Validation and standardization of DNA extraction and library construction methods for metagenomics-based human fecal microbiome measurements.

BACKGROUND: Validation and standardization of methodologies for microbial community measurements by high-throughput sequencing are needed to support human microbiome research and its industrialization. This study set out to establish standards-based solutions to improve the accuracy and reproducibility of metagenomics-based microbiome profiling of human fecal samples. RESULTS: In the first phase, we performed a head-to-head comparison of a wide range of protocols for DNA extraction and sequencing library construction using defined mock communities, to identify performant protocols and pinpoint sources of inaccuracy in quantification. In the second phase, we validated performant protocols with respect to their variability of measurement results within a single laboratory (that is, intermediate precision) as well as interlaboratory transferability and reproducibility through an industry-based collaborative study. We further ascertained the performance of our recommended protocols in the context of a community-wide interlaboratory study (that is, the MOSAIC Standards Challenge). Finally, we defined performance metrics to provide best practice guidance for improving measurement consistency across methods and laboratories. CONCLUSIONS: The validated protocols and methodological guidance for DNA extraction and library construction provided in this study expand current best practices for metagenomic analyses of human fecal microbiota. Uptake of our protocols and guidelines will improve the accuracy and comparability of metagenomics-based studies of the human microbiome, thereby facilitating development and commercialization of human microbiome-based products. Video Abstract.

Human papillomavirus types 52 and 58 are prevalent in uterine cervical squamous lesions from Japanese women.

Objective. To estimate the prevalence and genotypes of high-risk human papillomavirus (HPV) focusing HPV 16, 18, 52, and 58 in Japan. Methods. Liquid-base cytology specimens were collected from Japanese women (n = 11022), aged 14-98. After classifying cytodiagnosis, specimens were analyzed for HPV DNA by the multiplex polymerase chain reaction method, where 1195 specimens were positive for cervical smear, except adenomatous lesions. Result. HPV genotypes were detected in 9.5% of NILM and 72.2% of ASC-US or more cervical lesions. In positive cervical smears, HPV genotypes were HPV 52 at 26.6%, HPV 16 at 25.2%, HPV 58 at 21.8%, and HPV 18 at 7.1%. Most patients infected with HPV 16 were between 20-29 years old, decreasing with age thereafter. As for HPV 52 and 58, although the detection rate was high in 30- to 39-year-olds, it also was significant in the 50s and 60s age groups. Conclusion. In Japan, as a cause of abnormal cervical cytology, HPV52 and 58 are detected frequently in addition to HPV 16. In older age groups, HPV 52 and 58 detection rates were higher than that observed for HPV 16. After widespread current HPV vaccination, we still must be aware of HPV 52 and 58 infections.

Epigenetic alteration of DNA in mucosal wash fluid predicts invasiveness of colorectal tumors.

Although conventional colonoscopy is considered the gold standard for detecting colorectal tumors, accurate staging is often difficult because advanced histology may be present in small colorectal lesions. We collected DNA present in mucosal wash fluid from patients undergoing colonoscopy and then assessed the methylation levels of four genes frequently methylated in colorectal cancers to detect invasive tumors. We found that methylation levels in wash fluid were significantly higher in patients with invasive than those with noninvasive tumors. Cytologic and K-ras mutation analyses suggested that mucosal wash fluid from invasive tumors contained greater numbers of tumor cells than wash fluid from noninvasive tumors. Among the four genes, levels of mir-34b/c methylation had the greatest correlation with the invasion and showed the largest area under the receiver operating characteristic curve (AUC = 0.796). Using cutoff points of mir-34b/c methylation determined by efficiency considerations, the sensitivity/specificity were 0.861/0.657 for the 13.0% (high sensitivity) and 0.765/0.833 for the 17.8% (well-balanced) cutoffs. In the validation test set, the AUC was also very high (0.915), the sensitivity/specificity were 0.870/0.875 for 13.0% and 0.565/0.958 for 17.8%. Using the diagnostic tree constructed by an objective algorithm, the diagnostic accuracy of the invasiveness of colorectal cancer was 91.3% for the training set and 85.1% for the test set. Our results suggest that analysis of the methylation of DNA in mucosal wash fluid may be a good molecular marker for predicting the invasiveness of colorectal tumors.

A case of congenital toxoplasmosis whose mother demonstrated serum low IgG avidity and positive tests for multiplex-nested PCR in the amniotic fluid.

We encountered a woman whose infant developed congenital toxoplasmosis. Serum Toxoplasma gondii antibody titers (320x) at 12 weeks of gestation increased to 5120x at 25 weeks. Toxoplasma immunoglobulin M was 2.8 index, and immunoglobulin G avidity index was 23%. Cyclic administration of acetylspiramycin was maintained from 22 weeks until delivery. Multiplex-nested polymerase chain reaction of maternal blood and amniotic fluid at 28 weeks both tested positive for Toxoplasma DNA. A male neonate weighing 2916 g was born at 38 weeks via cesarean section. No abnormalities were detected by physical and funduscopic examinations, whereas a head computed tomography of the neonate revealed three independent intracranial calcifications. The infant underwent therapy with pyrimethamine and sulfadiazine for one year. Serum titers of Toxoplasma gondii antibodies were all less than cut-off values between 5 and 12 months after birth, but all increased up to positive levels 18 months after birth.

Genotyping of human papillomaviruses by a novel one-step typing method with multiplex PCR and clinical applications.

We describe here a rapid, high-throughput genotyping procedure that allows the simultaneous detection of 16 high- and low-risk genital human papillomavirus (HPV) types by multiplex PCR in a single reaction tube. Multiplex PCR is based on the amplification of HPV DNA by sets of HPV genotype-specific primers, and the genotypes of HPV are visually identified by the sizes of amplicons after they are separated by capillary electrophoresis. The procedure does not include a hybridization step with HPV-specific probes and is rapid and labor-saving. We detected all 16 HPV genotypes (types 16, 58, 52, 51, 56, 31, 18, 39, 66, 59, 6, 33, 30, 35, 45, and 11) with a high sensitivity and a high degree of reproducibility. By using this newly developed method, we conducted a pilot study to examine the correlation between the prevalence and genotype distributions of HPV and the cytological group classifications for 547 cervical samples. Compared with the group of samples considered normal (14.7%), there was a significant increase in the prevalence of HPV in women with atypical squamous cells of unknown significance (61.3%), low-grade intraepithelial lesions (75.8%), and high-grade intraepithelial lesions (HSILs) (82.2%). The prevalence and distribution of type 58 were correlated with cytological malignancies, with the highest prevalence in women with HSILs. In conclusion, the novel multiplex PCR method described appears to be highly suitable not only for the screening of cervical cancer precursor lesions but also for the characterization of genotype distributions in large-scale epidemiological studies and HPV vaccination trials.

Epidemiology of Epstein-Barr virus, cytomegalovirus, and Kaposi's sarcoma-associated herpesvirus infections in peripheral blood leukocytes revealed by a multiplex PCR assay.

A multiplex polymerase chain reaction (PCR) has been developed for the simultaneous detection of Epstein-Barr virus (EBV), cytomegalovirus (CMV), and Kaposi's sarcoma-associated herpesvirus (KSHV) in a clinical sample. Primers of multiplex PCR were designed to amplify specific regions of the EBV EBNA1, CMV IE2, and KSHV LANA genes. This multiplex PCR assay was found to have detection sensitivities of 1-10 copies of purified viral DNA cloned into the plasmid. To assess diagnostic and pre-clinical applications with this method, we utilized KSHV-positive primary effusion lymphoma (PEL) cells, EBV-positive Burkitt's lymphoma cells, CMV-infected fibroblast cells, and clinically prepared peripheral blood leukocytes (PBLs) that had been infected with viruses. We found that this multiplex PCR assay has high sensitivity and specificity for simultaneous detection of EBV, CMV, and KSHV genomes in a single amplification from a clinical material. Using this multiplex PCR assay, we investigated the prevalence of EBV, CMV, and KSHV in PBL samples from normal Japanese randomly selected. KSHV, EBV, and CMV genomes were detected in samples from 2 (0.2%), 377 (39.5%), and 27 (2.8%) of the 953 blood donors, respectively. Interestingly, both EBV and CMV genomes were detected in samples from all KSHV-positive donors.

Protective effects of heat shock protein 70 induced by geranylgeranylacetone on oxidative injury in rat intestinal epithelial cells.

OBJECTIVE: Geranylgeranylacetone (GGA), an anti-ulcer agent, has recently been demonstrated to protect a variety of cells and tissues via induction of heat shock protein (HSP)70 against numerous stresses. We investigated whether GGA induces HSP70 and protects against an oxidative stressor, monocrolamine (NH(2)Cl), in a rat intestinal epithelial cell line (IEC-18). MATERIAL AND METHODS: IEC-18 cells pretreated with GGA (0.1-10 microM) were subjected to injury induced by NH(2)Cl. Cell viability was assessed, and endogenous HSP70 levels were determined by enzyme-linked immunosorbent assay in IEC-18 cells. RESULTS: Treatment with GGA (0.1-10 microM) was found rapidly to elevate HSP70 levels and to protect against NH(2)Cl-induced injury in IEC-18 cells. Furthermore, quercetin, an inhibitor of HSP70 synthesis, diminished the protective effects of GGA in IEC-18 cells upon NH(2)Cl-caused injury. CONCLUSIONS: The results of this study suggest that GGA plays an important role in defense mechanisms against oxidative injury in the intestine, primarily via induction of HSP70.

Regulation of Toll-like receptor 4 expression in mouse colon by macrophage migration inhibitory factor.

Recent studies have indicated that macrophage migration inhibitory factor (MIF) and Toll-like receptor (TLR) play an important role in the regulation of innate immune responses. In this study, we investigated the effect of MIF on the expression of TLR4, a receptor that recognizes lipopolysaccharide, in colon using MIF-deficient mice. TLR4 mRNA expression in the colon tissues was determined by northern blot analysis. Western blot analysis and immunohistochemistry in the colon tissues were performed to evaluate the expression of TLR4 protein. The expressions of TLR4 mRNA and protein were remarkably down-regulated in colon tissues of MIF-deficient mice compared with wild-type mice and up-regulated by treatment with recombinant MIF. Immunohistochemical study revealed the presence of TLR4-positive staining in mononuclear cells in the lamina propria and intraepithelial mononuclear cells as well as weak staining in epithelial cells and crypts in colon tissues of wild-type mice. In contrast, MIF-deficient mice did not show TLR4-positive staining in the colonic mucosa. In MIF-deficient mice injected with recombinant mouse MIF (rMIF), TLR4-positive staining cells were observed in colon tissues similar to the findings in wild-type mice. Administration of dextran sulfate sodium (DSS) up-regulated the expression of TLR4 in the colons of WT mice but not in those of MIF-deficient mice. Furthermore, pretreatment with rMIF up-regulated the expression of TLR4 in response to DSS in MIF-deficient mice. Our results suggest that MIF affects the expression of TLR4 in mouse colon under both normal and colitic conditions.