The Tioman Island of Malaysia experienced acute muscular sarcocystosis outbreaks from 2011 to 2014. So far, a previous study based on the 18S rRNA gene sequencing has reported S. singaporensis, S. nesbitti and Sarcocystis sp. YLL-2013 in water samples acquired from the island, thus confirming the waterborne nature of this emerging parasitic disease. This study aimed to improve the detection methods for Sarcocystis, in order to have a clearer picture of the true diversity of Sarcocystis species in Tioman. A new primer set (28S R7F-28S R8 Deg R) was designed to amplify the 28S rRNA gene of Sarcocystis. Subsequently, Sarcocystidae was detected in 65.6% (21/32) of water samples and 28% (7/25) of soil samples acquired between 2014 and 2015 from Tioman. Next-generation sequencing (NGS) on 18 of the positive samples was then performed using amplicons generated from the same primer set. This yielded 53 potentially unique Sarcocystidae sequences (290 bp), of which nine of the most abundant, prevalent and unique sequences were named herein. In contrast, NGS of the 18S rRNA gene V9 hypervariable region of 10 selected samples detected only two Sarcocystis species (160 bp). S. mantioni was the most ubiquitous sequence found in this study.
Sarcocystosis outbreaks in Tioman and Pangkor islands of Malaysia between 2011 and 2014 have raised the need to improve Sarcocystis species detection from environmental samples. In-house works found that published primers amplifying the 18S rRNA gene of Sarcocystis either could not produce the target from environmental samples or produced Sarcocystis DNA sequence that was insufficient for species identification. Using the primer pair of 18S S5 F (published) and 28S R6 R (new), this study improved the PCR amplification of Sarcocystidae to overcome these two difficulties. The PCR product spanned from the 18S to 28S rRNA genes, providing more information for species identification. The long DNA sequence allowed comparison between the "Ident" and "Query Cover" sorting in GenBank identity matching. This revealed the ambiguity in identity matching caused by different lengths of reference DNA sequences, which is seldom discussed in the literature. Using the disparity index test, a measurement of homogeneity in nucleotide substitution pattern, it is shown that the internal transcribed spacer (ITS)1-5.8S-ITS2 and 28S genes are better than the 18S gene in indicating nucleotide variations, implying better potentials for species identification. The example given by the handful of Sarcocystidae long DNA sequences reported herein calls for the need to report DNA sequence from the 18S to the 28S rRNA genes for species identification, especially among emerging pathogens. DNA sequence reporting should include the hypervariable 5.8S and ITS2 regions where applicable, and not be limited to single gene, per the current general trend.
Today's workers are struggling to achieve a balance between their work and personal life roles because of both specific needs and limited resources. This study explored the socioecological factors that influence work-life balance (WLB) and how they operate. The relationships between WLB, subjective wellbeing, employee wellbeing and quality time allocation were examined. A total of 1063 responses were received, using an online survey. The results show that relational, community and societal factors directly influenced the individual factors and were indirectly associated with perceived WLB. Individual factors (i.e., personal feelings, behaviours and health) were found to be the crucial determinants of an individual's perceived WLB. It was found that WLB positively correlated with employee wellbeing and quality and quantity of personal life-time. Subjective wellbeing was found to be a significant moderator in the relationship between WLB and its outcomes. This study demonstrated the process of how workers determine their own WLB by applying the socioecological framework for categorising the determinants and suggested new avenues that improve the whole wellbeing of workers and also foster long-term development of organisations.
DNA samples selected for long read sequencing (LRS) are routinely required to be 'pure' with high DNA concentration. Hence the usefulness of samples with substandard DNA quality for LRS is unknown. We aim to perform de-novo assembly of Adenovirus sequenced from non-human primate (NHP) faeces using the Oxford Nanopore technologies (ONT), an LRS platform. Guided by initial conventional PCR screening, we performed ONT sequencing on 34 Adenovirus positive DNA samples, without prior selection based on faeces freshness level or DNA quality. Non-parametric correlations analysis showed that ONT sequencing outputs is not significantly associated (p > 0.05) with DNA concentrations, faeces freshness levels and the OD ratios of A260/A280 and A260/A230. This indicated that conventional DNA quality parameters may not be the most critical factors in determining the suitability of samples for ONT sequencing. A total of 61.76% (21/34) of the positive-by-PCR-screening samples yielded Adenovirus reads while 38.24% (13/34) did not in the PCR-free ONT workflow, although rarefaction analysis showed that sequencing saturation was achieved by all samples. Among the 21 samples with adenovirus reads, ten resulted in at least one Adenovirus contig by the Flye assembler while nine did not and two samples had only a single Adenovirus read. Identity similarity above 90% in conventional PCR screening may help in selecting ONT positive samples.