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.
The
-induced burden of gastric cancer varies based on geographical regions and ethnic grouping. Vietnam is a multiethnic country with the highest incidence of gastric cancer in Southeast Asia, but ...previous studies focused only on the Kinh ethnic group. A population-based cross-sectional study was conducted using 494 volunteers (18-78 years old), from 13 ethnic groups in Daklak and Lao Cai provinces, Vietnam.
status was determined by multiple tests (rapid urease test, culture, histology, and serology).
and
genotypes were determined by PCR-based sequencing. The overall
infection rate was 38.1%. Multivariate analysis showed that variations in geographical region, age, and ethnicity were independent factors associated with the risk of
acquisition. Therefore, multicenter, multiethnic, population based study is essential to assess the
prevalence and its burden in the general population. Only the E De ethnicity carried strains with Western-type CagA (82%) and exhibited significantly lower gastric mucosal inflammation compared to other ethnic groups. However, the histological scores of Western-type CagA and East-Asian-type CagA within the E De group showed no significant differences. Thus, in addition to bacterial virulence factors, host factors are likely to be important determinants for gastric mucosal inflammation and contribute to the Asian enigma.
Background and purpose Ischemic stroke (IS) is classified into clinical subtypes and likely influenced by various lipid components. Nevertheless, the roles of apolipoprotein A-I (apoA-I), ...apolipoprotein B (apoB), and apoB/apoA-I ratio in different IS subtypes remain underexplored. This study aimed to investigate the differential distribution of plasma apoA-I and apoB levels among IS subtypes and to evaluate the predictive value of the apoB/apoA-I ratio in assessing IS subtypes and disease severity. Methods In this study, 406 IS patients were categorized into three IS-subtypes based on clinical manifestations and imaging assessment, including intracranial atherosclerosis-related IS patients (ICAS, n = 193), extracranial atherosclerosis-related IS patients (ECAS, n = 111), and small artery occlusion-related IS patients (SAO, n = 102). Plasma apoA-I and apoB levels were measured upon hospital admission. Random forest (RF) models were performed to assess predictive values of these apolipoproteins apoB, apoA-I and their ratio in assessing IS subtype stratification and disease severity. Results Serum apoA-I levels were significantly lower in ICAS compared to ECAS and SAO patients ( p < 0.0001), while apoB levels were higher in ICAS patients ( p < 0.0001). The apoB/apoA-I ratio was significantly higher in ICAS compared to ECAS and SAO patients ( p < 0.0001). Correlation analyses found a significant correlation between the apoB/apoA-I ratio and conventional lipid components. Additionally, RF models and plots of variable importance and distribution of minimal depth revealed that the apoB/apoA-I ratio played the most influential predictor in predicting IS subtypes and stenosis severity. Conclusion Our study shows the differential distribution of apoA-I and apoB IS subtypes and reveals the significance of the apoB/apoA-I ratio in assessing IS subtypes and arterial stenosis severity. Further studies are warranted to validate these findings and enhance their clinical applicability.
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.
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.
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•A semi-nested, heptaplex RT-PCR assay for SARS-CoV-2 detection has been developed.•The complex melting spectrum was interpreted by an artificial intelligence algorithm.•The developed ...assay enables 96-sample pooled testing for increase of testing capacity.•About 8,000 pre-amplified samples could be screened in one realtime PCR run.
Asymptomatic transmission was found to be the Achilles’ heel of the symptom-based screening strategy, necessitating the implementation of mass testing to efficiently contain the transmission of COVID-19 pandemic. However, the global shortage of molecular reagents and the low throughput of available realtime PCR facilities were major limiting factors.
A novel semi-nested and heptaplex (7-plex) RT-PCR assay with melting analysis for detection of SARS-CoV-2 RNA has been established for either individual testing or 96-sample pooled testing. The complex melting spectrum collected from the heptaplex RT-PCR amplicons was interpreted with the support of an artificial intelligence algorithm for the detection of SARS-CoV-2 RNA. The analytical and clinical performance of the semi-nested RT-PCR assay was evaluated using RNAs synthesized in-vitro and those isolated from nasopharyngeal samples.
The LOD of the assay for individual testing was estimated to be 7.2 copies/reaction. Clinical performance evaluation indicated a sensitivity of 100% (95% CI: 97.83–100) and a specificity of 99.87% (95% CI: 99.55–99.98). More importantly, the assay supports a breakthrough sample pooling method, which makes possible parallel screening of up to 96 samples in one real-time PCR well without loss of sensitivity. As a result, up to 8,820 individual pre-amplified samples could be screened for SARS-CoV-2 within each 96-well plate of realtime PCR using the pooled testing procedure.
The novel semi-nested RT-PCR assay provides a solution for highly multiplex (7-plex) detection of SARS-CoV-2 and enables 96-sample pooled detection for increase of testing capacity.
.
Abstract
Aim
The aim of the present study is to compare the performance of 16S rRNA Nanopore sequencing and conventional culture in detecting infectious pathogens in patients with suspected ...meningitis in a resource-limited setting without extensive bioinformatics expertise.
Methods
DNA was isolated from the cerebrospinal fluid (CSF) of 30 patients with suspected bacterial meningitis. The isolated DNA was subjected to 16S sequencing using MinION™. The data were analysed in real time via the EPI2ME cloud platform. The Nanopore sequencing was done in parallel to routine microbiological diagnostics.
Results
Nanopore sequencing detected bacterial pathogens to species level in 13 of 30 (43%) samples. CSF culture showed 40% (12/30) positivity. In 21 of 30 patients (70%) with suspected bacterial meningitis, both methods yielded concordant results. About nine of 30 samples showed discordant results, of these five were false positive and four were false negative. In five of the culture negative results, nanopore sequencing was able to detect pathogen genome, due to the higher sensitivity of the molecular diagnostics. In two other samples, the CSF culture revealed Cryptococcus neoformans and Streptococcus pneumoniae, which were not detected by Nanopore sequencing. Overall, using both the cultures and 16S Nanopore sequencing, positivity rate increased from 40% (12/30) to 57% (17/30).
Conclusion
Next-generation sequencing could detect pathogens within six hours and could become an important tool for both pathogen screening and surveillance in low- and middle-income countries (LMICs) that do not have direct access to extensive bioinformatics expertise.
Blood cultures are commonly employed to identify bacterial pathogens causing sepsis. PCR assays to diagnose septicemia require extraction of bacterial DNA from blood samples and thus, delay the ...initiation of appropriate antimicrobial treatment. The presence of abundant human DNA may hamper the sensitivity of PCR in the detection of bacteria.
We used serial dilutions of E. Coli spiked pseudo-blood-sepsis samples to develop a simple method that combines the use of a polar detergent solvent and adjustment of the basic pH to remove human DNA. A 16S rRNA gene-based screening algorithm was established to differentiate Gram-positive and Gram-negative groups of bacteria and the family of Enterobacteriaceae. A stringent validation with appropriate controls was implemented. The method of human DNA removal was then applied on 194 sepsis blood samples and 44 cerebrospinal fluid (CSF) samples by real-time PCR.
This uncomplicated and straightforward approach allows to remove up to 98 % of human DNA from peripheral blood of septic patients. The inhibitory effect of human DNA is efficiently prevented and the detection limit of real-time PCR is increased to 10 E. Coli CFUs/ml. This sensitivity is 10 times higher compared to conventional real-time PCR assays. The classical blood culture detected 58/194 (30 %) of sepsis and 9/44 (21 %) of CSF samples. Out of the 194 blood samples tested, the conventional real-time PCR targeting 13 common sepsis causing pathogens correctly detected the bacterial DNA in 16/194 (8 %) only and 14/44 (32 %) in cerebrospinal fluid samples. Our newly established approach was able to provide correct diagnoses in 78 (40 %) of the 194 blood samples and in 14 (32 %) of the CSF samples. The combination of both blood cultures and our technique raised the rate of sepsis diagnoses to 112/194 (58 %). Of the total group tested positive, 46 (24 %) cases showed overlap with the classical methodology.
We report a simple optimized in-house protocol for removal of human DNA from blood sepsis samples as a pre-analytical tool to prepare DNA for subsequent PCR assays. With the detection increase of our in-house DNA removal approach, subsequent PCR assays can reach detection limits of 10 E. coli CFUs/ml and significantly improve the diagnostic rate in blood sepsis cases.
The incidence of gastric cancer in the Northern city, Hanoi is higher than in the Southern city, Ho Chi Minh, Vietnam. We previously reported that
m1 genotype might be responsible for the difference ...between the two cities, however, the study only included non-cancer patients. The aim of this study is to investigate the non-cardia gastric cancer characteristics and the role of
virulence on different non-cardia gastric cancer incidence between two cities in Vietnam.
We recruited 282 non-cardia gastric cancer patients that had undergone gastroscopy in two cities, Ho Chi Minh and Hanoi, Vietnam. Characteristics of non-cardia gastric cancer were late age of onset (mean age, 62.5 years), predominance in males (ratio of males/females; 3.9:1), diffuse type (55.3%), and high prevalence of
infection (79.4%).
infection and the
m1 genotype conferred an increased risk for GC (OR, 2.02; 95% CI 1.4-3.0;
= 0.0003 and OR, 2.7; 95% CI 1.5-4.7;
= 0.001, respectively). Interestingly, the presence of
m1 genotype in the gastric cancer group was significantly higher than that in the non-cancer group (68.8% vs 44.9%,
= 0.001) and the significant tendency still observed in Ho Chi Minh (67.6% vs 31.9%,
< 0.0001).
We first describe the characteristics of non-cardia gastric cancer in Vietnam.
infection was associated with the development of non-cardia GC.
m1 genotype might contribute to incidence differences between the two cities.
For the identification of bacterial pathogens, blood culture is still the gold standard diagnostic method. However, several disadvantages apply to blood cultures, such as time and rather large ...volumes of blood sample required. We have previously established an optimised multiplex real-time PCR method in order to diagnose bloodstream infections.
In the present study, we evaluated the diagnostic performance of this optimised multiplex RT-PCR in blood samples collected from 110 septicaemia patients enrolled at the 108 Military Central Hospital, Hanoi, Vietnam.
Positive results were obtained by blood culture, the Light Cylcler-based SeptiFast® assay and our multiplex RT-PCR in 35 (32%), 31 (28%), and 31 (28%) samples, respectively. Combined use of the three methods confirmed 50 (45.5%) positive cases of bloodstream infection, a rate significantly higher compared to the exclusive use of one of the three methods (P=0.052, 0.012 and 0.012, respectively). The sensitivity, specificity and area under the curve (AUC) of our assay were higher compared to that of the SeptiFast® assay (77.4%, 86.1% and 0.8 vs. 67.7%, 82.3% and 0.73, respectively). Combined use of blood culture and multiplex RT-PCR assay showed a superior diagnostic performance, as the sensitivity, specificity, and AUC reached 83.3%, 100%, and 0.95, respectively. The concordance between blood culture and the multiplex RT-PCR assay was highest for Klebsiella pneumonia (100%), followed by Streptococcus spp. (77.8%), Escherichia coli (66.7%), Staphylococcus spp. (50%) and Salmonella spp. (50%). In addition, the use of the newly established multiplex RT-PCR assay increased the spectrum of identifiable agents (Acintobacter baumannii, 1/32; Proteus mirabilis, 1/32).
The combination of culture and the multiplex RT-PCR assay provided an excellent diagnostic accomplishment and significantly supported the identification of causative pathogens in clinical samples obtained from septic patients.
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