RESUMO
Diagnosis of invasive pulmonary aspergillosis (IPA) remains a major challenge to clinical microbiology laboratories. We developed rapid and sensitive quantitative PCR (qPCR) assays for genus- and species-specific identification of Aspergillus infections by use of TaqMan technology. In order to validate these assays and understand their potential diagnostic utility, we then performed a blinded study of bronchoalveolar lavage (BAL) fluid specimens from well-characterized models of IPA with the four medically important species. A set of real-time qPCR primers and probes was developed by utilizing unique ITS1 regions for genus- and species-specific detection of the four most common medically important Aspergillus species (Aspergillus fumigatus, A. flavus, A. niger, and A. terreus). Pan-Aspergillus and species-specific qPCRs with BAL fluid were more sensitive than culture for detection of IPA caused by A. fumigatus in untreated (P < 0.0007) and treated (P ≤ 0.008) animals, respectively. For infections caused by A. terreus and A. niger, culture and PCR amplification from BAL fluid yielded similar sensitivities for untreated and treated animals. Pan-Aspergillus PCR was more sensitive than culture for detection of A. flavus in treated animals (P = 0.002). BAL fluid pan-Aspergillus and species-specific PCRs were comparable in sensitivity to BAL fluid galactomannan (GM) assay. The copy numbers from the qPCR assays correlated with quantitative cultures to determine the pulmonary residual fungal burdens in lung tissue. Pan-Aspergillus and species-specific qPCR assays may improve the rapid and accurate identification of IPA in immunocompromised patients.
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Aspergillus/isolamento & purificação , Aspergilose Pulmonar Invasiva/diagnóstico , Aspergilose Pulmonar Invasiva/microbiologia , Técnicas de Diagnóstico Molecular/métodos , Micologia/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Aspergillus/genética , Carga Bacteriana , Líquido da Lavagem Broncoalveolar/microbiologia , Primers do DNA/genética , DNA Espaçador Ribossômico/genética , Modelos Animais de Doenças , Feminino , Sondas de Oligonucleotídeos/genética , Coelhos , Sensibilidade e Especificidade , Fatores de TempoRESUMO
BACKGROUND: Human papilloma virus (HPV) infection has been shown to play an integral role in the development and prognosis of various head and neck cancers. Generational changes in sexual behavior may have led to an increased incidence of positivity in recent years. HPV positivity in both benign and malignant lesions of the sinonasal cavities has been shown in previous studies (estimates range from 20%-30% for malignancy). We intend to investigate if HPV positivity affected survival outcomes in our patient cohort. METHODS/MATERIALS: Twenty-six patients diagnosed pathologically for sinonasal squamous cell carcinoma (SCC) with available archived biopsy specimens were retrospectively analyzed to obtain HPV status using a real-time, multiplex polymerase chain reaction assay that detects and quantifies 15 known high-risk HPV types. Demographic information was collected, and survival analyses were performed using the Kaplan-Meier estimation. RESULTS: Sixteen of 26 (62%) SCC tumors in the patient cohort were positive for HPV DNA. HPV types 16 and 18 were the most common (n = 8 and 2, respectively), although a wide range of HPV types across the 15 tested were positive. Survival analyses showed a statistically significant survival advantage (median survival of 12 vs. 54 months) when accounting for HPV positivity using log-rank testing (P < 0.003). CONCLUSION: HPV positivity appears to be present in a significant proportion of squamous cell carcinoma cases of the nasal cavity. In our limited patient population there does appear to be a survival advantage to HPV positivity. Further prospective, multi-institutional trials with standardized treatment protocols are needed to elucidate the true impact of HPV positivity in this subset of head and neck cancers. LEVEL OF EVIDENCE: 4. Laryngoscope, 127:1600-1603, 2017.
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Carcinoma de Células Escamosas/terapia , Neoplasias Nasais/terapia , Infecções por Papillomavirus/terapia , Neoplasias dos Seios Paranasais/terapia , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/virologia , Estudos de Coortes , Feminino , Papillomavirus Humano 16 , Papillomavirus Humano 18 , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Nasais/mortalidade , Neoplasias Nasais/virologia , Infecções por Papillomavirus/mortalidade , Infecções por Papillomavirus/virologia , Neoplasias dos Seios Paranasais/mortalidade , Neoplasias dos Seios Paranasais/virologia , Estudos Retrospectivos , Análise de SobrevidaRESUMO
Which subtype(s) of high-risk human papillomavirus (hrHPV) are involved in squamous cell carcinoma (SCC) of the scrotum is unknown. Twenty-seven cases of SCC of the scrotum were retrieved, and all 15 subtypes of hrHPV and their viral loads were assessed using multiplex real-time polymerase chain reaction. The results were correlated with the histopathologic features, p16 expression, and in situ hybridization for hrHPV. hrHPV was identified in 18 (67%) of 27 of the cases, including HPV16 (n=8), HPV35 (n=7), HPV31 (n=5), HPV59 (n=5), HPV33 (n=3), HPV18 (n=2), HPV51 (n=2), HPV39 (n=1), HPV56 (n=1), and HPV82 (n=1). Of the 18 cases, 10 (56%) were infected by multiple hrHPV subtypes. In situ carcinomas had higher viral loads than invasive (50M versus 2M in average). The average age of HPV-positive and -negative cases was similar, 55 and 51, respectively. Of 11 cases of invasive carcinoma, 5 (45%) were positive for hrHPV versus 13 of 16 (81%) of in situ carcinomas. The highest proportion of hrHPV-positive cases was seen in basaloid type (7/7; 100%) and warty type (4/4; 100%), followed by usual type (7/16; 44%). Of 18 of the HPV-positive cases, 9 (50%) were also positive for p16 by immunohistochemistry and 6 of 18 (33%) were positive by in situ hybridization. Similar to SCC of the vulva and penis, the most frequently HPV-positive tumors are basaloid and warty types. However, a proportion of SCC usual type are also positive for hrHPV. Our results show that 8 (44%) of 18 of cases are associated with hrHPV subtypes other than 16 and 18. Additionally, 7 (70%) of 10 of hrHPV16/18-positive cases are coinfected with other subtypes.
Assuntos
Carcinoma in Situ/virologia , Carcinoma de Células Escamosas/virologia , Neoplasias dos Genitais Masculinos/virologia , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/virologia , Escroto/virologia , Adulto , Idoso , Biomarcadores Tumorais/análise , Carcinoma in Situ/química , Carcinoma in Situ/patologia , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/patologia , Inibidor p16 de Quinase Dependente de Ciclina/análise , DNA Viral/genética , Neoplasias dos Genitais Masculinos/química , Neoplasias dos Genitais Masculinos/patologia , Testes de DNA para Papilomavírus Humano , Humanos , Imuno-Histoquímica , Hibridização In Situ , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Multiplex , Papillomaviridae/genética , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , Escroto/patologia , Carga ViralRESUMO
BACKGROUND: Bangladesh, with a population of 160 million and nearly half being women, has the 4th highest rate of cervical carcinoma deaths in the world. It is projected that â¼500,000 of these women would die of this entirely preventable cancer by 2030. HPV vaccination is not widely offered in Bangladesh. This pilot study is designed to find out the prevalence of rare and multi-viral high-risk HPV (hrHPV) subtype(s) infection which may help strategize a large scale vaccination program in tackling cervical carcinoma in the country. METHODS: Forty cases of cervical High-Grade squamous intraepithelial lesion (HSIL) and Squamous cell carcinoma (SqCa) were collected. DNA was extracted from tissue representing HSIL and SqCa and multiplex PCR was run to identify all 15 hrHPV subtypes along with known positive controls. RESULTS: Of the total, 27 cases were biopsies/cones and 13 were hysterectomies including 5 HSIL and 35 SqCa. Infection caused by rare subtypes, hrHPV 45 and 52, were found in only two cases. Multi-subtype infection, detected in 28 % cases, was limited to HPV16/18 in all cases but one; one case showed hrHPV16/52 combination. CONCLUSION: A remarkable homogeneity of hrHPV 16 infection is noted in women with HSIL & SqCa in this country in these limited samples. This finding is in sharp contrast to the reports from western countries of frequent multi-viral and rare subtype hrHPV infection. This pilot study suggests that a vaccination program may be highly effective in controlling cervical cancer there. A larger study, however, is required to ratify the findings.
RESUMO
In an era where mutational profiles inform treatment options, it is critical to know the extent to which tumor biopsies represent the molecular profile of the primary and metastatic tumor. Head and neck squamous cell carcinoma (HNSCC) arise primarily in the mucosal lining of oral cavity and oropharynx. Despite aggressive therapy the 5-year survival rate is at 50%. The primary objective of this study is to characterize the degree of intratumor mutational heterogeneity in HNSCC. We used multi-region sequencing of paired primary and metastatic tumor DNA of 24 spatially distinct samples from seven patients with HNSCC of larynx, floor of the mouth (FOM) or oral tongue. Full length, in-depth sequencing of 202 genes implicated in cancer was carried out. Larynx and FOM tumors had more than 69.2% unique SNVs between the paired primary and metastatic lesions. In contrast, the oral tongue HNSCC had only 33.3% unique SNVs across multiple sites. In addition, HNSCC of the oral tongue had fewer mutations than larynx and FOM tumors. These findings were validated on the Affymetrix whole genome 6.0 array platform and were consistent with data from The Cancer Genome Atlas (TCGA). This is the first report demonstrating differences in mutational heterogeneity varying by subsite in HNSCC. The heterogeneity within laryngeal tumor specimens may lead to an underestimation of the genetic abnormalities within tumors and may foster resistance to standard treatment protocols. These findings are relevant to investigators and clinicians developing personalized cancer treatments based on identification of specific mutations in tumor biopsies.
Assuntos
Carcinoma de Células Escamosas/genética , Heterogeneidade Genética , Neoplasias de Cabeça e Pescoço/genética , Mutação , Adulto , Carcinoma de Células Escamosas/patologia , Variações do Número de Cópias de DNA , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Laríngeas/genética , Neoplasias Laríngeas/patologia , Perda de Heterozigosidade , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Neoplasias da Língua/genética , Neoplasias da Língua/patologiaRESUMO
The embryonic pancreas is thought to develop from pluripotent endodermal cells that give rise to endocrine and exocrine cells. A key guidance mechanism for pancreatic development has previously been found to be epithelial-mesenchymal interaction. Interactions within the epithelium, however, have not been well studied. Glucagon is the earliest peptide hormone present at appreciable levels in the developing pancreatic epithelium (embryonic day [E]-9.5 in mouse). Insulin accumulation begins slightly later (E11 in mouse), followed by a rapid accumulation during the "second wave" of insulin differentiation ( approximately E15). Here we found that blocking early expression and function of glucagon, but not GLP-1, an alternate gene product of preproglucagon mRNA, prevented insulin-positive differentiation in early embryonic (E11) pancreas. These results suggest a novel concept and a key role for glucagon in the paracrine induction of differentiation of other pancreatic components in the early embryonic pancreas.
Assuntos
Insulina/genética , Ilhotas Pancreáticas/embriologia , Pâncreas/embriologia , Animais , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Primers do DNA , Desenvolvimento Embrionário e Fetal , Feminino , Idade Gestacional , Glucagon/genética , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Camundongos , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Pâncreas/citologia , Reação em Cadeia da Polimerase , Gravidez , Proglucagon , Precursores de Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
High-risk human papillomavirus infection usually is seen at one anatomic site in an individual. Rarely, infection at multiple anatomic sites of the female lower genital tract in the same individual is encountered either simultaneously and/or at a later date. The current study identifies the various subtypes of high-risk human papillomavirus infection in these scenarios and analyzes the potential significance of these findings. High-risk human papillomavirus infection involving 22 anatomic sites from 7 individuals was identified after institutional review board approval. Residual paraffin-embedded tissue samples were retrieved, and all 15 high-risk human papillomavirus were identified and viral load quantified using multiplex real-time polymerase chain reaction-based method. Multiple high-risk human papillomavirus subtypes were identified in 32% of the samples and as many as 5 different subtypes of high-risk human papillomavirus infection in a single anatomic site. In general, each anatomic site has unique combination of viral subtypes, although one individual showed overlapping subtypes in the vagina, cervix, and vulvar samples. Higher viral load and rare subtypes are more frequent in younger patients and in dysplasia compared with carcinoma. Follow-up ranging from 3 to 84 months revealed persistent high-risk human papillomavirus infection in 60% of cases.
Assuntos
DNA Viral/genética , Neoplasias dos Genitais Femininos/diagnóstico , Testes de DNA para Papilomavírus Humano , Reação em Cadeia da Polimerase Multiplex , Papillomaviridae/genética , Infecções por Papillomavirus/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Neoplasias dos Genitais Femininos/patologia , Neoplasias dos Genitais Femininos/virologia , Humanos , Pessoa de Meia-Idade , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Valor Preditivo dos Testes , Prognóstico , Medição de Risco , Fatores de Risco , Fatores de Tempo , Carga Viral , Adulto JovemRESUMO
Human papillomavirus (HPV) 16 and 18 are the types most commonly found in cervical adenosquamous carcinoma. Multiple HPV types have been found in cervical adenocarcinoma but not in the adenosquamous variant. Type-specific detection of high-risk (HR) HPV allows the detection of co-infection by multiple HPV types and assessment of viral load per cell. Our aim was to identify and quantify all HR HPV types in cervical adenosquamous carcinoma and to correlate viral loads with prognosis-related histologic features. All 15 HR HPV types were tested for by multiplex real-time polymerase chain reaction, and standard curves were created for each type. Viral loads were determined retrospectively. Prognosis-related histologic features were correlated with specific HPV types and the viral loads. A total of 80% of the tumors examined expressed HPV. Types 16/18 were detected in 86% of these cases, whereas the remaining 14% of the positive cases were infected by other types. A single type of virus was detected in 67% of cases, 2 in 29%, and 3 in 4%. Poor prognostic features were seen in 84.6% of the tumors infected with HPV 16, 46% of those infected with HPV 18, and 100% of those infected with other types. As expected, HPV 16, HPV 18, or both were the most frequent viral types; HPV 73 was the next most frequent type. Multiple HPV types were detected in 33% of the tumors. Non-HPV 16/18 cases had low viral loads, but all of these had poor prognosis-related histologic features. Two of the three recurrent cases had multiple viral types.
Assuntos
Carcinoma Adenoescamoso/epidemiologia , Carcinoma Adenoescamoso/virologia , Infecções por Papillomavirus/epidemiologia , Infecções por Papillomavirus/virologia , Neoplasias do Colo do Útero/epidemiologia , Neoplasias do Colo do Útero/virologia , Adulto , Idoso , Colo do Útero/virologia , DNA Viral/análise , Feminino , Papillomavirus Humano 16 , Papillomavirus Humano 18 , Humanos , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Multiplex , Papillomaviridae/genética , Prevalência , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Carga ViralRESUMO
INTRODUCTION: High-risk (HR) human papillomavirus (HPV) testing is accepted as the standard of care for surveillance of cervical cancer. Its role in anal cancer is not clear. This study was therefore designed to determine if HR HPV genotyping is a useful adjunct in management of abnormal anal Papanicolaou (Pap) tests. MATERIALS AND METHODS: HR HPV genotyping and virus quantification was performed on 101 residual anal Pap test samples (28 negative, 25 atypical squamous cells of undetermined significance [ASC], 34 low-grade squamous intraepithelial lesion [LSIL], 6 atypical squamous cells of undetermined significance, cannot exclude high-grade squamous intraepithelial lesion, and 8 high-grade squamous intraepithelial lesion) using multiplex real-time polymerase chain reaction. Results were correlated with cytodiagnosis and follow-up. RESULTS: HR HPV was detected in 82% (50% negative, 84% ASC, and 100% LSIL and above) cases. Multiple genotypes were present in 71% of cases. Genotype number and viral load correlated with the degree of anal cytologic abnormality. HPV 16, 18, and 45 were the most frequent genotypes detected. The high frequency of HR HPV in abnormal anal cytologies limits its use as an adjunct test. Anal Pap test samples with anal intraepithelial neoplasia 2/3 (AIN 2/3) on follow-up were positive for HPV 16 and/or 18 (HPV 16/18+) in 80% of cases. We hypothesize that testing for HPV 16/18 on the ASC and LSIL cases would have detected AIN 2/3 with a sensitivity of 81%, specificity of 43%, positive predictive value of 39%, and negative predictive value of 83%. CONCLUSIONS: Our results with a small cohort suggest that genotyping for HPV 16/18 may be effective in identifying patients at high risk for anal cancer and in reducing the number of anoscopy referrals. Prospective studies with follow-up are warranted.
RESUMO
Loop-mediated isothermal amplification (LAMP) is a novel method for rapid amplification of DNA. Its advantages include rapidity and minimal equipment requirement. The LAMP assay was developed for BK virus (BKV), which is a leading cause of morbidity in renal transplant recipients. The characteristics of the assay, including its specificity and sensitivity, were evaluated. BKV LAMP was performed using various incubation times with a variety of specimens, including unprocessed urine and plasma samples. A ladder pattern on gel electrophoresis, typical of successful LAMP reactions, was observed specifically only for BKV and not for other viruses. The sensitivity of the assay with 1 h of incubation was 100 copies/tube of a cloned BKV fragment. Additionally, a positive reaction was visually ascertained by a simple color reaction using SYBR green dye. BKV LAMP was also successful for urine and plasma specimens without the need for DNA extraction. Due to its simplicity and specificity, the LAMP assay can potentially be developed for "point of care" screening of BKV.
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Vírus BK/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Sequência de Bases , DNA Viral , Genes Virais , Humanos , Transplante de Rim/efeitos adversos , Infecções por Polyomavirus/diagnóstico , Infecções por Polyomavirus/virologia , Sensibilidade e Especificidade , Fatores de Tempo , Infecções Tumorais por Vírus/diagnóstico , Infecções Tumorais por Vírus/virologiaRESUMO
Human herpesvirus (HHV)--6 infections are ubiquitous, but infection or reactivation under immunocompromised conditions, such as bone marrow or solid organ transplantation, can often result in serious clinical manifestations. Two HHV-6 subtypes are known. Most primary HHV-6 infections are caused by subtype 6B, but little information is available about the prevalence, distribution, and clinical divergence of 6A and 6B. To study this, we have developed a highly sensitive and specific real-time polymerase chain reaction (PCR) assay that can detect, quantitate, and reliably differentiate HHV-6A and -6B in clinical specimens. Exploiting a single-base variation in the DNA polymerase gene of these respective subtypes, we used melting curve analysis for subtype discrimination. Moreover, this assay's ability to discriminate HHV-6 subtypes was confirmed by PCR/restriction fragment length polymorphism analysis of the HHV-6 large tegument protein gene and PCR amplicon size-discrimination analysis of the HHV-6 immediate-early gene. Using this assay, we present our findings about the prevalence and distribution of these subtypes in bone marrow transplant patients. Of 803 plasma specimens tested from 353 patients, 136 specimens (17%) from 60 patients were determined to be HHV-6 positive. We analyzed these HHV-6--positive patients for subtype identification by using our newly developed assay and determined that 58 patients (97%) were HHV-6B positive and 2 patients (3%) were HHV-6A positive. No patient was coinfected with both subtypes. This assay can be a sensitive, genotype-specific, rapid method to reliably diagnose life-threatening HHV-6 infections in immunocompromised patients and can be useful in guiding and monitoring specific therapy.
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Transplante de Medula Óssea , DNA Viral/sangue , Herpesvirus Humano 6/genética , Reação em Cadeia da Polimerase , Infecções por Roseolovirus/sangue , Herpesvirus Humano 6/classificação , Humanos , Reação em Cadeia da Polimerase/métodos , Estudos Retrospectivos , Infecções por Roseolovirus/genética , Sensibilidade e EspecificidadeRESUMO
Mouse pancreatic development is critically dependent on epithelial-mesenchymal interactions. The pancreas differs from other epithelial-mesenchymal organs in that the epithelium gives rise to both epithelial exocrine cells and non-epithelial endocrine cells. We studied the nature of the interactions between the epithelium and mesenchyme with respect to the decision between exocrine and endocrine lineages. We show here a tripartite influence of mesenchyme on the developing epithelium. First, close proximity or contact of mesenchyme with the epithelium induces exocrine differentiation. Second, this mesenchymal proximity to the epithelium suppresses endocrine differentiation. Third, mesenchyme has an overall enhancing effect on the degree of insulin differentiation, suggesting a pro-endocrine effect in those epithelial cells at a distance from the mesenchyme. Proximity or contact between the mesenchyme and epithelium appeared to be necessary for the pro-exocrine effects of mesenchyme. We found that, in a co-culture system, NIH3T3 cells were able to substitute for mesenchyme in exocrine induction as well as in both the endocrine induction and endocrine inhibition, implying that the responsible molecules are not unique to pancreatic mesenchyme. Laminin appears to be a key molecule mediating the epithelial-mesenchymal interactions that lead to exocrine differentiation, since inhibition of laminin expression resulted in blockage of the pro-exocrine induction of mesenchyme.
Assuntos
Diferenciação Celular/fisiologia , Mesoderma/metabolismo , Pâncreas/embriologia , Animais , Epitélio/embriologia , Epitélio/metabolismo , Técnicas In Vitro , Ilhotas Pancreáticas/embriologia , Ilhotas Pancreáticas/metabolismo , Laminina/metabolismo , Camundongos , Células NIH 3T3 , Pâncreas/metabolismoRESUMO
The embryogenesis of tracheoesophageal anomalies remains controversial. The purpose of this study was to better define the embryogenesis of developing esophageal atresia with tracheoesophageal fistula (EA/TEF), with specific attention to the controversial issue of whether a discontinuity exists in the foregut during its development of EA/TEF. Pregnant outbred rats were injected with adriamycin (2 mg/kg i.p.) on days 6-9 of gestation (E6-E9). At E12.5 and 13.5, microdissection of the entire foregut was performed. Foreguts were examined by phase microscopy, and serial, precisely transverse sections were created for hematoxylin and eosin (H&E) staining. Gross microdissection of the developing foregut at E12.5 (n = 9) revealed a blind-ending, bulbous fistula tract arising from the middle branch of the tracheal trifurcation (as seen by direct and phase microscopy). No connection with the gut could be appreciated at E12.5, but by E13.5 (n = 10) there was an obvious connection between the fistula and the stomach. Serial H&E transverse sections also demonstrated a blind-ending fistula tract arising from the trachea at E12.5. This fistula tract was clearly discontinuous from the developing stomach, which appeared much further caudal to the end of the fistula tract. These results strongly support a model of experimental TEF wherein the fistula tract arises from a trifurcation of the trachea, and (only during a specific gestational window between days 12.5 and 13.5) there is discontinuity between the fistula tract and the stomach. By day 13.5, the fistula joins with the stomach anlage. These observations in the developing EA/TEF should help to resolve the controversy about the mechanism of EA/TEF formation.