Drug Susceptibility Research Articles

Dynamics and Evolution of Donor-derived Cytomegalovirus Infection in 3 Solid Organ Transplant Recipients With the Same Multiorgan Donor.

Cytomegalovirus (CMV) infection poses a significant risk to immunosuppressed transplant recipients, manifesting through primary infection, reinfection, or reactivation. We analyzed the emergence of drug resistance in CMV infection in 3 patients who were later found to have received an allograft from a shared, deceased donor. The seronegative transplant recipients developed symptomatic CMV infections after bowel/pancreas, kidney, or lung transplantation. Prospective Sanger sequencing was used to identify mutations in the viral DNA polymerase (DP) and protein kinase (PK). DP and PK variants were retrospectively quantified by targeted next-generation sequencing. The impact of the novel DP-A505G substitution on drug susceptibility was assessed using a recombinant virus. Whole-genome sequencing of clinical CMV samples was enabled through target DNA enrichment. The DP-A505G substitution was found in all patient samples and could be associated with a natural polymorphism. A subsequent review of the patients' clinical histories revealed that they had all received organs from a single donor. The CMV infection exhibited divergent evolution among the patients: patient 1 developed resistance to ganciclovir and foscarnet because of 2 DP mutations (V715M and V781I), patient 2 showed no genotypic resistance, and patient 3 developed ganciclovir (PK-L595S) and maribavir resistance (PK-T409M). Interpatient variation across the entire CMV genome was minimal, with viral samples clustering in phylogenetic analysis. All 3 transplant recipients were infected with the same donor-derived CMV strain and readily developed different drug susceptibility profiles. This underscores the importance of judicious antiviral drug use and surveillance in preventing antiviral resistance emergence.

IGFBP7+ subpopulation and IGFBP7 risk score in astrocytoma: insights from scRNA-Seq and bulk RNA-Seq.

Glioma is the predominant malignant brain tumor that lacks effective treatment options due to its shielding by the blood-brain barrier (BBB). Astrocytes play a role in the development of glioma, yet the diverse cellular composition of astrocytoma has not been thoroughly researched. We examined the internal diversity of seven distinct astrocytoma subgroups through single-cell RNA sequencing (scRNA-seq), pinpointed crucial subgroups using CytoTRACE, monocle2 pseudotime analysis, and slingshot pseudotime analysis, employed various techniques to identify critical subgroups, and delved into cellular communication analysis. Then, we combined the clinical information of GBM patients and used bulk RNA sequencing (bulk RNA-seq) to analyze the prognostic impact of the relevant molecules on GBM patients, and we performed in vitro experiments for validation. The analysis of the current study revealed that C0 IGFBP7+ Glioma cells were a noteworthy subpopulation of astrocytoma, influencing the differentiation and progression of astrocytoma. A predictive model was developed to categorize patients into high- and low-scoring groups based on the IGFBP7 Risk Score (IGRS), with survival analysis revealing a poorer prognosis for the high-IGRS group. Analysis of immune cell infiltration, identification of genes with differential expression, various enrichment analyses, assessment of copy number variations, and evaluation of drug susceptibility were conducted, all of which highlighted their significant influence on the prognosis of astrocytoma. This research enhances comprehension of the diverse cell composition of astrocytoma, delves into the various factors impacting the prognosis of astrocytoma, and offers fresh perspectives on treating glioma.

A rapid and simple modified nitrate reductase assay for testing first and second-line antituberculosis drug susceptibilities in Mycobacterium tuberculosis isolates

In this study, we developed a modified NRA (MONRA) to determine the first and second-line drug susceptibilities of 5 reference ATCC strains and 42 clinical M. tuberculosis isolates. Unlike conventional NRA, which is often performed in solid media or 7H9 broth, the MONRA is performed in a different medium, AYC.2.1 broth, using lyophilized antibiotic tubes to determine drug susceptibility. The MONRA results were compared with BACTEC MGIT 960 method as the reference method for first-line drugs and conventional NRA performed in 7H9 broth for second-line drugs. The agreement between the MONRA and the reference method was determined as 97.62, 100, 97.62, and 100 % for streptomycin, isoniazid, rifampicin, and ethambutol, respectively. When the results were compared with convantional NRA, the agreement was determined as 100 % for all second-line antibiotics including levofloxacin, ofloxacin, and kanamycin. MONRA has the potential to eliminate challenges in implementing drug susceptibility testing in resource-limited settings.

Changes in Ergosterol Biosynthesis Alter the Response to Cycloheximide, 4-Nitroquinoline-N-Oxide, Weak Organic Acids, and Virulence in Candida glabrata.

The ERG6 gene encodes the sterol C24-methyltransferase converting zymosterol to fecosterol in the ergosterol biosynthetic pathway. Here, we extend the results of functional analysis of the CgERG6 gene, which was previously shown to modulate drug susceptibility in Candida glabrata mutant cells, by demonstrating that its deletion leads to increased susceptibility to cycloheximide, 4-nitroquinoline-N-oxide and weak organic acids, and such effects are associated with attenuated virulence. Together with abrogated efflux of drug substrates by CgCdr1p and CgPdr12p, the Cgerg6Δ mutation leads to reduced cell surface hydrophobicity and decreased virulence of the mutant cells of C. glabrata. The absence of CgErg6p impacts the lipid organization and function of the plasma membrane, resulting in non-specific permeability and abrogation of normal function of membrane-bound proteins accompanied by decreased virulence in Cgerg6Δ cells. Galleria mellonella larvae were used as a non-vertebrate animal host model to determine differences in the virulence potential of C. glabrata strains (parental strain and the Cgerg6Δ deletion mutant). We found that Cgerg6Δ mutant strain attenuated in virulence caused 25-30% survival of larvae compared with parental strain.

Synergistic antimicrobial activity of alpha-linolenic acid in combination with tetracycline or florfenicol against multidrug-resistant Salmonella typhimurium

Salmonella is a major foodborne pathogen that can be transmitted from livestock and poultry to humans through the food chain. Due to the widespread use of antibiotics, antibiotic resistance Salmonella has become an important factor threatening food safety. Combining antibiotic and non-antibiotic agents is a promising approach to address the widespread emergence of antibiotic-resistant pathogens.In this study, we investigated the antibiotic resistance profile and molecular characterization of different serotypes of Salmonella isolated from large-scale egg farms using drug susceptibility testing and whole genome sequencing. The synergistic effect of alpha-linolenic acid (ALA) with antibiotics was evaluated using the checkerboard test and time-kill curve. The molecular mechanism of α-linolenic acid synergism was explored using biochemical assays, pull-down assays, and molecular docking. In vivo efficacy of ALA in combination with florfenicol (FFC) or tetracycline (TET) against multidrug-resistant (MDR) Salmonella enterica subsp. enterica serovar typhimurium was also investigated using a mouse model.We found that ALA reduced the minimum inhibitory concentration (MIC) of tetracycline and florfenicol in all strains tested. When ALA (512 mg/L) was combined with florfenicol (32 mg/L) or tetracycline (16 mg/L), we observed disruption of cell membrane integrity, increased outer membrane permeability, lowered cell membrane potential, and inhibition of proton-drive-dependent efflux pumps. The synergistic treatment also inhibited biofilm production and promoted oxidative damage. These changes together led to an increase in bacterial antibiotic susceptibility. The improved efficacy of ALA combination treatment with antibiotics was validated in the mouse model. Molecular docking results indicate that ALA can bind to membrane proteins via hydrogen bonding. Our findings demonstrated that combined treatment using ALA and antibiotics is effective in preventing infections involving MDR bacteria. Our results are of great significance for the scientific and effective prevention and control of antibiotic resistance Salmonella, as well as ensuring food safety.

Pan-cancer analysis of ADAR1 with its prognostic relevance in low-grade glioma

ADAR1, known as the primary enzyme for adenosine-to-inosine RNA editing, has recently been implicated in cancer development through both RNA editing-dependent and −independent pathways. These discoveries suggest that ADAR1′s functions may extend beyond our current understanding. A pan-cancer analysis offers a unique opportunity to identify both common and distinct mechanisms across various cancers, thereby advancing personalized medicine. Low-grade glioma (LGG), characterized by a diverse group of tumor cells, presents a challenge in risk stratification, leading to significant variations in treatment approaches. Recently discovered molecular alterations in LGG have helped to refine the stratification of of these tumors and offered novel targets for predicting likely outcomes. This study aims to provide a detailed analysis of ADAR mRNA across multiple cancers, emphasizing its prognostic significance in LGG. We observed inconsistent mRNA and consistent protein expression patterns of ADAR1/ADAR in pan-cancer analyses that across tumor types. ADAR mRNA expression did not always correlate with ADAR1 protein expression. Nevertheless, the transcript levels correlated significantly with genetic alterations, tumor mutation burden, microsatellite instability, overall survival, recurrence-free survival, immune marker presence, immune infiltration, and the survival of patients undergoing immunotherapy in select cancers. Furthermore, ADAR and its top 50 associated genes were primarily involved in mRNA-related events, as identified through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. Utilizing the Cox proportional hazards model, we developed a 3-gene signature (ADAR, HNRNPK, and SMG7), which effectively stratified patients into high- and low-risk groups, with high-risk patients exhibiting poorer overall survival, higher tumor grades, and a greater number of non-codeletions. Overall, this signature was inversely related to immune infiltration across cancers. Transcription factor SPI1 and miR-206, potential upstream regulators of the signature genes, were closely linked to patient survival in LGG. The promoter regions of these genes were hypermethylated, further associating them with patient outcomes. Additionally, these genes displayed consistent drug susceptibility patterns. In conclusion, our findings reveal multiple aspects of ADAR1′s role in cancer and underscore its prognostic value in LGG, offering insights into potential therapeutic targets and strategies.

Oxidative stress gene signature construction to identify subtypes and prognosis of patients with lung adenocarcinoma

BackgroundAlthough oxidative stress and malignancies are intimately connected, it is unknown how lung adenocarcinoma (LUAD) is affected by oxidative stress response-related genes (OSRGs).Our goal in this work was to create a genetic signature based on OSRGs that might both predict prognosis and hint to potential treatment options for LUAD. MethodsClinicopathological and transcriptome information on LUAD patients was obtained from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. A model for predicting risk was created using LASSO regression. The TCGA, GSE72094, and GSE41271 cohorts all demonstrated the risk model's prediction ability. Immune cell infiltration was measured using the CIBERSORT method, and the TIDE platform was implemented to evaluate the therapeutic efficacy of immune checkpoint inhibition (ICI). Chemotherapy sensitivity was predicted using drug activity data by the Genomics of Drug Sensitivity. An investigation into gene expression was conducted using qRT-PCR. CCK-8 and transwell assays were employed to look into how DKK1 affected the migration and proliferation of LUAD cells. ResultsA gene signature consisting of ANLN, FAM83A, DKK1, LOXL2, RHOV, IGFBP1, CCR2, GNG7, and C11orf16 was efficiently determined and used to calculate a patient-specific risk score, this functioned as a stand-alone biomarker for prediction. Correlations were found between risk scores and immune cell infiltration frequency, ICI therapy response rate, estimated chemotherapeutic drug susceptibility and autophagy-related genes.Furthermore, DKK1 knockdown reduced the ability of LUAD cells to multiply and migrate. ConclusionOur thorough transcriptome study of OSRGs generated a biological framework effective in forecasting outcome and responsiveness to therapy in LUAD patients.

HPV-ferroptosis related genes as biomarkers to predict the prognosis of cervical cancer

BackgroundFerroptosis can be used as a powerful predictor of cancer prognosis. HPV persistent infection is the main cause of cervical cancer, so it is very important to improve the prognosis of patients. Therefore, it is necessary to explore the value of HPV-ferroptosis related genes as prognostic biomarkers of cervical cancer patients.MethodsIn this study, differentially expressed HPV-ferroptosis related genes were obtained from GSE7410, HPV gene set crossed with iron death genes. Five HPV-ferroptosis related genes with prognostic features were finally identified: CYBB, VEGFA, CKB, EFNA1 and HELLS. Multifactorial Cox regression was applied to establish and validate the prognostic model, and drug susceptibility and immune infiltration analyses were also performed.ResultsThe prognostic model was validated in the training set (TCGA) and validation set (GSE44001). Kaplan–Meier curves reveal significant differences in overall survival (OS) between high-risk and low-risk groups. Receiver operating characteristic (ROC) curve reflects the stability and accuracy of the prognostic model established in this study. In terms of immune function, T cell costimulation was better in the low-risk group than in the high-risk group (P < 0.01). The therapeutic effects of cisplatin, paclitaxel, docetaxel and cyclophosphamide, commonly used chemotherapy drugs for cervical cancer, are better in the high-risk group than in the low-risk group (P < 0.001).ConclusionHPV-ferroptosis related gene prognostic model not only has good stability and accuracy in predicting the prognosis of cervical cancer patients, but also has certain guiding value for clinicians in terms of drug sensitivity and immune microenvironment.

Age and gender in drug resistance tuberculosis: A cross-sectional case study at a national tuberculosis reference hospital in Nigeria

ObjectivesDrug resistance in tuberculosis (TB) is a very important public health threat that should not be ignored. Understanding the gender, age, and characteristics of individuals affected by TB (without HIV, diabetes, or hepatitis B complications), particularly in terms of drug resistance or susceptibility, is crucial for effective prevention and management strategies, as most studies focus on TB/HIV co-infection. MethodsA cross-sectional case study of age and gender was carried out in 140 individuals grouped into drug-resistant tuberculosis (DR-TB), drug-susceptible tuberculosis (DS-TB), and apparently healthy controls (AHCs). Data collection was through medical records and a structured questionnaire. Statistical analyses compared age, gender, and selected risk factors across the groups. ResultsThe mean age of the DR-TB group was 32 years (SD ≈ 2). A total of 80.0% were ≤40 years of age and four times more likely to have DR-TB; 55% were male, with 1.22 times more likelihood of DR-TB in males. The mean age of the DS-TB group was 34 years (SD ≈ 12); 66.7% were ≤40 years old. The odds ratio of DS-TB in males was 2.16. Only 10% of DR-TB enrolees had BCG scars compared with 65% AHCs. A high percentage of the DR-TB group reported handling raw meat (75%) and drinking unpasteurised milk (70%) compared with the DS-TB group. ConclusionsThe observed gender disparities and age-related factors, particularly among the DR-TB group, highlight the importance of considering age and gender factors in DR-TB prevention, diagnosis, and treatment. Our findings also highlight the need to bridge gaps in awareness as well as for the prevention of zoonotic TB and issues around effective BCG vaccination and coverage.

Comparative Evaluation of the Antibiotic Resistance Profile of Staphylococcus aureus Isolated From Breeders and Livestock.

Animals are a potential source of Methicillin Resistant Staphylococcus aureus. This study evaluated the antibiotics susceptibility pattern of S. aureus isolates from breeders and livestock. S. aureus strains were isolated from 180 livestock and 48 livestock farmers and identified using standard methods. Antibiotic susceptibility profiles and MRSA status were determined via disk diffusion susceptibility method. Among farm workers, 37.5% were colonized by S. aureus, with pig farm workers exhibiting the highest prevalence (56.2%), cattle herders (37.5%), and goat farm workers (18.7%). MRSA carriage among livestock isolates was 41.3%, while, six isolates from the poultry farm worker were MRSA, representing a carriage of 33.3%. Drug susceptibility profiles revealed differential patterns between isolates from breeders and animals. Gentamicin and levofloxacin demonstrated higher efficacy against farm worker isolates compared to animal isolates. Resistance to cefuroxime was higher among animal isolates (84.1%) as against the 66.7% for the breeders. The identification of multidrug-resistant S. aureus strains underscores the risk posed to humans in contact with animals. These findings stress the importance of monitoring and managing MRSA transmission between animals and humans.

Optimizing Antibiotic Treatment for Urinary Tract Infections Secondary to Escherichia coli in Elderly Diabetic Patients: Considering Age and Blood Glucose Control

Introduction: This study aimed to examine the susceptibility profile of Escherichia coli in urinary tract infections (UTIs) among elderly diabetic patients to support judicious and evidence-based antibiotic use. Methods: From January 2021 to December 2022, urine culture results were analyzed to determine the distribution of pathogens, especially E. coli, and their drug susceptibility. Results: E. coli infection was the most prevalent infection in elderly diabetic patients with UTIs, accounting for 32.6% of cases. Moreover, this bacterium’s multiple resistance rate (38.3%) was significantly higher than other bacteria’s multiple resistance rate (χ2 = 81.644, p < 0.05). Compared to older diabetic patients with optimal glucose control (HbA1c ≤7.0%), patients with poor glycemic control (HbA1c >7.0%) had lower resistance rates to lactams, and urine pH values were higher (p < 0.05). Conclusion: The most common cause of UTIs is E. coli, with advanced age and diabetes being the main risk factors. To optimize UTI treatment safety and efficacy, antibiotics should be administered based on the patient’s age and blood glucose control.

The catalogue of Mycobacterium tuberculosis mutations associated with drug resistance to 12 drugs in China from a nationwide survey: a genomic analysis

WHO issued the first edition catalogue of Mycobacterium tuberculosis complex (MTBC) mutations associated with drug resistance in 2021. However, country-specific issues might lead to arising complex and additional drug-resistant mutations. We aimed to fully reflect the characteristics of drug resistance mutations in China. We analysed MTBC isolates from the nationwide drug-resistant tuberculosis surveillance with 70counties in 31provinces, municipalities, and autonomous regions in China. Three types of MYCOTB plates were used to perform drug susceptibility testing for 12antibiotics (rifampicin, isoniazid, ethambutol, levofloxacin, moxifloxacin, amikacin, kanamycin, ethionamide, clofazimine, linezolid, delamanid, and bedaquiline). Mutations were divided into five groups according to their odds ratios, positive predictive values, false discovery rate-corrected p values, and 95% CIs: (1) associated with resistance; (2) associated with resistance-interim; (3) uncertain significance; (4) not associated with resistance-interim; and (5) not associated with resistance. The Wilcoxon rank-sum and Kruskal-Wallis tests were used to quantify the association between mutations and minimum inhibitory concentrations (MICs). Our dataset was compared with the first edition of the WHO catalogue. We analysed 10 146MTBC isolates, of which 9071 (89·4%) isolates were included in the final analysis. 744(8·2%) isolates were resistant to rifampicin and 1339 (14·8%) to isoniazid. 208 (1·9%) of 11 065mutations were classified as associated with resistance or associated with resistance-interim. 33 (97·1%) of 34mutations in group 1and 92 (52·9%) of 174in group 2also appeared in groups 1or 2of the WHO catalogue. Of 81indel mutations in group 2, 15 (18·5%) were in the WHO catalogue. The newly discovered mutation gyrA_Ala288Asp was associated with levofloxacin resistance. MIC values for rifampicin, isoniazid, moxifloxacin, and levofloxacin corresponding to resistance mutations in group 1were significantly different (p<0·0001), and 12 high-level resistance mutations were detected. 61mutations in group 3occurred as solo in at least five phenotypically susceptible isolates, but with MIC values moderately higher than other susceptible isolates. Among 945phenotypically resistant but genotypically susceptible isolates, 433 (45·8%) were mutated for at least one efflux pump gene. Our analysis reflects the complexity of drug resistance mutations in China and suggests that indel mutations, efflux pump genes, protein structure, and MICs should be fully considered in the WHO catalogue, especially in countries with a high tuberculosis burden. National Key Research and Development Program of China and the Science and Technology Major Project of Tibetan Autonomous Region of China.

A Comparison of Sanger Sequencing and Amplicon-Based Next Generation Sequencing Approaches for the Detection of HIV-1 Drug Resistance Mutations.

Next-generation sequencing (NGS) kits are needed to finalise the transition from Sanger sequencing to NGS in HIV-1 genotypic drug resistance testing. We compared a homemade NGS amplicon-based protocol and the AD4SEQ HIV-1 Solution v2 (AD4SEQ) NGS kit from Arrow Diagnostics for identifying resistance-associated mutations (RAMs) above the 5% threshold in 28 plasma samples where Sanger sequencing previously detected at least one RAM. The samples had a median 4.8 log [IQR 4.4-5.2] HIV-1 RNA copies/mL and were mostly subtype B (61%) and CRF02_AG (14%). Homemade NGS had a lower rate of samples with low-coverage regions (2/28) compared with AD4SEQ (13/28) (p < 0.001). Homemade NGS and AD4SEQ identified additional mutations with respect to Sanger sequencing in 13/28 and 9/28 samples, respectively. However, there were two and eight cases where mutations detected by Sanger sequencing were missed by homemade NGS and AD4SEQ-SmartVir, respectively. The discrepancies between NGS and Sanger sequencing resulted in a few minor differences in drug susceptibility interpretation, mostly for NNRTIs. Both the NGS systems identified additional mutations with respect to Sanger sequencing, and the agreement between them was fair. However, AD4SEQ should benefit from technical adjustments allowing higher sequence coverage.

Prevalence and Occurrence of Type 1 Fimbriae in Klebsiella pneumoniae

Background: Klebsiella pneumoniae causes both community acquired and nosocomial infections. The various virulence factors have been well characterised in K. pneumoniae includes: capsule, lipopolysaccharides, siderophores and fimbriae. FimH 1, encoding for fimbriae and mediate adhesion. Aim and objective: The aim of the study was to determine drug susceptibility and the prevalence of FimH 1 genes among clinical isolates of K. pneumoniae. Materials and methods: A total of 200 isolates collected over a period of one year, were included in this study. The source of the isolates were urine (n=74), respiratory (n=73), exudates (n=50) and blood (n=3). For all the isolates antimicrobial susceptibility testing by disc diffusion was done. Polymerase chain reaction was performed for the detection of FimH 1 gene. Results: The susceptibility of the study isolates to different classes of antimicrobial agent was: meropenem (75%), amikacin (69%), piperacillin/tazobactam (67.5%), ciprofloxacin (59%), and cefotaxime (53.5%). FimH-1 gene was detected in 55% of the total isolates. Conclusion: FimH 1 was not a major mediator associated with adherence in this study. Detection of virulence gene such as type 1 fimbriae will help to understand their occurrence in different strains of K. pneumoniae and how they function in different host environments. Most of the isolates were resistant to third generation of cephalosporins. Knowing the prevalence of antimicrobial resistance helps to formulate infection control practices and formulating antimicrobial therapy. Keywords: Klebsiella pneumoniae, virulence gene, fimbrial adhesin, type 1 fimbriae, antimicrobial susceptibility, disc diffusion.

Construction and validation of a TAMRGs prognostic signature for gliomas by integrated analysis of scRNA and bulk RNA sequencing data

BackgroundThis study aimed to construct and validate a prognostic model based on tumor associated macrophage-related genes (TAMRGs) by integrating single-cell RNA sequencing (scRNA-seq) and bulk RNA sequencing (bulk RNA-seq) data. MethodsThe scRNA-seq data of three inhouse glioma tissues were used to identify the tumor-associated macrophages (TAMs) marker genes, the DEGs from the The Cancer Genome Atlas (TCGA) − Genotype-Tissue Expression (GTEx) dataset were used to further select TAMs marker genes. Subsequently, a TAMRG-score was constructed by Least absolute shrinkage and selection operator (LASSO) regression and multivariate Cox regression analysis in the TCGA dataset and validated in the Chinese Glioma Genome Atlas (CGGA) dataset. ResultsWe identified 186 TAMs marker genes, and a total of 6 optimal prognostic genes including CKS2, LITAF, CTSB, TWISTNB, PPIF and G0S2 were selected to construct a TAMRG-score. The high TAMRG-score was significantly associated with worse prognosis (log-rank test, P<0.001). Moreover, the TAMRG-score outperformed the other three models with AUC of 0.808. Immune cell infiltration, TME scores, immune checkpoints, TMB and drug susceptibility were significantly different between TAMRG-score groups. In addition, a nomogram were constructed by combing the TAMRG-score and clinical information (Age, Grade, IDH mutation and 1p19q codeletion) to predict the survival of glioma patients with AUC of 0.909 for 1-year survival. ConclusionThe high TAMRG-score group was associated with a poor prognosis. A nomogram by incorporating TMARG-score could precisely predict glioma survival, and provide evidence for personalized treatment of glioma.

Holistic understanding of trimethoprim resistance in Streptococcus pneumoniae using an integrative approach of genome-wide association study, resistance reconstruction, and machine learning.

In the age of next-generation sequencing (NGS), while data-driven methods such as genome-wide association study (GWAS) and machine learning (ML) excel at finding patterns, functional validation can be challenging due to the high numbers of candidate variants. We designed an integrative approach combining a GWAS on S. pneumoniae clinical isolates, followed by whole-genome transformation coupled with NGS to functionally characterize a large set of GWAS candidates. Our study validated several phenotypic folA mutations beyond the standard Ile100Leu mutation, and showed that the overexpression of the sulA locus produces trimethoprim (TMP) resistance in Streptococcus pneumoniae. These validated loci, when used to build ML models, were found to be the best inputs for predicting TMP minimal inhibitory concentrations. Integrative approaches can bridge the genotype-phenotype gap by biological insights that can be incorporated in ML models for accurate prediction of drug susceptibility.

Evaluation of mycobacterial microscopy and culture results from clinical samples: A five-year analysis.

Ehrlich-Ziehl-Neelsen (EZN) staining and culture methods are often used to diagnose tuberculosis. This study aimed to determine the acidfast bacteria (AFS) positivity rates in various clinical samples sent to our laboratory over five years and the growth and resistance rates in two different (solid and liquid) cultures and compare them with the data from Türkiye and the world. A total of 62.456 clinic samples were accepted in the microbiology laboratory between 2019 and 2024. The mycobacterial culture was performed by searching for acid-resistant bacilli microscopically and parallel inoculation media [solid Löwenstein-Jensen (L-J) and MGIT 960 liquid]. Those growing in the MGIT 960 system were identified using BD MGIT TBC Identification test kits that detect the MPT64 antigen. AFS and MPT64 antigenpositive samples were identified as Mycobacterium tuberculosis complex (MTBC) while AFS-positive samples and MPT64 antigen-negative results were classified as non-tuberculous mycobacteria (NTM). Drug susceptibility testing was performed with the BACTEC MGIT 960 SIRE kit. Susceptibility to NTM samples was not performed. Out of a total of 120.829 samples, 95.101 were lung samples and 25.728 were extrapulmonary samples. AFS positivity was detected in 2961 (2.4%) samples. MTBC grew in 6854 (5.6%) samples, and NTM grew in 1506 (1.24%) samples. Contamination was detected in 7171 (5.9%) media. Two thousand one hundred and sixty-nine susceptibility tests were performed. Considering antibiotic resistance rates, isoniazid resistance was detected in 154 (7%), rifampicin resistance in 140 (6.4%), ethambutol resistance in 18 (0.8%), and streptomycin resistance in 120 (0.5%) samples. All four-drug resistance was observed in 91 (4.1%) samples. AFP positivity and resistance rates for rifampicin have decreased significantly, while there have been no significant changes in NTM rates over the years. When our data was determined, the sensitivity of microscopy was low. It is understood that mycobacterial culture and microscopy must be evaluated together to exclude tuberculosis infection. The high mycobacterial culture positivity rate, which is 5.6%, is due to the high number of follow-up patients and new referrals. It is seen that the change in sensitivity rates is due to the period of the COVID-19 epidemic, and it is similar to World Health Organization (WHO) data.

Comparison of targeted next-generation sequencing and traditional microbial culture in the diagnosis of pulmonary infections

This study investigated the diagnostic potential of targeted next-generation sequencing (tNGS) for pulmonary infections. The positivity rate of tNGS was significantly higher than that of traditional microbial culture (92.6 % vs 25.2 %, χ2 = 378.272, P < 0.001). The proportion of two or more species of pathogens detected using tNGS exceeded that detected using microbial culture (χ2 = 337.283, P < 0.001). There were inconsistencies between the results of the tNGS antibiotic resistance gene and the drug susceptibility test resistance phenotype. The tNGS technique demonstrates rapid and effective capabilities in identifying bacteria, fungi, viruses, and specific pathogens, with a detection sensitivity that surpasses that of conventional culture methodologies. Microbial drug resistance genotypes detected by tNGS cannot accurately predict drug resistance phenotypes and require further improvement or integration with traditional microbial culture to establish a foundation for effective clinical treatment.

Diagnostic accuracy of LiquidArray MTB-XDR VER1.0 for the detection of Mycobacterium tuberculosis complex, fluoroquinolone, amikacin, ethambutol, and linezolid susceptibility.

Drug susceptibility testing (DST) is essential for effectively starting people on effective tuberculosis (TB) regimens. No accuracy data exists for the new high-throughput LiquidArray MTB-XDR (LA-XDR) test, which detects Mycobacterium tuberculosis complex (MTBC) and susceptibility to the fluoroquinolones, amikacin, ethambutol, and linezolid (the latter two drugs have no rapid molecular DSTs available). We enrolled (n=720) people with presumptive TB who provided two sputa for Xpert MTB/RIF Ultra and culture (MTBC reference standard). Phenotypic DST and Sanger sequencing served as a composite reference standard. Manual FluoroLyse and automated GenoXtract-fleXT (fleXT) DNA extraction methods were compared. For MTBC, LA-XDR using fleXT-extracted or FluoroLyse-extracted DNA had similar sensitivities (85-87%; which improved upon eluate retesting) and specificities (99%). Drug susceptibility sensitivities varied: 94% (86, 98) for fluoroquinolones, 64% (45, 80) for amikacin, and 88% (79, 93) for ethambutol (specificities 97-100%). LA-XDR detected 86% (6/7) phenotypically resistant linezolid isolates. LA-XDR with fleXT had indeterminate proportions of 8% (21/251) for fluoroquinolones, 1% (2/251) for ethambutol, 25% (63/251) for amikacin, and 37% (93/251) for linezolid. In a hypothetical population of 100 smear-negative fluoroquinolones-resistant cases, 24% (24/100) could be missed due to an unsuccessful result (1 fleXT error and, for LA-XDR, 2 invalid results, 15 MTBC-negative, 6 fluoroquinolone-indeterminate, 1 false-susceptible). LA-XDR met the minimum WHO target product profile for a next-generation sputum-based moderate complexity DST with high sensitivity for fluoroquinolones and ethambutol resistance, moderate sensitivity for amikacin resistance, and promise for linezolid resistance, for which more data are needed. Improved MTBC detection would reduce missed resistance.

Ex vivo susceptibilities to ganaplacide and diversity in potential resistance mediators in Ugandan Plasmodium falciparum isolates.

Novel antimalarials are urgently needed to combat rising resistance to available drugs. The imidazolopiperazine ganaplacide is a promising drug candidate, but decreased susceptibility of laboratory strains has been linked to polymorphisms in the Plasmodium falciparum cyclic amine resistance locus (PfCARL), acetyl-CoA transporter (PfACT), and UDP-galactose transporter (PfUGT). To characterize parasites causing disease in Africa, we assessed ex vivo drug susceptibilities to ganaplacide in 750 P. falciparum isolates collected in Uganda from 2017 to 2023. Drug susceptibilities were assessed using a 72-hour SYBR Green growth inhibition assay. The median IC50 for ganaplacide was 13.8 nM, but some isolates had up to 31-fold higher IC50s (31/750 with IC50 > 100 nM). To assess genotype-phenotype associations, we sequenced genes potentially mediating altered ganaplacide susceptibility in the isolates using molecular inversion probe and dideoxy sequencing methods. PfCARL was highly polymorphic, with eight mutations present in >5% of isolates. None of these eight mutations had previously been selected in laboratory strains with in vitro drug pressure and none were found to be significantly associated with decreased ganaplacide susceptibility. Mutations in PfACT and PfUGT were found in ≤5% of isolates, except for two frequent (>20%) mutations in PfACT; one mutation in PfACT (I140V) was associated with a modest decrease in susceptibility. Overall, Ugandan P. falciparum isolates were mostly highly susceptible to ganaplacide. Known resistance mediators were polymorphic, but mutations previously selected with in vitro drug pressure were not seen, and mutations identified in the Ugandan isolates were generally not associated with decreased ganaplacide susceptibility.