Low-pass genome sequencing versus chromosomal microarray analysis: implementation in prenatal diagnosis.

PURPOSE: Emerging studies suggest that low-pass genome sequencing (GS) provides additional diagnostic yield of clinically significant copy-number variants (CNVs) compared with chromosomal microarray analysis (CMA). However, a prospective back-to-back comparison evaluating accuracy, efficacy, and incremental yield of low-pass GS compared with CMA is warranted. METHODS: A total of 1023 women undergoing prenatal diagnosis were enrolled. Each sample was subjected to low-pass GS and CMA for CNV analysis in parallel. CNVs were classified according to guidelines of the American College of Medical Genetics and Genomics. RESULTS: Low-pass GS not only identified all 124 numerical disorders or pathogenic or likely pathogenic (P/LP) CNVs detected by CMA in 121 cases (11.8%, 121/1023), but also defined 17 additional and clinically relevant P/LP CNVs in 17 cases (1.7%, 17/1023). In addition, low-pass GS significantly reduced the technical repeat rate from 4.6% (47/1023) for CMA to 0.5% (5/1023) and required less DNA (50 ng) as input. CONCLUSION: In the context of prenatal diagnosis, low-pass GS identified additional and clinically significant information with enhanced resolution and increased sensitivity of detecting mosaicism as compared with the CMA platform used. This study provides strong evidence for applying low-pass GS as an alternative prenatal diagnostic test.

The effects of perineal disinfection on infant's oral microflora after transvaginal examination during delivery.

BACKGROUND: Early life microflora is an important determinant of immune and metabolic development and may have lasting consequences. However, the mode of delivery and the effect of povidone iodine disinfection on neonatal oral microflora colonization are still unclear. The objective of the study was to understand the effects of the use of polyvidone iodine on infant's oral microflora after transvaginal examination during delivery, provided data support for the establishment of neonatal oral microflora health. METHODS: A total of 20 cases of full-term neonatal delivered in October 2017 in Shenzhen Bao'an Maternity and Child Health Hospital through vaginal delivery. These neonates were randomly divided into two groups, the conventional disinfection group and the non-disinfection group. Simultaneously, 10 infants with elective cesarean section were taken as comparison. With Illumina MiSeq platform, 16S rRNA V3-V4 sequencing method was used to analyze bacterial DNA of oral secretions. RESULTS: At the phylum level, compared to the non-disinfection group, higher relative abundance of Bacteroidetes and Proteobacteria, and lower proportion of Firmicutes were observed in the cesarean section group and the disinfection group. As main composition of phylum Firmicutes, genus Lactobacillus presented extremely low in the cesarean section group and the disinfection group, whereas it was the absolute dominant bacteria in the non-disinfection group. Compared with the caesarean section group, only Lactobacillus increased in majority of the non-disinfection group. There was no increase in Lactobacillus in the disinfection group, but Prevotella, Escherichia-Shigella, Staphyloccus, and Klebsiella increased significantly. Through KEGG pathway analysis, we found that there were more harmful pathways such as staphylococcus aureus infection, viral myocarditis and sporulation in the disinfection group. CONCLUSIONS: The mode of delivery affects the infant's Lactobacillus obtained from the mother. Moreover, vulvar disinfection played an important part in the colonization of neonatal oral microbiota. And the impact of the first oral colonizers on infant health needs further follow-up investigations.

An experimental study of ovarian cancer imaging and therapy by paclitaxel-loaded phase-transformation lipid nanoparticles combined with low-intensity focused ultrasound.

Drug-loaded phase-transformation lipid nanoparticles (NPs) combined with low-intensity focused ultrasound (LIFU) for ultrasound molecular imaging and therapy, which is a very promising drug carrier and can provide both physical and chemical therapeutics, simultaneously. We successfully prepared the paclitaxel (PTX) loaded anti-LHRHR targeted phase-transformation lipid nanoparticles (PTX-anti-LHRHR-PTNPs) for ovarian cancer in this study combined with LIFU has the following characteristics: On the one hand, it showed smaller size and greater stability than blood cells, which significantly prolonged its half-life in the body, and can actively target ovarian cancer OVCAR-3 cells, and smoothly penetrate the endothelial gap into the tumor site for specifically killing the ovarian cancer cells. Thereby, the special drug carrier improved the therapeutic effect and reduced toxic and side effects, maximized the protection of normal tissues and minimized adverse reactions. On the other hand, PTX-anti-LHRHR-PTNPs can be targeted to focus after being injected intravenously and remain in the tumor target tissue for a long time. At the same time, liquid-gas phase-transformation can occur under LIFU triggering, resulting in more ideal and sustained ultrasound imaging effects. Then acoustic contrast agent is used to develop the molecular level of ultrasound scattering, so as to evaluate the diseased tissue from the molecular level.

[Effects of hepatitis B virus on human semen parameters and sperm DNA integrity].

OBJECTIVE: To investigate the effects of hepatitis B virus (HBV) in semen on human semen parameters and sperm DNA integrity. METHODS: We detected HBV DNA in the semen samples of 153 HBsAg-seropositive patients by real-time fluorescence quantitative PCR and calculated the sperm nuclear DNA fragmentation index (DFI) by sperm chromatin dispersion (SCD) assay. We compared the semen parameters between the HBV DNA-positive group (A, n = 43) and HBV DNA-negative group (B, n = 110) and analyzed the correlation of sperm DFI with the number of HBV DNA copies in the semen. RESULTS: HBV DNA was detected in 43 (28.1%) of the 153 semen samples. No statistically significant differences were observed in age, semen volume and sperm concentration between groups A and B (P >0.05). Compared with group B, group A showed significantly decreased sperm viability ([58.0 +/- 18.8]% vs [51.4 +/-17.1]%, P<0.05), progressively motile sperm ([29.6 +/- 13.3]% vs [24.5 +/- 10.1]%, P<0.05), average straight-line velocity ([23.7 +/- 4.0] microm/s vs [19.9 +/- 4.5 ] microm/s, P<0.01) and average path velocity ([26.5 +/- 7.0] microm/s vs [23.4 +/- 5.3] microm/s, P<0.01), but remarkably decreased sperm DFI ([19.3 +/- 8.0]% vs [24.2 +/- 9.4]%, P<0.01). The number of HBV DNA copies in semen exhibited a significant positive correlation with sperm DFI (r = 0.819, P < 0.01). CONCLUSION: HBV DNA in semen is not significantly associated with the number of sperm, but may affect sperm viability, velocity and DFI. There is a load-effect relationship between the number of HBV DNA copies in semen and sperm nuclear DNA integrity.

Comparison of two capillary gel electrophoresis systems for Clostridium difficile ribotyping, using a panel of ribotype 027 isolates and whole-genome sequences as a reference standard.

PCR ribotyping is the most commonly used Clostridium difficile genotyping method, but its utility is limited by lack of standardization. In this study, we analyzed four published whole genomes and tested an international collection of 21 well-characterized C. difficile ribotype 027 isolates as the basis for comparison of two capillary gel electrophoresis (CGE)-based ribotyping methods. There were unexpected differences between the 16S-23S rRNA intergenic spacer region (ISR) allelic profiles of the four ribotype 027 genomes, but six bands were identified in all four and a seventh in three genomes. All seven bands and another, not identified in any of the whole genomes, were found in all 21 isolates. We compared sequencer-based CGE (SCGE) with three different primer pairs to the Qiagen QIAxcel CGE (QCGE) platform. Deviations from individual reference/consensus band sizes were smaller for SCGE (0 to 0.2 bp) than for QCGE (4.2 to 9.5 bp). Compared with QCGE, SCGE more readily distinguished bands of similar length (more discriminatory), detected bands of larger size and lower intensity (more sensitive), and assigned band sizes more accurately and reproducibly, making it more suitable for standardization. Specifically, QCGE failed to identify the largest ISR amplicon. Based on several criteria, we recommend the primer set 16S-USA/23S-USA for use in a proposed standard SCGE method. Similar differences between SCGE and QCGE were found on testing of 14 isolates of four other C. difficile ribotypes. Based on our results, ISR profiles based on accurate sequencer-based band lengths would be preferable to agarose gel-based banding patterns for the assignment of ribotypes.

Epidemiologic features of enterovirus associated with hand, foot and mouth disease in 2013 and 2014 in Shenzhen, China.

Hand, foot and mouth disease (HFMD) is responsible for a heavy economic and social burden in the Asia-Pacific region. Previous studies have shown that coxsackievirus A6 (CVA6) and coxsackievirus A10 (CVA10) have become the predominant agents of HFMD in mainland China in recent years, replacing enterovirus 71 (EV71) and coxsackievirus A16 (CVA16), although it is unclear if this is consistent throughout China. In this study, samples from 253 HFMD cases were collected in Shenzhen, China, from May 2013 through April 2014 to identify the etiological agent of HFMD. In total, 64.8% (164/253) of HFMD cases were enterovirus positive, in which 81.1% (133/164) were determined to be CVA6. The phylogenetic tree of the partial viral protein 1 sequence showed that the CVA6 isolates were divided into four clusters (Clusters A to D), and cluster D was further divided into four sub-clusters (Clusters D1 to D4). The 133 CVA6 samples isolated in our study were classified into cluster D4, in which the first identified sequence was isolated in Shenzhen in 2008. This study demonstrated that the CVA6 cluster D4, which is predominantly circulating in HFMD in mainland China, may have originated from a local strain identified in 2008 in Shenzhen.

[Diagnostic value of p16INK4a in squamous intraepithelial lesion in gynecologic cytology].

OBJECTIVE: To study the diagnostic value of p16(INK4a) in squamous intraepithelial lesion in gynecologic cytology and its relationship with types of human papillomavirus (HPV). METHODS: 88 liquid-based gynecologic cytology cases with histologic correlation, including 20 cases of cervicitis, 18 cases of low-grade squamous intraepithelial lesion (LSIL), 34 cases of high-grade squamous intraepithelial lesion (HSIL) and 16 cases of squamous cell carcinoma (SCC), were enrolled into the study. Immunocytochemistry for p16(INK4a) protein and polymerase chain reaction-based HPV DNA testing (for HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 and 68, as well as HPV types 6, 11, 42, 43 and 44) were performed. RESULTS: The rate of expression of p16(INK4a) protein was 0, 27.8%, 100% and 100% in the cervicitis group, LSIL group, HSIL group and SCC group, respectively. The expression was significantly higher in the latter 3 groups than that in the cervicitis group (P < 0.01). Besides, the expression was significantly higher in cases associated with high-risk HPV genotypes (96.4%) than in cases associated with low-risk HPV genotypes (7.7%). CONCLUSION: p16(INK4a) is a valuable biomarker for detection of HPV-related dysplastic squamous cells, with high sensitivity and specificity.

Hypermethylated ERG as a cell-free fetal DNA biomarker for non-invasive prenatal testing of Down syndrome.

BACKGROUND: Previous reports have shown that the ERG gene is hypermethylated in the placenta and hypomethylated in maternal blood cells. In this study, we explore the feasibility of hypermethylated ERG as a cell-free fetal (cff) DNA biomarker for non-invasive prenatal testing (NIPT) of Down syndrome. METHODS: We randomly selected 90 healthy pregnant women, including 30 cases at each trimester of pregnancy. In addition, 15 pregnant women were recruited as the case group whose fetuses had been confirmed to have trisomy 21 by amniotic fluid analysis at 18th to 26th week gestation. Using HpaII, MspІ to digest cell-free maternal plasma DNA, we performed SYBR Green PCR to detect methylated sites of ERG sequences, and analyzed the concentrations of cff DNA in maternal plasma in different gestational trimesters and the case group. RESULTS: The ERG median concentrations of the maternal plasma after Hpa II digestion (LG copies/ml) in first, second and third-trimesters were 5.38, 6.10, and 7.04, respectively (Kruskal-Wallis, P<0.01); and that in the trisomy 21 case group was 6.85, which was higher than the second-trimester (Mann-Whitney, P<0.01). CONCLUSIONS: The study demonstrated that ERG gene is hypermethylated in cff DNA but hypomethylated in maternal DNA; and the median concentration of ERG gene in the trisomy 21 case group is higher than that in the gestational trimester matched normal group. ERG gene, as a fetal DNA biomarker, may be useful for NIPT of Down syndrome.

Multiplex PCR targeting slpA: a rapid screening method to predict common Clostridium difficile ribotypes among fluoroquinolone resistant clinical strains.

AIMS: Based on the relationship between Clostridium difficile surface layer protein A (slpA) sequence types (STs) and PCR-ribotypes (RTs), a multiplex polymerase chain reaction (mPCR) assay was developed to rapidly confirm C. difficile toxigenicity and, simultaneously, to identify any of five slpA STs, gr, hr, fr, gc8 and 078, that usually correspond with globally distributed RTs, 001, 014, 017, 027 and 078, respectively. METHODS: The mPCR, containing five slpA type-specific primers, was developed using 46 well-characterised C. difficile reference strains, representing 11 slpA STs, and validated by testing 90C. difficile clinical isolates. RESULTS: The slpA mPCR correctly identified the five slpA STs without cross-reactions. A much higher proportion of moxifloxacin resistant (32/39; 82%) than susceptible (12/51; 24%) clinical isolates were slpA typeable (χ=30.3, p<0.0001), even when RT027 isolates were excluded [10/17 (59%) versus 12/51 (24%); χ=7.3, p=0.0071<0.01]. slpA mPCR correctly predicted the RTs of all 39 isolates that belonged to the five targeted RTs. CONCLUSION: slpA mPCR is simple, rapid and inexpensive. It can provisionally identify five globally significant, highly transmissible RTs, particularly among moxifloxacin resistant C. difficile isolates, and could be easily modified to include a broader range of slpA sequence types, based on local requirements.