Molecular Markers Of Resistance Research Articles

Fine mapping and analysis of a candidate gene controlling Phytophthora blight resistance in cucumber.

Cucumber blight is a destructive disease. The best way to control this disease is resistance breeding. This study focuses on disease resistance gene mapping and molecular marker development. We used the resistant cucumber, JSH, and susceptible cucumber, B80, as parents to construct F2 populations. Bulked segregant analysis (BSA) combined with specific length amplified fragment sequencing (SLAF-seq) were used, from which we developed cleaved amplified polymorphic sequence (CAPs) markers to map the resistance gene. Resistance in F2 individuals showed a segregation ratio of resistance:susceptibility close to 3:1. The gene in JSH resistant cucumber was mapped to an interval of 9.25 kb, and sequencing results for the three genes in the mapped region revealed three mutations at base sites 225, 302, and 591 in the coding region of Csa5G139130 between JSH and B80, but no mutations in coding regions of Csa5G139140 and Csa5G139150. The mutations caused changes in amino acids 75 and 101 of the protein encoded by Csa5G139130, suggesting that Csa5G139130 is the most likely resistance candidate gene. We developed a molecular marker, CAPs-4, as a closely linked marker for the cucumber blight resistance gene. This is the first report on mapping of a cucumber blight resistance gene and will provideg a useful marker for molecular breeding of cucumber resistance to Phytophthora blight.

Systematic Review and Geospatial Modeling of Molecular Markers of Resistance to Artemisinins and Sulfadoxine-Pyrimethamine in Plasmodium falciparum in India.

Surveillance for genetic markers of resistance can provide valuable information on the likely efficacy of antimalarials but needs to be targeted to ensure optimal use of resources. We conducted a systematic search and review of publications in seven databases to compile resistance marker data from studies in India. The sample collection from the studies identified from this search was conducted between 1994 and 2020, and these studies were published between 1994 and 2022. In all, Plasmodium falciparum Kelch13 (PfK13), P. falciparum dihydropteroate synthase, and P. falciparum dihydrofolate reductase (PfDHPS) genotype data from 2,953, 4,148, and 4,222 blood samples from patients with laboratory-confirmed malaria, respectively, were extracted from these publications and uploaded onto the WorldWide Antimalarial Resistance Network molecular surveyors. These data were fed into hierarchical geostatistical models to produce maps with a predicted prevalence of the PfK13 and PfDHPS markers, and of the associated uncertainty. Zones with a predicted PfDHPS 540E prevalence of >15% were identified in central, eastern, and northeastern India. The predicted prevalence of PfK13 mutants was nonzero at only a few locations, but were within or adjacent to the zones with >15% prevalence of PfDHPS 540E. There may be a greater probability of artesunate-sulfadoxine-pyrimethamine failures in these regions, but these predictions need confirmation. This work can be applied in India and elsewhere to help identify the treatments most likely to be effective for malaria elimination.

Trends of Plasmodium falciparum molecular markers associated with resistance to artemisinins and reduced susceptibility to lumefantrine in Mainland Tanzania from 2016 to 2021

BackgroundTherapeutic efficacy studies (TESs) and detection of molecular markers of drug resistance are recommended by the World Health Organization (WHO) to monitor the efficacy of artemisinin-based combination therapy (ACT). This study assessed the trends of molecular markers of artemisinin resistance and/or reduced susceptibility to lumefantrine using samples collected in TES conducted in Mainland Tanzania from 2016 to 2021.MethodsA total of 2,015 samples were collected during TES of artemether-lumefantrine at eight sentinel sites (in Kigoma, Mbeya, Morogoro, Mtwara, Mwanza, Pwani, Tabora, and Tanga regions) between 2016 and 2021. Photo-induced electron transfer polymerase chain reaction (PET-PCR) was used to confirm presence of malaria parasites before capillary sequencing, which targeted two genes: Plasmodium falciparum kelch 13 propeller domain (k13) and P. falciparum multidrug resistance 1 (pfmdr1).ResultsSequencing success was ≥ 87.8%, and 1,724/1,769 (97.5%) k13 wild-type samples were detected. Thirty-seven (2.1%) samples had synonymous mutations and only eight (0.4%) had non-synonymous mutations in the k13 gene; seven of these were not validated by the WHO as molecular markers of resistance. One sample from Morogoro in 2020 had a k13 R622I mutation, which is a validated marker of artemisinin partial resistance. For pfmdr1, all except two samples carried N86 (wild-type), while mutations at Y184F increased from 33.9% in 2016 to about 60.5% in 2021, and only four samples (0.2%) had D1246Y mutations. pfmdr1 haplotypes were reported in 1,711 samples, with 985 (57.6%) NYD, 720 (42.1%) NFD, and six (0.4%) carrying minor haplotypes (three with NYY, 0.2%; YFD in two, 0.1%; and NFY in one sample, 0.1%). Between 2016 and 2021, NYD decreased from 66.1% to 45.2%, while NFD increased from 38.5% to 54.7%.ConclusionThis is the first report of the R622I (k13 validated mutation) in Tanzania. N86 and D1246 were nearly fixed, while increases in Y184F mutations and NFD haplotype were observed between 2016 and 2021. Despite the reports of artemisinin partial resistance in Rwanda and Uganda, this study did not report any other validated mutations in these study sites in Tanzania apart from R622I suggesting that intensified surveillance is urgently needed to monitor trends of drug resistance markers and their impact on the performance of ACT.

Whole genome sequencing identifies novel mutations in malaria parasites resistant to artesunate (ATN) and to ATN + mefloquine combination.

The global evolution of resistance to Artemisinin-based Combination Therapies (ACTs) by malaria parasites, will severely undermine our ability to control this devastating disease. Here, we have used whole genome sequencing to characterize the genetic variation in the experimentally evolved Plasmodium chabaudi parasite clone AS-ATNMF1, which is resistant to artesunate + mefloquine. Five novel single nucleotide polymorphisms (SNPs) were identified, one of which was a previously undescribed E738K mutation in a 26S proteasome subunit that was selected for under artesunate pressure (in AS-ATN) and retained in AS-ATNMF1. The wild type and mutated three-dimensional (3D) structure models and molecular dynamics simulations of the P. falciparum 26S proteasome subunit Rpn2 suggested that the E738K mutation could change the toroidal proteasome/cyclosome domain organization and change the recognition of ubiquitinated proteins. The mutation in the 26S proteasome subunit may therefore contribute to altering oxidation-dependent ubiquitination of the MDR-1 and/or K13 proteins and/or other targets, resulting in changes in protein turnover. In light of the alarming increase in resistance to artemisin derivatives and ACT partner drugs in natural parasite populations, our results shed new light on the biology of resistance and provide information on novel molecular markers of resistance that may be tested (and potentially validated) in the field.

Genomic surveillance of Anopheles mosquitoes on the Bijagós Archipelago using custom targeted amplicon sequencing identifies mutations associated with insecticide resistance

BackgroundInsecticide resistance is reducing the efficacy of vector control interventions, consequently threatening efforts to control vector-borne diseases, including malaria. Investigating the prevalence of molecular markers of resistance is a useful tool for monitoring the spread of insecticide resistance in disease vectors. The Bijagós Archipelago (Bijagós) in Guinea-Bissau is a region of stable malaria transmission where insecticide-treated nets are the mainstay for malaria control. However, the prevalence of molecular markers of insecticide resistance in malaria vectors is not well understood.MethodsA total of 214 Anopheles mosquitoes were analysed from 13 islands across the Bijagós. These mosquitoes were collected using CDC light traps in November 2019, during the peak malaria transmission season. High-throughput multiplex amplicon sequencing was used to investigate the prevalence of 17 different molecular markers associated with insecticide resistance in four genes: vgsc, rdl, ace1 and gste2.ResultsOf the 17 screened mutations, four were identified in mosquitoes from the Bijagós: vgsc L995F (12.2%), N1570Y (6.2%) and A1746S (0.7%) and rdl A269G (1.1%). This study is the first to report the L995F knock-down resistance (kdr)-west allele in Anopheles melas on the Archipelago. An additional eight non-synonymous single-nucleotide polymorphisms were identified across the four genes which have not been described previously. The prevalences of the vgsc L995F and N1570Y mutations were higher on Bubaque Island than on the other islands in this study; Bubaque is the most populous island in the archipelago, with the greatest population mobility and connection to continental Guinea-Bissau.ConclusionsThis study provides the first surveillance data for genetic markers present in malaria vectors from islands across the Bijagós Archipelago. Overall prevalence of insecticide resistance mutations was found to be low. However, the identification of the vgsc L995F and N1570Y mutations associated with pyrethroid resistance warrants further monitoring. This is particularly important as the mainstay of malaria control on the islands is the use of pyrethroid insecticide-treated nets.Graphical

Seasonal malaria chemoprevention in a context of high presumed sulfadoxine-pyrimethamine resistance: malaria morbidity and molecular drug resistance profiles in South Sudan

BackgroundSeasonal malaria chemoprevention (SMC) using sulfadoxine-pyrimethamine plus amodiaquine (SP-AQ), is a community-based malaria preventive strategy commonly used in the Sahel region of sub-Saharan Africa. However, to date it has not been implemented in East Africa due to high SP resistance levels. This paper is a report on the implementation of SMC outside of the Sahel in an environment with a high level of presumed SP-resistance: five cycles of SMC using SPAQ were administered to children 3–59 months during a period of high malaria transmission (July–December 2019) in 21 villages in South Sudan.MethodsA population-based SMC coverage survey was combined with a longitudinal time series analysis of health facility and community health data measured after each SMC cycle. SMC campaign effectiveness was assessed by Poisson model. SPAQ molecular resistance markers were additionally analysed from dried blood spots from malaria confirmed patients.ResultsIncidence of uncomplicated malaria was reduced from 6.6 per 100 to an average of 3.2 per 100 after SMC administration (mean reduction: 53%) and incidence of severe malaria showed a reduction from 21 per 10,000 before SMC campaign to a mean of 3.3 per 10,000 after each cycle (mean reduction: 84%) in the target group when compared to before the SMC campaign. The most prevalent molecular haplotype associated with SP resistance was the IRNGE haplotype (quintuple mutant, with 51I/59R/108N mutation in pfdhfr + 437G/540E in pfdhps). In contrast, there was a low frequency of AQ resistance markers and haplotypes resistant to both drugs combined (< 2%).ConclusionsThe SMC campaign was effective and could be used as an additional preventive tool in seasonal malaria settings outside of the Sahel, especially in areas where access to health care is unstable. Malaria case load reduction was observed despite the high level of resistance to SP.

EP12.01-29 Study on the Molecular Markers of Resistance in the First-line Treatment of NSCLC with EGFR Positive by Aumolertinib

EP12.01-29 Study on the Molecular Markers of Resistance in the First-line Treatment of NSCLC with EGFR Positive by Aumolertinib

Prevalence of molecular markers of resistance to sulfadoxine–pyrimethamine before and after community delivery of intermittent preventive treatment of malaria in pregnancy in sub-Saharan Africa: a multi-country evaluation

The effectiveness of community delivery of intermittent preventive treatment (C-IPT) of malaria in pregnancy (IPTp) with sulfadoxine-pyrimethamine has been evaluated in selected areas of the Democratic Republic of the Congo, Madagascar, Mozambique, and Nigeria. We aimed to assess the effect of C-IPTp on the potential development of Plasmodium falciparum resistance to sulfadoxine-pyrimethamine, since it could threaten the effectiveness of this strategy. Health facility-based cross-sectional surveys were conducted at baseline and 3 years after C-IPTp implementation in two neighbouring areas per country, one with C-IPTp intervention, and one without, in the four project countries. Dried blood spots from children under five years of age with clinical malaria were collected. Sulfadoxine-pyrimethamine resistance-associated mutations of the P falciparum dhfr (Asn51Ile/Cys59Arg/Ser108Asn/Ile164Leu) and dhps (Ile431Val/Ser436Ala/Ala437Gly/Lys540Glu/Ala581Gly/Ala613Ser) genes were analysed. 2536 children were recruited between June 19 and Oct 10, 2018, during baseline surveys. Endline surveys were conducted among 2447 children between July 26 and Nov 30, 2021. In the Democratic Republic of the Congo, the dhfr/dhps IRNI/ISGEAA inferred haplotype remained lower than 10%, from 2% (5 of 296) at baseline to 8% (24 of 292) at endline, and from 3% (9 of 300) at baseline to 6% (18 of 309) at endline surveys in intervention and non-intervention areas respectively with no significant difference in the change between the areas. In Mozambique, the prevalence of this haplotype remained stable at over 60% (194 [64%] of 302 at baseline to 194 [64%] of 303 at endline, and 187 [61%] of 306 at baseline to 183 [61%] of 301 in endline surveys, in non-intervention and intervention areas respectively). No isolates harbouring the dhps ISGEAA genotype were found in Nigeria. In Madagascar, only five isolates with this haplotype were found in the non-intervention area (2 [>1%] of 300 at baseline and 3 [1%] of 300 at endline surveys). No isolates were found carrying the dhps ISGEGA genotype. C-IPTp did not increase the prevalence of molecular markers associated with sulfadoxine-pyrimethamine resistance after three years of programme implementation. These findings reinforce C-IPTp as a strategy to optimise the control of malaria during pregnancy, and support the WHO guidelines for prevention of malaria in pregnancy. UNITAID [2017-13-TIPTOP].

Surveillance of drug resistance molecular markers in Plasmodium vivax before and after introduction of dihydroartemisinin and piperaquine in Thailand: 2009-2019.

Resistance to antimalarial drugs is a serious issue around the world. Widespread Plasmodium vivax and P. falciparum coinfections are commonly found in Thailand. Dihydroartemisinin and piperaquine (DHA-PPQ) have been used as first-line treatments for P. falciparum since 2015, and chloroquine (CQ) and primaquine (PQ) have remained first-line drugs for P. vivax for more than 60 years. Coinfections may lead parasites to evolve with regard to genetics under selective drug pressure. This study is aimed at investigating genes linked to antimalarial resistance in P. vivax before and after introduction of DHA-PPQ as a new drug regimen in Thailand. A total of 400 P. vivax isolates were collected from samples along the Thai-Myanmar and Thai-Malaysian borders before (2009-2015) and after (2016-2019) introduction of DHA-PPQ. Genomic DNA of P. vivax was obtained and subjected to analysis of five drug resistance-associated genes (Pvdhfr, Pvdhps, Pvmdr1, Pvcrt-o, and PvK12) by nested polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and nucleotide sequencing. A high prevalence of Pvdhfr was found in both endemic areas over the period. The quadruple (57I/58R/61M/117T) Pvdhfr haplotype was predominant in both periods in both endemic areas. Although the wild-type haplotype of Pvdhps was predominant in Thai-Malaysian isolates in both periods, a single mutant haplotype (383G) was dominant in Thai-Myanmar isolates during both periods. A low prevalence of the Pvmdr1 976F mutation was found in both periods among Thai-Myanmar isolates. A significant decrease in Pvmdr1 976F was identified in Thai-Malaysian isolates from the second period (p < 0.01). Only one nonsynonymous mutation of Pvcrt-o (193E) and one synonymous mutation of PvK12 (R584) were detected in four isolates (4.7%) and one isolate (0.5%) in the first period among Thai-Myanmar isolates, respectively. Thus, with limited clinical efficacy data, the low prevalence of drug-resistance markers may suggest that there is a low prevalence of P. vivax-resistant strains and that the current drug regimen for P. vivax is still effective for treating this P. vivax parasite population. Continued surveillance of antimalarial drug resistance markers and monitoring of clinical drug efficacy should be conducted for epidemiological and policy implications.

Molecular characterization of resistance to immune checkpoint inhibitor and chemotherapy treatment in advanced non-small cell lung cancer.

9034 Background: Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and a leading cause of cancer death. Standard of care (SOC) treatment for advanced NSCLC patients include immune checkpoint inhibitors (ICI) and/or chemotherapy (CT). However, the majority of patients do not respond to SOC therapy. Mechanisms of resistance and how they differ across treatments are not fully understood. We characterized molecular markers of resistance for advanced NSCLC patients with ICI monotherapy, CT, or combination ICI-CT. Methods: Advanced stage III or IV NSCLC patients with adenocarcinoma histology who received at least 21 days of ICI monotherapy, CT, or combination ICI-CT were identified from a de-identified, multimodal real-world database from Tempus Labs, Inc. All patients had a tissue biopsy taken within 100 days prior to the specified therapy start and all samples underwent next generation sequencing with the Tempus xT assays (DNA-seq of 596-648 genes at 500x coverage and whole transcriptome RNA-seq). Resistance to therapy was defined as having time to next treatment &lt; 90 days, or disease progression or death within 120 days of therapy initiation. We characterized the clinical and molecular characteristics of patients and compared differential expression of genes by resistance status for each cohort. Results: A total of 365 patients were included in the study (ICI = 121, CT = 122, ICI-CT = 122). Across cohorts, 30% of patients were identified as having resistance to treatment (ICI = 35%, CT = 23%, ICI-CT = 32%). 53% of patients were &gt; 65 years (median [range] = 67 [25- 88]), and 53% were males. Most patients were white (63%) and had a history of tobacco use (85% current or former smoker). At the time of biopsy, most patients were diagnosed with metastatic NSCLC (Stage IV = 73%) and had received no previous lines of therapy (92%). The alteration frequencies of known NSCLC oncogenic driver mutations including TP53 (65%), KRAS (42%), STK11 (14%), KEAP1 (11%), and EGFR (9%) were broadly consistent with previous characterizations of NSCLC. Validated biomarkers of response to ICI treatment such as Tumor Mutation Burden, IFN-γ signaling, PD-1, and PD-L1 expression were negatively associated with resistance to ICI treatment. Across the ICI, ICI-CT, and CT cohorts, 253, 153, and 178 genes, respectively, were significantly upregulated (FDR &lt; 0.05, LogFC &gt; 1) in patients with resistance to treatment. Of the 550 distinct genes correlated to resistance across treatments, 95% were unique to each cohort, and only 32 genes (5%) were shared across more than one treatment cohort. Conclusions: Distinct molecular characteristics were associated with resistance to ICI and/or CT treatment. This suggests that mechanisms of resistance to ICI monotherapy, ICI-CT, or CT could be separate and unique. Further research can provide valuable insights for novel therapies or combinations.

Prevalence of mutations in the cysteine desulfurase IscS (Pfnfs1) gene in recurrent Plasmodium falciparum infections following artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) treatment in Matayos, Western Kenya

BackgroundMalaria remains a public health concern globally. Resistance to anti-malarial drugs has consistently threatened the gains in controlling the malaria parasites. Currently, artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) are the treatment regimens against Plasmodium falciparum infections in many African countries, including Kenya. Recurrent infections have been reported in patients treated with AL or DP, suggesting the possibility of reinfection or parasite recrudescence associated with the development of resistance against the two therapies. The Plasmodium falciparum cysteine desulfurase IscS (Pfnfs1) K65 selection marker has previously been associated with decreased lumefantrine susceptibility. This study evaluated the frequency of the Pfnfs1 K65 resistance marker and associated K65Q resistant allele in recurrent infections collected from P. falciparum-infected individuals living in Matayos, Busia County, in western Kenya.MethodsArchived dried blood spots (DBS) of patients with recurrent malaria infection on clinical follow-up days after treatment with either AL or DP were used in the study. After extraction of genomic DNA, PCR amplification and sequencing analysis were employed to determine the frequencies of the Pfnfs1 K65 resistance marker and K65Q mutant allele in the recurrent infections. Plasmodium falciparum msp1 and P. falciparum msp2 genetic markers were used to distinguish recrudescent infections from new infections.ResultsThe K65 wild-type allele was detected at a frequency of 41% while the K65Q mutant allele was detected at a frequency of 22% in the recurrent samples. 58% of the samples containing the K65 wild-type allele were AL treated samples and while 42% were DP treated samples. 79% of the samples with the K65Q mutation were AL treated samples and 21% were DP treated samples. The K65 wild-type allele was detected in three recrudescent infections (100%) identified from the AL treated samples. The K65 wild-type allele was detected in two recrudescent DP treated samples (67%) while the K65Q mutant allele was identified in one DP treated (33%) recrudescent sample.ConclusionsThe data demonstrate a higher frequency of the K65 resistance marker in patients with recurrent infection during the study period. The study underscores the need for consistent monitoring of molecular markers of resistance in regions of high malaria transmission.

Recent Advances in Chemotherapeutics for Leishmaniasis: Importance of the Cellular Biochemistry of the Parasite and Its Molecular Interaction with the Host.

Leishmaniasis, a category 1 neglected protozoan disease caused by a kinetoplastid pathogen called Leishmania, is transmitted through dipteran insect vectors (phlebotomine, sand flies) in three main clinical forms: fatal visceral leishmaniasis, self-healing cutaneous leishmaniasis, and mucocutaneous leishmaniasis. Generic pentavalent antimonials have long been the drug of choice against leishmaniasis; however, their success is plagued with limitations such as drug resistance and severe side effects, which makes them redundant as frontline therapy for endemic visceral leishmaniasis. Alternative therapeutic regimens based on amphotericin B, miltefosine, and paromomycin have also been approved. Due to the unavailability of human vaccines, first-line chemotherapies such as pentavalent antimonials, pentamidine, and amphotericin B are the only options to treat infected individuals. The higher toxicity, adverse effects, and perceived cost of these pharmaceutics, coupled with the emergence of parasite resistance and disease relapse, makes it urgent to identify new, rationalized drug targets for the improvement in disease management and palliative care for patients. This has become an emergent need and more relevant due to the lack of information on validated molecular resistance markers for the monitoring and surveillance of changes in drug sensitivity and resistance. The present study reviewed the recent advances in chemotherapeutic regimens by targeting novel drugs using several strategies including bioinformatics to gain new insight into leishmaniasis. Leishmania has unique enzymes and biochemical pathways that are distinct from those of its mammalian hosts. In light of the limited number of available antileishmanial drugs, the identification of novel drug targets and studying the molecular and cellular aspects of these drugs in the parasite and its host is critical to design specific inhibitors targeting and controlling the parasite. The biochemical characterization of unique Leishmania-specific enzymes can be used as tools to read through possible drug targets. In this review, we discuss relevant metabolic pathways and novel drugs that are unique, essential, and linked to the survival of the parasite based on bioinformatics and cellular and biochemical analyses.

Drug Resistance Molecular Markers of Plasmodium falciparum and Severity of Malaria in Febrile Children in the Sentinel Site for Malaria Surveillance of Melen in Gabon: Additional Data from the Plasmodium Diversity Network African Network.

The objective of this study was to analyze the relationship between the frequency of artemisinin-based combination (ACT) drug resistance molecular markers and clinical forms of P. falciparum malaria and parasitemia. A cross-sectional study was carried out between January and April 2014 at the Operational Clinical Research Unit of Melen in febrile children aged 12 to 240 months with a Plasmodium sp. infection. A total of 3 mL of peripheral blood collected from an EDTA tube was used for leukocyte depletion. DNA mutation detection was performed by next generation sequencing (NGS). A total of 1075 patients were screened for malaria. Among them, 384 had a Plasmodium infection. P. falciparum mono-infection was found in 98.9% of the patients. Pfcrt-326T mutation was found in all isolates, while 37.9% had Pfmdr2-484I mutant allele. The highest median parasite densities were found in patients infected by parasites carrying the CVIET haplotype of the Pfcrt gene. The different genetic profiles found here, and their variations according to clinical and biological signs of severe malaria, are additional arguments for the surveillance of P. falciparum strains.

QTL analysis of drought tolerance traits in rice during the vegetative growth period

Drought affects the growth and development of rice from direct seeding or transplanting to maturity in northern China. It is important to carry out QTL mapping for drought tolerance to improve rice breeding. In this study, a rice RIL population consisting of 253 families was constructed by crossing Miyang 23 with Jileng 1. The leaf rolling index (LRI), leaf withering degree (LWD) and leaf chlorophyll content (LCC) were measured over three years, and QTLs for these traits were mapped. The results showed that LRI, LWD and LCC were all quantitative traits controlled by multiple genes. A total of 28 QTLs related to three drought-resistant indices were detected; four QTLs controlling LRI and LWD were detected on Chromosome 1. qLRI1-1 and qLWD1-4 were located in the interval of AH01001843-RM302, explaining 10.30% and 4.90% of the phenotypic variation, respectively; qLWD1-2B and qLRI-1JC were both located in RM315-S01167A, and qLWD1-2B was explained 11.15% of the phenotypic variation, while qLRI-1JC explained 7.96% of the phenotypic variation. The two QTLs located on Chromosome 5, qLWD5-1B and qLRI5, were located in CMB0526.3486-RM87. qLWD5-1B explained 6.93% of the phenotypic variation, while qLRI5 explained 8.79%-9.13% of the phenotypic variation. The two QTLs detected on Chromosome 12, qLWD12 and qLRI12, were located in the S12099-CMB1226.0 interval and explained 4.59%-7.25% of the phenotypic variation. The above QTLs, including qLWD1-2B, qLRI1-1JC, qLWD5-1B and qLRI5, have been detected many times. Future work should involve implementing in-depth fine mapping of these QTLs and using the identified markers in the drought resistance molecular marker selection breeding of rice.

Preparing for the Next Influenza Season: Monitoring the Emergence and Spread of Antiviral Resistance.

The relaxation of pandemic restrictions in 2022 has led to a reemergence of respiratory virus circulation worldwide and anticipation of substantial influenza waves for the 2022/2023 Northern Hemisphere winter. Therefore, the antiviral susceptibility profiles of human influenza viruses circulating in Germany were characterized. Between October 2019 (week 40/2019) and March 2022 (week 12/2022), nasal swabs from untreated patients with acute respiratory symptoms were collected in the national German influenza surveillance system. A total of 598 influenza viruses were isolated and analyzed for susceptibility to oseltamivir, zanamivir and peramivir, using a neuraminidase (NA) inhibition assay. In addition, next-generation sequencing was applied to assess molecular markers of resistance to NA, cap-dependent endonuclease (PA) and M2 ion channel inhibitors (NAI, PAI, M2I) in 367 primary clinical samples. Furthermore, a genotyping assay based on RT-PCR and pyrosequencing to rapidly assess the molecular resistance marker PA-I38X in PA genes was designed and established. While NAI resistance in the strict sense, defined by a ≥ 10-fold (influenza A) or ≥5-fold (influenza B) increase of NAI IC50, was not detected, a subtype A(H1N1)pdm09 isolate displayed 2.3- to 7.5-fold IC50 increase for all three NAI. This isolate carried the NA-S247N substitution, which is known to enhance NAI resistance induced by NA-H275Y. All sequenced influenza A viruses carried the M2-S31N substitution, which confers resistance to M2I. Of note, one A(H3N2) virus displayed the PA-I38M substitution, which is associated with reduced susceptibility to the PAI baloxavir marboxil. Pyrosequencing analysis confirmed these findings in the original clinical specimen and in cultured virus isolate, suggesting sufficient replicative fitness of this virus mutant. Over the last three influenza seasons, the vast majority of influenza viruses in this national-level sentinel were susceptible to NAIs and PAIs. These findings support the use of antivirals in the upcoming influenza season.

Different In Vitro Drug Susceptibility Profile of Plasmodium falciparum Isolates from Two Adjacent Areas of Northeast Myanmar and Molecular Markers for Drug Resistance.

The Greater Mekong Subregion (GMS) is the epicenter of antimalarial drug resistance. We determined in vitro susceptibilities to 11 drugs of culture-adapted Plasmodium falciparum isolates from adjacent areas (Laiza and Muse) along the China−Myanmar border. Parasites from this region were highly resistant to chloroquine and pyrimethamine but relatively sensitive to other antimalarial drugs. Consistently, the Dd2-like pfcrt mutations were fixed or almost fixed in both parasite populations, and new mutations mediating piperaquine resistance were not identified. Similarly, several mutations related to pfdhfr and pfdhps were also highly prevalent. Despite their geographical proximity, malaria parasites from Laiza showed significantly higher in vitro resistance to artemisinin derivatives, naphthoquine, pyronaridine, lumefantrine, and pyrimethamine than parasites from Muse. Likewise, the pfdhfr N51I, pfdhps A581G, pfmrp1 H785N, and pfk13 F446I mutations were significantly more frequent in Laiza than in Muse (p < 0.05). For the pfmdr1 mutations, Y184F was found only in Laiza (70%), whereas F1226Y was identified only in Muse (31.8%). Parasite isolates from Laiza showed a median RSA value of 5.0%, significantly higher than the 2.4% in Muse. Altogether, P. falciparum parasite populations from neighboring regions in the GMS may diverge substantially in their resistance to several antimalarial drugs. This information about different parasite populations will guide antimalarial treatment policies to effectively manage drug resistance during malaria elimination.

The dynamic changes of genes revealed that persistently overexpressed genes drive the evolution of cyflumetofen resistance in Tetranychus cinnabarinus.

Changes in gene expression are associated with the evolution of pesticide resistance in arthropods. In this study, transcriptome sequencing was performed in 3 different resistance levels (low, L; medium, M; and high, H) of cyflumetofen-resistant strain (YN-CyR). A total of 1685 genes, including 97 detoxification enzyme genes, were upregulated in all 3 stages, of which 192 genes, including 11 detoxification enzyme genes, showed a continuous increase in expression level with resistance development (L to H). RNA interference experiments showed that overexpression of 7 genes (CYP392A1, TcGSTd05, CCE06, CYP389A1, TcGSTz01, CCE59, and CYP389C2) is involved in the development of cyflumetofen resistance in Tetranychus cinnabarinus. The recombinant CYP392A1 can effectively metabolize cyflumetofen, while CCE06 can bind and sequester cyflumetofen in vitro. We compared 2 methods for rapid screening of resistance molecular markers, including short-term induction and 1-time high-dose selection. Two detoxification enzyme genes were upregulated in the field susceptible strain (YN-S) by induction with 20% lethal concentration (LC20 ) of cyflumetofen. However, 16 detoxification enzyme genes were upregulated by 1-time selection with LC80 of cyflumetofen. Interestingly, the 16 genes were overexpressed in all 3 resistance stages. These results indicated that 1685 genes that were upregulated at the L stage constituted the basis of cyflumetofen resistance, of which 192 genes in which upregulation continued to increase were the main driving force for the development of resistance. Moreover, the 1-time high-dose selection is an efficient way to rapidly obtain the resistance-related genes that can aid in the development of resistance markers and resistance management in mites.

Molecular assays for determining sulphadoxine-pyrimethamine drug resistance in India: a systematic review.

A plethora of studies analyse the molecular markers of drug resistance and hence help in guiding the evidence-based malaria treatment policies in India. For reporting mutations, a number of techniques including DNA sequencing, restriction-fragment length polymorphism and mutation-specific polymerase chain reaction have been employed across numerous studies, including variations in the methodology used. However, there is no sufficient data from India comparing these methods as well as report the prevalence of polymorphisms in SP drug resistance molecular markers independently using such methods. Therefore, all data from Indian studies available for molecular marker studies of Plasmodium falciparum drug resistance to sulphadoxine-pyrimethamine was gathered, and a systematic review was performed. This systematic review identifies the molecular methods in use in India and compares each method for detecting sulphadoxine-pyrimethamine drug resistance marker. To delay the spread of drug-resistant parasite strains, a simplified and standardized molecular method is much needed which can be obtained by analysing the performance of each method in use and answering the necessity of newer methodological approaches.

The prevalence of molecular markers of resistance to sulfadoxine-pyrimethamine among pregnant women at first antenatal clinic attendance and delivery in the forest-savannah area of Ghana.

Intermittent preventive treatment during pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) is used to prevent malaria and associated unfavorable maternal and foetal outcomes in pregnancy in moderate to high malaria transmission areas. Effectiveness of IPTp-SP is, however, threatened by mutations in the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes which confer resistance to pyrimethamine and sulfadoxine, respectively. This study determined the prevalence of molecular markers of SP resistance among pregnant women in a high malaria transmission area in the forest-savannah area of Ghana. Genomic DNA was extracted from 286 P. falciparum-positive dried blood spots obtained from pregnant women aged ≥18 years (255 at first Antenatal Care (ANC) clinic visit and 31 at delivery from 2017 to 2019) using Chelex 100. Mutations in Pfdhfr and Pfdhps genes were detected using molecular inversion probes and next generation sequencing. In the Pfdhfr gene, single nucleotide polymorphisms (SNPs) were detected in 83.1% (157/189), 92.0% (173/188) and 91.0% (171/188) at codons 51, 59, and 108 respectively in samples collected at first ANC visit, while SNPs were detected in 96.6 (28/29), 96.6% (28/29) and 96.8% (30/31) in isolates collected at delivery. The Pfdhfr triple mutant N51I, C59R and S108N (IRN) was carried by 80.5% (128/159) and 96.5% (28/29) of the typed isolates collected at ANC visit and at delivery respectively. In the Pfdhps gene, SNPs were detected in 0.6% (1/174), 76.2% (138/181), 33.2% (60/181), 1.2% (2/174), 0% (0/183), and 16.6% (27/173) at codons 431, 436, 437, 540, 581 and 613 respectively in samples collected at ANC, and 0% (0/25), 72% (18/25), 40% (10/25), 3.6% (1/25), 0% (0/29) and 7.4% (2/27) in samples collected at delivery. Quadruple mutant Pfdhfr N51I, C59R, and S108N + Pfdhps A437G (IRN-GK) was present in 25.8% (33/128) and 34.8% (8/23) of isolates at ANC and at delivery respectively. Quintuple mutant alleles Pfdhfr N51I, C59R, and S108N + Pfdhps A437G and K540E (IRN-GE) were detected in 0.8% (1/128) and 4.4% (1/23) of samples collected at ANC and at delivery respectively. No mutations were identified at Pfdhfr codons 16 or 164 or Pfdhps 581. There is a high prevalence of Pfdhfr triple mutant P. falciparum infections among pregnant women in the study area. However, prevalence of the combined Pfdhfr/Pfdhps quadruple and quintuple mutants IRN-GK and IRN-GE respectively prior to commencement of IPTp-SP were low, and no Pfdhps A581G mutant was detected, indicating that SP is still likely to be efficacious for IPTp-SP in the forest-savannah area in the middle belt of Ghana.

Screening a collection of local and foreign varieties of Solanum lycopersicum L. in Kazakhstan for genetic markers of resistance against three tomato viruses

The tomato is one of the most important vegetable crops. The successful development of tomato cultures in Kazakhstan depends on the implementation of intensive agricultural methods, including breeding and selecting for new tomato varieties resistant to plant pathogens. Common tomato viruses, although not detected in our country to date, may potentially have a deleterious impact on agriculture if allowed to spread. The implementation of tomato breeding programs based on molecular markers of resistance is therefore an important preventive measure for protecting the agriculture and food safety of Kazakhstan. In the present work, we used nine molecular markers associated with resistance to three tomato viruses, i.e., tomato mosaic virus (ToMV), tomato spot wilt virus (TSWV), and tomato yellow leaf curl virus (TYLCV), to test the local breeding collection for the presence of genetic resistance factors. Two tomato varieties, ‘Zhiraf’ (Russia) and ‘Sunnik’ (Armenia), were revealed to possess the resistant allele marker PrRuG86-151 against ToMV; three hybrid forms had the same allele in the heterozygous state. One hybrid, based on the ‘Mirsini’ F1 variety from the Netherlands, had resistance to TSWV, which was confirmed by four markers: NCSw003, NCSw007, NCSw011, and NCSw012. Two cultivars, ‘Nicola’ and ‘Malinovyi Slon’ (Russia), and the local hybrid based on ‘Yarkiy Rumyanets’ had two to three resistant alleles of markers based on locus Tm-3 of resistance to TYLCV. The obtained results have demonstrated that the collection of tomato varieties involved in breeding programs in Kazakhstan lacks well-known genetic resistance factors to the considered tomato viruses. Thus, the prospective breeding programs require introduction of known resistant genetic resources to establish resistance to viruses using marker-assisted selection.