Drug Resistance Screening Research Articles

Methylation and ovarian cancer: Can DNA methylation be of diagnostic use? (Review)

Ovarian cancer is a silent killer and, due to late diagnosis and frequent chemo resistance in patients, the primary cause of fatality amongst the various types of gynecological cancer. The discovery of a specific and sensitive biomarker for ovarian cancer could improve early diagnosis, thereby saving lives. Biomarkers could also improve treatment, by predicting which patients will benefit from specific treatment strategies. DNA methylation is an epigenetic mechanism, and 'methylation imbalance' is characteristic of cancer. Previous research suggests that changes in DNA methylation can be used diagnostically, and that they may predict resistance to treatment. This paper gives an up-to-date overview of research investigating the potential of DNA methylation-based markers for diagnostics, prognostics, screening and prediction of drug resistance for ovarian cancer patients. DNA methylation cancer-biomarkers may be useful for cancer treatment, particularly since they are chemically stable and since cancer-associated changes in methylation typically precedes tumor growth. DNA methylation markers could improve diagnosis and treatment and might even be used for screening in the future. Furthermore, DNA methylation biomarkers could facilitate the development of precision medicine. However, at this point no biomarkers for ovarian cancer have a sufficient combination of sensitivity and specificity in a clinical setting. A reason for this is that most studies have focused on a single or a few methylation sites. More large screenings and genome-wide studies must be performed to increase the chance of identifying a DNA methylation marker which can identify ovarian cancer.

A First Insight into the katG and rpoB Gene Mutations of Multidrug-Resistant Mycobacterium tuberculosis Strains from Ecuador.

The aim of this study was to characterize the most frequent mutations associated with rifampicin (RIF) and isoniazid (INH) resistance of Mycobacterium tuberculosis isolates from Ecuador. Sequence analysis of 40 strains, resistant for the tuberculosis drugs INH, RIF, or for both showed that of the 31 strains with resistance to INH, 20 strains (64.5%) carried a mutation in the katG gene (codon 315). Eight INH-resistant strains carried a mutation in the katG gene at codon 463. This katG463 mutation, considered a phylogenetic marker, was exclusively found in INH-resistant strains and not in 121 INH-susceptible strains. Of the 35 strains resistant to RIF, 33 (93.9%) had mutations in the hot spot region of the rpoB gene, predominantly in codons 531, 516, and 526. Our results show that sequence-based detection for drug resistance of the katG will identify, respectively, 64.5% or, considering katG463 as a marker, 90.3% of the INH-resistant strains. Sequencing of the hot spot region of the rpoB gene will detect 94.3% of the RIF drug-resistant isolates in Ecuador. This is appropriate for fast screening for drug resistance with the GeneXpert MTB/RIF assay or by direct sequencing of a part of the genes katG and rpoB of PCR products obtained from DNA isolation from primary cultures.

Pretreatment human immunodeficiency virus type 1 (HIV-1) drug resistance in transmission clusters of the Cologne-Bonn region, Germany

ObjectivesIn Germany, previous reports have demonstrated transmitted human immunodeficiency virus type 1 (HIV-1) drug-resistance mutations (DRM) in 11% of newly diagnosed individuals, highlighting the importance of drug-resistance screening before the initiation of antiretroviral therapy (ART). Here, we sought to understand the molecular epidemiology of HIV DRM transmission in the Cologne-Bonn region of Germany, given one of the highest rates of new HIV diagnoses in western Europe (13.7 per 100 000 habitants). MethodsWe analysed 714 HIV-1 ART-naive infected individuals diagnosed at the University Hospitals Cologne and Bonn between 2001 and 2016. Screening for DRM was performed according to the Stanford University Genotypic Resistance Interpretation. Shared DRM were defined as any DRM present in genetically linked individuals (<1.5% genetic distance). Phylogenetic and network analyses were performed to infer putative relationships and shared DRM. ResultsThe prevalence of any DRM at time of diagnosis was 17.2% (123/714 participants). Genetic transmission network analyses showed comparable frequencies of DRM in clustering versus non-clustering individuals (17.1% (85/497) versus 17.5% (38/217)). The observed rate of DRM in the region was higher than previous reports 10.8% (87/809) (p < 0.001), revealing the need to reduce onward transmission in this area. Genetically linked individuals harbouring shared DRM were more likely to live in suburban areas (24/38) than in central Cologne (1/38) (p < 0.001). ConclusionThe rate of DRM was exceptionally high. Network analysis elucidated frequent cases of shared DRM among genetically linked individuals, revealing the potential spread of DRM and the need to prevent onward transmission of DRM in the Cologne-Bonn area.

Abstract B04: Direct downregulation of MYC in AML cells using promoter G-quadruplex interacting small molecules

Abstract Acute myeloid leukemia (AML) is a disease that can affect both children and adults and is characterized by complex and heterogeneous genomic changes, including MYC overexpression driven in part by the bromodomain (BRD) and extraterminal (BET) protein family of epigenetic readers. Current limitations in treatment approaches, including protein overexpression and mutations that confer resistance to therapy, lead to a wide range of response variability to conventional therapy, excessive treatment-related toxicity, and an overall poor outcome. A new therapeutic strategy involves stabilizing the MYC promoter G-quadruplex to downregulate expression. We hypothesize that direct downregulation of MYC in AML cells using MYC-promoter G-quadruplex interacting drugs (GQID) is a feasible novel approach in targeted therapy of AML. Treatment of AML cell lines KG-1a and UT-7epo with the MYC-promoter GQID, GQC-05 resulted in decreased MYC expression and induced a more significant decrease in c-Myc protein than treatment with the BET inhibitor (+)-JQ1. GQC-05 also decreased cell viability and increased apoptosis. RNA-seq of GQC-05-treated KG-1a cells identified 947 significantly differentially expressed genes compared to DMSO. The expression of several c-MYC target genes (including E2F1, GNL3, HSPA8, MAT2A, RCC1, SHMT1, and TFRC) was significantly decreased concomitant with MYC downregulation. The 947 genes were compared with genes with putative quadruplex sequences (PQS) from the literature. Of the 17 genes in common, MYC, NOTCH1, PIM1, and RHOU are significantly downregulated following GQC-05 treatment. These findings were validated by Q-RT-PCR in KG-1a and several other AML cell lines. Drug sensitivity and resistance (DSR) screening on a panel of AML cell lines using a library of 55 anticancer and targeted agents identified a range of sensitivities to GQC-05 (AUC range: 1.65-2.99; DMSO AUC ~ 4). These were compared to microarray gene-expression profiles and, as expected, GQC-05 sensitivity was not correlated with MYC expression. Nevertheless, ANOVA identified 1,049 significantly differentially expressed probe sets between the most sensitive vs. the least sensitive cell lines. Functional annotation revealed over-representation of cadherin, Wnt, and p53 signaling pathways as well over genes involved in hemostasis and hematopoietic cell lineage. In conclusion, directly targeting the MYC promoter G-quadruplex in AML cells leads to knockdown of MYC expression and induces apoptosis. These results further support the development of GQID for targeting key genetic drivers in AML, and lay the groundwork for advances in treatment of other cancers driven by G-quadruplex regulated oncogenes. Citation Format: Megan A. Turnidge, Daniel H. Wai, Apurvi Patel, Justin J. Montoya, David W. Lee, Laurence H. Hurley, Vijay Gokhale, Robert J. Arceci, David O. Azorsa. Direct downregulation of MYC in AML cells using promoter G-quadruplex interacting small molecules [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B04.

Abstract 4644: Patient-derived 3D tumor cultures for clinical diagnostics and pre-clinical drug development

Abstract Ex vivo cultures of patient-derived tumor enable functional testing of treatment options and optimization of pre-clinical drug development. Patient biopsies or tumor from patient-derived xenografts (PDX) mouse models are a valuable source of human tumor material that can be cultured in 3D for screening of drug efficacy, drug resistance and cellular processes such as proliferation, survival and invasion. A high throughput approach using 384 well plates enables evaluation of multiple drug treatments and dose ranges in parallel. After compound exposure, cultures are fixed and stained with cellular markers. 3D image stack acquisition is followed by ultra-high content multiparametric analysis using the OMiner platform to profile drug responses and quantify tumor spheroid volume, apoptosis and tumor invasion. Dissected PDX material and tumor biopsy material, were used to establish 3D tumor cultures derived from various indications, including breast, ovarian, cervix, endometrium, stomach, pancreatic, colon, bladder and lung cancer. These were exposed to standard-of-care chemotherapeutics (e.g. 5-FU, taxanes, platinum compounds, anthracyclines, alkylating agents), small molecules (e.g. erlotinib, lapatinib, trametinib), targeted therapies (PARPi; niraparib, olaparib, rucaparib), antibodies (e.g. cetuximab, trastuzumab) and antibody-drug-conjugate (ADC, T-DM1) dose ranges. The tumor culture response was measured, generating dose-dependent profiles for relevant features. Patient-derived 3D tumor cultures were tested with standard-of-care and novel therapeutic agents and high content analysis was used to evaluate drug sensitivity. This method enables both the in vitro selection of drug candidates in a pre-clinical setting as well as efficient selection of PDX models for in vivo follow-up in the same tumor. This highly translational in vitro-in vivo PDX pipeline is expected to reduce attrition and increase efficiency in early drug-discovery. Correlation of drug sensitivity in 3D cultures from fresh patient tumor biopsies, on the other hand, can be used for development of predictive diagnostics and also provides a unique source of patient material for drug discovery and development. Citation Format: Thomas Dijkmans, Sander Basten, Bram Herpers, Kuan Yan, Torsten Giesemann, Julia Schueler, Willemijn Vader, Leo Price. Patient-derived 3D tumor cultures for clinical diagnostics and pre-clinical drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4644.

Resistance screening and trend analysis of imported falciparum malaria in NSW, Australia (2010 to 2016).

BackgroundThe World Health Organization currently recommends artemisinin (along with a partner drug) as the global frontline treatment for Plasmodium falciparum malaria. Artemisinin resistant P. falciparum are now found throughout the greater Mekong subregion of South East Asia. Several polymorphisms in the parasite’s kelch gene have been demonstrated to confer artemisinin resistance. While genotypes within the greater Mekong subregion are thoroughly examined in the literature, P. falciparum populations within several areas that do not (yet) have endemic resistance are underrepresented.ResultsThis investigation characterised the Pfkelch13 propeller domains from 153 blood samples of 140 imported cases of P. falciparum malaria in New South Wales from 2010 to 2016. A low level of propeller domain diversity was observed, including the C580Y coding mutation most strongly associated with artemisinin resistance in South East Asia. The resistance genotype was found in a sample originating in Papua New Guinea, where this mutation, or artemisinin treatment failure, have not been previously reported. Sequencing a panel of geographically informative polymorphisms within the organellar genomes identified the C580Y parasite as having Oceanic origins. Patient data analysis revealed that New South Wales, Australia, P. falciparum malaria cases often originated from regions with limited drug resistance screening.ConclusionsThe C580Y finding from outside of the greater Mekong subregion supports the consensus to upscale molecular surveillance of artemisinin resistance outside of South East Asia. The genetic screening results identify a risk of importing resistant falciparum malaria to Australia, supporting an ongoing surveillance protocol to pre-empt treatment failure and contribute to global data gathering.

Clinical significance of miRNA - 106a in non-small cell lung cancer patients who received cisplatin combined with gemcitabine chemotherapy.

Objective:Research has demonstrated that microRNA (miR)-106a is related to cisplatin resistance. We investigated the expression of miR-106a in the serum of patients with non-small cell lung cancer (NSCLC) and their sensitivity to chemotherapy by cisplatin combined with gemcitabine.Methods:Eighty-five NSCLC patients, who completed four cycles of gemcitabine and cisplatin chemotherapy, volunteered for this study and their serum samples were collected. Serum samples from 60 healthy subjects were used as controls. Real-time quantitative polymerase chain reaction (real-time qPCR) was used to quantify the level of miR-106a in the serum. Demographic and survival data of these patients were collected for the analysis.Results:The expression of miR-106a in the serum of NSCLC patients was significantly higher than that of healthy subjects (P < 0.001). The expression of miR-106a was not correlated with patients' gender, age, tumor size, lymphatic metastasis, and pathological types; but was correlated with patients' tumor staging ( P = 0.003). After chemotherapy, serum miR-106a expression decreased in patients. The decrease in miR-106a expression in the chemotherapy-sensitive group was much higher than that in the chemotherapy-resistant group. Survival analysis shows that NSCLC patients with high expression of miR-106a have a poorer prognosis. The overall survival of NSCLC patients in the chemotherapy-sensitive group was significantly higher than that in the chemotherapy-resistant group. Conclusions:High expression of miR-106a may be involved in the development of NSCLC. MiR-106a has significance in the prognosis of NSCLC. The level of miR-106a in the serum can be a useful parameter in screening for drug resistance during cisplatin-based chemotherapy.

Increasing Access to Tuberculosis Services in Ethiopia: Findings From a Patient-Pathway Analysis.

BackgroundIn Ethiopia, extensive scale-up of the availability of health extension workers (HEWs) at the community level has been credited with increased identification and referral of patients with presumptive tuberculosis, which has contributed to increased tuberculosis case notification and better treatment outcomes. However, nearly 30% of Ethiopia’s estimated 191000 patients with tuberculosis remained unnotified in 2015. A better understanding of patient care-seeking practices may inform future government action to reach all patients with tuberculosis.MethodsA patient-pathway analysis was completed to assess the alignment between patient care initiation and the availability of diagnostic and treatment services at the national level.ResultsMore than one third of patients initiated care with HEWs, who refer patients to health centers for diagnosis. An additional one third of patients initiated care at health centers. Of those health centers, >80% had microscopy services, but few had access to Xpert. Despite an extensive microscopy and radiography network at middle levels of the health system, a quarter of all notified patients with tuberculosis had no bacteriological confirmation of disease. While 30% of patients reported receiving some form of care from the private sector, private-sector facilities, especially pharmacies, were not widely accessed for tuberculosis diagnosis.DiscussionThe availability of HEWs can increase access to tuberculosis diagnostic and treatment support services, particularly for rural populations. Continued strengthening of referral systems from HEWs and health posts are needed to enable consistent and timely access to Xpert as an initial diagnostic test and to drug resistance screening.

MTBDRplus for the rapid diagnosis of ocular tuberculosis and screening of drug resistance.

PurposeTimely diagnosis of intraocular tuberculosis (IOTB) along with detection of drug resistance can save many eyes from visual impairment. With the growing incidence of IOTB and rising drug resistance, a reliable diagnostic platform for simultaneous detection of the agent and mutated gene is urgently needed. The MTBDRplus assay was evaluated directly on vitreous fluid samples for the same.Patients and methodsIn a prospective study, The MTBDRplus assay was performed on 127 vitreous fluid samples (77 'study group' comprising cases of presumed ocular tuberculosis and 50 'control group' cases of disease controls (n=25) and non-uveitic controls (n=25)). All samples positive by MTBDRplus assay were subjected to gene sequencing to confirm the mutations for rifampicin and isoniazid resistance.ResultsThe MTBDRplus assay produced a sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 36.36%, 100%, 100%, and 50.50%, respectively, for the detection of IOTB. Among the 28 cases from study group that were positive by MTBDRplus assay, rifampicin resistance was reported in six and isoniazid resistance in two cases. On sequencing of rpoB and katG gene, one case of false rifampicin-resistant by MTBDRplus was found. The other resistant isolates showed concordant mutations between MTBDRplus assay and sequencing.ConclusionThe MTBDRplus assay is an effective tool for the rapid diagnosis of IOTB along with detection of drug resistance, thereby improving the outcome in IOTB.

Plasmid-based high-resolution melting analysis for accurate detection of rpoB mutations in Mycobacterium tuberculosis isolates from Moroccan patients

BackgroundRapid diagnosis of drug resistance in tuberculosis (TB) is pivotal for the timely initiation of effective antibiotic treatment to prevent the spread of drug-resistant strains. The development of low-cost, rapid and robust methods for drug-resistant TB detection is highly desirable for resource-limited settings.MethodsWe report the use of an in house plasmid-based quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis for the detection of mutations related to rifampicin-resistant Mycobacterium tuberculosis (MTB) in clinical isolates from Moroccan patients. Five recombinant plasmids containing predominant mutations (S531L, S531W, H526Y and D516V) and the wild-type sequence of the Rifampicin Resistance-Determining Region (RRDR) have been used as controls to screen 45 rifampicin-resistant and 22 rifampicin-susceptible MTB isolates.ResultsThe sensitivity and the specificity of the qPCR-HRM analysis were 88.8% and 100% respectively as compared to rifampicin Drug Susceptibility Testing (DST). The results of qPCR-HRM and DNA sequencing had a concordance of 100%.ConclusionOur qPCR-HRM assay is a sensitive, accurate and cost-effective assay for the high-throughput screening of mutation-based drug resistance in TB reference laboratories.

Abstract 4077: Application of drug sensitivity and resistance testing to identify novel therapeutic agents for Down syndrome patients with AML

Abstract Patients with Down’s syndrome who develop acute myeloid leukemia (AML) are at increased risk of toxicity secondary to chemotherapy regimens. These patients are harder to treat with conventional therapies at relapse. Overall, the use of targeted drug therapy is rising in the pediatric oncology field. Development of drug sensitivity and resistance test allows identification of specific targeted therapies for patient’s with difficult to treat leukemia. Our laboratory is working on the development of a drug sensitivity and resistance screening assay that would provide functionally relevant drug response data of the patient’s leukemia cells. Development of this assay included the preparation of pre-dosed drug library plates allowing for rapid initiation of the assay. AML cells are then added to the plate and treated for 72 hours after which cell viability was determined. Previous work in our laboratory tested a library of 56 anti-cancer and targeted agents on a panel of 16 AML cell lines that included two Down’s syndrome AML cell lines CMK and CMY. Drug dose response data including IC50 values and Area Under the Curve (AUC) were calculated for each cell line. Hierarchical clustering was performed to determine similar responses between cell lines. Down’s syndrome cell lines, CMK and CMY were found to be resistant to most conventional chemotherapeutic agents, but susceptible to targeted therapies such as Navitoclax and 17-AAG. We further tested several cell lines with the drug sensitivity and resistance assay under hypoxic conditions. Our screening results showed improved sensitivity of the CMK and CMY cells to Navitoclax and 17-AAG under normoxic and hypoxic conditions. Validation of the screening results was done using a 10-concentration drug dose response assay. Future studies will explore the mechanism by which the Down’s syndrome AML cells have increased sensitivity to these targeted agents under both normoxic and hypoxic conditions. Furthermore, we will investigate the activity of combinations of these targeted therapies with conventional therapies. The results from these studies could lead to the establishment of improved therapeutic regimens for Down’s syndrome patients with AML. Citation Format: Apurvi R. Patel, Justin J. Montoya, Daniel H. Wai, David W. Lee, Robert J. Arceci, David O. Azorsa. Application of drug sensitivity and resistance testing to identify novel therapeutic agents for Down syndrome patients with AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4077. doi:10.1158/1538-7445.AM2017-4077

Neural network models for supporting drug and multidrug resistant tuberculosis screening diagnosis

Tuberculosis (TB) is the leading cause of global mortality among communicable diseases. The diagnosis of Drug-Resistant Tuberculosis (DR-TB) demands even more attention, leading to longer treatments and higher deceased rates. All diagnostic methods available have deficiencies in their detection rates, time release results, or have a higher cost and need a complex infrastructure to setup. New molecular diagnostics, such as the Xpert MTB/RIF assay, have great potential for revolutionizing the diagnosis of Rifampicin Resistance (RR). But, a positive RR result with this test should be carefully interpreted and take into consideration the risk of Multidrug-Resistant TB (MDR-TB) according to its prevalence, locally. Therefore, the development of screening approaches for DR/MDR-TB suspects would help to identify those should be tested by Xpert MTB/RIF. This work develops Artificial Neural Network (ANN) models considering data from presumed DR/MDR-TB subjects according to the National Guidelines at Rio de Janeiro/Brazil, attended in reference centers in Rio de Janeiro, from Feb 2011 and May 2013. Subjects aged 18 years or older, and results were compared with models based on Classification And Regression Trees (CART). Practical operation at different epidemiological scenarios are considered by constructing models using different variable selection criteria, so that environments with low resource conditions can be assisted. Among 280 presumed DR-TB cases included, 38 were DR-TB, 48-MDR, 32-Drug-Sensitive and 162 with no TB. Between DR-TB and non DR-TB, the sensitivity and specificity reached 95.1%(±5.0) and 85.0%(±4.9), respectively. The promising results of clinical score for DR/MDR-TB diagnosis indicate that this approach may be used in the evaluation of presumed DR/MDR-TB.

Establishment and characterization of a platinum- and paclitaxel-resistant high grade serous ovarian carcinoma cell line.

High grade serous ovarian cancer (HGSOC) patients have a high recurrence rate after surgery and adjuvant chemotherapy due to inherent or acquired drug resistance. Cell lines derived from HGSOC tumors that are resistant to chemotherapeutic agents represent useful pre-clinical models for drug discovery. Here, we describe establishment of a human ovarian carcinoma cell line, which we term WHIRC01, from a patient-derived mouse xenograft established from a chemorefractory HGSOC patient who did not respond to carboplatin and paclitaxel therapy. This newly derived cell line is platinum- and paclitaxel-resistant with cisplatin, carboplatin, and paclitaxel half-maximal lethal doses of 15, 130, and 20µM, respectively. Molecular characterization of this cell line was performed using targeted DNA exome sequencing, transcriptomics (RNA-seq), and mass spectrometry-based proteomic analyses. Results from exomic sequencing revealed mutations in TP53 consistent with HGSOC. Transcriptomic and proteomic analyses of WHIRC01 showed high level of alpha-enolase and vimentin, which are associated with cell migration and epithelial-mesenchymal transition. WHIRC01 represents a chemorefractory human HGSOC cell line model with a comprehensive molecular profile to aid future investigations of drug resistance mechanisms and screening of chemotherapeutic agents.

Challenges of research and development on antimalarial medicinal products in China: a bibliometric analysis and systematic review.

The advancement in any antimalarial medicinal product including vaccines, drugs and diagnostics will have a vital influence on malaria elimination in China and on the global malaria control framework. This study aimed to identify research progress and challenges in China, hoping to better facilitate domestic elimination and for China to be more effectively involved in global malaria research and development. A systematic search was conducted for research articles published from 2005 to 2014 in PubMed, CNKI and Wanfang using terms including malaria, diagnosis, drugs and vaccines. In total, 4259 articles from PubMed and 561 references from Chinese databases were included and categorized by topic. The literature from PubMed was clustered and seven antimalarial medicinal product research hotspots were identified; including drug resistance, diagnostic tests and vaccine antigen screening. The reports related to drugs accounted for the largest proportion in PubMed (57%) and Chinese studies (51%) while references associated with diagnostics accounted for the lowest proportion, 10% in PubMed and 14% in Chinese studies. Despite continuous effort in malaria research and development, there exist gaps in progressive discoveries on malaria diagnostics and drugs in China. Successive focus on antimalarial medicinal products is essential to facilitate malaria control in China and worldwide.

Molecular screening for primary drug resistance in M. leprae from newly diagnosed leprosy cases from India

Background: India records an Annual New Case Detection Rate (ANCDR) of 9.98 per 100,000 population, with a range of 0.42–81.7, across its various states. Although there is not much evidence available for presence of primary drug resistance in leprosy, it is essential to know such information on a periodic basis, in such high endemic regions, with increasing incidence of new leprosy cases. Objective: The study was undertaken to screen for Mycobacterium leprae drug resistance to dapsone, rifampicin and ofloxacin by PCR sequencing of folP1, rpoB, and gyrA genes respectively. Methods: Prospective community based study conducted during 2011–14, with samples collected from two districts- Adilabad and Hyderabad, Andhra Pradesh (presently Telangana state), Sonepur and Koraput, of Odisha state of India, for molecular drug resistance analysis of M. leprae. PCR and gene sequencing was carried out on the Slit Skin Samples SSS) collected from the enrolled subjects. An attempt was made to screen M. leprae PCR as a bacteriological tool for diagnosing leprosy. Results: During the study period 234 new leprosy cases were enrolled out of which 74% cases were multibacillary (MB) and 26% were paucibacillary (PB). 20% of the 214 specimens tested were positive for PCR of fol P1, 11%, positive for rpoB and 35% for gyrA. None of the isolates demonstrated drug resistance. Conclusion: Study findings indicate the absence of drug resistance in new leprosy patients and also suggest reintroduction of skin smear examination in to routine practice. PCR with some improvements may also be included for specific diagnosis of leprosy.

Abstract 4766: Establishment of drug sensitivity and resistance testing using focused drug libraries to develop targeted therapies for patients with leukemia

Abstract Despite major advancements in pediatric leukemia therapy, toxicities and morbidity from chemotherapy regimens have been detrimental. Advances in genomic research as well as precision medicine have helped identify specific mutations that may be susceptible to targeted therapy in leukemic patients. Overall, the use of targeted drug therapy is rising in the pediatric oncology field. To complement genomic analysis of leukemia patients, our laboratory is working on the development of a drug sensitivity and resistance screening library, that would provide functionally relevant drug response data of the patient's leukemia cells. Development of this assay included the preparation of pre-dosed library plates allowing for rapid initiation of the assay. Cells were treated for 72 hours and cell viability was determined. Preliminary work in our laboratory used a library of 56 anti-cancer and targeted agents, which were tested on 16 AML cell lines and 10 ALL cell lines. Biological replicates were preformed for each cell line and drug response data in the form of IC50s and Area Under the Curve (AUC) were calculated. Hierarchical clustering was performed to determine similar responses between cell lines. Our results identified several cell lines that showed hypersensitivity to specific drugs. Among them, the AML cell line KG-1a showed sensitivity to three FGFR inhibitors including Ponatinib, which was previously reported. Validation of the screening results was done using a 10-concentration drug dose response assay. Further testing will include expansion of our drug library and application to ex vivo patient samples. The results from these studies can lead to the establishment of a drug sensitivity and resistance assay that could provide specifically engineered therapies for each patient. This form of personalized therapy would lead to diminished toxicities from systemic therapies as well as allow increased cure rates. Citation Format: Apurvi Patel, Justin J. Montoya, Daniel H. Wai, Robert J. Arceci, David O. Azorsa. Establishment of drug sensitivity and resistance testing using focused drug libraries to develop targeted therapies for patients with leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4766.

Generation and application of mammalian haploid embryonic stem cells.

Haploid cells contain one set of chromosomes and are amenable for genetic analyses. In mammals, haploidy exists only in gametes. An intriguing question is whether haploid cells can be derived from gametes. Recently, by application of haploid cell enrichment using fluorescence-activated cell sorting, stable haploid embryonic stem cells (haESCs) have been successfully derived from oocyte-derived parthenogenetic and sperm-derived androgenetic embryos from several species. Whilst both parthenogenetic and androgenetic (AG)-haESCs enable whole-genome genetic screening at the cellular level, such as screening of drug resistance or disease-related genes, AG-haESCs, after intracytoplasmic injection into oocytes, can also be used to produce alive semi-cloned mice. Nevertheless, one major drawback associated with wild-type AG-haESCs is the very low birth rate of healthy semi-cloned mice. Of interest, after inhibiting the expression of two paternally imprinted genes (H19 and Gtl2) in AG-haESCs by removal of their differentially DNA methylated regions, double-knockout AG-haESCs can efficiently and stably support the generation of healthy semi-cloned pups. Importantly, double-knockout AG-haESCs are feasible for multiple genetic manipulations, followed by efficient generation of semi-cloned mice carrying multiple genetic traits; thus they could be used to validate candidate loci that have been identified in genome-wide association studies of multigenic diseases by generation of mouse models carrying multiple alterations. Of note, by combining a CRISPR-Cas9 library and double-knockout AG-haESCs, semi-cloned mice carrying different mutant genes can be efficiently generated in one step, enabling functional mutagenic screening in mice. HaESCs, therefore, provide a powerful tool for genetic analyses in mammals at both the cellular and organismal levels.

Clotrimazole Drug Resistance in Candida glabrata Clinical Isolates Correlates with Increased Expression of the Drug:H(+) Antiporters CgAqr1, CgTpo1_1, CgTpo3, and CgQdr2.

For years, antifungal drug resistance in Candida species has been associated to the expression of ATP-Binding Cassette (ABC) multidrug transporters. More recently, a few drug efflux pumps from the Drug:H+ Antiporter (DHA) family have also been shown to play a role in this process, although to date only the Candida albicans Mdr1 transporter has been demonstrated to be relevant in the clinical acquisition of antifungal drug resistance. This work provides evidence to suggest the involvement of the C. glabrata DHA transporters CgAqr1, CgQdr2, CgTpo1_1, and CgTpo3 in the clinical acquisition of clotrimazole drug resistance. A screening for azole drug resistance in 138 C. glabrata clinical isolates, from patients attending two major Hospitals in Portugal, was performed. Based on this screening, 10 clotrimazole susceptible and 10 clotrimazole resistant isolates were selected for further analysis. The transcript levels of CgAQR1, CgQDR2, CgTPO1_1, and CgTPO3 were found to be significantly up-regulated in resistant isolates when compared to the susceptible ones, with a level of correlation that was found to be similar to that of CgCDR2, an ABC gene known to be involved in the clinical acquisition of resistance. As a proof-of-concept experiment, the CgTPO3 gene was deleted in an azole resistant C. glabrata isolate, exhibiting high levels of expression of this gene. The deletion of CgTPO3 in this isolate was found to lead to decreased resistance to clotrimazole and fluconazole, and increased accumulation of azole drugs, thus suggesting the involvement of this transporter in the manifestation of azole resistance.

Evaluation of the in vitro expression of ATP binding-cassette (ABC) proteins in an Ixodes ricinus cell line exposed to ivermectin.

BackgroundTicks are among the most important vectors of pathogens causing human and animal disease. Acaricides are used to control tick infestation, although there are increasing reports of resistance. Recently, over-expression of ATP-binding cassette (ABC) transporter proteins (P-glycoproteins, PgP) has been implicated in resistance to the acaricide ivermectin in the ticks Rhipicephalus (Boophilus) microplus and Rhipicephalus sanguineus sensu lato. Ixodid tick cell lines have been used to investigate drug resistance mechanisms. The aim of the present study was to evaluate expression of several PgPs in the Ixodes ricinus-derived cell line IRE/CTVM19 and to determine modulation of expression following treatment with ivermectin.FindingsIRE/CTVM19 cells were treated with different concentrations of ivermectin (0, 11, 22 or 33 μM) and incubated for 10 days. Evaluation of viability and relative expression of ABCB1, ABCB6, ABCB8 and ABCB10 genes were carried out at day 10 post treatment. Cell viability ranged between 84 % and 92 % with no significant differences between untreated and treated cells. qRT-PCR showed that ABC pump expression was not significantly modulated by ivermectin treatment. Expression of the ABCB8 PgP subfamily revealed a biphasic trend, based on the ivermectin concentration. ABCB6 and ABCB10 gene expression was not modulated by ivermectin treatment and ABCB1 expression was not detected.ConclusionsThis is the first report of PgP expression in an I. ricinus-derived tick cell line. Development of an in vitro model for the study of acaricide resistance mechanisms would greatly facilitate screening for drug resistance in ticks.

RpoB gene high-resolution melt curve analysis: a rapid approach for diagnosis and screening of drug resistance in tuberculous meningitis

Timely and rapid diagnosis of multidrug resistance in tuberculous meningitis (TBM) is a challenge both for a microbiologist and neurologist. The present study was conducted to evaluate role of real-time polymerase chain reaction (PCR) using rpoB, IS6110, and MPB64 as targets in diagnosis of TBM in 110 patients and subsequent high-resolution melt (HRM) curve analysis of rpoB gene amplicons for screening of drug resistance. The sensitivity of smear, culture, and real-time PCR was 1.8%, 10.9%, and 83.63%, respectively. All 120 control patients showed negative results. With HRM rpoB analysis, rifampicin resistance was detected in 3 out of 110 cases of TBM (3.33%). Subsequently, results of HRM analysis were confirmed by rpoB gene sequencing, and mutations were observed at 516 (2 patients) and 531 (1 patient) codons, respectively. rpoB HRM analysis can be a promising tool for rapid diagnosis and screening of drug resistance in TBM patients in 90minutes.