Molecular Detection Of Mutations Research Articles

Analysing the Impact of Bedaquiline Resistance in Fluoroquinolone-Resistant Clinical Isolates and Investigating the Molecular Interactions of BDQ with the atpE Gene

The progress of the tuberculosis eradication programme is hindered by the global health issue of drug-resistant tuberculosis, which is a significant threat to the population. Resistance-associated mutations vary geographically, so investigate the prevalence of resistance to fluoroquinolone (anti-TB) drugs and its association with resistance-related mutations in clinical isolates of Tamilnadu (north zone) and Puducherry was carried out. And the resultant fluoroquinolone drug-resistant tuberculosis strains will be subjected to the molecular analysis of bedaquiline resistance to study the mutation in the atpE gene. The study includes 430 specimens received at Intermediate Reference Laboratory, State TB training and Demonstration Centre in the Government Hospital for Chest Diseases for Molecular testing between January 2020 and December 2021. Samples were analysed by GenoType MTBDRslV.2 for the molecular detection of mutations in the gyrA and gyrB genes. FQ resistance was observed in 32 samples (7.4%), most of which were naïve and previous treatment failure cases. Twenty-five isolates had mutations in DNA gyrase subunit -A (gyrA), while mutations in gyrB were observed in only 7 isolates. Mutational analysis revealed that the mutations mainly alter codon 94 (replacing aspartic acid with glycine, D94G), and 90 (replacing alanine with valine, A90V). In Isoniazid Resistance, MDR, and treatment failure cases, the resistance to Fluoroquinolones was most commonly associated with the D94G mutation. A molecular stimulation study evaluates the effect of BDQ on atpE gene by docking, using AutoDock 4.2, showing hydrogen, hydrophobic and electrostatic interactions.

Molecular characterization correlates of different metastasis in advanced colorectal cancer.

e15565 Background: As we all know that patients with advanced colorectal cancer (CRC) are often diagnosed with liver-only metastases (LM), pulmonary-only metastases (PM) and widespread metastasis (WM). Various methods have also been applied to molecular mutation detection in CRC, However, the differences in molecular mutation profiles between the different metastatic lesions are unclear. Methods: In the period of September 2019 to January 2022, 376 LM, 111 PM and 116 WM were included in this retrospective analysis. The average age of patients with LM, PM and WM were respectively 61, 63 and 63 (range, 21-86, 39-95, 29-83). The proportion of male patients accounted for about 60% of LM (246/376), PM (69/111) and WM (74/116) . In this study, we retrospectively analyzed the single nucleotide variants (SNV), copy number variation (CNV) and fusion variants, detected in patients with different metastatic lesions by using next-generation sequencing (NGS). Pearson’s chi-square test was used to test differences in mutation frequency. P-values < 0.05 were considered significant. SPSS 26.0 was used for the statistical analyses. Results: In LM, PM and WM analyzed cohorts, SNV was detected in 370, 108 and 114 patients. CNV was detected in 225, 58 and 60 patients, and fusions were detected in 25,7 and 9 patients. In LM, PM and WM analyzed cohorts, the genes with the highest mutation frequency were APC (11.60%, 8.73%, 6.40%), TP53 (8.90%, 7.70%, 5.29%), KRAS (5.90%, 6.67%, 5.03%) . In addition, the SMAD4 mutation frequency was 2.13%, 0.82% and 1.04%, respectively. APC and SMAD4 mutation frequency was higher in LM than PM ( P= 0.012, P= 0.007) and WM ( P < 0.0001, P= 0.008). 1026, 221 and 281 CNV were detected in LM, PM and WM, respectively. The genes with the highest mutation frequency were MYC (43%, 7.24%, 4.63%), MET (5.95%, 7.69%, 5.34%), EGFR (5.36%, 7.24%, 4.63%) and FLT3 (4.39%, 4.98%, 6.05%) in LM, PM and WM. The SMAD4 mutation frequency were 0.58% and 1.07% in LM and WM, which was not detected in PM. No significant difference was found in those CNVs. No characteristic fusions were found in the total analyzed cohorts. Conclusions: The SNV and CNV molecular mutation profiles were similar in LM, PM and WM. However, the genes with higher mutation probability show higher mutation frequency in LM. Compared with PM, SNV of SMAD4 displays a higher mutation frequency in LM. It has been previously reported that LM may display poorer overall survival (OS) compared with PM. Considering the effect of SMAD4 on the prognosis of CRC, this study may provide a possible analysis of the poor prognosis of LM patients. However, there is still a need for more clinical research to support these results.

Risk Factors and Treatment Outcome Analysis Associated with Second-Line Drug-Resistant Tuberculosis

The present study aimed at analyzing the treatment outcomes and risk factors associated with fluoroquinolone drug resistance having mutations in the gyrA and gyrB genes. A total of 258 pulmonary tuberculosis samples with first-line drug-resistant (H, R, or HR) were subjected to GenoType MTBDRsl assay for the molecular detection of mutations. Among the 258 samples, 251 were drug-resistant tuberculosis and seven were sensitive to all first-line TB drugs. Out of 251 DR-TB cases, 42 cases were MDR TB, 200 were INH mono-resistant and nine cases were RIF mono-resistant tuberculosis. Out of 251 DR-TB cases performed with a MTBDRsl assay, 14 had Pre-XDR-FQ, one patient had pre-XDR-SLID, one had extensively drug-resistant tuberculosis (XDR-TB) and 235 cases were sensitive to both FQ and SLID drugs. The study group had a mean average of 42.7 ± 16.4 years. The overall successful treatment outcomes among the MDR, INH mono-resistant, and pre-XRD patients were 70.6%, 82.0%, and 51%, respectively. The percentage of risk for the unfavorable outcomes in the pre-XDR, INH -mono-resistant, and XDR cases were 113.84% increased risk with RR 2.14; 95% CI 0.7821–5.8468. The independent risk factor associated with the unfavorable outcomes to failure was 77.78% increased risk with RR 1.78; 95% CI 0.3375–9.3655. Logistic regression analysis revealed that the percentage relative risk among MDR-TB patients for gender, male (RR: 1.85), age ≥ 61 years (RR: 1.96), and diabetics (RR: 1.05) were 84.62%, 95.83%, and 4.76%, respectively. The independent risk factors associated with INH mono-resistant cases of age 16–60 (RR: 1.86), ≥61 year (RR: 1.18), and treated cases (RR: 5.06). This study presaged the significant risk of INH mono-resistant, pre-XDR, and MDR among males, young adults, diabetics, and patients with previous treatment failure. Timely identification of high-risk patients will give pronounced advantages to control drug resistance tuberculosis diseases.

Molecular Detection of Mutations in the Propeller Domain of Kelch 13 and pfmdr1 Copy Number Variation in Plasmodium falciparum Isolates from Thailand Collected from 2002 to 2007.

We determined the prevalence of Kelch 13 mutations and pfmdr1 copy number in samples collected from the Thailand-Myanmar border, the Thailand-Cambodia border, and southern Thailand from 2002 to 2007. C580Y was the most prevalent in Trat (Thailand-Cambodia border) and Ranong (Thailand-Myanmar border) at 42% (24/57) and 13% (6/48), respectively. Less predominant mutations were also identified including R539T (7%, 4/57) and Y493H (2%, 1/57) in Trat, P574L (6%, 3/48) and P553L (2%, 1/48) in Ranong, and N537I and D452E (7%, 1/15) in Sangkhlaburi (Thailand-Myanmar border). Samples from Mae sot (33%, 11/33) harbored the highest percentage of multiple pfmdr1 copies, followed by Trat (18%, 10/57), Chiang Dao in 2003 (13%, 4/30), Phang Nga (5%, 2/44), and Chiang Dao in 2002 (4%, 1/26). This retrospective study provides geographic diversity of K13 and pfmdr1 copies and the emergence of these molecular markers in Thailand, an important background information for future surveillance in the region.

Can We Improve Antifungal Susceptibility Testing?

Systemic antifungal agents are increasingly used for prevention or treatment of invasive fungal infections, whose prognosis remains poor. At the same time, emergence of resistant or even multi-resistant strains is of concern as the antifungal arsenal is limited. Antifungal susceptibility testing (AFST) is therefore of key importance for patient management and antifungal stewardship. Current AFST methods, including reference and commercial types, are based on growth inhibition in the presence of an antifungal, in liquid or solid media. They usually enable Minimal Inhibitory Concentrations (MIC) to be determined with direct clinical application. However, they are limited by a high turnaround time (TAT). Several innovative methods are currently under development to improve AFST. Techniques based on MALDI-TOF are promising with short TAT, but still need extensive clinical validation. Flow cytometry and computed imaging techniques detecting cellular responses to antifungal stress other than growth inhibition are also of interest. Finally, molecular detection of mutations associated with antifungal resistance is an intriguing alternative to standard AFST, already used in routine microbiology labs for detection of azole resistance in Aspergillus and even directly from samples. It is still restricted to known mutations. The development of Next Generation Sequencing (NGS) and whole-genome approaches may overcome this limitation in the near future. While promising approaches are under development, they are not perfect and the ideal AFST technique (user-friendly, reproducible, low-cost, fast and accurate) still needs to be set up routinely in clinical laboratories.

Relation between mutations in the 5′ UTR of ANKRD26 gene and inherited thrombocytopenia with predisposition to myeloid malignancies. An Egyptian study

Inherited thrombocytopenias are a heterogeneous group of diseases characterized by a reduced number of platelets and a bleeding tendency that ranges from very mild to life threatening especially in surgery. Mutations in the 5′ untranslated region (UTR) of Ankirin repeat domain 26 (ANKRD26) are responsible for autosomal-dominant form of thrombocytopenia, that is known as ANKRD26-related thrombocytopenia (ANKRD26 RT), characterized by a moderate thrombocytopenia with mild propensity to bleeding and predisposition to hematological malignancies including AML and MDS. We included 90 unrelated patients with inherited thrombocytopenia. In addition, we investigated 45 patients with ITP. Peripheral blood and bone marrow samples were collected and examined and molecular detection of mutations in the 5︡ UTR of ANKRD26 gene was performed for all the patients. Also, screening of the mutation and development of myeloid malignancies in the extended series of the affected subjects was done. ANKRD26 mutations were identified in 10% of the patients with inherited thrombocytopenia. The most common types were c.128 G > A and c.127A>T, while no mutations were found in the ITP group. In those affected, the median number of platelets was 69 x109/L (43–106) with normal MPV in most of the patients (9.4–11.6). There was a statistically significant increase in the unexpected high frequency of myeloid malignancies in the extended series of the mutated subjects compared with the ITP group-extended series (P < .001). So, we can conclude that ANKRD26 RT is associated with increased risk for developing myeloid malignancies and ANKRD26 mutations can represent a valuable tool for making therapeutic decisions.

Fluoroquinolone resistance and mutational profile of gyrA in pulmonary MDR tuberculosis patients

BackgroundFluoroquinolones (FQs) are potential drugs that inhibit DNA synthesis and are used in the treatment of multidrug-resistant tuberculosis (TB) and short-term anti-TB regimens. In recent years, a high proportion of FQ resistance has been observed in Mycobacterium tuberculosis isolates. The development of FQ resistance in multidrug-resistant TB negatively impacts patient treatment outcome and is a serious threat to control of TB.MethodsThe study included a total of 562 samples from patients with pulmonary TB that had been on anti-tuberculosis therapy. MTBDRsl assays were performed for the molecular detection of mutations. Sequence analysis was performed for the characterization and mutational profiling of FQ-resistant isolates.ResultsFQ resistance was observed in 104 samples (18.5%), most of which were previously treated and treatment failure cases. A total of 102 isolates had mutations in DNA gyrase subunit A (gyrA), while mutations in gyrB were observed in only two isolates. Mutational analysis revealed that the mutations mostly alter codons 94 (replacing aspartic acid with glycine, D94G) and 90 (replacing alanine with valine, A90V). In MDR and treatment failure cases, resistance to FQs was most commonly associated with the D94G mutation. In contract, a high proportion of A90V mutations were observed in isolates that were newly diagnosed.ConclusionThe findings suggest that genotypic assays for FQ resistance should be carried out at the time of initial diagnosis, before starting treatment, in order to rule out mutations that impact the potential use of FQs in treatment and to control drug resistance.

Molecular detection of mutations in rpoB gene involved in rifampin resistance in leprosy patients from Côte d'Ivoire

The control measures recommended for treatment of leprosy with multidrug therapy should limit spread of drug-resistant strains; however, rifampicin resistance continues to be reported in several countries. Resistance to anti-infectious used in multidrug therapy has not yet been molecularly established in Cote d'Ivoire. This preliminary study presents results of evaluation of level rifampicin resistance in Cote d'Ivoire. For each case of clinically confirmed multibacillary leprosy (new or relapsed cases) a dermal juice sample was examined by microscopy after Ziehl-Neelsen staining and a specific PCR targeting RLEP repeat sequence. 48 isolates were used to perform amplification of the rpoB gene involved in rifampicin resistance. The origin of the resistance was then investigated after sequencing and mutations analysis. Thus, 24 isolates (50 %) had mutations, 11 (22. 91 %) of which had mutations conferring rifampicin resistance. The mutations correlated with resistance were as follows: Gln438Val (9. 09 %), Asp441Asn (18. 18 %), Ser456Leu (45. 50 %) and Ser456Phe (27. 27 %) in 9 health districts in Cote d'Ivoire. These mutations were observed in both relapsed and new cases. In conclusion, cases of rifampicin resistance exist in Cote d'Ivoire and an active surveillance should be carried out to monitor the evolution of this resistance. In addition, this information can be used to control the spread of drug-resistant M. leprae strains.

Molecular detection of mutations in rpoB gene involved in rifampin resistance in leprosy patients from Côte d'Ivoire

Molecular detection of mutations in rpoB gene involved in rifampin resistance in leprosy patients from Côte d'Ivoire

Molecular Detection of Mutations in mtCOX1 Gene in Iraqi Patients with Aortic and Mitral Valve Diseases

Many evidences suggest that aortic and mitral valve diseases are not a direct result of aging but may be linked to various genetic factors. This study was designed to determine the potential role of mutations in certain mitochondrial genes and their association with aortic and mitral valve disease. The study included 31 patients , 16 with aortic valve defect and 15 mitral valve patients in addition to 20 healthy volunteers as comparative group. The results of the molecular analysis showed that there were 11 mutations of those with an aortic valve, seven silent mutations and two mutations recorded for the first time in the present study at the sites m.6922 G>T; p.W340L, m.6690 G> C; p.G263R and deletion mutation at m.6936 delA site recorded at the clinical variation site with accession number SCV000845763 and one insertion mutation at m.6908 insG site and registered with accession number SCV000845764. The results of the study recorded 12 mutations of the mitral valve eight silent mutations and the other significant mutations at sites m.6253 T>C; p.M117T, m.6366G>A; p.V155I, m.6690G> C; p.G263R and deletion mutation m.6607 insT site registered with the accession number SCV000852048.

Molecular Detection of Mutations in mtCOX1 Gene in Iraqi Patients with Aortic and Mitral Valve Diseases

Many evidences suggest that aortic and mitral valve diseases are not a direct result of aging but may be linked to various genetic factors. This study was designed to determine the potential role of mutations in certain mitochondrial genes and their association with aortic and mitral valve disease. The study included 31 patients , 16 with aortic valve defect and 15 mitral valve patients in addition to 20 healthy volunteers as comparative group. The results of the molecular analysis showed that there were 11 mutations of those with an aortic valve, seven silent mutations and two mutations recorded for the first time in the present study at the sites m.6922 G>T; p.W340L, m.6690 G> C; p.G263R and deletion mutation at m.6936 delA site recorded at the clinical variation site with accession number SCV000845763 and one insertion mutation at m.6908 insG site and registered with accession number SCV000845764. The results of the study recorded 12 mutations of the mitral valve eight silent mutations and the other significant mutations at sites m.6253 T>C; p.M117T, m.6366G>A; p.V155I, m.6690G> C; p.G263R and deletion mutation m.6607 insT site registered with the accession number SCV000852048.

Urine Biopsy-Liquid Gold for Molecular Detection and Surveillance of Bladder Cancer.

With recent advancements in a non-invasive approach to cancer diagnosis and surveillance, the term “liquid biopsy” has gained traction but is currently limited by technological challenges in identifying and isolating circulating tumor cells (CTCs), proteins, cell-free DNA (cfDNA), or other nucleic acids. Tumor tissue biopsy, especially in genitourinary (GU) system is sometimes inadequate and requires invasive surgical options, especially for upper tract urothelial cancer. Urine can prove to be “liquid gold” since it may be a more abundant source of tumor-derived material without the background noise; however, urine DNA (uDNA) may be associated with low mutant allele fraction (MAF). Molecular detection of mutations in uDNA requires a sensitive and accurate method of analysis that allows a high depth of sequencing while minimizing artifacts. Several sequencing approaches to address this hurdle using enhanced library preparation techniques such as Tagged amplicon deep sequencing (TAm-Seq), Safe-SeqS, FAST-SeqS, and CAPP-Seq approaches have been developed. Urine biopsy utilizing next-generation sequencing (NGS) can prove useful at all stages of urologic malignancy care, where urine can be collected to aid in clinical decision making through the identification of commonly known mutations, and potentially reduce or avoid all forms of invasive procedures.

A Study on Molecular Detection of Mutations Associated with Second Line Anti-tuberculosis Drug Resistance

Background: Efficient tuberculosis (TB) control is based on an early diagnosis followed by the rapid identification of drug resistance, in order to treat patients adequately, break the chain of transmission, and avoid the spread of resistant strains. Multidrug-resistant (MDR) Mycobacterium tuberculosis have emerged worldwide and seriously threaten TB control and prevention programs. At the same time, the emergence of extensively drug-resistant tuberculosis (XDR TB) has also become an important global health problem.&#x0D; Objectives: To detect common gene mutation pattern associated with drug resistance against second line anti-tuberculosis drugs in TB patients by SL-LPA as a rapid and early diagnostic test.&#x0D; Materials and Methods:total 652 sputum samples were received from 30 districts at culture district laboratory, Jamnagar from October 2018 to December 2018, and were included in the study. Second line Second line- line probe assay (SL-LPA) was used to detect mutations associated with resistance for anti-tuberculosis drugs.&#x0D; Results: Out of the 652 samples analyzed for mutations associated with second line anti-tuberculosisdrug resistance, 43% of the samples exhibited various forms of mutations. Out of these samples, mutations associated with gyrA gene were detected in 36.6% samples, gyrB gene mutation in 0.7% samples, rrs gene mutation in 4.4% samples and eis gene mutation in 1.2% samples.&#x0D; Conclusion: The present study provide information on the mutation pattern of drug resistant strains present in the geographical area and help to provide the basis for effective strategies for control of drug resistant TB in this region. It also re-emphasizes the importance of second line LPA which can diagnose TB and drug resistance in a single day and allows earlier administration of appropriate treatment as compared to culture result which take 1-2 months.

Progress in Non-invasive Detection of EGFR Mutation in Non-small Cell Lung Cancer

在过去的十年里，癌症患者的管理模式已经逐渐转向为基于分子突变检测的个体化模式。表皮生长因子受体（epidermal growth factor receptor, EGFR）基因突变是非小细胞肺癌（non-small cell lung cancer, NSCLC）的重要驱动因素，针对EGFR的靶向治疗和传统化疗相比，显示出显著的安全性和有效性。然而，并不是所有的EGFR突变患者都可接受EGFR靶向治疗，不同的突变类型往往预示着不同的临床结局，如敏感性突变EGFR 19-Del、L858R和耐药性突变20ins。此外，如今已经开发出第三代TKI药物Osimertinib（AZD9291）和Rociletinib（CO-1686）可使因EGFR T790M突变，导致初代TKI耐药的患者进一步获益。因此，治疗前了解患者EGFR突变状态，治疗过程中持续监测耐药基因EGFR T790M突变情况，对NSCLC患者靶向药物的管理有着重要的意义。最近几年来，“液体活检”技术得到快速的发展，让我们看到采用非侵入性方法以实时监测耐药性突变成为现实的可能。在本综述中，我们回顾了NSCLC中检测EGFR突变的多种非侵入性检测技术在不同液体样本的临床应用。

Molecular Profile and Cytomorphological Manifestation of Isolated Y Loss in Myelodysplastic Syndromes

Introduction: Loss of the Y-chromosome (LOY) is frequent in myelodysplastic syndromes (MDS) and observed as a single aberration in 3-4% of male MDS patients (pts). It is often clonal and not only age associated and confers a very good prognosis and a very low risk for leukemic transformation (Greenberg et al, Blood 2012; Schanz et al, JCO 2012). But LOY does not prove a hematologic disease per se (Arber et al, Blood 2016). To facilitate a better discrimination between age-related and clonal LOY, the aim of this study was to identify molecular mutations and cytomorphological features which might be characteristic for MDS with isolated LOY.Methods: We included 291 pts in our analysis. The cohort comprised 199 pts with normal karyotype (NK) and morphologically proven MDS (excess blasts (EB) in 77/199 (38%) pts) and 92 pts with LOY. NK was defined by 20 normal metaphases or at least 10 normal metaphases and normal fluorescence in situ hybridization (FISH, Tab.1). Results from mutational analysis were available for all pts with NK and for 61 pts with LOY as single cytogenetic aberration in ≥3 metaphases. Seventeen core genes (Tab.2) were sequenced in all 260 pts by Sanger and/or next generation sequencing (NGS). In 134 pts further 28 genes (Tab.2) were analyzed using one of two NGS panels. In addition to these myeloid genes, the second NGS panel covered single nucleotide polymorphisms on the Y-chromosome which enabled determination of the LOY clone size. Detailed cytomorphology for the evaluation of dysplasia was performed by two experts (UG, UB) as previously described (Germing et al, Leuk Res 2012) in 41 pts, including pts with small LOY clones and with cytogenetic sub-clones.Results: Sequencing of 40 pts with LOY and morphologically proven dysplasia showed higher frequencies of mutations in TET2 (epigenetic regulator), ZRSR2 (splicing factor, located at Xp22.2), and CBL (kinase signaling) compared to MDS with NK (Fig.1). Amongst others, mutations in IDH1/2 (epigenetic regulators) and RUNX1 (transcription factor) were rare in MDS with LOY (Fig.1). The total number of mutated core genes did not significantly differ between MDS with LOY and MDS with NK and no EB (p=0.54), but it was significantly higher in MDS with NK and EB (p=0.014, Fig.2). To distinguish between LOY as ancestral or secondary mutation we sequenced 12 pts with MDS and cases we included as clonal cytopenia of undetermined significance (CCUS, pts with cytopenia(s) and molecular mutation and/or LOY≥75% of metaphases) (Wiktor et al, GCC 2000) using the second NGS panel that allowed determination of LOY clone size and detection of molecular mutations. Thereby, we identified four pts where LOY was most likely the founder aberration, two pts with LOY as secondary aberration in addition to ancestral molecular mutations, and six pts with co-dominance of LOY and a molecular mutation (Fig.3). Finally, we aimed to evaluate if the cut off of LOY≥75% (Wiktor et al, GCC 2000) can distinguish between age-related and clonal LOY in our cohort. In 41 pts analyzed in more detail, peripheral blood counts (hemoglobin: mean 10.4 vs. 9.7 g/dL; white blood count: 4.9 vs. 6.0x10(9)/L, platelets: 163 vs. 198x10(9)/L) and dysplasia of the individual cell lines (erythro-, granulo-, megakaryopoesis) did not differ significantly between LOY≥75% and <75%. Likewise, the VAF of molecular mutations did not significantly differ between MDS and pts without dysplasia according to LOY≥75% and <75%. Notably, for pts with LOY≥75% the number of mutated core genes was significantly higher in MDS compared to pts without dysplasia (p=0.024, Fig.4).Conclusions: Cytopenia with isolated LOY seems to comprise a heterogeneous mixture of cytomorphologic and molecular subtypes. We identified pts where LOY definitely is part of the MDS clone that also harbors molecular mutations, indicating that LOY is not only age-associated in these cases. The low total number of mutated genes corresponds to the previously described favorable prognosis of isolated LOY (Schanz et al, JCO 2012; Greenberg et al, Blood 2012). Our data support the current guidelines that LOY does not prove a hematologic disease in the absence of diagnostic morphologic features. In these cases LOY might fulfill the requirements of a clonal mutation as described for clonal hematopoiesis of indeterminate potential (CHIP) or CCUS. In summary, our data suggest that MDS with LOY shows a characteristic molecular mutation profile. [Display omitted] DisclosuresGerming:Janssen: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Molecular detection of mutations involved in Helicobacter pylori antibiotic resistance in Algeria.

In Algeria, there are limited data regarding the pattern of Helicobacter pylori primary antibiotic resistance. The aim of this study was to evaluate the primary resistance of H. pylori to clarithromycin, ciprofloxacin, tetracycline and rifampicin and to determine the molecular mechanisms involved in the resistance. Two hundred and seventy Algerian adults who had never received H. pylori treatment were enrolled in this study. Human biopsies were obtained for culture and antimicrobial susceptibility testing was performed by Etest for clarithromycin, ciprofloxacin, tetracycline and rifampicin. Real-time fluorescence resonance energy transfer (FRET)-PCR was also performed in all cases to assess primary clarithromycin resistance and point mutations involved, real-time PCR was used to detect mutations involved in tetracycline primary resistance and sequencing of the QRDR of gyrA was performed to detect mutations involved in quinolone resistance. No resistance to rifampicin was detected. Resistance to clarithromycin and ciprofloxacin was found in 29.7% and 17.9%, respectively. Results of real-time FRET-PCR showed that A2143G was the most frequent point mutation, A2142C was not found and 42 patients (15.5%) were infected by both resistant and susceptible genotypes. Only two isolates were resistant to tetracycline and exhibited an A926G mutation. Four mutations were found to be responsible for resistance to ciprofloxacin [N87K (44.73%), D91N (23.68%), N87I (18.42%) and D91G (7.89%)]. Local data regarding the primary resistance of H. pylori to clarithromycin, ciprofloxacin, tetracycline and rifampicin and the main genetic mutations involved in the resistance are necessary for a periodic evaluation of antibiotic consumption and new therapeutic strategies in Algeria.

Liquid Biopsy as Surrogate to Tissue in Lung Cancer for Molecular Profiling: A Meta-analysis

Background: The accurate microscopic diagnosis of lung cancer has become insufficient due to the concept of personalized medicine. Tissue samples are used not only for microscopic diagnosis but also for the assessment of the different targets. Biopsies are performed in 80% of the patients and they are not sufficient for molecular diagnosis in 30% of the cases. Liquid biopsy (LB) has been reported as a possible surrogate to tissue samples and has been introduced in the management scheme of the patients since 2014. We aimed to highlight the diagnostic value of liquid biopsy in assessing the molecular profile of non small cell carcinomas in comparison with tissue biopsy.Methods: We retracted eligible articles from PubMed, Embase and Cochrane databases. We calculated the pooled sensitivity (SEN), specificity (SPE), positive likelihood ratio (PLR), negative likelihood ratio (NLR) and diagnostic odds ratio (DOR). A summary receiver operating characteristic curve (SROC) and area under curve (AUC) were used to evaluate the overall diagnostic performance using the Meta-Disc software 5.1.32. The heterogeneity was assessed using I square statistics. A meta-regression was performed in case of heterogeneity. In case of absence of covariates, a sensitivity analysis was done in order to assess publications that induced a statistical bias.Results: 39 eligible studies involving 4782 patients were included. The overall statistical studies showed heterogeneity in the SEN, SPE, PLR, NLR and DOR. No threshold effect was revealed. The meta-regression incorporating the ethnicity, the test, the technique used in tissue and plasma and the use of plasma or serum as covariates showed no impact of these factors. A sensitivity analysis allowed achieving the homogeneity in the SPE and DOR. The overall pooled SEN and SPE were 0.61 and 0.95 respectively. The PLR was 9.51, the NLR was 0.45 and DOR was 24.58. The SROC curve with AUC of 0,93 indicated that the liquid biopsy is capable of identifying wild type samples from mutated ones with a relatively high accuracy.Conclusion: This meta-analysis suggested that detection of molecular mutations by cfDNA is of adequate diagnostic accuracy in association to tissues. The high specificity and the moderate sensitivity highlight the value of LB as a screening test

Molecular basis of antifungal drug resistance in yeasts

Besides inherent differences in in vitro susceptibilities, clinically-relevant yeast species may acquire resistance upon exposure to most antifungal drugs used in the clinic. In recent years, major fundamental research studies have been conducted to improve our understanding of the molecular basis of antifungal resistance. This topic is of major interest as antifungal resistance in yeast is clearly evolving and is correlated with clinical failure. This minireview is an overview of the most recent findings about key molecular mechanisms evolving in human pathogenic yeasts, particularly Candida spp., in the context of antifungal drug resistance. Also included are the methods currently available for in vitro antifungal susceptibility testing and for molecular detection of mutations associated with resistance. Finally, the genetic drivers of antifungal resistance are discussed in light of the spectra of multidrug resistance as observed in Candida glabrata.

Culture-Based Methods and Molecular Tools for Azole-Resistant Aspergillus fumigatus Detection in a Belgian University Hospital.

Azole-resistant Aspergillus fumigatus is an increasing worldwide problem with major clinical implications. Surveillance is warranted to guide clinicians to provide optimal treatment to patients. To investigate azole resistance in clinical Aspergillus isolates in our institution, a Belgian university hospital, we conducted a laboratory-based surveillance between June 2015 and October 2016. Two different approaches were used: a prospective culture-based surveillance using VIPcheck on unselected A. fumigatus (n = 109 patients, including 19 patients with proven or probable invasive aspergillosis [IA]), followed by molecular detection of mutations conferring azole resistance, and a retrospective detection of azole-resistant A. fumigatus in bronchoalveolar lavage fluid using the commercially available AsperGenius PCR (n = 100 patients, including 29 patients with proven or probable IA). By VIPcheck, 25 azole-resistant A. fumigatus specimens were isolated from 14 patients (12.8%). Of these 14 patients, only 2 had proven or probable IA (10.5%). Mutations at the cyp51A gene were observed in 23 of the 25 A. fumigatus isolates; TR34/L98H was the most prevalent mutation (46.7%), followed by TR46/Y121F/T289A (26.7%). Twenty-seven (27%) patients were positive for the presence of Aspergillus species by AsperGenius PCR. A. fumigatus was detected by AsperGenius in 20 patients, and 3 of these patients carried cyp51A mutations. Two patients had proven or probable IA and cyp51A mutation (11.7%). Our study has shown that the detection of azole-resistant A. fumigatus in clinical isolates was a frequent finding in our institution. Hence, a rapid method for resistance detection may be useful to improve patient management. Centers that care for immunocompromised patients should perform routine surveillance to determine their local epidemiology.

Molecular Detection of Mutations within the Quinolone Resistance-Determining Regions in Non Typhoidal Salmonella Isolates from Malaysia

Introduction: The efficacy of chemotherapy in bacteraemia caused by non-typhoidal Salmonella (NTS) is compromised by antibiotic resistance. Objective: This study was undertaken to describe the mechanism of resistance among clinical NTS isolates. Materials & Methodology: Thirty of NTS were isolated from blood (n = 19), stool (n = 10) and bronchioalveolar lavage (BAL; n = 1) respectively. These isolates were tested for susceptibility testing by disc diffusion method against ampicillin, gentamicin, tetracycline, co-trimoxazole, nalidixic acid, ciprofloxacin and ceftriaxone. Epsilometer tests (E-test) for nalidixic acid and ciprofloxacin were performed for nalidixic acid resistant isolates by disc diffusion method. DNA sequencing was carried out on six of the nalidixic acid resistant Salmonella Enteritidis isolates to identify mutations within quinolones resistance determining regions (QRDR) of gyrA, gyrB, parC and parE genes. Results: Resistance rates of NTS isolates from blood, stool, and BAL were respectively 37%, 20% and 0% for ampicillin, 79%, 40% and 0% for tetracycline, 32%, 40% and 0% for co-trimoxazole, 37%, 10% and 100% for nalidixic acid. Eight isolates were resistant to nalidixic acid and had exhibited reduced susceptibility towards ciprofloxacin by E-test. Mutation within QRDR was detected in gyrA gene (n = 6; Asp 47 → His [3], Asp 51 → Asn [1], Asp 73 → Gly [1], and Gly 48 → Asp [1]) and double mutation was detected in parE gene (n = 3; Gly 48 → Asp [3], Glu 82 → Ser [3]). Out of six isolates, three isolates were found to have both gyrA and parE gene mutations. Conclusions: There was no mutation observed in gyrB and parC gene. Mutation in gyrA gene was sufficient to induce decreased susceptibility to ciprofloxacin. Variation in amino acid sequences are novel, while detection of other gene mutation was uncommon.