Data_Sheet_1_A population-based study on prevalence and predisposing risk factors of infant functional gastrointestinal disorders in a single center in Southern Fujian.doc

Background and aimThe prevalence of infant functional gastrointestinal disorders (FGIDs) varies across different areas but is largely unknown in southern Fujian. The aim of this study is to evaluate the prevalence of infant FGIDs in southern Fujian according to Rome IV diagnostic criteria.MethodsA cross-sectional prospective questionnaire-based survey was conducted among healthy infants between 0 and 3 months of age in southern Fujian. A total of 1,006 infants who received a physical examination from October 2017 to October 2018 were recruited in this study. Parents or caregivers provided demographic information and completed the questionnaire on gastrointestinal symptoms for infants. Infants with FGIDs were diagnosed using the Rome IV criteria.ResultsBased on the Rome IV criteria, the prevalence of having a FGID in infants is 58.3% (586/1,006). The most common FGIDs in infants were regurgitation (45.7%, 460/1,006), followed by difficult defecation (3.6%, 36/1,006), functional constipation (3.2%, 32/1,006), and colic (2.4%, 24/1,006). No infants fulfilled diagnostic criteria for rumination syndrome and cyclic vomiting syndrome. Among the infants with FGIDs, 457 cases (78.0%, 457/586) were found with single FGID. Combined FGIDs were diagnosed in 129 (22.0%, 129/586) infants; of whom, 21.2% (124/586) had double disorders and 0.9% (5/586) had triple disorders. The most common combined FGIDs were regurgitation and difficult defecation (12.8%), followed by regurgitation and colic (2.4%). Risk factor analysis revealed that younger paternal age (B = 0.424, P = 0.004), paternal history of FGIDs (B = 0.821, P = 0.000), maternal history of FGIDs (B = 0.427, P = 0.012), and probiotics received in infant (B = 0.324, P = 0.032) were associated with an increased risk of infant FGIDs, whereas vitamin D supplementation after birth (B = −0.690, P = 0.000) can reduce the risk of developing FGIDs.ConclusionFGIDs are common in infants living in southern Fujian according to Rome IV diagnostic criteria. The most common FGIDs in infants were regurgitation, difficult defecation, and functional constipation. Factors including younger paternal age, parental history of FGIDs, and the probiotic supplementation in infant showed a significant association with infant FGIDs. Whereas, vitamin D supplementation in infant was found to be a protective factor against FGIDs.</p

Establishment of Real Time Allele Specific Locked Nucleic Acid Quantitative PCR for Detection of HBV YIDD (ATT) Mutation and Evaluation of Its Application

<div><p>Background</p><p>Long-term use of nucleos(t)ide analogues can increase risk of HBV drug-resistance mutations. The rtM204I (ATT coding for isoleucine) is one of the most important resistance mutation sites. Establishing a simple, rapid, reliable and highly sensitive assay to detect the resistant mutants as early as possible is of great clinical significance.</p><p>Methods</p><p>Recombinant plasmids for HBV YMDD (tyrosine-methionine-aspartate-aspartate) and YIDD (tyrosine-isoleucine-aspartate-aspartate) were constructed by TA cloning. Real time allele specific locked nucleic acid quantitative PCR (RT-AS-LNA-qPCR) with SYBR Green I was established by LNA-modified primers and evaluated with standard recombinant plasmids, clinical templates (the clinical wild type and mutant HBV DNA mixture) and 102 serum samples from nucleos(t)ide analogues-experienced patients. The serum samples from a chronic hepatitis B (CHB) patient firstly received LMV mono therapy and then switched to LMV + ADV combined therapy were also dynamically analyzed for 10 times.</p><p>Results</p><p>The linear range of the assay was between 1×10⁹ copies/μl and 1×10² copies/μl. The low detection limit was 1×10¹ copies/μl. Sensitivity of the assay were 10⁻⁶, 10⁻⁴ and 10⁻² in the wild-type background of 1×10⁹ copies/μl, 1×10⁷ copies/μl and 1×10⁵ copies/μl, respectively. The sensitivity of the assay in detection of clinical samples was 0.03%. The complete coincidence rate between RT-AS-LNA-qPCR and direct sequencing was 91.2% (93/102), partial coincidence rate was 8.8% (9/102), and no complete discordance was observed. The two assays showed a high concordance (<i>Kappa</i> = 0.676, <i>P</i> = 0.000). Minor variants can be detected 18 weeks earlier than the rebound of HBV DNA load and alanine aminotransferase level.</p><p>Conclusions</p><p>A rapid, cost-effective, high sensitive, specific and reliable method of RT-AS-LNA-qPCR with SYBR Green I for early and absolute quantification of HBV YIDD (ATT coding for isoleucine) variants was established, which can provide valuable information for clinical antiretroviral regimens.</p></div

The correlation between actual and calculated proportion of mutant plasmid DNA at the concentration of 1×10⁷ copies/μl.

<p><i>R²</i> = 0.9907, <i>P</i><0.0001.</p

Dynamics of HBV YMDD, YIDD DNA levels and serum ALT level during LMV and LMV + ADV therapy in a CHB patient with breakthrough hepatitis.

<p>Dynamics of HBV YMDD, YIDD DNA levels and serum ALT level during LMV and LMV + ADV therapy in a CHB patient with breakthrough hepatitis.</p

Clinical templates with different proportions of mutant DNA were tested by mutant specific primer set.

<p>Amplification plot with different colors represented different proportions of mutant DNA which were illustrated in the figure. The Ct of no-template control was undetectable.</p

Amplification plot and standard curve of RT-AS-LNA-qPCR for pMD-18-YMDD.

<p>(A) Amplification plot with different colors represented different concentrations of pMD-18-YMDD plasmids which were illustrated in the figure. (B) Standard curve of pMD-18-YMDD: Y = -3.478X+41.654 (<i>R²</i> = 0.999).</p

Primers for RT-AS-LNA-qPCR.

<p>KF/KF_n indicate the common forward primers to all reactions. L1/L2/L2_l/L1_n/L2_n indicate specific RT-AS-LNA-qPCR reverse primers. M indicates A/C; K indicates G/T; W indicates A/T; +A/+C indicate LNA nucleosides.</p

Detection sensitivity comparison of RT-AS-LNA-qPCR and sequencing on clinical samples containing different proportions of rtM204I variant.

<p>M: Methionine; I: isoleucine; -: not performed; NTC: no-template control; N: undetectable.</p

Concordance between RT-AS-LNA-qPCR and direct sequencing.

<p>Complete concordant results are shown in bold. Partial concordant results are shown in italics.</p

Partial concordant results between RT-AS-LNA-qPCR and direct sequencing.

<p>Partial concordant results between RT-AS-LNA-qPCR and direct sequencing.</p