Early Detection Of Drug Resistance Research Articles

Targeted sequencing from cerebrospinal fluid for rapid identification of drug-resistant tuberculous meningitis.

Mortality from tuberculous meningitis (TBM) remains around 30%, with most deaths occurring within 2 months of starting treatment. Mortality from drug-resistant strains is higher still, making early detection of drug resistance (DR) essential. Targeted next-generation sequencing (tNGS) produces high read depths, allowing the detection of DR-associated alleles with low frequencies. We applied Deeplex Myc-TB-a tNGS assay-to cerebrospinal fluid (CSF) samples from 72 adults with microbiologically confirmed TBM and compared its genomic drug susceptibility predictions to a composite reference standard of phenotypic susceptibility testing (pDST) and whole genome sequencing, as well as to clinical outcomes. Deeplex detected Mycobacterium tuberculosis complex DNA in 24/72 (33.3%) CSF samples and generated full DR reports for 22/24 (91.7%). The read depth generated by Deeplex correlated with semi-quantitative results from MTB/RIF Xpert. Alleles with <20% frequency were seen at canonical loci associated with first-line DR. Disregarding these low-frequency alleles, Deeplex had 100% concordance with the composite reference standard for all drugs except pyrazinamide and streptomycin. Three patients had positive CSF cultures after 30 days of treatment; reference tests and Deeplex identified isoniazid resistance in two, and Deeplex alone identified low-frequency rifampin resistance alleles in one. Five patients died, of whom one had pDST-identified pyrazinamide resistance. tNGS on CSF can rapidly and accurately detect drug-resistant TBM, but its application is limited to those with higher bacterial loads. In those with lower bacterial burdens, alternative approaches need to be developed for both diagnosis and resistance detection.

Strengthening the Diagnosis of Drug-Resistant Tuberculosis Using NGS-Based Approaches and Bioinformatics Pipelines for Data Analysis in India.

In India, drug-resistant tuberculosis (DR-TB) is a major public health issue and a significant challenge to stop TB program. An estimated 27% of new TB cases and 44% of previously treated TB cases are resistant to at least one anti-TB drug. The conventional methods for DR-TB diagnosis are time-consuming and have limitations, leading to delays in treatment initiation and the spread of the disease. Next-generation sequencing (NGS) based approaches have emerged as a promising tool for diagnosing DR-TB, simultaneously offering rapid and accurate detection of resistance mutations in multiple genes. NGS-based approaches generate a large amount of data, which requires efficient and reliable bioinformatics pipelines for data analysis. TBProfiler and Mykrobe are the bioinformatics pipelines that have been created to analyze NGS data for the diagnosis of DR-TB. These pipelines use reference-based and machine-learning approaches to detect resistance mutations and predict drug susceptibility, enabling clinicians to make informed treatment decisions. Implementing NGS-based approaches and bioinformatics pipelines for DR-TB diagnosis can potentially improve patient outcomes by facilitating early detection of drug resistance and guiding personalized treatment regimens. However, the widespread adoption of these approaches in India faces several challenges, including high costs, limited infrastructure, and a lack of trained personnel. Addressing these challenges requires concerted effort to ensure equitable access to and effective implementation of these innovative technologies.

A risk assessment framework for multidrug-resistant Staphylococcus aureus using machine learning and mass spectrometry technology.

The emergence of multidrug-resistant bacteria is a critical global crisis that poses a serious threat to public health, particularly with the rise of multidrug-resistant Staphylococcus aureus. Accurate assessment of drug resistance is essential for appropriate treatment and prevention of transmission of these deadly pathogens. Early detection of drug resistance in patients is critical for providing timely treatment and reducing the spread of multidrug-resistant bacteria. This study aims to develop a novel risk assessment framework for S. aureus that can accurately determine the resistance to multiple antibiotics. The comprehensive 7-year study involved ˃20000 isolates with susceptibility testing profiles of six antibiotics. By incorporating mass spectrometry and machine learning, the study was able to predict the susceptibility to four different antibiotics with high accuracy. To validate the accuracy of our models, we externally tested on an independent cohort and achieved impressive results with an area under the receiver operating characteristic curve of 0. 94, 0.90, 0.86 and 0.91, and an area under the precision-recall curve of 0.93, 0.87, 0.87 and 0.81, respectively, for oxacillin, clindamycin, erythromycin and trimethoprim-sulfamethoxazole. In addition, the framework evaluated the level of multidrug resistance of the isolates by using the predicted drug resistance probabilities, interpreting them in the context of a multidrug resistance risk score and analyzing the performance contribution of different sample groups. The results of this study provide an efficient method for early antibiotic decision-making and a better understanding of the multidrug resistance risk of S. aureus.

Optimizing Antiviral Dosing for HSV and CMV Treatment in Immunocompromised Patients.

Herpes simplex virus (HSV) and cytomegalovirus (CMV) are DNA viruses that are common among humans. Severely immunocompromised patients are at increased risk of developing HSV or CMV disease due to a weakened immune system. Antiviral therapy can be challenging because these drugs have a narrow therapeutic window and show significant pharmacokinetic variability. Above that, immunocompromised patients have various comorbidities like impaired renal function and are exposed to polypharmacy. This scoping review discusses the current pharmacokinetic (PK) and pharmacodynamic (PD) knowledge of antiviral drugs for HSV and CMV treatment in immunocompromised patients. HSV and CMV treatment guidelines are discussed, and multiple treatment interventions are proposed: early detection of drug resistance; optimization of dose to target concentration by therapeutic drug monitoring (TDM) of nucleoside analogs; the introduction of new antiviral drugs; alternation between compounds with different toxicity profiles; and combinations of synergistic antiviral drugs. This research will also serve as guidance for future research, which should focus on prospective evaluation of the benefit of each of these interventions in randomized controlled trials.

Therapeutic efficacies of artemether-lumefantrine and dihydroartemisinin-piperaquine for the treatment of uncomplicated Plasmodium falciparum and chloroquine and dihydroartemisinin-piperaquine for uncomplicated Plasmodium vivax infection in Ethiopia

BackgroundRoutine monitoring of anti-malarial drugs is recommended for early detection of drug resistance and to inform national malaria treatment guidelines. In Ethiopia, the national treatment guidelines employ a species-specific approach. Artemether-lumefantrine (AL) and chloroquine (CQ) are the first-line schizonticidal treatments for Plasmodium falciparum and Plasmodium vivax, respectively. The National Malaria Control and Elimination Programme in Ethiopia is considering dihydroartemisinin-piperaquine (DHA/PPQ) as an alternative regimen for P. falciparum and P. vivax.MethodsThe study assessed the clinical and parasitological efficacy of AL, CQ, and DHA/PPQ in four arms. Patients over 6 months and less than 18 years of age with uncomplicated malaria mono-infection were recruited and allocated to AL against P. falciparum and CQ against P. vivax. Patients 18 years or older with uncomplicated malaria mono-infection were recruited and randomized to AL or dihydroartemisinin-piperaquine (DHA/PPQ) against P. falciparum and CQ or DHA/PPQ for P. vivax. Patients were followed up for 28 (for CQ and AL) or 42 days (for DHA/PPQ) according to the WHO recommendations. Polymerase chain reaction (PCR)-corrected and uncorrected estimates were analysed by Kaplan Meier survival analysis and per protocol methods.ResultsA total of 379 patients were enroled in four arms (n = 106, AL-P. falciparum; n = 75, DHA/PPQ- P. falciparum; n = 142, CQ-P. vivax; n = 56, DHA/PPQ-P. vivax). High PCR-corrected adequate clinical and parasitological response (ACPR) rates were observed at the primary end points of 28 days for AL and CQ and 42 days for DHA/PPQ. ACPR rates were 100% in AL-Pf (95% CI: 96–100), 98% in CQ-P. vivax (95% CI: 95–100) at 28 days, and 100% in the DHA/PPQ arms for both P. falciparum and P. vivax at 42 days. For secondary endpoints, by day three 99% of AL-P. falciparum patients (n = 101) cleared parasites and 100% were afebrile. For all other arms, 100% of patients cleared parasites and were afebrile by day three. No serious adverse events were reported.ConclusionThis study demonstrated high therapeutic efficacy for the anti-malarial drugs currently used by the malaria control programme in Ethiopia and provides information on the efficacy of DHA/PPQ for the treatment of P. falciparum and P. vivax as an alternative option.

Differentiating between drug-sensitive and drug-resistant tuberculosis with machine learning for clinical and radiological features

Tuberculosis (TB) drug resistance is a worldwide public health problem that threatens progress made in TB care and control. Early detection of drug resistance is important for disease control, with discrimination between drug-resistant TB (DR-TB) and drug-sensitive TB (DS-TB) still being an open problem. The objective of this work is to investigate the relevance of readily available clinical data and data derived from chest X-rays (CXRs) in DR-TB prediction and to investigate the possibility of applying machine learning techniques to selected clinical and radiological features for discrimination between DR-TB and DS-TB. We hypothesize that the number of sextants affected by abnormalities such as nodule, cavity, collapse and infiltrate may serve as a radiological feature for DR-TB identification, and that both clinical and radiological features are important factors for machine classification of DR-TB and DS-TB. We use data from the NIAID TB Portals program (https://tbportals.niaid.nih.gov), 1,455 DR-TB cases and 782 DS-TB cases from 11 countries. We first select three clinical features and 26 radiological features from the dataset. Then, we perform Pearson's chi-squared test to analyze the significance of the selected clinical and radiological features. Finally, we train machine classifiers based on different features and evaluate their ability to differentiate between DR-TB and DS-TB. Pearson's chi-squared test shows that two clinical features and 23 radiological features are statistically significant regarding DR-TB vs. DS-TB. A ten-fold cross-validation using a support vector machine shows that automatic discrimination between DR-TB and DS-TB achieves an average accuracy of 72.34% and an average AUC value of 78.42%, when combing all 25 statistically significant features. Our study suggests that the number of affected lung sextants can be used for predicting DR-TB, and that automatic discrimination between DR-TB and DS-TB is possible, with a combination of clinical features and radiological features providing the best performance.

Socio-Demographic and Clinical Profile of Drug Resistant Tuberculosis Patients in a Tertiary Care Centre of Kolkata

Background: Drug resistant tuberculosis is a major public health threat that hinders progress in tuberculosis control worldwide. In 2019, India contributed highest cases of TB (27%) Worldwide. In 2019, 3.3% of new TB cases and 18% of previously treated cases had MDR/RRTB worldwide and India (27%) had the largest share of the global burden. This study was done to find out the sociodemographic and clinical profile of drug resistant TB patients. Methodology: It is an observational descriptive study with cross-sectional design, conducted at the drug resistant tuberculosis centre of R. G. Kar Medical College of Kolkata, West Bengal. Study Population consisted of the patients who were registered for the DR-TB regimen. Study duration was July 2016 –March 2017. Pre-tested, semi-structured schedule was designed to capture their sociodemographic profile, treatment history, clinical findings and available medical records. Data were compiled in MS Excel and analyzed in IBM SPSS 23.0. Results: Out of 159 cases, 27% patients were between 21-30 years. Males were predominant (68.6%). Most patients (56.6%) were underweight (BMI&lt;18.5kg/m2). HIV seropositivity was found in 4 (2.5%) cases. Among all cases, 81.1% had history of taking ATD and 34.4% was cured in previous episode of treatment. The commonest associated comorbidity was DM (15.7%). Rifampicin resistance (93.1%) was most common followed by Isoniazid (8.2%). Pallor was found among 94.3% patients. Most patients had bilateral (62.3%) and moderately extensive (57.2%) lesions in chest x-ray. Conclusion: DR-TB control should focus adequately on younger age group as numbers of resistance is increasing among them. Relapse in previously cured cases was found to be major contributor of DR-TB suspect cases. Though, Rifampicin resistance was so common but prevalence of resistance to 2nd line drugs is still low. Focus should be given on early detection of drug resistance in all TB cases and improvement of nutritional status of the TB patient.

Early detection of drug-resistant Streptococcus pneumoniae and Haemophilus influenzae by quantitative flow cytometry

Early detection of drug resistance contributes to combating drug-resistant bacteria and improving patient outcomes. Microbial testing in the laboratory is essential for treating infectious diseases because it can provide critical information related to identifying pathogenic bacteria and their resistance profiles. Despite these clinical requirements, conventional phenotypic testing is time-consuming. Additionally, recent rapid drug resistance tests are not compatible with fastidious bacteria such as Streptococcus and Haemophilus species. In this study, we validated the feasibility of direct bacteria counting using highly sensitive quantitative flow cytometry. Furthermore, by combining flow cytometry and a nucleic acid intercalator, we constructed a highly sensitive method for counting viable fastidious bacteria. These are inherently difficult to measure due to interfering substances from nutrients contained in the medium. Based on the conventional broth microdilution method, our method acquired a few microliter samples in a time series from the same microplate well to exclude the growth curve inconsistency between the samples. Fluorescent staining and flow cytometry measurements were completed within 10 min. Therefore, this approach enabled us to determine antimicrobial resistance for these bacteria within a few hours. Highly sensitive quantitative flow cytometry presents a novel avenue for conducting rapid antimicrobial susceptibility tests.

MfloDx® - an innovative technology for MDR-TB antibiotic resistance profiling

Aims &amp; objectives: The aim of this work was to evaluate the performance of mfloDx® MDR-TB assay kit on DNA extracted from 100 pulmonary sputum samples. mfloDx® MDR-TB is a qualitative test that can identify bacteria belonging to the Mycobacterium tuberculosis complex (MTC) as well as robustly identify single-nucleotide variants of the most common mutations in multidrug resistant M. tuberculosis DNA. The current assay can detect the most prevalent mutations causing resistance to Rifampicin (RIF) and Isoniazid (INH). Methods: Genes of interest were selectively enriched and targeted using amplifiable padlock probes (PLP). PLPs that recognize mutation or wild type were ligated upon specific recognition of the correct target sequence. Once PLPs were circularized, they underwent rolling circle amplification. Polymers resulting from this amplification were monomerized and subsequently visualized on disposable cassettes. The developed bands allowed for accurate genotyping of eleven resistance-related mutations, as well as respective wild type alleles and MTC identification. Results: From 100 samples; 81 were successfully genotyped, 16 were negative for MTC, two were inconclusive and one ran out during analysis. Among the genotyped samples; 46 were resistant to both RIF and INH, eight samples were resistant only to INH, three samples were resistant only to RIF. The remaining samples were susceptible to both drugs. Clinical sensitivity and specificity were calculated based on phenotypic drug susceptibility test results (MGIT culture) as reference. Sensitivity was 89.6% for RIF and 92% for INH resistance. Specificity was 95.5% for RIF and 89.5% for INH susceptibility. Conclusions: In conclusion, mfloDx® MDR-TB offers a reliable and affordable rapid drug-susceptibility testing for MDR-TB with potential implications in early detection of drug-resistance in resource-limited, high burden TB and MDR-TB settings. It enables clinicians to ensure the timely administration of effective drug therapy which will not only reduce the risk of treatment failure but also limit the further development and spread of dug resistant TB.

Detection of Antibiotic-Resistance by MALDI-TOF Mass Spectrometry: An Expanding Area.

Several MALDI-TOF MS-based methods have been proposed for rapid detection of antimicrobial resistance. The most widely studied methods include assessment of β-lactamase activity by visualizing the hydrolysis of the β-lactam ring, detection of biomarkers responsible for or correlated with drug-resistance/non-susceptibility, and the comparison of proteomic profiles of bacteria incubated with or without antimicrobial drugs. Antimicrobial-resistance to a number of antibiotics belonging to different classes has been successfully tested by MALDI-TOF MS in a variety of clinically relevant bacterial species including members of Enterobacteriaceae family, non-fermenting Gram-negative bacteria, Gram-positive cocci, anaerobic bacteria and mycobacteria, opening this field to further clinically important developments. Early detection of drug-resistance by MALDI-TOF MS can be particularly helpful for clinicians to streamline the antibiotic therapy for a better outcome of patients with systemic infection, in all cases where a prompt and effective antibiotic treatment is essential to preserve organ function and/or patient survival.

Rapid and Simple Detection of Isoniazid-Resistant Mycobacterium tuberculosis Utilizing a DNA Chromatography-Based Technique.

Despite the availability of anti-tuberculosis drugs, the treatment of tuberculosis has been complicated by drug-resistant tuberculosis. The early detection of drug resistance makes early treatment possible. However, the available tools are mainly for rifampicin resistance detection, and the existing isoniazid resistance detection method is expensive, highly technical, and complicated, making it unsustainable for use in developing nations. This study aimed to develop a simple, rapid, and low-cost diagnostic kit for isoniazid-resistant tuberculosis using the single-stranded tag hybridization method to target an isoniazid resistance-conferring mutation. Specificity and sensitivity were assessed using DNA extracted from 49 isoniazid-resistant and 41 isoniazid-susceptible Mycobacterium tuberculosis clinical isolates cultured in mycobacterial growth indicator tubes. Positive signals were observed on mutant and wild-type lines with 100% sensitivity and specificity compared with Sanger sequencing results. In contrast, no positive signal was observed for non-tuberculosis mycobacteria. The detection limit of this method was 103 CFU or less. The STH-PAS system for isoniazid-resistant M. tuberculosis detection developed in this study offers a better alternative to conventional phenotypic isoniazid resistance determination, which will be of both clinical and epidemiological significance in resource-limited nations.

Predictive utility of fluorescent microscopy for detection of drug resistance in patient of pulmonary tuberculosis

Tuberculosis (TB) is a global health problem with 480000 new cases being reported annually, 9 percent of which exhibit extensive drug resistant TB. With 9.6 cases per lac population early detection of drug resistance at peripheral levels is must. The study was planned to use Fluorescent microscopy (FM) with Fluorescein Diacetate Ethidium Bromide (FDA/EB) stain to evaluate the viability of the mycobacteria to predict the drug resistance. This cross-sectional analytical study was carried out on all sputum smear positive patients after obtaining the Institutional Ethics Committee clearance over a period of 3 months. Samples were obtained on day 0, 3, 7 and 14. After staining with the working solution, observation was made by two independent observers for viable verses non-viable mycobacteria. FDA will stain the live bacilli and fluoresce green whereas dead bacilli lack the esterase activity and are counterstained by ethidium bromide and appear red. Data was maintained in MS Excel and analysed using tests of proportion and significance. A total of 30 participants were included based on the inclusion criteria. There was a loss of follow up from 14 patients. 4/16 (25%) patients were found be harbor drug resistant mycobacteria strains bases on decreasing ratio of viable to non-viable bacteria on followup. Results when compared with gold standard of Line Probe Assay (LiPA) was found to be highly significant with Chi square value of 11.73 and p value &amp;#60;0.001This study establishes the utility of fluorescent microscopy using FDA and EB to detect drug resistance in patients of pulmonary tuberculosis by evaluation of viable and non-viable at peripheral microscopic centers Fluorescent Microscopy with FDA/EB staining holds a potential to predict drug resistance in sputum smear positive patients at peripheral designated microscopic centers which will reduce financial as well as human resource burden.

Tuberculosis, an old disease, still present in Romania

Tuberculosis is a chronic infection, most often affecting the lungs, which usually manifests after a latency period from primary infection with Mycobacterium tuberculosis. Symptoms are generally nonspecific, with fever, cough, weight loss and malaise. The diagnosis is based on microscopic examination of sputum smear and rapid diagnostic molecular tests, which are increasingly used today. Genotypic tests for establishing the strain involved and phenotypic antibiograms for early detection of drug resistance should guide the initiation of treatment but are still expensive. Treatment of active tuberculosis is done with combination of antimycobacterial drugs, administered for at least 6 months. The antituberculosis treatment has several purposes: to cure the patient, to reduce the risk of recurrence, to prevent the installation of chemo-resistance, to prevent complications and to reduce mortality, as well as to limit the spread of the infection. Drug combinations are used to prevent the development of resistance. The administration is long-lasting in order to achieve the sterilization of foci that are difficult to access by medicines, ensuring healing and relapse prevention. Generally, standard pharmacological protocols are used. In order to increase the adherence to the treatment and its completion, often the anti-tuberculosis treatment is done under direct observation, in what is called directly observed therapy. Undesirable effects of anti-tuberculosis drugs should be detected early and managed appropriately. Recently, many cases of tuberculosis are resistant to the first-line drugs isoniazid and rifampicin (multidrug-resistant tuberculosis), or to these drugs, fluoroquinolones and at least one injectable antimycobacterial drugs (extensively drug-resistant tuberculosis). Especially the treatment of the latter is difficult to do, because there are not currently too many therapeutic options. That is why it is important to detect the resistance early and to establish the appropriate treatment. Treatment of latent tuberculosis usually involves the administration of isoniazid for 9 months. BCG vaccination is an active immunization method used in countries with high incidence of tuberculosis (Romania being the country of the European Union with the highest incidence of tuberculosis), protecting mainly against miliary tuberculosis, a spread form of tuberculosis, severe especially in children.

Association Between the Phenotype and Genotype of Isoniazid Resistance Among Mycobacterium tuberculosis Isolates in Thailand.

PurposeThe emergence of isoniazid-resistant tuberculosis (HR-TB) is a global public health problem, causing treatment failure and high mortality rates. This study aimed to determine the minimal inhibitory concentration (MIC) of isoniazid and detect the gene mutation in HR-TB and any association between the level of isoniazid resistance and gene mutation.MethodsWe collected 74 clinical HR-TB isolates from two tertiary-care centers in Thailand. MICs were established using broth macrodilution. A line probe assay (LPA) was used to detect gene mutations that confer resistance to isoniazid, rifampicin, aminoglycosides, and fluoroquinolones.ResultsSixty-one (82.4%) isolates were monoresistant to isoniazid and 44 (72.1%) were highly resistant to isoniazid. From the clinical isolates, the range of isoniazid MICs was 0.4–16 μg/mL. The katG S315T gene mutation was the prominent mutation in both isoniazid-monoresistant TB (70.5%) and multidrug-resistant TB (72.7%) isolates. The positive predictive value (PPV) of katG was 100% in detecting high levels of isoniazid resistance. The PPV of the inhA mutation was 93.8% in detecting low levels of isoniazid resistance. Five isolates (6.8%) exhibited low-level phenotypic resistance, whereas an LPA failed to detect an isoniazid gene mutation. Our study found one HR-TB isolate with a gyrA fluoroquinolone-resistant gene mutation.ConclusionMost HR-TB isolates had high isoniazid-resistance levels associated with the katG gene mutation. High-dose isoniazid should be used with caution in patients with HR-TB. Early detection of drug resistance by genotypic assay can help determine an appropriate regimen.

Detection of Second Line Drug Resistance among Drug Resistant Mycobacterium Tuberculosis Isolates in Botswana.

The emergence and transmission of multidrug resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis (M.tb) strains is a threat to global tuberculosis (TB) control. The early detection of drug resistance is critical for patient management. The aim of this study was to determine the proportion of isolates with additional second-line resistance among rifampicin and isoniazid resistant and MDR-TB isolates. A total of 66 M.tb isolates received at the National Tuberculosis Reference Laboratory between March 2012 and October 2013 with resistance to isoniazid, rifampicin or both were analyzed in this study. The genotypes of the M.tb isolates were determined by spoligotyping and second-line drug susceptibility testing was done using the Hain Genotype MTBDRsl line probe assay version 2.0. The treatment outcomes were defined according to the Botswana national and World Health Organization (WHO) guidelines. Of the 57 isolates analyzed, 33 (58%) were MDR-TB, 4 (7%) were additionally resistant to flouroquinolones and 3 (5%) were resistant to both fluoroquinolones and second-line injectable drugs. The most common fluoroquinolone resistance-conferring mutation detected was gyrA A90V. All XDR-TB cases remained smear or culture positive throughout the treatment. Our study findings indicate the importance of monitoring drug resistant TB cases to ensure rapid detection of second-line drug resistance.

A circulating tumor cell specific RNA assay for assessment of androgen receptor signaling inhibitor sensitivity in metastatic castration-resistant prostate cancer.

5059 Background: Our objective is to develop a circulating tumor cell (CTC)-RNA assay for characterizing clinically relevant RNA signatures for the assessment of androgen receptor signaling inhibitors (ARSIs) sensitivity in metastatic castration-resistant prostate cancer (mCRPC) patients. Methods: We developed NanoVelcro CTC-RNA Assay by combining Thermoresponsive(TR)-NanoVelcro CTC purification system with NanoString nCounter platform for CTC purification and RNA analysis. Based on the well-validated, tissue-based Prostate Cancer Classification System (PCS), we selected the most aggressive and ARSI-resistant subtype- the PCS1, for CTC analysis. We applied a rigorous bioinformatic process to develop a CTC-PCS1 panel that is specific to PC CTCs. We validated NanoVelcro CTC-RNA Assay and CTC-PCS1 panel with PC cell lines to demonstrate sensitivity and specificity of the PCS1 Z score (the likelihood estimate of the PCS1 subtype) for identifying PCS1 subtype and ARSI resistance. We then selected 31 blood samples from 23 PC patients receiving ARSIs to test in our assay. The PCS1 Z score of each sample was computed and compared with ARSI treatment sensitivity. Results: We established a 16-gene CTC-PCS1 panel that consists of CTC-specific RNA signatures. The validation studies using PC cell lines showed that the assay can detect the RNA transcripts with high sensitivity and scalability in the range of 1-100 cells. We also showed that the genes in CTC-PCS1 panel is highly expressed in PC cells. We further demonstrated that the CTC-PCS1 panel is highly specific in identifying PCS1-like samples, and the high PCS1 Z score is associated with ARSI resistance. In patient bloods, ARSI-resistant samples (ARSI-R, n=14) had significantly higher PCS1 Z scores as compared with ARSI-sensitive samples (ARSI-S, n=17) (Rank-sum test, P=0.003). In 8 patients who were initially sensitive to ARSI (ARSI-S) and later developed resistance (ARSI-R), we found that the PCS1 Z score increased from the time of ARSI-S to the time of ARSI-R (Pairwise T-test, P=0.016). Conclusions: Using our new methodology, we developed a first-in-class CTC-RNA assay and demonstrated the feasibility of transforming clinically-relevant tissue-based RNA profiling into CTC tests. This approach allows for detecting RNA expression relevant to clinical drug resistance in a non-invasive fashion, which can facilitate patient-specific treatment selection and early detection of drug resistance- a goal in precision oncology.

A Circulating Tumor Cell-RNA Assay for Assessment of Androgen Receptor Signaling Inhibitor Sensitivity in Metastatic Castration-Resistant Prostate Cancer.

Rationale: Our objective was to develop a circulating tumor cell (CTC)-RNA assay for characterizing clinically relevant RNA signatures for the assessment of androgen receptor signaling inhibitor (ARSI) sensitivity in metastatic castration-resistant prostate cancer (mCRPC) patients.Methods: We developed the NanoVelcro CTC-RNA assay by combining the Thermoresponsive (TR)-NanoVelcro CTC purification system with the NanoString nCounter platform for cellular purification and RNA analysis. Based on the well-validated, tissue-based Prostate Cancer Classification System (PCS), we focus on the most aggressive and ARSI-resistant PCS subtype, i.e., PCS1, for CTC analysis. We applied a rigorous bioinformatic process to develop the CTC-PCS1 panel that consists of prostate cancer (PCa) CTC-specific RNA signature with minimal expression in background white blood cells (WBCs). We validated the NanoVelcro CTC-RNA assay and the CTC-PCS1 panel with well-characterized PCa cell lines to demonstrate the sensitivity and dynamic range of the assay, as well as the specificity of the PCS1 Z score (the likelihood estimate of the PCS1 subtype) for identifying PCS1 subtype and ARSI resistance. We then selected 31 blood samples from 23 PCa patients receiving ARSIs to test in our assay. The PCS1 Z scores of each sample were computed and compared with ARSI treatment sensitivity.Results: The validation studies using PCa cell line samples showed that the NanoVelcro CTC-RNA assay can detect the RNA transcripts in the CTC-PCS1 panel with high sensitivity and linearity in the dynamic range of 5-100 cells. We also showed that the genes in CTC-PCS1 panel are highly expressed in PCa cell lines and lowly expressed in background WBCs. Using the artificial CTC samples simulating the blood sample conditions, we further demonstrated that the CTC-PCS1 panel is highly specific in identifying PCS1-like samples, and the high PCS1 Z score is associated with ARSI resistance samples. In patient bloods, ARSI-resistant samples (ARSI-R, n=14) had significantly higher PCS1 Z scores as compared with ARSI-sensitive samples (ARSI-S, n=17) (Rank-sum test, P=0.003). In the analysis of 8 patients who were initially sensitive to ARSI (ARSI-S) and later developed resistance (ARSI-R), we found that the PCS1 Z score increased from the time of ARSI-S to the time of ARSI-R (Pairwise T-test, P=0.016).Conclusions: Using our new methodology, we developed a first-in-class CTC-RNA assay and demonstrated the feasibility of transforming clinically-relevant tissue-based RNA profiling such as PCS into CTC tests. This approach allows for detecting RNA expression relevant to clinical drug resistance in a non-invasive fashion, which can facilitate patient-specific treatment selection and early detection of drug resistance, a goal in precision oncology.

MR-Diffusion characteristics of cerebral alveolar echinococcosis and its role in differentiating cerebral alveolar echinococcosis from metastases

BackgroundAlmost all of the cases of cerebral alveolar echinococcos are showed T2WI hypointensity, with “coal-like” changes; as multiple and T2WI hypointensity of nodular lesions on magnetic resonance imaging (MRI). These imaging findings were also observed in other lesions like metastases. PurposeTo study the MR Diffusion-weighted imaging characteristics of cerebral alveolar echinococcosis and its role in differentiating cerebral alveolar echinococcosis from metastases. Material and methodsA total of thirty-two cerebral alveolar echinococcosis (CAE) and metastases (16 CAE and 16 metastases) were evaluated by conventional and diffusion MRI, respectively. The mean of apparent diffusion coefficient (ADC) and exponential apparent diffusion coefficient (eADC) values from the determined ROI of the solid region, the peripheral edema and control group were calculated. ResultsA total of thirty-two cerebral alveolar echinococcosis (CAE) and brain metastases (BM) (16 CAE and 16 metastases) were nodular lesions. The results showed that in solid regions, the mean ADC values of CAE (1.17 ± 0.06) were higher than BM (0.90 ± 0.07) lesions, while the mean eADC values were lower (CAE 0.31 ± 0.02, BM 0.41 ± 0.03). In the peripheral edematous region, the mean ADC values of CAE were higher than BM lesions, but there were no significant difference, whereas, the mean eADC values were opposite. ConclusionDiffusion MR is a useful tool for the assessment of cerebral alveolar echinococcosis and can help differentiate them from metastases. It also has a potential role in monitoring therapy and early detection of drug resistance.

Dynamic monitoring of EGFR mutations in circulating cell-free DNA for EGFR-mutant metastatic patients with lung cancer: Early detection of drug resistance and prognostic significance.

Detecting genetic mutations in circulating cell-free DNA (cfDNA) is a promising approach of liquid biopsy. Between June 2014 and May 2015, 168 plasma samples were collected monthly from 20 patients with metastatic lung adenocarcinoma with epidermal growth factor receptor (EGFR) mutation receiving gefitinib therapy. Clinically relevant EGFR mutations, including exon 19 deletion, L858R and T790M, were quantified using droplet digital polymerase chain reaction. In baseline samples, 19 (95.0%) patients had the same mutation with the matched tumors, and pretreatment T790M mutations were also detected in 3 (15.0%) patients. The dynamics of EGFR mutations were generally associated with treatment response for patients with or without measurable disease. For patients with immeasurable tumor deposits, monitoring disease evolution using cfDNA-based mutation quantification appeared to be more reliable compared with measuring the diameters of target tumor lesions. In addition, molecular progressive disease, defined as a ≥20% increase of EGFR mutation concentration compared with the lowest concentration recorded during treatment, was tracked up to 8 months prior to objective progression. In survival analysis, sex (P=0.005), pretreatment T790M mutation status (P=0.006), T790M mutation status at the disease progression (P=0.043) and growth rate of EGFR mutations (P=0.023), had a significant impact on median progression-free survival. In conclusion, dynamic monitoring of EGFR mutations in cfDNA is feasible and appears to be useful in early prediction of drug resistance for patients with lung cancer receiving EGFR tyrosine kinase inhibitors.

Tuberculosis of the lymph nodes: Many facets, many hues

Lymph node tuberculosis (LNTB) is one of the most common extrapulmonary manifestations of tuberculosis. A high index of suspicion is needed for diagnosis of tuberculous lymphadenitis, which is known to mimic numerous pathological conditions. Availability of molecular technology has improved the ease of diagnosis. Molecular techniques are also useful for the early detection of drug resistance. With the advent of endobronchial ultrasound in the last decade, the diagnosis of mediastinal tuberculosis has also improved. Management of LNTB often has difficulties. However, most cases can be managed medically and surgical intervention is rarely required. The treatment is similar to pulmonary tuberculosis. However, paradoxical reaction, which is observed in 10–15% of immunocompetent and about 50% of human immunodeficiency virus positive patients needs a special mention for an appropriate management of LNTB.