Analysis of the Knockdown Resistance Locus (kdr) in Anopheles stephensi, An. arabiensis, and Culex pipiens s.l. for Insight Into the Evolution of Target-site Pyrethroid Resistance in Eastern Ethiopia

The malaria vector, Anopheles stephensi, which is typically restricted to South Asia and the Middle East, was recently detected in the Horn of Africa. Addressing the spread of this vector could involve integrated vector control that considers the status of insecticide resistance of multiple vector species in the region. Previous reports indicate that the knockdown resistance mutations (kdr) in the voltage-gated sodium channel (vgsc) are absent in both pyrethroid-resistant and pyrethroid-sensitive An. stephensi in eastern Ethiopia; however, similar information about other vector species in the same areas is limited. In this study, kdr and the neighboring intron were analyzed in An. stephensi, An. arabiensis, and Culex pipiens s.l. collected between 2016 and 2017 to determine the evolutionary history of kdr in eastern Ethiopia. A sequence analysis revealed that all of Cx. pipiens s.l. (N = 42) and 71.6% of the An. arabiensis (N = 67) carried kdr L1014F, which is known to confer target-site pyrethroid resistance. Intronic variation was only observed in An. stephensi (six segregating sites, three haplotypes), which was previously shown to have no kdr mutations. In addition, no evidence of non-neutral evolutionary processes was detected at the An. stephensi kdr intron, thereby further supporting the target-site mechanism not being a major resistance mechanism in this An. stephensi population. Overall, these results show key differences in the evolution of target-site pyrethroid/dichlorodiphenyltrichloroethane resistance mutations in populations of vector species from the same region. Variations in insecticide resistance mechanism profiles between eastern Ethiopian mosquito vectors may lead to different responses to insecticides used in integrated vector control.

Role of ctDNA in Breast Cancer

Breast cancer is currently classified by immunohistochemistry. However, technological advances in the detection of circulating tumor DNA (ctDNA) have made new options available for diagnosis, classification, biological knowledge, and treatment selection. Breast cancer is a heterogeneous disease and ctDNA can accurately reflect this heterogeneity, allowing us to detect, monitor, and understand the evolution of the disease. Breast cancer patients have higher levels of circulating DNA than healthy subjects, and ctDNA can be used for different objectives at different timepoints of the disease, ranging from screening and early detection to monitoring for resistance mutations in advanced disease. In early breast cancer, ctDNA clearance has been associated with higher rates of complete pathological response after neoadjuvant treatment and with fewer recurrences after radical treatments. In metastatic disease, ctDNA can help select the optimal sequencing of treatments. In the future, thanks to new bioinformatics tools, the use of ctDNA in breast cancer will become more frequent, enhancing our knowledge of the biology of tumors. Moreover, deep learning algorithms may also be able to predict breast cancer evolution or treatment sensitivity. In the coming years, continued research and the improvement of liquid biopsy techniques will be key to the implementation of ctDNA analysis in routine clinical practice.

Effects of the Pathological E200K Mutation on Human Prion Protein: A Computational screening and Molecular Dynamic approach

The Human Prion protein gene (PRNP) is mapped to short arm of chromosome 20 (20pter-12). Prion disease is associated with mutations in the Prion Protein encoding gene sequence. The mutations that occur in the prion protein could be divided into two types based on their influence on pathogenic potential: 1. Mutations that cause disease. 2. Disease-resistance mutations. Earlier studies found that the mutation G127V in the PRNP increases protein stability, whereas the mutation E200K, which has the highest mutation rate in the Prion protein, causes Creutzfeldt–Jakob disease (CJD) in humans and induces protein aggregation. We used a variety of bioinformatic algorithms, including SIFT, PolyPhen, I-Mutant, PhD-SNP, and SNP&GO, to predict the association of the E200K mutation with Prion disease. MD simulation is performed and graphs for RMSD, RMSF, Rg, DSSP, PCA, porcupine and FEL are generated to confirm and prove the stability of the wild type and mutant protein structures. The protein is analyzed for aggregation, and the results indicates more fluctuations in the protein structure during the simulation by the E200K mutation, however the G127V mutation makes protein structure stable against aggregation during the simulation.

Rapid Detection of Quinolone Resistance Mutations in gyrA of Helicobacter pylori by Real-Time PCR

The treatment of infections by the gastric pathogen Helicobacter pylori (H. pylori) has become more difficult due to increased rates of resistances against various antibiotics. Typically, atriple therapy, employing a combination of at least two antibiotics and a proton pump inhibitor, is used to cure H. pylori infections. In case of first-line therapy failure, quinolones are commonly applied in a second-line therapy. To prevent second-line treatment failures, we developed an improved method to detect the most common quinolone-resistance mutations located in the quinolone-resistance-determining region (QRDR) of the bacterial gyrA gene. Biopsy material from the gastric mucosa of infected patients was used to identify quinolone-resistant strains before the onset of drug administration. Two different wild-type and six mutant QRDR sequences were included. Melting curve analyses were performed with corresponding gyrA plasmid DNAs using a real-time polymerase chain reaction (RT-PCR) assay. By applying a combination of only two different fluorescent probes, this assay allows wild-type sequences to be unambiguously distinguished from all known mutant QRDR sequences of H. pylori. Next, the Tm values of patient DNAs were established, and the genotypes were confirmed by sequencing. Thus, quinolone-resistant H. pylori strains can be easily and quickly diagnosed before treatment, which will help to avoid the administration of ineffective drug regimes.

Resting Behavior of Malaria Vectors in Ghana and Its Implication on Vector Control.

Background: In Sub-Saharan Africa, there is widespread use of long-lasting insecticidal nets (LLINs) and Indoor residual spraying (IRS) to help control the density of malaria vectors and decrease the incidence of malaria in communities. An understanding of the interactions between increased insecticide use and resting behaviour patterns of malaria mosquitoes is important for an effective vector control programme. This study was carried out to investigate the resting behavior, host preference and infection with Plasmodium falciparum of malaria vectors in Ghana in the context of increasing insecticide resistance in malaria vectors in sub-saharan Africa.Methods: Indoor and outdoor resting Anopheline mosquitoes were sampled during the dry and rainy seasons in five sites that were in 3 ecological landscapes [Sahel savannah (Kpalsogou, Pagaza, Libga), Coastal savannah (Anyakpor) and Forest (Konongo) zones] using pyrethrum spray catches (PSC), mechanical aspiration (Prokopack) for indoor collections, pit shelter and Prokopack for outdoor collections. PCR based molecular diagnostics were used to determine mosquito speciation, genotype for knockdown resistance mutations (L1014S and L1014F), G119S Ace-1 mutation, specific host blood meal origins and sporozoite infection in field collected mosquitoes.Results: Anopheles gambiae s. l. was the predominant species (89.95%, n = 1,718), followed by An. rufipes (8.48%, n=162), and An. funestus s. l. (1.57%, n = 30). Sibling species of the Anopheles gambiae revealed An. coluzzii accounted for 63% (95% CI: 57.10 – 68.91), followed by An. gambiae s. s [27% (95% CI: 21.66 – 32.55)], and An. arabiensis [9% (95% CI: 6.22 – 13.57)]. The mean resting density of An. gambiae s. l. was higher outdoors (79.63%; 1,368/1,718) than indoors (20.37%; 350/1,718) (z = -4.815, p< 0.0001). The kdr west L1014F and the Ace-1 mutations were highest in indoor resting An. coluzzii and An. gambiae in the sahel-savannah sites compared to the forest and coastal savannah sites. Overall, the blood meal analyses revealed a large proportion of the malaria vectors preferred feeding on humans (70.2 %) than animals (29.8%) in all sites. The sporozoite rates was only detected in indoor resting An. coluzzii from the sahel savannah (5.0%) and forest (2.5%) zones.Conclusion: The study reports high outdoor resting densities of An. gambiae and An. coluzzii with high kdr west mutation frequencies, and persistence of malaria transmission indoors despite the use of LLINs and IRS. Continuous monitoring of changes in resting behavior of mosquitoes and implementation of complementary malaria control interventions are needed to target outdoor resting Anopheles mosquitoes in Ghana.

Epidemiology and Transmitted HIV-1 Drug Resistance among Treatment-Naïve Individuals in Israel, 2010–2018

Despite the low prevalence of HIV-1 in Israel, continuous waves of immigration may have impacted the local epidemic. We characterized all people diagnosed with HIV-1 in Israel in 2010–2018. The demographics and clinical data of all individuals (n = 3639) newly diagnosed with HIV-1 were retrieved. Subtypes, transmitted drug-resistance mutations (TDRM), and phylogenetic relations, were determined in >50% of them. In 39.1%, HIV-1 transmission was through heterosexual contact; 34.3% were men who have sex with men (MSM); and 10.4% were people who inject drugs. Many (>65%) were immigrants. Israeli-born individuals were mostly (78.3%) MSM, whereas only 9% of those born in Sub-Saharan Africa (SSA), Eastern Europe and Central Asia (EEU/CA), were MSM. The proportion of individuals from SSA decreased through the years 2010–2018 (21.1% in 2010–2012; 16.8% in 2016–2018) whereas those from EEU/CA increased significantly (21% in 2010–2012; 27.8% in 2016–2018, p < 0.001). TDRM were identified in 12.1%; 3.7, 3.3 and 6.6% had protease inhibitors (PI), nucleotide reverse transcriptase inhibitors (NRTI), and non-nucleoside reverse transcriptase inhibitors (NNRTI) TDRM, respectively, with the overall proportion remaining stable in the studied years. None had integrase TDRM. Subtype B was present in 43.9%, subtype A in 25.2% (A6 in 22.8 and A1 in 2.4%) and subtype C in 17.1% of individuals. Most MSM had subtype B. Subtype C carriers formed small clusters (with one unexpected MSM cluster), A1 formed a cluster mainly of locally-born patients with NNRTI mutations, and A6 formed a looser cluster of individuals mainly from EEU. Israelis, <50 years old, carrying A1, had the highest risk for having TDRM. In conclusion, an increase in immigrants from EEU/CA and a decrease in those from SSA characterized the HIV-1 epidemic in 2010–2018. Baseline resistance testing should still be recommended to identify TDRM, and improve surveillance and care.

Revisiting Antibiotic Resistance: Mechanistic Foundations to Evolutionary Outlook

Antibiotics are the pivotal pillar of contemporary healthcare and have contributed towards its advancement over the decades. Antibiotic resistance emerged as a critical warning to public wellbeing because of unsuccessful management efforts. Resistance is a natural adaptive tool that offers selection pressure to bacteria, and hence cannot be stopped entirely but rather be slowed down. Antibiotic resistance mutations mostly diminish bacterial reproductive fitness in an environment without antibiotics; however, a fraction of resistant populations ‘accidentally’ emerge as the fittest and thrive in a specific environmental condition, thus favouring the origin of a successful resistant clone. Therefore, despite the time-to-time amendment of treatment regimens, antibiotic resistance has evolved relentlessly. According to the World Health Organization (WHO), we are rapidly approaching a ‘post-antibiotic’ era. The knowledge gap about antibiotic resistance and room for progress is evident and unified combating strategies to mitigate the inadvertent trends of resistance seem to be lacking. Hence, a comprehensive understanding of the genetic and evolutionary foundations of antibiotic resistance will be efficacious to implement policies to force-stop the emergence of resistant bacteria and treat already emerged ones. Prediction of possible evolutionary lineages of resistant bacteria could offer an unswerving impact in precision medicine. In this review, we will discuss the key molecular mechanisms of resistance development in clinical settings and their spontaneous evolution.

Structural basis for potent antibody neutralization of SARS-CoV-2 variants including B.1.1.529

With B.1.1.529 SARS-CoV-2 variant's rapid spread and substantially increased resistance to neutralization by vaccinee and convalescent sera, monoclonal antibodies with potent neutralization are eagerly sought. To provide insight into effective neutralization, we determined cryo-EM structures and evaluated potent receptor-binding domain (RBD) antibodies for their ability to bind and neutralize this new variant. B.1.1.529 RBD mutations altered 16% of the RBD surface, clustering on a ridge of this domain proximal to the ACE2-binding surface and reducing binding of most antibodies. Significant inhibitory activity was retained, however, by select monoclonal antibodies including A19-58.1, B1-182.1, COV2-2196, S2E12, A19-46.1, S309 and LY-CoV1404, which accommodated these changes and neutralized B.1.1.529 with IC50s between 5.1-281 ng/ml, and we identified combinations of antibodies with potent synergistic neutralization. Structure-function analyses delineated the impact of resistance mutations and revealed structural mechanisms for maintenance of potent neutralization against emerging variants.