Sepsis is an acute, often fatal syndrome that requires early diagnosis and proper treatment. Blood culture (BC) is the gold standard for the identification of pathogens, however it has marked ...limitations, including that it is time-consuming (delaying treatment) and can only detect microbes that readily grow under culture conditions. Alternatively, non-culture-based methodologies like polymerase chain reaction (PCR) are faster but also have limitations; e.g., the reaction is often inhibited by the abundance of human DNA and thus can only detect limited known target pathogens. In our previous publication, we have demonstrated a proof-of-concept of a simple pre-analytical tool to remove human DNA from patients' blood specimens, hence allowing downstream PCRs to detect rare bacterial genetic materials. In the current study, we reported a better performance of a novel prototype diagnosis kit named Sepsis@Quick that combines human DNA removal step with real-time PCRs compared to blood-culture for identifying sepsis causative bacteria. Our data showed that Sepsis@Quick is superior to blood culture in which the novel diagnostic kit could identify more pathogens and even polymicrobial infection, faster and less influenced by the empirical administration of broad spectrum antibiotic therapy (single administration or combination of cephalosporin III and fluoroquinolon). Additionally, for the first time, we demonstrated that positive results achieved by Sepsis@Quick are significantly associated with a reduction of sepsis-related mortality.
Telomerase reverse-transcriptase (TERT) gene promoter mutations in circulating cell-free DNA (cfDNA) as well as the levels of circulating microRNA-122 (miR-122) have been reported as potential ...noninvasive biomarkers for several. This study evaluates the diagnostic performance of potent biomarker-based panels composing of serological AFP, miR-122 and circulating TERT promoter mutations for screening HBV-related HCC. TERT promoter mutations (C228T and C250T) and miR-122 expression were assessed in the plasma samples from 249 patients with HBV-related liver diseases by nested PCR and qRT-PCR assays, respectively. The diagnostic values of TERT promoter mutations, miR-122 expression and biomarker-based panels were assessed by computation of the area under the curve (AUC). Nested-PCR assays were optimized to detect C228T and C250T mutations in TERT promoter with detection limit of 1%. The common hotspot C228T was observed in 22 HCC cases. The triple combinatory panel (AFP@TERT@miR-122) acquired the best diagnostic value to distinguish HCC from CHB (AUC = 0.98), LC (AUC = 0.88) or non-HCC (LC + CHB, AUC = 0.94) compared to the performance of double combinations or single biomarkers, respectively. Notably, among patients with AFP levels≤20 ng/μl, the double combination panel (TERT@miR-122) retains satisfactory diagnostic performance in discriminating HCC from the others (HCC vs. CHB, AUC = 0.96; HCC vs. LC, AUC = 0.88, HCC vs. non-HCC, AUC = 0.94). The triple combination panel AFP@TERT@miR-122 shows a better diagnostic performance for screening HCC in HBV patients, regardless of AFP levels. The newly established panels can be a potential application in clinical practice in Vietnamese setting.
Early diagnosis, precise antimicrobial treatment and subsequent patient stratification can improve sepsis outcomes. Circulating biomarkers such as plasma microRNAs (miRNAs) have proven to be ...surrogates for diagnosis, severity and case management of infections. The expression of four selected miRNAs (miR-146-3p, miR-147b, miR-155 and miR-223) was validated for their prognostic and diagnostic potential in a clinically defined cohort of patients with sepsis and septic shock.
The expression of plasma miRNAs was quantified by quantitative PCR (qPCR) in patients with bacterial sepsis (n = 78), in patients with septic shock (n = 52) and in patients with dengue haemorrhagic fever (DHF; n = 69) and in healthy controls (n = 82).
The expression of studied miRNA was significantly increased in patients with bacterial sepsis and septic shock. The plasma miR-147b was able to differentiate bacterial sepsis from non-sepsis and septic shock (AUC = 0.77 and 0.8, respectively, p≤ 0.05), while the combination of plasma miR-147b and procalcitonin (PCT) predicted septic shock (AUC = 0.86, p≤ 0.05).
The plasma miR-147b may be an useful biomarker independently or in combination with PCT to support clinical diagnosis of sepsis and equally prognosis of patients with septic shock.
Circulating microRNAs (miRNA) are biomarkers for several neoplastic diseases, including hepatocellular carcinoma (HCC). We performed a literature search, followed by experimental screening and ...validation in order to establish a miRNA panel in combination with the assessment of alpha-fetoprotein (AFP) levels and to evaluate its performance in HCC diagnostics.
Expression of miRNAs was quantified by quantitative PCR (qPCR) in 406 serum samples from 118 Vietnamese patients with hepatitis B (HBV)-related HCC, 69 patients with HBV-related liver cirrhosis (LC), 100 chronic hepatitis B (CHB) patients and 119 healthy controls (HC).
Three miRNAs (mir-21, mir-122, mir-192) were expressed differentially among the studied subgroups and positively correlated with AFP levels. The individual miRNAs mir-21, mir-122, mir192 or the triplex miRNA panel showed high diagnostic accuracy for HCC (HCC vs. CHB, AUC = 0.906; HCC vs. CHB+LC, AUC = 0.81; HCC vs. CHB+LC+HC, AUC = 0.854). When AFP levels were ≤20ng/ml, the triplex miRNA panel still was accurate in distinguishing HCC from the other conditions (CHB, AUC = 0.922; CHB+LC, AUC = 0.836; CHB+LC+HC, AUC = 0.862). When AFP levels were used in combination with the triplex miRNA panel, the diagnostic performance was significantly improved in discriminating HCC from the other groups (LC, AUC = 0.887; CHB, AUC = 0.948; CHB+LC, AUC = 0.887).
The three miRNAs mir-21, mir-122, mir-192, together with AFP, are biomarkers that may be applied to improve diagnostics of HCC in HBV patients, especially in HBV-related LC patients with normal AFP levels or HCC patients with small tumor sizes.
Loop isothermal amplification (LAMP) has recently been proposed as a point-of-care diagnostic tool to detect acute infectious pathogens; however, this technique embeds risk of generating ...false-positive results. Whereas, with abilities to accurately recognize specific sequence, the CRISPR/Cas12a can forms complexes with cognate RNA sensors and cleave pathogen's DNA targets complimerntary to its cognate RNA, afterward acquiring the collateral activity to unbiasedly cut nearby off-target fragments. Therefore, if relevant fluorescent-quencher-nucleic probes are present in the reaction, the non-specific cleavage of probes releases fluorescences and establish diagnostic read-outs.
The MetA gene of N. meningitidis was selected as target to optimize the LAMP reaction, whereas pseudo-dilution series of N. meningitidis gemonics DNA was used to establish the detection limit of LAMP/Cas12a combination assay. The diagnostic performance of established LAMP/Cas12a combination assay was validated in comparation with standard real-time PCR on 51 CSF samples (14 N. meningitidis confirmed patients and 37 control subjects).
In relevant biochemical conditions, CRISPR-Cas12a and LAMP can work synchronously to accurately identify genetics materials of Nesseria menitigistis at the level 40 copies/reaction less than 2 h.
In properly optimized conditions, the CRISPR-Cas12a system helps to alleviate false positive result hence enhancing the specificity of the LAMP assays.
Neisseria meningitidis can cause life-threatening meningococcal meningitis and meningococcemia. Old standard microbiological results from CSF/blood cultures are time consuming. This study aimed to ...combine the sensitivity of loop-mediated isothermal nucleic acid amplification (LAMP) with the specificity of CRISPR/Cas12a cleavage to demonstrate a reliable diagnostic assay for rapid detection of N. meningitidis.
A total of n = 139 samples were collected from patients with suspected meningococcal disease and were used for evaluation. The extracted DNA was subjected to qualitative real-time PCR, targeting capsular transporter gene (ctrA) of N. meningitidis. LAMP-specific primer pairs, also targeting the ctrA, were designed and the LAMP products were subjected to CRISPR/Cas12 cleavage reaction. the readout was on a lateral flow strip. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of LAMP-CRISPR/Cas was compared with real-time PCR assays. The limit of detection (LOD) was established with serial dilutions of the target N. meningitidis DNA and calculated by Probit regression analysis.
Six LAMP assay-specific primers were developed targeting the ctrA gene of N. meningitidis, which is conserved in all meningococcal serogroups. The LAMP primers did not amplify DNA from other bacterial DNA tested, showing 100% specificity. The use of 0.4 M betaine increased the sensitivity and stability of the reaction. LAMP-CRISPR/Cas detected meningococcal serogroups (B, C, W). The assay showed no cross-reactivity and was specific for N. meningitidis. The LOD was 74 (95% CI: 47-311) N. meningitidis copies. The LAMP-CRISPR/Cas performed well compared to the gold standard. In the 139 samples from suspected patients, the sensitivity and specificity of the test were 91% and 99% respectively.
This developed and optimized method can complement for the available gold standard for the timely diagnosis of meningococcal meningitis and meningococcemia.
Vitamin D derivatives and their receptor (VDR) are potent modulators of immune responses in various diseases including malignancies as well as in metabolic and infectious disorders. The impact of ...vitamin D receptor polymorphisms on clinical outcomes of hepatitis B virus (HBV) infection is not well understood. This study aims to investigate the potential role of VDR polymorphisms (TaqI, FokI, ApaI, and BsmI) in Vietnamese HBV infected patients and to correlate these polymorphisms with the progression of HBV-related liver disease.
Four hundred forty-three HBV infected patients of the three clinically well-defined subgroups chronic hepatitis B (CHB, n = 183), liver cirrhosis (LC, n = 89) and hepatocellular carcinoma (HCC, n = 171) and 238 healthy individuals (HC) were enrolled. VDR polymorphisms were genotyped by DNA sequencing and in-house validated ARMS assays. Logistic regression models were applied in order to determine the association of VDR polymorphisms with manifest HBV infection as well as with progression of related liver diseases mulin different genetic models.
The VDR ApaI CA genotype was less frequent in HCC than in CHB patients in different genetic models (codominant model, OR = 0.5, 95%CI = 0.3-0.84, P = 0.004; dominant model, OR = 0.46, 95%CI = 0.27-0.76, P = 0.0023). In the recessive model, the genotype ApaI AA was found more frequently among HCC compared to CHB patients (OR = 2.56, 95%CI = 1.01-6.48, P = 0.04). Similarly, the ApaI CA genotype was less frequent in HCC than in non-HCC group codominant model, OR = 0.6, 95%CI = 0.4-0.98, dominant model, P = 0.04 and OR = 0.6, 95%CI = 0.38-0.90, P = 0.017). The ApaI genotypes CA and AA was significantly associated with higher levels of liver enzymes, bilirubin, and HBV DNA (P < 0.05). No association between TaqI, FokI and BsmI polymorphisms and any clinical outcome as well as liver disease progression was found.
Among the four investigated VDR polymorphisms, ApaI is associated with clinical outcome and liver disease progression in Vietnamese HBV infected patients.
The inhibitory effects of programmed cell death 1/programmed cell death ligand 1 (PD-1/PD-L1) modulates T-cell depletion. T-cell depletion is one of the key mechanisms of hepatitis B virus (HBV) ...persistence, in particular liver disease progression and the development of hepatocellular carcinoma (HCC). This case-control study aimed to understand the significance of PD-1 polymorphisms (PD-1.5 and PD-1.9) association with HBV infection risk and HBV-induced liver disease progression. Genotyping of PD-1.5 and PD-1.9 variants was performed by direct Sanger sequencing in 682 HBV-infected patients including chronic hepatitis (CHB, n = 193), liver cirrhosis (LC, n = 183), hepatocellular carcinoma (HCC, n = 306) and 283 healthy controls (HC). To analyze the association of PD-1 variants with liver disease progression, a binary logistic regression, adjusted for age and gender, was performed using different genetic models. The PD-1.9 T allele and PD-1.9 TT genotype are significantly associated with increased risk of LC, HCC, and LC + HCC. The frequencies of PD-1.5 TT genotype and PD-1.5 T allele are significantly higher in HCC compared to LC patients. The haplotype CT (PD-1.5 C and PD-1.9 T) was significantly associated with increased risk of LC, HCC, and LC + HCC. In addition, the TC (PD-1.5 T and PD-1.9 C) haplotype was associated with the risk of HCC compared to non-HCC. The PD-1.5 CC, PD-1.9 TT, genotype, and the CC (PD-1.5 C and PD-1.9) haplotype are associated with unfavorable laboratory parameters in chronic hepatitis B patients. PD-1.5 and PD1.9 are useful prognostic predictors for HBV infection risk and liver disease progression.
As an immune modulator, vitamin D is involved in various pathophysiological mechanisms in a plethora of diseases. This study aims to correlate the vitamin D deficiency status and clinical progression ...of liver diseases associated with hepatitis B virus (HBV) infection in patients in Vietnam and to compare it to healthy controls.
We quantified the levels of total vitamin D 25-(OH) D2 and D3 in serum samples from 400 HBV patients (chronic hepatitis B infection CHB, n = 165; HBV-associated liver cirrhosis LC, n = 127; HBV-associated hepatocellular carcinoma HCC, n = 108) and 122 unrelated healthy controls (HC). Univariate and multivariate analyses were performed in order to determine the association between vitamin D levels and distinct clinical parameters.
The prevalence of vitamin D inadequacy (<30 ng/mL) was high among healthy individuals (81.7 %) as well as in HBV patients (84.3 %). Vitamin D deficiency (<20 ng/ml) or severe deficiency (<10 ng/ml) was observed more frequently among HBV patients (52 %) and subgroups (CHB, 47.8 %; LC, 54.4 %; HCC, 55.3 %) compared to the control group (32.5 %) (P < 0.001). Vitamin D levels and HBV-DNA load were strongly and inversely correlated (rho = -0.57, P < 0.0001). Multivariate regression analysis also revealed an independent association of HBV-DNA loads with low vitamin D levels (P = 0.0004). In addition, reduced vitamin D levels were associated with significant clinical progression of LC (Child-Pugh C versus Child-Pugh A, P = 0.0018; Child-Pugh C versus Child-Pugh B, P = 0.016).
Vitamin D deficiency was observed in the majority of HBV-infected patients and associated with adverse clinical outcomes. Our findings suggest that substitution of vitamin D may be a supportive option in the treatment of chronic liver diseases, in particular of HBV-associated disorders.
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