An isothermal CRISPR- based lateral flow assay for detection of Neisseria meningitidis.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: This developed and optimized method can complement for the available gold standard for the timely diagnosis of meningococcal meningitis and meningococcemia.

Association Between Soluble Notch Ligand Delta-like Ligand 1 and Bleeding Complications in Patients With Dengue Fever Infection.

Bleeding associated with endothelial damage is a key feature of severe dengue fever. In the current study, we investigated whether Notch ligands were associated with bleeding in 115 patients with confirmed dengue infection in Vietnam. Soluble Notch ligands were determined by means of enzyme-linked immunosorbent assay. Seventeen of 115 patients (14.8%) experienced bleeding manifestations. High soluble delta-like ligand 1 (sDLL1) plasma levels was associated with bleeding (median, 15 674 vs 7117 pg/mL; P < .001). Receiver operating characteristic (ROC) curve analysis demonstrated that sDLL1 had the best test performance (area under the ROC curve, 0.852), with 88% sensitivity and 84% specificity. The combination with alanine aminotransferase and aspartate aminotransferase slightly increased sDLL1 performance. sDLL1 may be useful to guide clinical management of patients with patients in endemic settings.

CRISPR-Cas12a combination to alleviate the false-positive in loop-mediated isothermal amplification-based diagnosis of Neisseria meningitidis.

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: In properly optimized conditions, the CRISPR-Cas12a system helps to alleviate false positive result hence enhancing the specificity of the LAMP assays.

Diagnosis of pathogens causing bacterial meningitis using Nanopore sequencing in a resource-limited setting.

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.

Molecular detection of blaCTX-M gene to predict phenotypic cephalosporin resistance and clinical outcome of Escherichia coli bloodstream infections in Vietnam.

BACKGROUND: Blood stream infections (BSI) caused by Extended Spectrum Beta-Lactamases (ESBLs) producing Enterobacteriaceae is a clinical challenge leading to high mortality, especially in developing countries. In this study, we sought to describe the epidemiology of ESBL-producing Escherichia coli strains isolated from Vietnamese individuals with BSI, to investigate the concordance of genotypic-phenotypic resistance, and clinical outcome of ESBL E. coli BSI. METHODS: A total of 459 hospitalized patients with BSI were screened between October 2014 and May 2016. 115 E. coli strains from 115 BSI patients were isolated and tested for antibiotic resistance using the VITEK®2 system. The ESBL phenotype was determined by double disk diffusion method following the guideline of Clinical and Laboratory Standards Institute. Screening for beta-lactamase (ESBL and carbapenemase) genes was performed using a multiplex-PCR assay. RESULTS: 58% (67/115) of the E. coli strains were ESBL-producers and all were susceptible to both imipenem and meropenem. Resistance to third-generation cephalosporin was common, 70% (81/115) were cefotaxime-resistant and 45% (52/115) were ceftazidime-resistant. blaCTX-M was the most common ESBL gene detected (70%; 80/115) The sensitivity and specificity of blaCTX-M-detection to predict the ESBL phenotype was 87% (76-93% 95% CI) and 54% (39-48% 95% CI), respectively. 28%% (22/80) of blaCTX-M were classified as non-ESBL producers by phenotypic testing for ESBL production. The detection of blaCTX-M in ESBL-negative E. coli BSI was associated with fatal clinical outcome (27%; 6/22 versus 8%; 2/26, p = 0.07). CONCLUSION: A high prevalence of ESBL-producing E. coli isolates harbouring blaCTX-M was observed in BSI patients in Vietnam. The genotypic detection of blaCTX-M may have added benefit in optimizing and guiding empirical antibiotic therapy of E. coli BSI to improve clinical outcome.

White Paper: Bridging the gap between human and animal surveillance data, antibiotic policy and stewardship in the hospital sector-practical guidance from the JPIAMR ARCH and COMBACTE-MAGNET EPI-Net networks.

BACKGROUND: Antimicrobial surveillance and antimicrobial stewardship (AMS) are essential pillars in the fight against antimicrobial resistance (AMR), but practical guidance on how surveillance data should be linked to AMS activities is lacking. This issue is particularly complex in the hospital setting due to structural heterogeneity of hospital facilities and services. The JPIAMR ARCH and COMBACTE-MAGNET EPI-Net networks have joined efforts to formulate a set of target actions for linking surveillance data with AMS activities. METHODS: A scoping review of the literature was carried out addressing research questions on three areas: (i) AMS leadership and accountability; (ii) antimicrobial usage and AMS; (iii) AMR and AMS. Consensus on the target actions was reached through a RAND-modified Delphi process involving over 40 experts in different fields from 18 countries. RESULTS: Evidence was retrieved from 51 documents. Initially 38 targets were proposed, differentiated as essential or desirable according to clinical relevance, feasibility and applicability to settings and resources. In the first consultation round, preliminary agreement was reached for 32 targets. Following a second consultation, 27 targets were approved, 11 were deleted and 4 were suggested for rephrasing, leading to a final approved list of 34 target actions in the form of a practical checklist. CONCLUSIONS: This White Paper provides a pragmatic and flexible tool to guide the development of calibrated hospital-surveillance-based AMS interventions. The strength of this tool is that it is a comprehensive perspective that takes into account the hospital patient case-mix and the related epidemiology, which ultimately drives antimicrobial usage, and the feasibility in low-resource settings.

Upregulation of Enzymes involved in ISGylation and Ubiquitination in patients with hepatocellular carcinoma.

Background: ISGylation is the conjugation of ISG15 with target proteins. ISGylation occurs through an enzymatic cascade, which is similar to that of ubiquitination. Through ISGylation, ISG15 can bind to proteins involved in cell proliferation and differentiation, thus promoting genesis and progression of malignancies. The present study aims to investigate expression of genes involved in ISGylation and ubiquitination in patients with hepatocellular carcinoma and to correlate gene expression with clinical laboratory parameters of these patients. Methods: mRNA expression of genes encoding enzymes involved in the ISGylation process (EFP, HERC5, UBA1, UBC and USP18) was evaluated by quantitative real-time PCR in 38 pairs of tumour and adjacent non-tumour tissues from patients with hepatocellular carcinoma and correlated with distinct clinical laboratory parameters. Results: Relative mRNA expression of EFP, HERC5, UBA1 and USP18 was significantly higher in tumour tissues compared to adjacent non-tumour tissues (P=0.006; 0.012; 0.02 and 0.039, respectively). The correlation pattern of mRNA expression between genes in the tumours differed from the pattern in adjacent non-tumour tissues. Relative expression of EFP, HERC5 and UBA1 in adjacent non-tumour tissues was positively associated with direct bilirubin levels (Spearman's rho=0.31, 0.33 and 0.45; P=0.06, 0.05 and 0.01, respectively) and relative expression of USP18 in adjacent non-tumour tissues correlated negatively with ALT levels (Spearman's rho= -0.33, P=0.03). Conclusions: EFP, HERC5, UBA1, and USP18 genes are upregulated in tumour tissues of patients with HCC and, thus, may be associated with the pathogenesis of hepatocellular carcinoma.

Rapid, low cost and sensitive detection of Calreticulin mutations by a PCR based amplicon length differentiation assay for diagnosis of myeloproliferative neoplasms.

BACKGROUND: Calreticulin (CALR) gene mutations are currently recommended as biomarkers in diagnosis of patients with myeloproliferative neoplasms (MPN) with Jak2 V617F negative phenotype. Our aim was to establish a rapid, low cost and sensitive assay for identification of CALR gene mutations and to validate the diagnostic performance of the established assay in a patient cohort with different clinical MPN phenotypes. METHODS: One hundred five Philadelphia-negative MPN patients, including polycythemia vera (PV), essential thrombocythaemia (ET), and primary myelofibrosis (PMF) were initially screened for JAK2 mutations by amplification-refractory mutation system (ARMS-PCR) methodology and were further subjected to detection of CALR gene mutations by our in-house assay, a PCR based amplicon length differentiation assay (PCR-ALDA). The PCR-ALDA methodology was compared with real time PCR and Sanger sequencing methods. Furthermore, the analytical sensitivity of the assay was established. RESULTS: PCR - ALDA approach was able to detect and discriminate the pseudo-positive samples containing more than 1% CALR mutant alleles. CALR mutations were not detected in 63 Jak2 V617F positive cases in all three methods. In contrast, amongst 42 Jak2 V617F negative cases, both PCR-ALDA and Sanger sequencing coherently identified 12 CALR mutants compared to 10 CALR mutants detected by real-time PCR method. CONCLUSION: PCR-ALDA can be utilized as an easy-to-use, rapid, low cost and sensitive tool in the detection of CALR mutations in Philadelphia-negative MPN patients.

Vitamin D receptor ApaI polymorphism associated with progression of liver disease in Vietnamese patients chronically infected with hepatitis B virus.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Among the four investigated VDR polymorphisms, ApaI is associated with clinical outcome and liver disease progression in Vietnamese HBV infected patients.

Soluble fibrinogen-like protein 2 levels in patients with hepatitis B virus-related liver diseases.

BACKGROUND: Clinical progression of HBV-related liver diseases is largely associated with the activity of HBV-specific T cells. Soluble fibrinogen-like protein 2 (sFGL2), mainly secreted by T cells, is an important effector molecule of the immune system. METHODS: sFGL2 levels were determined by ELISA assays in sera of 296 HBV patients clinically classified into the subgroups of acute hepatitis B (AHB), chronic hepatitis B (CHB), liver cirrhosis (LC), hepatocellular carcinoma (HCC) and patients with LC plus HCC. As control group, 158 healthy individuals were included. FGL2 mRNA was quantified by qRT-PCR in 32 pairs of tumor and adjacent non-tumor liver tissues. RESULTS: sFGL2 levels were elevated in HBV patients compared to healthy controls (P <  0.0001). In the patient group, sFGL2 levels were increased in AHB compared to CHB patients (P = 0.017). sFGL2 levels were higher in LC patients compared to those without LC (P = 0.006) and were increased according to the development of cirrhosis as staged by Child-Pugh scores (P = 0.024). Similarly, HCC patients had increased sFGL2 levels compared to CHB patients (P = 0.033) and FGL2 mRNA was up-regulated in tumor tissues compared to adjacent non-tumor tissues (P = 0.043). In addition, sFGL2 levels were positively correlated with HBV-DNA loads and AST (Spearman's rho = 0.21, 0.25 and P = 0.006, 0.023, respectively), but reversely correlated with platelet counts and albumin levels (Spearman's rho = - 0.27, - 0.24 and P = 0.014, 0.033, respectively). CONCLUSIONS: sFGL2 levels are induced by HBV infection and correlated with the progression and clinical outcome of HBV-related liver diseases. Thus, sFGL2 may serve as a potential indicator for HBV-related liver diseases.

Enrichment of bacterial DNA for the diagnosis of blood stream infections.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Association of vitamin D deficiency with hepatitis B virus - related liver diseases.

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Soluble MICB protein levels and platelet counts during hepatitis B virus infection and response to hepatocellular carcinoma treatment.

BACKGROUND: The human major histocompatibility complex class I polypeptide-related sequence B (MICB) is a protein that modulates the NK and T cell activation through the NKG2D receptor and is related to several diseases including cancer. METHODS: The study investigated the prognostic role of soluble MICB (sMICB) protein in the progression of HBV-related liver diseases and to HBV-related HCC treatment. The sMICB serum levels were measured in 266 chronic HBV-infected Vietnamese patients and in healthy controls, and correlated with clinical and laboratory parameters and with therapeutic interventions for HBV-related HCC. RESULTS: Significant differences in both clinical and laboratory parameters were observed among the patient groups with different stages of hepatitis. The platelet counts were significantly decreased with disease progression (P < 0.001). The sMICB serum levels were significantly increased in HBV patients compared to healthy controls (P < 0.0001). Among the patients with different stages of hepatitis, asymptomatic individuals (ASYM) revealed higher sMICB serum levels while liver cirrhosis (LC) patients revealed lower sMICB serum levels (P < 0.0001) compared to other patient groups. Notably, the sMICB serum levels were decreased in treated HCC patient group compared to not-treated HCC patient group (P = 0.05). Additionally, the sMICB levels were significantly correlated with platelet counts in ASYM and HCC patients (r = -0.37, P = 0.009; and r = 0.22, P = 0.025, respectively). CONCLUSIONS: Our results demonstrate a potential role of sMICB serum levels and platelet counts during immune response to the HBV infection, liver disease progression and response to the HCC treatment.

Simple multiplex PCR assays to detect common pathogens and associated genes encoding for acquired extended spectrum betalactamases (ESBL) or carbapenemases from surgical site specimens in Vietnam.

UNLABELLED: Surgical site infection (SSI) is common in Vietnamese post-operative patients. It contributes to increased morbidity, mortality, hospitalization time and health care expenditure. Bacterial culture is considered the gold standard procedure to identify SSI pathogens and antibiotic resistant properties; however, it can detect microbes that can readily grow and is time-consuming. We propose optimized multiplex PCR assays to diagnose the most relevant microbes and associated genes encoding for acquired extended spectrum betalactamases (ESBL) or carbapenemases from Vietnamese patients with SSI in a hospital setting in Hanoi. METHODS: Ninety-one patients (n = 91) were collected in order to identify microbial pathogens and associated genes encoding for acquired extended spectrum betalactamases (ESBL) or carbapenemases by both conventional bacterial culture and in-house multiplex PCR assays. RESULT AND CONCLUSION: The novel in-house multiplex PCR assays are comparable to the bacterial culture approach in screening for common pathogens causing SSI and for relevant genotypes conferring betalactam/carbapenem resistance for bacteria. This is the first report of Turkey-specific ESBL gene (PER-1) and two Oxacilinase families (Oxa23 and Oxa 58) in Vietnam.

Identification of breast cancer-associated PIK3CA H1047R mutation in blood circulation using an asymmetric PCR assay.

PURPOSE: To establish a highly sensitive and specific approach for the detection of circulating PIK3CA H1047R mutation in breast cancer (BC) patients and to investigate the association between the prevalence of PIK3CA H1047R mutation and clinical presentations. METHODS: A proper blocker was designed in an allele-specific manner and optimized for PCR-based identification of the PIK3CA H1047R mutation. The established technique was validated in cell-free DNA samples from 196 recruited BC patients. RESULTS: The allele-specific PCR assay with a properly designed blocker was able to detect the H1047R mutant variant with 0.01%. By applying the newly established assay, 62 cases (31.6% of the total recruited cases) were found to carry a blood-circulating H1047R mutant. Wherein, the detected mutant rates increased with disease stages from 2/18 (11.1%) of stage I to 17/71 (23.9%) of stage II, 20/53 (37.7%) of stage III, and 23/31 (42.6%) of stage IV (p = 0.025), respectively. Higher frequencies of H1047R mutation were associated with late-stage (p = 0.033) or recurrence (p = 0.045) or metastatic patients (p = 0.049) as well as radiation-treated human epidermal growth factor receptor 2 (HER2) positive BC (p = 0.004). PIK3CA mutant carriers were frequently observed in patients under the age of 50 who had liver-metastasized or brain metastases or lymph node-invaded (p < 0.05). CONCLUSION: A novel allele-specific PCR assay with high sensitivity was established successfully for the detection of the PIK3CA H1047R mutation in clinical practice.

Markers of prolonged hospitalisation in severe dengue.

BACKGROUND: Dengue is one of the most common diseases in the tropics and subtropics. Whilst mortality is a rare event when adequate supportive care can be provided, a large number of patients get hospitalised with dengue every year that places a heavy burden on local health systems. A better understanding of the support required at the time of hospitalisation is therefore of critical importance for healthcare planning, especially when resources are limited during major outbreaks. METHODS: Here we performed a retrospective analysis of clinical data from over 1500 individuals hospitalised with dengue in Vietnam between 2017 and 2019. Using a broad panel of potential biomarkers, we sought to evaluate robust predictors of prolonged hospitalisation periods. RESULTS: Our analyses revealed a lead-time bias, whereby early admission to hospital correlates with longer hospital stays - irrespective of disease severity. Importantly, taking into account the symptom duration prior to hospitalisation significantly affects observed associations between hospitalisation length and previously reported risk markers of prolonged stays, which themselves showed marked inter-annual variations. Once corrected for symptom duration, age, temperature at admission and elevated neutrophil-to-lymphocyte ratio were found predictive of longer hospitalisation periods. CONCLUSION: This study demonstrates that the time since dengue symptom onset is one of the most significant predictors for the length of hospital stays, independent of the assigned severity score. Pre-hospital symptom durations need to be accounted for to evaluate clinically relevant biomarkers of dengue hospitalisation trajectories.

Characterization of zoonotic hepatitis E virus in domestic pigs and wild boar in Vietnam: Implications for public health.

Vietnam's unprecedented demand for meat from livestock, including pigs and farmed wildlife, underscores the importance of understanding zoonotic reservoirs for hepatitis E virus (HEV). This study aimed to identify and characterize circulating zoonotic HEV in domestic pigs and wild boar to understand genotype frequencies, transmission dynamics, and associated human health burdens. Rectal swabs, feces, and liver samples from 415 pigs and 102 wild boars were collected across various farms and slaughterhouses in central and southern Vietnam and screened for HEV RNA using nested PCR. HEV RNA-positive samples underwent sanger sequencing and genotyping. Overall, 10% (n = 54/517) of samples were HEV RNA-positive, with wild boars exhibiting the highest HEV positivity rate at 25%, followed by domestic pigs at 7%. Southern Vietnam showed a higher HEV RNA positivity rate (20%) compared to central Vietnam (7%). Notably, rectal swabs demonstrated the highest positivity rate (15%), followed by feces (8%) and liver (4%). HEV-3a was the predominant genotype at 85%, followed by HEV-4b at 9% and HEV-3f at 6%. While HEV-3a was distributed across both central and southern Vietnam, HEV-3f was exclusively detected in central Vietnam, and HEV-4b was identified in wild boar in southern Vietnam. These findings underscore the substantial prevalence of HEV in wild boars, emphasizing their potential as crucial zoonotic reservoirs alongside domestic pigs. Further investigations involving occupationally exposed individuals in high-prevalence areas are warranted to evaluate the human health impact of zoonotic hepatitis E and inform preventive measures. Regular epidemiological studies are imperative for assessing the prevalence and transmission of zoonotic HEV infections among common reservoirs, thereby aiding in the prevention of spillover events within the community.

Evaluation of screening algorithms to detect rectal colonization with carbapenemase-producing Enterobacterales in a resource-limited setting.

Objectives: To improve and rationalize the detection of carbapenemase-producing Enterobacterales (CPE) in rectal swabs in a high-prevalence and resource-constrained setting, addressing surveillance challenges typically encountered in laboratories with limited resources. Methods: A point prevalence survey (PPS) was conducted on 15 August 2022, in a provincial children's hospital in northern Vietnam. Rectal swab samples of all admitted children were collected and plated on a selective medium for carbapenem-resistant Enterobacterales (CRE). Species identification and antimicrobial susceptibility testing (AST) were performed by MALDI-TOF, and VITEK2 XL and interpreted according to CLSI breakpoints (2022). Carbapenemases were detected by the carbapenem inactivation method (CIM) and quantitative real-time PCR (qRT-PCR). Results: Rectal swab samples were obtained from 376 patients. Of 178 isolates growing on the CRE screening agar, 140 isolates were confirmed as Enterobacterales of which 118 (84.3%) isolates were resistant to meropenem and/or ertapenem. CIM and PCR showed that 90/118 (76.3%) were carbapenemase producers. Overall, 83/367 (22.6%) were colonized by CPE. Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae complex were the most common CPE detected, with NDM as the predominant carbapenemase (78/90; 86.7%). Phenotypic resistance to meropenem was the best predictor of CPE production (sensitivity 85.6%, specificity 100%) compared with ertapenem resistance (95.6% sensitivity, 36% specificity). CIM was 100% concordant with PCR in detecting carbapenemases. Conclusions: These findings underscore the effectiveness of meropenem resistance as a robust indicator of the production of carbapenemases and the reliability of the CIM method to detect such carbapenemases in resource-limited settings where the performance of molecular methods is not possible.

High Hepatitis E Virus (HEV) Seroprevalence and No Evidence of HEV Viraemia in Vietnamese Blood Donors.

The prevalence of hepatitis E virus (HEV) in the Vietnamese population remains underestimated. The aim of the present study was to investigate the seroprevalence of HEV IgG/IgM antibodies and the presence of HEV RNA in blood donors as a part of epidemiological surveillance for transfusion-transmitted viruses. Serum samples from blood donors (n = 553) were analysed for markers of past (anti-HEV IgG) and recent/ongoing (anti-HEV IgM) HEV infections. In addition, all serum samples were subsequently tested for HEV RNA positivity. The overall prevalence of anti-HEV IgG was 26.8% (n = 148/553), while the seroprevalence of anti-HEV IgM was 0.5% (n = 3/553). Anti-HEV IgG seroprevalence in male and female donors was similar (27.1% and 25.5%, respectively). A higher risk of hepatitis E exposure was observed with increasing age. None of the blood donors were HEV RNA positive, and there was no evidence of HEV viraemia. Although the absence of HEV viraemia in blood donors from Northern Vietnam is encouraging, further epidemiological surveillance in other geographical regions is warranted to rule out transfusion-transmitted HEV.