High Content and High Throughout Phenotypic Assay for the Hourly Resolution of the Malaria Parasite Erythrocytic Cycle

AbstractOver the last 20 years increased funding for malaria research has resulted in very significant technical advances to study the biology of Plasmodium species. High throughput phenotypic assays have been developed to screen millions of compounds and identify small molecules with antiparasitic activity. At the same time, advances in malaria genetic have greatly facilitated the generation of genetically modified parasites, and whole genome genetic screens are now feasible in Plasmodium species. Finally, there has been an increased interest to study malaria parasites at the population level, in particular in the area of drug resistance. Drug resistant field isolates have been collected around the world, and drug resistant strains are routinely generated in the lab to study the mechanisms of drug resistance. As a result, one of the current bottlenecks in malaria research is our ability to quickly characterize the phenotype associated with compound treatment or genetic modification, or to quickly compare differences in intracellular development between strains. Here, we present a high content/high throughput phenotypic assay that combines highly selective RNA, DNA, and RBC membrane dyes to provide hourly resolution of the full erythrocytic cycle for both P. falciparum and P. knowlesi. A flow cytometry assay allows the analysis of samples in a 384-well format and a quick way to determine the parasite developmental stage. On the other hand, the fluorescence microscopy format allows for a detailed visualization of parasite morphology. Finally, using open source software we have developed protocols for the automated cluster analysis of microscopy images. This assay can be applied to any Plasmodium species, requires very little amount of sample, is performed with fixed cells, and is easily scalable. Overall, we believe this assay will be a great tool for the malaria community to study Plasmodium species.

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Primary HIV drug resistance among newly HIV type-1 diagnosed patients in St. Petersburg

HIV Infection and Immunosuppressive Disorders ◽

10.22328/2077-9828-2021-13-1-70-79 ◽

2021 ◽

Vol 13 (1) ◽

pp. 70-79

Author(s):

Thierry Ingabire ◽

A. V. Semenov ◽

E. V. Esaulenko ◽

E. B. Zueva ◽

A. N. Schemelev ◽

...

Keyword(s):

Drug Resistance ◽

Reverse Transcriptase ◽

Direct Sequencing ◽

Population Level ◽

Multidrug Resistant ◽

Antiretroviral Drugs ◽

Drug Resistant ◽

Resistant Virus ◽

Primary Drug Resistance ◽

Hiv 1

There is concern that the widespread use of antiretroviral drugs (ARV) to treat human immunodeficiency virus 1 (HIV-1) infection may result in the emergence of transmission of drug-resistant virus among persons newly infected with HIV-1. Russia is one of a growing number of countries in the world where drug-resistant HIV is becoming a serious health problem because it has the potential to compromise the efficacy of antiretroviral therapy (ART) at the population level.Materials and methods. We performed a genetic analysis of the HIV-1 plasma derived pol gene among the newly diagnosed ART-naïve HIV-1 infected patients during the period from November 2018 to October 2019 in the St. Petersburg Clinical Infectious Diseases Hospital named after S.P. Botkin. We used reverse transcriptase polymerase chain reaction (RT-PCR) followed by direct sequencing of PCR products to determine the prevalence of primary drug resistance (PDR) conferring mutations. HIV-1 genotypes were determined by phylogenetic analysis.Results. The predominant HIV-1 subtype was A1 (87.2%), followed by B (11.8%) and CRF06_cpx (1%). The overall prevalence of PDR was 11%. Virus with known resistance-conferring mutations to any nucleoside reverse transcriptase inhibitors (NRTIs) was found in 8 individuals, to any non NRTIs in 5 subjects, and to any protease inhibitors in 1 case. Multidrug-resistant virus was identified in 2 individuals (2%).Conclusion. The distribution of HIV-1 genotypes in St. Petersburg, Russia is diverse. The emerging prevalence of PDR in ART-naïve patients demonstrates the significance of constant monitoring due to the challenges it presents towards treatment.

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Accuracy of an amplicon-sequencing nanopore approach to identify variants in tuberculosis drug-resistance-associated genes

Microbial Genomics ◽

10.1099/mgen.0.000740 ◽

2021 ◽

Vol 7 (12) ◽

Author(s):

Carla Mariner-Llicer ◽

Galo A. Goig ◽

Laura Zaragoza-Infante ◽

Manuela Torres-Puente ◽

Luis Villamayor ◽

...

Keyword(s):

Drug Resistance ◽

Type Species ◽

Variant Calling ◽

Targeted Sequencing ◽

Clinical Samples ◽

Drug Resistant ◽

Content Type ◽

Link Type ◽

Resistant Strains ◽

Drug Resistant Strains

A rapid and accurate diagnostic assay represents an important means to detect Mycobacterium tuberculosis , identify drug-resistant strains and ensure treatment success. Currently employed techniques to diagnose drug-resistant tuberculosis include slow phenotypic tests or more rapid molecular assays that evaluate a limited range of drugs. Whole-genome-sequencing-based approaches can detect known drug-resistance-conferring mutations and novel variations; however, the dependence on growing samples in culture, and the associated delays in achieving results, represents a significant limitation. As an alternative, targeted sequencing strategies can be directly performed on clinical samples at high throughput. This study proposes a targeted sequencing assay to rapidly detect drug-resistant strains of M. tuberculosis using the Nanopore MinION sequencing platform. We designed a single-tube assay that targets nine genes associated with drug resistance to seven drugs and two phylogenetic-determining regions to determine strain lineage and tested it in nine clinical isolates and six sputa. The study’s main aim is to calibrate MinNION variant calling to detect drug-resistance-associated mutations with different frequencies to match the accuracy of Illumina (the current gold-standard sequencing technology) from both culture and sputum samples. After calibrating Nanopore MinION variant calling, we demonstrated 100% agreement between Illumina WGS and our MinION set up to detect known drug resistance and phylogenetic variants in our dataset. Importantly, other variants in the amplicons are also detected, decreasing the recall. We identify minority variants and insertions/deletions as crucial bioinformatics challenges to fully reproduce Illumina WGS results.

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Snapshot of Mycobacterium tuberculosis Phylogenetics from an Indian State of Arunachal Pradesh Bordering China

10.20944/preprints202112.0283.v1 ◽

2021 ◽

Author(s):

Rashmi S Mudliar ◽

Umay Kulsum ◽

Syed Beenish Rufai ◽

Mika Umpo ◽

Moi Nyori ◽

...

Keyword(s):

Drug Resistance ◽

Mycobacterium Tuberculosis ◽

Drug Resistant ◽

Second Line ◽

Arunachal Pradesh ◽

Tuberculosis Programme ◽

Indian State ◽

Northeastern State ◽

Resistant Strains ◽

Drug Resistant Strains

Uncontrolled transmission of Mycobacterium tuberculosis (M. tuberculosis, MTB) drug resistant strains is a challenge to control efforts of global tuberculosis programme. Due to increasing multi-drug resistant (MDR) cases in Arunachal Pradesh, a northeastern state of India, the tracking and tracing of these resistant MTB strains is crucial for infection control and spread of drug resistance. This study aims to correlate the phenotypic DST, genomic DST (gDST) and phylogenetic analysis of MDR-MTB strains in the region. Of total 200 suspected MDR-MTB isolates, 125(62.5%) were identified as MTB. MGIT-960 SIRE DST detected 71/125(56.8%) isolates as MDR/RR-MTB of which 22(30.9%) were detected resistant to second line drugs. Whole genome sequencing of 65 isolates and their gDST found Ser315Thr mutation in katG (35/45;77.8%) and Ser531Leu mutation in rpoB (21/41;51.2%) associated with drug resistance. SNP barcoding categorized the dataset with Lineage2 (41;63.1%) being predominant followed by Lineage3 (10;15.4%), Lineage1 (8;12.3%) and Lineage4 (6;9.2%) respectively. Phylogenetic assignment by cgMLST gave insights of two Beijing sub-lineages viz; 2.2.1 (SNP difference &lt; 19) and 2.2.1.2 (SNP difference &lt; 9) associated with recent ongoing transmission in Arunachal Pradesh. This study provides first insight in identifying the ongoing transmission of two virulent Beijing sub-lineages associated with TB drug resistance.

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Activities of tubercular inflammation (on the morphological data) with different duration in the patients with drug-resistant pulmonary tuberculosis

Journal of New Medical Technologies eJournal ◽

10.12737/18556 ◽

2016 ◽

Vol 10 (1) ◽

pp. 0-0

Author(s):

Елипашев ◽

A. Elipashev ◽

Никольский ◽

V. Nikolskiy ◽

Шпрыков ◽

...

Keyword(s):

Drug Resistance ◽

High Sensitivity ◽

Morphological Data ◽

Control Group ◽

Drug Resistant ◽

Specific Therapy ◽

Resistant Tuberculosis ◽

Number Of Patients ◽

Resistant Strains ◽

Drug Resistant Strains

The purpose of this research was to determine the dependence of the tubercular inflammation activity of varying duration of the disease and drug resistance. Morphological activity of inflammation in 161 patients with drug-resistant and 149 patients with retaining its high sensitivity was studied. Morphological assessment of the activity of specific changes in tuberculosis was carried out according to the B.M. Ariel classification (1998). It was revealed at morphologic study of resection material that the greatest activity of specific inflammation and its prevalence outside the main lesion was in the group of patients limited with drug resistance tuberculosis. It was noted the prevalence of IV-V degree of morphological activity of tubercular process in the study group by 3 times over the control group with disease duration of more than 1 year. Predominance of widespread active specific changes (IV degree) was determined in 2 times for the first educed patients of basic group with drug-resistant above a control group. This is due to increasing the number of patients with cavernous and fibro-cavernous tuberculosis.Thus, it is necessary to operate patients with drug-resistant tuberculosis as soon as possi-ble after adequate specific therapy and the presence of the signs of stabilization process, because as the full stabilization of tuberculosis process did not achieve according to the morphological study of surgical specimens in the preoperative period. Further specific therapy becomes futile due to the rise of drug resistance, the emer-gence of new drug-resistant strains of Mycobacterium tuberculosis.

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Transferred drug-resistance in Eimeria maxima

Parasitology ◽

10.1017/s0031182000047168 ◽

1975 ◽

Vol 71 (3) ◽

pp. 385-392 ◽

Cited By ~ 24

Author(s):

L. P. Joyner ◽

C. C. Norton

Keyword(s):

Drug Resistance ◽

The Other ◽

Drug Resistant ◽

Resistance Factors ◽

Eimeria Maxima ◽

Series Of Experiments ◽

Resistant Strains ◽

Drug Resistant Strains

A series of experiments is described in which two drug-resistant strains of Eimeria maxima were passaged together in untreated chicks. The resultant oocysts were then inoculated into chicks treated with both drugs. When strains resistant to methyl benzoquate and sulphaquinoxaline or clopidol and sulphaquinoxaline were used the resultant infections were not controlled by the double treatment, indicating the acquisition of resistance factors by one strain from the other. When strains resistant to clopidol and methyl benzoquate were used the phenomenon was not observed.

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Change of drug resistance patterns and genetic properties of R plasmids inSalmonella typhimuriumof bovine origin isolated from 1970 to 1979 in northern Japan

Journal of Hygiene ◽

10.1017/s0022172400069473 ◽

1981 ◽

Vol 87 (2) ◽

pp. 257-269 ◽

Cited By ~ 9

Author(s):

S. Makino ◽

N. Ishiguro ◽

G. Sato ◽

N. Seno

Keyword(s):

Drug Resistance ◽

Mercury Resistance ◽

Drug Resistant ◽

Fertility Inhibition ◽

Resistance Patterns ◽

Clonal Distribution ◽

R Plasmids ◽

Resistant Strains ◽

Drug Resistant Strains ◽

Northern Japan

SummaryA total of 321Salmonella typhimuriumstrains of bovine origin obtained in northern Japan during the period 1970–1979 were tested for drug resistance and detection of conjugative R plasmids. Three hundred and eighteen (99·1 %) of these strains were resistant to one or more drugs. The isolation frequency of multiply drug-resistant strains tended to increase year by year. Two hundred and thirty-seven (74·5%) of these resistant strains carried conjugative R plasmids. A total of 308 R plasmids including 174 (56·5 %) thermosensitive (ts) R plasmids were derived from the 237 drug-resistant strains, indicating that 71 (30·0%) strains have two different conjugative R plasmids in a single host cell. Of the 308 R plasmids examined for fertility inhibition (fi), 167 ts and 131 non-ts R plasmids werefi−. Of the 60 ts R plasmids examined for incompatibility, 50 were classified into H1 group and 10 into H2 group. Of the 52 non-ts R plasmids examined, 35 were classified into the Iα group and the remaining plasmids were untypable in our tests. Mercury resistance marker was found in about 20% of H1 R plasmids coding for multiresistance, and all of H2 R plasmids coded for resistance to tellurite. The clonal distribution of anS. typhimuriumstrain which carried an H1 R plasmid coding for resistance to six drugs and mercury was recognized in 1978 and 1979.

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Increase of Virulence and Its Phenotypic Traits in Drug-Resistant Strains of Candida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01223-07 ◽

2008 ◽

Vol 52 (3) ◽

pp. 927-936 ◽

Cited By ~ 39

Author(s):

Letizia Angiolella ◽

Anna Rita Stringaro ◽

Flavia De Bernardis ◽

Brunella Posteraro ◽

Mariantonietta Bonito ◽

...

Keyword(s):

Drug Resistance ◽

Candida Albicans ◽

Biological Properties ◽

Antifungal Drugs ◽

Infection Model ◽

Phenotypic Traits ◽

Homozygous Mutation ◽

Drug Resistant ◽

Resistant Strains ◽

Drug Resistant Strains

ABSTRACT There is concern about the rise of antifungal drug resistance, but little is known about comparative biological properties and pathogenicity of drug-resistant strains. We generated fluconazole (FLC; CO23RFLC)- or micafungin (FK; CO23RFK)-resistant strains of Candida albicans by treating a FLC- and FK-susceptible strain of this fungus (CO23S) with stepwise-increasing concentrations of either drug. Molecular analyses showed that CO23RFLC had acquired markedly increased expression of the drug-resistance efflux pump encoded by the MDR1 gene, whereas CO23RFK had a homozygous mutation in the FSK1 gene. These genetic modifications did not alter to any extent the growth capacity of the drug-resistant strains in vitro, either at 28°C or at 37°C, but markedly increased their experimental pathogenicity in a systemic mouse infection model, as assessed by the overall mortality and target organ invasion. Interestingly, no apparent increase in the vaginopathic potential of the strains was observed with an estrogen-dependent rat vaginal infection. The increased pathogenicity of drug-resistant strains for systemic infection was associated with a number of biochemical and physiological changes, including (i) marked cellular alterations associated with a different expression and content of major cell wall polysaccharides, (ii) more rapid and extensive hypha formation in both liquid and solid media, and (iii) increased adherence to plastic and a propensity for biofilm formation. Overall, our data demonstrate that experimentally induced resistance to antifungal drugs, irrespective of drug family, can substantially divert C. albicans biology, affecting in particular biological properties of potential relevance for deep-seated candidiasis.

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Potential Therapeutic Targets for Combination Antibody Therapy against Pseudomonas aeruginosa Infections

Antibiotics ◽

10.3390/antibiotics10121530 ◽

2021 ◽

Vol 10 (12) ◽

pp. 1530

Author(s):

Luke L. Proctor ◽

Whitney L. Ward ◽

Conner S. Roggy ◽

Alexandra G. Koontz ◽

Katie M. Clark ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Drug Resistance ◽

Drug Targets ◽

Antimicrobial Agents ◽

Antibody Therapy ◽

Therapeutic Targets ◽

Multidrug Resistant ◽

Drug Resistant ◽

Future Drug ◽

Drug Resistant Strains

Despite advances in antimicrobial therapy and even the advent of some effective vaccines, Pseudomonas aeruginosa (P. aeruginosa) remains a significant cause of infectious disease, primarily due to antibiotic resistance. Although P. aeruginosa is commonly treatable with readily available therapeutics, these therapies are not always efficacious, particularly for certain classes of patients (e.g., cystic fibrosis (CF)) and for drug-resistant strains. Multi-drug resistant P. aeruginosa infections are listed on both the CDC’s and WHO’s list of serious worldwide threats. This increasing emergence of drug resistance and prevalence of P. aeruginosa highlights the need to identify new therapeutic strategies. Combinations of monoclonal antibodies against different targets and epitopes have demonstrated synergistic efficacy with each other as well as in combination with antimicrobial agents typically used to treat these infections. Such a strategy has reduced the ability of infectious agents to develop resistance. This manuscript details the development of potential therapeutic targets for polyclonal antibody therapies to combat the emergence of multidrug-resistant P. aeruginosa infections. In particular, potential drug targets for combinational immunotherapy against P. aeruginosa are identified to combat current and future drug resistance.

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Screening for community-acquired strains of methicillinresistant Staphylococcus aureus susceptible to extracts of Centaurea nigrescens

The Journal of Phytopharmacology ◽

10.31254/phyto.2018.7312 ◽

2018 ◽

Vol 7 (3) ◽

pp. 298-304

Author(s):

Luke G Huggins ◽

◽

Kathryn D Robinson ◽

Kyra P Lasko ◽

Lauren B Clower ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Drug Resistance ◽

Efflux Pump ◽

The United States ◽

Drug Efflux ◽

Drug Resistant ◽

Plant Metabolites ◽

Import And Export ◽

Mrsa Strains ◽

Drug Resistant Strains

The rates of infection by community-acquired multi-drug resistant Staphylococcus aureus have risen dramatically over fifteen years in the United States. Community-acquired multi-drug resistant Staphylococcus aureus is responsible for rapidly progressive diseases, including necrotizing pneumonia, severe sepsis, and necrotizing fasciitis. Consequently, novel antibacterial strategies are needed to combat the rising antibiotic resistance seen in community-acquired multi-drug resistant strains. We have screened the Nebraska Transposon Mutant Library for MRSA strains that are either susceptible or resistant to methanol extracts of Centaurea nigrescens leaves and flowers. 10 strains containing mutations affecting transporter proteins were identified as having either significant resistance or susceptibility to Centaurea extract. Insertions in two different drug efflux transporter families have been identified. The EmrB/QacA drug resistance transporter subfamily is a multi-drug efflux pump responsible for the export of toxic molecules from bacteria and yeast. The ABC transporters are involved in drug import and export. These results confirm the effectiveness of the screen as a means for identifying drug-resistance genes affected by the C. nigrescens methanolic extract and suggest a role for drug efflux proteins in the resistance of S. aureus community-acquired multi-drug resistant Staphylococcus aureus to antibacterial plant metabolites

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