containing sites of mutations that confer resistance to lamivudine. We compared the efficacy and usefulness of the RFMP assay with a commercial assay using a reverse hybridization line probe technology, namely INNO-LiPA HBV DR (referred to henceforth as the LiPA assay), for the detection of lamivudine-resistant HBV mutants. A total of 60 patient samples were analysed for the presence of mutations at rtL1 80M and rtM204I/V of HBV polymerase by the LiPA and RFMP assays. The ability to detect mutations at rtM204I/V was compared with defined mixtures of wild-type and mutant HBV cloned in plasmids at relative concentrations ranging from 1-25%. Concordance between methods was found to be 95.0% (57/60) when only the presence of resistance mutations was considered, regardless of quasispecies. In three cases, additional minor populations of resistant viruses were identified by RFMP. Defined mixtures were consistently successfully identified at a 1% relative concentration of mutant versus wild-type viruses by the RFMP assay and 4% by the LiPA assay. The RFMP assay proved to be an accurate and reliable tool for detection of lamivudine-resistant mutations and was more sensitive than the LiPA assay in detecting mixtures of mutant and wild-type viruses. The improved sensitivity of the RPMP assay can help monitor drug resistance as it develops, enabling early intervention and prevention.
Assuntos
Antivirais/uso terapêutico , Farmacorresistência Viral , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/tratamento farmacológico , Lamivudina/uso terapêutico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , DNA Viral , Monitoramento de Medicamentos , Variação Genética , Vírus da Hepatite B/genética , Humanos , Mutação , Polimorfismo de Fragmento de RestriçãoRESUMO
BACKGROUND/AIMS: Recent studies have shown that the genotype of hepatitis B virus (HBV) may correlate with the disease natural history and treatment outcome. However, several of these studies used low sensitivity assays in a small number of patients, and this has precluded an accurate evaluation of Korean HBV genotypes. We analyzed Korean HBV genotypes in a large population by employing a new technology, restriction fragment mass polymorphism (RFMP) using MALDI-TOF mass spectrometry, in a sensitive and specific manner. METHODS: Between February 1995 and December 2003, a total of 475 patients with chronic HBV infection were enrolled. The assay is based on the mass measurement of oligonucleotides having genotypic variations of the S gene. Clinical features including the virologic status and disease progression were also evaluated. RESULTS: The median age of the total patients was 35.5 years. Out of 475 patients, there were 162 (34.1%) inactive carriers, 172 (36.2%) had chronic hepatitis, 77 (16.2%) had liver cirrhosis and 64 (13.5%) had hepatocellular carcinoma (HCC). There were 454 patients (95.6%) with genotype C, 4 patients (0.8%) with genotype A, 16 patients (3.4%) with the mixed A and C genotype [7 patients (1.4%) with A
Assuntos
Vírus da Hepatite B/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Adolescente , Adulto , Idoso , Feminino , Genótipo , Vírus da Hepatite B/classificação , Hepatite B Crônica/virologia , Humanos , Coreia (Geográfico) , Masculino , Pessoa de Meia-Idade , Polimorfismo de Fragmento de RestriçãoRESUMO
BACKGROUND/AIMS: Mutations in hepatitis B virus (HBV) to lamivudine resistance that arise during prolonged treatment frequently cause amino acid substitutions in the YMDD motif of HBV DNA polymerase. Current methods of detecting such variants are time-consuming, labor intensive, and unsuitable for screening large numbers of samples. Here, we describe the development of a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) genotyping assay suitable for detecting HBV variants in a sensitive and specific manner. METHODS: The assay is based on PCR amplification and mass measurement of oligonucleotides containing sites of mutation of the YMDD motif. RESULTS: The MALDI-TOF MS-based genotyping assay is sufficiently sensitive to detect as few as 100 copies of HBV genome per millilitre of serum, with superior specificity for determining mixtures of wild-type and variant viruses. When sera from 40 patients were analyzed, the MALDI-TOF MS-based assay correctly identified known viral variants and additional viral quasi-species not detected by previous methods, as well as their relative abundance. CONCLUSIONS: The sensitivity, accuracy and amenability to high-throughput analysis makes the MALDI-TOF MS-based assay suitable for mass screening of HBV infected patients receiving lamivudine, and can help provide further understanding of disease progression and response to therapy.