A novel Enterovirus 96 circulating in China causes hand, foot, and mouth disease.

Enterovirus 96 (EV-96) is a recently described member of the species Enterovirus C and is associated with paralysis and myelitis. In this study, using metagenomic sequencing, we identified a new enterovirus 96 strain (EV-96-SZ/GD/CHN/2014) as the sole pathogen causing hand, foot, and mouth disease (HFMD). A genomic comparison showed that EV-96-SZ/GD/CHN/2014 is most similar to the EV-96-05517 strain (85% identity), which has also been detected in Guangdong Province. This is the first time that metagenomic sequencing has been used to identify an EV-96 strain shown to be associated with HFMD.

First report of human salivirus/klassevirus in respiratory specimens of a child with fatal adenovirus infection.

Adenovirus is a leading cause of respiratory infection in children. Salivirus/klassevirus was first identified as an etiologic agent of gastroenteritis and was never reported in respiratory infection cases. The case being discussed here caught our attention because, although it is a common respiratory infection, it was fatal, while similar cases were mild. In order to find potential causes in the fatal case, we describe the clinical diagnosis and treatment, the sequencing analysis of the salivirus/klassevirus, and the co-infectious adenovirus. Metagenomics sequencing was conducted on the samples from a nasopharyngeal swab of the children with adenovirus infection. Sequences were assembled using IDBA-ud (1.1.1); phylogenetic analysis was performed using MEGA 5.2. RT-PCR and quantitative PCR were performed to verify the existence of the virus in the samples. A nearly full genome of this new virus strain was obtained with 7633 nt encoding a polyprotein of 2331 aa. Meanwhile, it was detected specifically in the nasopharyngeal swab by RT-PCR. Further, homology analysis indicated that the virus has a closer relationship with Salivirus A strain in Shanghai (GU245894). Our study reports the first case of Human salivirus/klassevirus in respiratory specimens of a child with fatal adenovirus infection in Shenzhen, China. The finding and investigation of the virus will provide more useful information for the clinical diagnosis of unexplained lethal infection and expand our knowledge of the new family, salivirus/klassevirus in picornavirus.

Genetic association of lipid metabolism related SNPs with myocardial infarction in the Pakistani population.

Myocardial infarction (MI) is the major cardiovascular disease. This can be caused by mutual interaction of environmental and genetic factors. The current study was designed to investigate the role of lipid metabolism related genetic polymorphisms with the onset of MI in Punjabi population of Pakistan. A total of 384 subjects was studied from April 2011 to July 2012. To determine the genetic associations with MI, the single nucleotide polymorphisms (SNPs) were genotyped by sequencing, as well as one label extension method. Out of eight SNPs in four candidate genes, seven genetic variants were significantly (P < 0.05) associated with elevated risk of MI. In current study two SNPs rs662799 risk allele G (P = 0.03) and rs3135506 risk allele C (P = 0.05) of APOA5 were found to be associated with significant higher risk of triglyceride levels, irrespective of age, sex, obesity, diabetes, hypertension and smoking. Gene variants (rs1558861, rs662799 and rs10750097) in APOA5 showed almost complete linkage disequilibrium and their minor allele frequencies (0.34, 0.28, and 0.41 respectively) were more prevalent (P < 0.05) in cases than controls. We further revealed risk haplotypes (C-T-G-A, G-C-A-G; P = 0.001) and protective haplotypes (G-T-A-G, C-C-G-A; P = 0.005) between these four SNPs for the progression of MI. Current study confirms the correlation between lipid metabolism related SNPs with MI and supports the role of APOA5 in raising plasma triglyceride levels in Pakistanis. However further studies are needed for delineating the role of these SNPs.

Genetic association of IDE, POU2F1, PON1, IL1α and IL1ß with type 2 diabetes in Pakistani population.

A number of genes are known to be involved in glucose homeostasis. Mutations and polymorphisms in candidate genes may effect insulin production, action or resistance. This study was designed to report the association of genetic polymorphism with the type 2 diabetes (T2D) in Pakistani population. A total of 458 subjects (case n=288, control n=170) participated in the study. Nine single nucleotide polymorphisms were investigated in genes IDE (rs6583813 C>T, rs7910977 C>T), POU2F1 (rs3767434 A>T, rs10918682 A>T, rs2146727 A>G), WFS1 (rs734312 A>G), PON1 (rs854560 T>A), IL1α (rs1800587 C>T) and IL1ß (rs1143634 C>T). Genotyping was performed by DNA sequencing after nested polymerase chain reaction of targeted regions. Results indicated that rs7910977 in IDE showed significant association with the development of T2D [P=0.012, OR 1.677 (95% CI 1.112-2.438)]. The rs10918682 in POU2F1 was associated with T2D [P<0.001, OR 3.606 (95% CI 2.165-6.005)]. The rs854560 in PON1 was associated with incidences of T2D and increased the risk of cardiovascular complications [P=0.031, OR 0.663 (95% CI 0.455-0.965)] in diabetics. The rs734312 from WFS1 gene was associated with diabetes at genotype level (P<0.01). Haplotype analysis of rs1800587-rs1143634 depicted CC haplotype increased the susceptibility to diabetes (P<0.05). Haplotype GAA from rs2146727-10918682-rs3767434 was protective against diabetes (P<0.01) and GGA exhibited the association with T2D (P<0.01). Haplotype CT from rs6583813-rs7910977 was protective against diabetes (P=0.02). Our study provided evidence to IDE, PON1, WFS1, POU2F1, IL1α and IL1ß associated with T2D in Pakistanis.

A rapid fluorescence polarization-based method for genotypic detection of drug resistance in Mycobacterium tuberculosis.

Rapid detection of drug-resistant Mycobacterium tuberculosis is critical to the effective early treatment and prevention of the transmission of tuberculosis. However, conventional drug susceptibility tests for M. tuberculosis require up to several weeks. In the present study, the One Label Extension genotyping method was adapted for rapid detection of drug resistance-associated sequence variations in six genes of M. tuberculosis, viz. rpoB, rpsL, rrs, embB, katG, or inhA. The method utilizes polymerase chain reaction amplified fragments of the drug resistant genes as reaction templates, and proceeds with template-directed primer extension incorporating a fluorescence-labeled nucleotide, which is then measured by fluorescence polarization. A total of 121 M. tuberculosis isolates from clinical sputum specimens were examined by this genotyping method and verified by direct sequencing of polymerase chain reaction amplicons harboring previously reported mutational sites associated with M. tuberculosis drug resistance. Based on phenotyping results obtained from microbiology-based drug susceptibility tests, the sensitivity, specificity, and test efficiency estimated for One Label Extension assays were respectively 83.9 %, 95.5 %, and 92.4 % with ropB in rifampin resistance, 67.3 %, 97.1 %, and 84.3 % with rpsL and rrs in streptomycin resistance, 60.0 %, 96.0 %, and 91.4 % with embB in ethambutol resistance, 68.4 %, 94.9 %, and 86.3 % with inhA and katG in isoniazid resistance, and 74.1 %, 98.9 %, and 93.2 % in multiple drug resistance defined as resistance to at least both isoniazid and rifampin. In conclusion, examination of clinical sputum specimens by One Label Extension based genotyping provides a valid method for the rapid molecular detection of drug-resistant M. tuberculosis.