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.

Aetiologies and clinical presentation of central nervous system infections in Vietnamese patients: a prospective study.

Knowledge of the clinical presentation of central nervous system (CNS) infections and the causative pathogens is crucial for appropriate diagnosis and rapid initiation of appropriate treatment to prevent severe neurological sequelae. The aim of this study is to understand the aetiology of CNS infections based on the clinical presentation of Vietnamese patients. A prospective hospital-based cohort study was conducted between May 2014 and May 2017. We screened 137 patients with clinically suspected CNS infection for fungal, bacterial and viral pathogens using their cerebrospinal fluid (CSF) and blood cultures. In addition, DNA or RNA extracted from CSF samples were subjected to nucleic acid testing (NAT) with a selective panel of bacterial, viral and fungal pathogens. At least one pathogen could be detected in 41% (n = 56) of the patients. The main pathogens causing CNS infections were Streptococcus suis (n = 16; 12%) and Neisseria meningitidis (n = 9; 7%), followed by Herpes simplex virus 1/2 (n = 4; 3%) and Klebsiella pneumoniae (n = 4; 3%). Other pathogens were only identified in a few cases. Patients with bacterial CNS infections were significantly older, had a worse outcome, a lower Glasgow Coma Scale (GCS), a higher rate of speech impairment and neck stiffness than patients with viral or tuberculous CNS infections. In northern Vietnam, adults are mostly affected by bacterial CNS infections, which have a severe clinical course and worse outcomes compared to viral or tuberculous CNS infections. Clinicians should be aware of the regional occurrence of pathogens to initiate rapid and appropriate diagnosis and treatment.

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.

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.

Circulating miR-147b as a diagnostic marker for patients with bacterial sepsis and septic shock.

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

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.

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.

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.

Interferon-stimulated gene 20 kDa protein serum levels and clinical outcome of hepatitis B virus-related liver diseases.

Interferon-stimulated gene 20 kDa protein (ISG20) with 3' to 5' exonuclease activity mainly targeting single-stranded RNA plays an important role in immune responses against various infectious pathogens, including hepatitis viruses. ISG20 levels were measured by ELISA assays in sera of 339 hepatitis B-virus (HBV) infected patients and 71 healthy individuals and were correlated with clinical and laboratory parameters. ISG20 mRNA was quantified by qRT-PCR in 30 pairs of hepatocellular carcinoma (HCC) tumour and adjacent non-tumour liver tissues. ISG20 levels were significantly elevated in HBV patients compared to healthy controls (P<0.0001). In the patient group, varying ISG20 levels were associated with different forms of HBV-related liver diseases. ISG20 levels were higher in patients with HCC compared to those without HCC (P<0.0001), and increased according to the stages of HCC (P<0.0001). ISG20 mRNA expression was up-regulated in tumour tissues compared to the expression in adjacent non-tumour tissues (P=0.017). Importantly, ISG20 levels were strongly correlated with the levels of AST, ALT, total and direct bilirubin among HCC patients (Pearson's r = 0.43, 0.35, 0.34, 0.3; P<0.0001, respectively). Although differences between liver cirrhosis (LC) and non-LC patients were not observed, ISG20 levels were elevated according to the progression of cirrhosis in patients with LC plus HCC (P=0.005). In conclusions, ISG20 levels are induced by HBV infection and significantly associated with progression and clinical outcome of HBV-related liver diseases, especially in patients with HCC. ISG20 might be a potential indicator for liver injury and the clinical outcome in HBV-related HCC.

Clinical utility of an optimised multiplex real-time PCR assay for the identification of pathogens causing sepsis in Vietnamese patients.

INTRODUCTION: 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. MATERIAL AND METHODS: 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. RESULTS: 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). CONCLUSION: 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.

Significance of nucleic acid testing in diagnosis and treatment of post-neurosurgical meningitis caused by multidrug-resistant Acinetobacter baumannii: a case report.

BACKGROUND: Neurosurgery may pose the risk of patients' developing nosocomial meningitis caused by infection with hospital pathogens. Rapid detection of the causative pathogens is essential for selecting the appropriate antibiotic treatment. However, the classical culture-based detection of bacterial infection is time-consuming and often fails to establish the correct diagnosis. Molecular techniques offer improved diagnostic means to guide the proper antibiotic therapy. CASE PRESENTATION: A 32-year-old Vietnamese man underwent neurosurgery and subsequently developed meningitis. The classical bacterial culture method failed to detect any infectious agents, whereas polymerase chain reaction-based assays identified Acinetobacter baumannii as the causative pathogen. In addition, detection of the acquired extended-spectrum beta-lactamase gene VEB and carbapenem resistance genes NDM-1 and IMP suggested that the isolated A. baumannii strain was multidrug resistant. Upon the establishment of the correct diagnosis, an adequate treatment regimen was chosen and he recovered completely. CONCLUSIONS: This case report demonstrates the usefulness of the molecular approach as an important addendum and alternative to culture-based diagnosis in order to detect the pathogen causative for meningitis, including the indicators for resistance.

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.

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.