Emergence of artemisinin-resistant Plasmodium falciparum with kelch13 C580Y mutations on the island of New Guinea

The rapid and aggressive spread of artemisinin-resistant Plasmodium falciparum carrying the C580Y mutation in the kelch13 gene is a growing threat to malaria elimination in Southeast Asia, but there is no evidence of their spread to other regions. We conducted cross-sectional surveys in 2016 and 2017 at two clinics in Wewak, Papua New Guinea (PNG) where we identified three infections caused by C580Y mutants among 239 genotyped clinical samples. One of these mutants exhibited the highest survival rate (6.8%) among all parasites surveyed in ring-stage survival assays (RSA) for artemisinin. Analyses of kelch13 flanking regions, and comparisons of deep sequencing data from 389 clinical samples from PNG, Indonesian Papua and Western Cambodia, suggested an independent origin of the Wewak C580Y mutation, showing that the mutants possess several distinctive genetic features. Identity by descent (IBD) showed that multiple portions of the mutants’ genomes share a common origin with parasites found in Indonesian Papua, comprising several mutations within genes previously associated with drug resistance, such as mdr1, ferredoxin, atg18 and pnp. These findings suggest that a P. falciparum lineage circulating on the island of New Guinea has gradually acquired a complex ensemble of variants, including kelch13 C580Y, which have affected the parasites’ drug sensitivity. This worrying development reinforces the need for increased surveillance of the evolving parasite populations on the island, to contain the spread of resistance.     

Simplified Paper Format for Detecting HIV Drug Resistance in Clinical Specimens by Oligonucleotide Ligation

Human immunodeficiency virus (HIV) is a chronic infection that can be managed by antiretroviral treatment (ART). However, periods of suboptimal viral suppression during lifelong ART can select for HIV drug resistant (DR) variants. Transmission of drug resistant virus can lessen or abrogate ART efficacy. Therefore, testing of individuals for drug resistance prior to initiation of treatment is recommended to ensure effective ART. Sensitive and inexpensive HIV genotyping methods are needed in low-resource settings where most HIV infections occur. The oligonucleotide ligation assay (OLA) is a sensitive point mutation assay for detection of drug resistance mutations in HIV pol. The current OLA involves four main steps from sample to analysis: (1) lysis and/or nucleic acid extraction, (2) amplification of HIV RNA or DNA, (3) ligation of oligonucleotide probes designed to detect single nucleotide mutations that confer HIV drug resistance, and (4) analysis via oligonucleotide surface capture, denaturation, and detection (CDD). The relative complexity of these steps has limited its adoption in resource-limited laboratories. Here we describe a simplification of the 2.5-hour plate-format CDD to a 45-minute paper-format CDD that eliminates the need for a plate reader. Analysis of mutations at four HIV-1 DR codons (K103N, Y181C, M184V, and G190A) in 26 blood specimens showed a strong correlation of the ratios of mutant signal to total signal between the paper CDD and the plate CDD. The assay described makes the OLA easier to perform in low resource laboratories.     

Structural mapping of Kelch13 mutations associated with artemisinin resistance in malaria

Mutations in Plasmodium falciparum gene kelch13 (pfkelch13) are strongly and causally associated with resistance to anti-malarial drug artemisinin, but their effects on PfKelch13 structure and function remain unclear. Utilizing the publicly available three-dimensional structure of PfKech13 (PDB ID: 4yy8), we find that most of the mutations in its propeller domain occur in two spatial clusters. Of these, one cluster is enriched in surface exposed residues which may drive PfKelch13-centered protein interactions, and the second cluster mostly contains residues which are buried and whose mutations may destabilize PfKelch13 structure. The most prevalent resistant mutations C580Y and Y493H are distal from the above two clusters. The C580Y mutation creates sterically unfavourable contacts while Y493H possibly alters the hydrophobic core of the propeller domain. These analyses will facilitate further experimental studies aimed at understanding how mutations in pfkelch13 lead to artemisinin resistance.     

Genetic background and PfKelch13 affect artemisinin susceptibility of PfCoronin mutants in Plasmodium falciparum

Malaria continues to impose a significant health burden in the continent of Africa with 213 million cases in 2018 alone, representing 93% of cases worldwide. Because of high transmission of malaria within the continent, the selection pressures to develop drug resistance in African parasites are distinct compared to the rest of the world. In light of the spread of resistance to artemisinin conferred by the C580Y mutation in the PfKelch13 propeller domain in Southeast Asia, and its independent emergence in South America, it is important to study genetic determinants of resistance in the African context using African parasites. Through in vitro evolution of Senegalese parasites, we had previously generated the artemisinin-resistant parasites Pikine_R and Thiès_R and established pfcoronin mutations to be sufficient to confer artemisinin resistance in the standard ring-stage survival assay (RSA). In the current study, we used genetic analysis of revertants to demonstrate pfcoronin to be the major driver of elevated RSA in the artemisinin-resistant parasites Pikine_R and Thiès_R evolved in vitro. We interrogated the role of a second gene PF3D7_1433800, which also had mutations in both the Pikine_R and Thiès_R selected lines, but found no evidence of a contribution to reduced susceptibility in the RSA survival assay. Nevertheless, our genetic analysis demonstrates that parasite genetic background is important in the level of pfcoronin mediated RSA survival, and therefore we cannot rule out a role for PF3D7_1433800 in other genetic backgrounds. Finally, we tested the potential synergy between the mutations of pfcoronin and pfkelch13 through the generation of single and double mutants in the Pikine genetic background and found that the contribution of pfcoronin to reduced susceptibility is masked by the presence of pfkelch13. This phenomenon was also observed in the 3D7 background, suggesting that pfcoronin may mediate its effects via the same pathway as pfkelch13. Investigating the biology of proteins containing the beta-propeller domain could further elucidate the different pathways that the parasite could use to attain resistance.     

Selection and Spread of Artemisinin-Resistant Alleles in Thailand Prior to the Global Artemisinin Resistance Containment Campaign

The recent emergence of artemisinin resistance in the Greater Mekong Subregion poses a major threat to the global effort to control malaria. Tracking the spread and evolution of artemisinin-resistant parasites is critical in aiding efforts to contain the spread of resistance. A total of 417 patient samples from the year 2007, collected during malaria surveillance studies across ten provinces in Thailand, were genotyped for the candidate Plasmodium falciparum molecular marker of artemisinin resistance K13. Parasite genotypes were examined for K13 propeller mutations associated with artemisinin resistance, signatures of positive selection, and for evidence of whether artemisinin-resistant alleles arose independently across Thailand. A total of seven K13 mutant alleles were found (N458Y, R539T, E556D, P574L, R575K, C580Y, S621F). Notably, the R575K and S621F mutations have previously not been reported in Thailand. The most prevalent artemisinin resistance-associated K13 mutation, C580Y, carried two distinct haplotype profiles that were separated based on geography, along the Thai-Cambodia and Thai-Myanmar borders. It appears these two haplotypes may have independent evolutionary origins. In summary, parasites with K13 propeller mutations associated with artemisinin resistance were widely present along the Thai-Cambodia and Thai-Myanmar borders prior to the implementation of the artemisinin resistance containment project in the region.     

Incidence of Cyp51 A Key Mutations in Aspergillus fumigatus—A Study on Primary Clinical Samples of Immunocompromised Patients in the Period of 1995–2013

As the incidence of azole resistance in Aspergillus fumigatus is rising and the diagnosis of invasive aspergillosis (IA) in immunocompromised patients is rarely based on positive culture yield, we screened our Aspergillus DNA sample collection for the occurrence of azole resistance mediating cyp51 A key mutations. Using two established, a modified and a novel polymerase chain reaction (PCR) assays followed by DNA sequence analysis to detect the most frequent mutations in the A. fumigatus cyp51 A gene conferring azole resistance (TR34 (tandem repeat), L98H and M220 alterations). We analyzed two itraconazole and voriconazole and two multi-azole resistant clinical isolates and screened 181 DNA aliquots derived from clinical samples (blood, bronchoalveolar lavage (BAL), biopsies, cerebrospinal fluid (CSF)) of 155 immunocompromised patients of our Aspergillus DNA sample collection, previously tested positive for Aspergillus DNA and collected between 1995 and 2013. Using a novel PCR assay for the detection of the cyp51 A 46 bp tandem repeat (TR46) directly from clinical samples, we found the alteration in a TR46/Y121F/T289A positive clinical isolate. Fifty stored DNA aliquots from clinical samples were TR46 negative. DNA sequence analysis revealed a single L98H mutation in 2010, two times the L98H alteration combined with TR34 in 2011 and 2012 and a so far unknown N90K mutation in 1998. In addition, four clinical isolates were tested positive for the TR34/L98H combination in the year 2012. We consider our assay of epidemiological relevance to detect A. fumigatus azole resistance in culture-negative clinical samples of immunocompromised patients; a prospective study is ongoing.     

Rapid 5′ Nuclease (TaqMan) Assay for Detection of Virulent Strains of Yersinia enterocolitica

We have developed a rapid procedure for the detection of virulent Yersinia enterocolitica in ground pork by combining a previously described PCR with fluorescent dye technologies. The detection method, known as the fluorogenic 5′ nuclease assay (TaqMan), produces results by measuring the fluorescence produced during PCR amplification, requiring no post-PCR processing. The specificity of the chromosomal yst gene-based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 nonpathogenic serotypes of Y. enterocolitica, other species of Yersinia (Y. aldovae, Y. pseudotuberculosis, Y. mollaretti, Y. intermedia, Y. bercovieri, Y. ruckeri, Y. frederiksenii, and Y. kristensenii), and other enteric bacteria (Escherichia, Salmonella, Citrobacter, and Flavobacterium). The assay was 100% specific in identifying the pathogenic strains of Y. enterocolitica. The sensitivity of the assay was found to be ≥10² CFU/ml in pure cultures and ≥10³ CFU/g in spiked ground pork samples. Results of the assay with food enrichments prespiked with Y. enterocolitica serotypes O:3 and O:9 were comparable to standard culture results. Of the 100 field samples (ground pork) tested, 35 were positive for virulent Y. enterocolitica with both 5′ nuclease assay and conventional virulence tests. After overnight enrichment the entire assay, including DNA extraction, amplification, and detection, could be completed within 5 h.     

Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR

Background

Yersinia pestis, the agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Timely and reliable detection of such a dangerous pathogen is of critical importance. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics.

Methodology/Principal Findings

The objective of this work was to develop an alternative to conventional phage lysis tests – a rapid and highly sensitive method of indirect detection of live Y. pestis cells based on quantitative real-time PCR (qPCR) monitoring of amplification of reporter Y. pestis-specific bacteriophages. Plague diagnostic phages ϕA1122 and L-413C were shown to be highly effective diagnostic tools for the detection and identification of Y. pestis by using qPCR with primers specific for phage DNA. The template DNA extraction step that usually precedes qPCR was omitted. ϕA1122-specific qPCR enabled the detection of an initial bacterial concentration of 10³ CFU/ml (equivalent to as few as one Y. pestis cell per 1-µl sample) in four hours. L-413C-mediated detection of Y. pestis was less sensitive (up to 100 bacteria per sample) but more specific, and thus we propose parallel qPCR for the two phages as a rapid and reliable method of Y. pestis identification. Importantly, ϕA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR.

Conclusions/Significance

Thus, we developed a novel assay for detection and identification of Y. pestis using amplification of specific phages monitored by qPCR. The method is simple, rapid, highly sensitive, and specific and allows the detection of only live bacteria.     

The HIV-1 Integrase Mutations Y143C/R Are an Alternative Pathway for Resistance to Raltegravir and Impact the Enzyme Functions

Resistance to HIV-1 integrase (IN) inhibitor raltegravir (RAL), is encoded by mutations in the IN region of the pol gene. The emergence of the N155H mutation was replaced by a pattern including the Y143R/C/H mutations in three patients with anti-HIV treatment failure. Cloning analysis of the IN gene showed an independent selection of the mutations at loci 155 and 143. Characterization of the phenotypic evolution showed that the switch from N155H to Y143C/R was linked to an increase in resistance to RAL. Wild-type (WT) IN and IN with mutations Y143C or Y143R were assayed in vitro in 3′end-processing, strand transfer and concerted integration assays. Activities of mutants were moderately impaired for 3′end-processing and severely affected for strand transfer. Concerted integration assay demonstrated a decrease in mutant activities using an uncleaved substrate. With 3′end-processing assay, IC₅₀ were 0.4 µM, 0.9 µM (FC = 2.25) and 1.2 µM (FC = 3) for WT, IN Y143C and IN Y143R, respectively. An FC of 2 was observed only for IN Y143R in the strand transfer assay. In concerted integration, integrases were less sensitive to RAL than in ST or 3′P but mutants were more resistant to RAL than WT.     

The synergy of germline C634Y and V292M RET mutations in a northern Chinese family with multiple endocrine neoplasia type 2A

Genetic analysis for germline mutations of RET proto‐oncogene has provided a basis for individual management of medullary thyroid carcinoma (MTC) and pheochromocytoma. Most of compound mutations have more aggressive phenotypes than single point mutations, but the compound C634Y/V292M variant in MTC has never been reported. Thus, we retrospectively investigated synergistic effect of C634Y and V292M RET germline mutations in family members with multiple endocrine neoplasia type 2A. Nine of 14 family members in a northern Chinese family underwent RET mutation screening using next‐generation sequencing and PCR followed by direct bidirectional DNA sequencing. Clinical features of nine individuals were retrospectively carefully reviewed. In vitro, the scratch‐wound assay was used to investigate the difference between the cells carrying different mutations. We find no patients died of MTC. All 3 carriers of the V292M variant were asymptomatic and did not have biochemical or structural evidence of disease (age: 82, 62 and 58). Among 4 C634Y mutation carriers, 2 patients had elevated calcitonin with the highest (156 pg/mL) in an 87‐year‐old male. Two carriers of compound C634Y/V292M trans variant had bilateral MTC with pheochromocytoma or lymph node metastasis (age: 54 and 41 years, respectively). Further, the compound C634Y/V292M variant had a faster migration rate than either single point mutation in vitro (P < .05). In conclusion, the V292M RET variant could be classified as ‘likely benign’ according to ACMG (2015). The compound variant V292M/C634Y was associated with both more aggressive clinical phenotype and faster cell growth in vitro than was either single mutation.     

Clinical Evaluation of an Affordable Qualitative Viral Failure Assay for HIV Using Dried Blood Spots in Uganda

Background

WHO recommends regular viral load (VL) monitoring of patients on antiretroviral therapy (ART) for timely detection of virological failure, prevention of acquired HIV drug resistance (HIVDR) and avoiding unnecessary switching to second-line ART. However, the cost and complexity of routine VL testing remains prohibitive in most resource limited settings (RLS). We evaluated a simple, low–cost, qualitative viral–failure assay (VFA) on dried blood spots (DBS) in three clinical settings in Uganda.

Methods

We conducted a cross–sectional diagnostic accuracy study in three HIV/AIDS treatment centres at the Joint Clinical Research Centre in Uganda. The VFA employs semi-quantitative detection of HIV–1 RNA amplified from the LTR gene. We used paired dry blood spot (DBS) and plasma with the COBASAmpliPrep/COBASTaqMan, Roche version 2 (VL_ref) as the reference assay. We used the VFA at two thresholds of viral load, (>5,000 or >1,000 copies/ml).

Results

496 paired VFA and VL_ref results were available for comparative analysis. Overall, VFA demonstrated 78.4% sensitivity, (95% CI: 69.7%–87.1%), 93% specificity (95% CI: 89.7%–96.4%), 89.3% accuracy (95% CI: 85%–92%) and an agreement kappa = 0.72 as compared to the VL_ref. The predictive values of positivity and negativity among patients on ART for >12 months were 72.7% and 99.3%, respectively.

Conclusions

VFA allowed 89% of correct classification of VF. Only 11% of the patients were misclassified with the potential of unnecessary or late switch to second–line ART. Our findings present an opportunity to roll out simple and affordable VL monitoring for HIV–1 treatment in RLS.     

Natural Transformation-Mediated Transfer of Erythromycin Resistance in Campylobacter coli Strains from Turkeys and Swine

Erythromycin resistance in Campylobacter coli from meat animals is frequently encountered and could represent a substantial barrier to antibiotic treatment of human infections. Erythromycin resistance in this organism has been associated with a point mutation (A2075G) in the 23S rRNA gene. However, the mechanisms responsible for possible dissemination of erythromycin resistance in C. coli remain poorly understood. In this study, we investigated transformation-mediated acquisition of erythromycin resistance by genotypically diverse C. coli strains from turkeys and swine, with total genomic DNA from erythromycin-resistant C. coli of either turkey or swine origin used as a donor. Overall, transformation to erythromycin resistance was significantly more frequent in C. coli strains from turkeys than in swine-derived strains (P < 0.01). The frequency of transformation to erythromycin resistance was 10⁻⁵ to 10⁻⁶ for turkey-derived strains but 10⁻⁷ or less for C. coli from swine. Transformants harbored the point mutation A2075G in the 23S rRNA gene, as did the erythromycin-resistant strains used as DNA donors. Erythromycin resistance was stable in transformants following serial transfers in the absence of the antibiotic, and most transformants had high MICs (>256 μg/ml), as did the C. coli donor strains. In contrast to the results obtained with transformation, spontaneous mutants had relatively low erythromycin MICs (32 to 64 μg/ml) and lacked the A2075G mutation in the 23S rRNA gene. These findings suggest that natural transformation has the potential to contribute to the dissemination of high-level resistance to erythromycin among C. coli strains colonizing meat animals.     

Highly sensitive detection and quantification of the pathogen Yersinia ruckeri in fish tissues by using real-time PCR

Yersinia ruckeri is the causative agent of enteric redmouth diseases (ERM) and one of the major bacterial pathogens causing losses in salmonid aquaculture. Since recent ERM vaccine breakdowns have been described mostly attributed to emergence of Y. ruckeri biotype 2 strains, rapid, reproducible, and sensitive methods for detection are needed. In this study, a real-time polymerase chain reaction (PCR) primer/probe set based on recombination protein A (recA) gene was designed and optimized to improve the detection of Y. ruckeri. The primer/probe set proved to have a 100 % analytical specificity and a sensitivity of 1.8 ag μl^?1, equivalent to 1.7 colony-forming units (CFU)?ml^?1, for purified DNA, 3.4 CFU g^?1 for seeded liver, kidney, and spleen tissues, and 0.34 CFU/100 μl^?1 for seeded blood, respectively. The assay was highly reproducible with low variation coefficient values for intra- and inter-run experiments (2.9 % and 9.5 %, respectively). Following optimization, the assay was used to detect changes in the bacterial load during experimental infection. Rainbow trout (Onchorhynchus mykiss) were exposed to two strains of Y. ruckeri (biotype 1 and biotype 2) by intraperitoneal inoculation. Internal organs (liver, kidney, spleen) and blood were biopsied from dead fish daily for 15 days to quantify copies of pathogen DNA per gram of tissue. The findings showed the efficacy of this real-time PCR assay to quantify Y. ruckeri cells in the fish tissues and also confirmed this assay as a non-lethal method for the detection of this pathogen in blood samples.     

Development of a Highly Sensitive Loop-Mediated Isothermal Amplification (LAMP) Method for the Detection of Loa loa

The filarial parasite Loa loa, the causative agent of loiasis, is endemic in Central and Western Africa infecting 3–13 million people. L. loa has been associated with fatal encephalopathic reactions in high Loa-infected individuals receiving ivermectin during mass drug administration programs for the control of onchocerciasis and lymphatic filariasis. In endemic areas, the only diagnostic method routinely used is the microscopic examination of mid-day blood samples by thick blood film. Improved methods for detection of L. loa are needed in endemic regions with limited resources, where delayed diagnosis results in high mortality. We have investigated the use of a loop-mediated isothermal amplification (LAMP) assay to facilitate rapid, inexpensive, molecular diagnosis of loiasis. Primers for LAMP were designed from a species-specific repetitive DNA sequence from L. loa retrieved from GenBank. Genomic DNA of a L. loa adult worm was used to optimize the LAMP conditions using a thermocycler or a conventional heating block. Amplification of DNA in the LAMP mixture was visually inspected for turbidity as well as addition of fluorescent dye. LAMP specificity was evaluated using DNA from other parasites; sensitivity was evaluated using DNA from L. loa 10-fold serially diluted. Simulated human blood samples spiked with DNA from L. loa were also tested for sensitivity. Upon addition of fluorescent dye, all positive reactions turned green while the negative controls remained orange under ambient light. After electrophoresis on agarose gels, a ladder of multiple bands of different sizes could be observed in positive samples. The detection limit of the assay was found to be as little as 0.5 ag of L. loa genomic DNA when using a heating block. We have designed, for the first time, a highly sensitive LAMP assay for the detection of L. loa which is potentially adaptable for field diagnosis and disease surveillance in loiasis-endemic areas.     

Novel point mutations in the ERG11 gene in clinical isolates of azole resistant Candida species

The azoles are the class of medications most commonly used to fight infections caused by Candida sp. Typically, resistance can be attributed to mutations in ERG11 gene (CYP51) which encodes the cytochrome P450 14α-demethylase, the primary target for the activity of azoles. The objective of this study was to identify mutations in the coding region of theERG11 gene in clinical isolates of Candidaspecies known to be resistant to azoles. We identified three new synonymous mutations in the ERG11 gene in the isolates of Candida glabrata (C108G, C423T and A1581G) and two new nonsynonymous mutations in the isolates of Candida krusei - A497C (Y166S) and G1570A (G524R). The functional consequence of these nonsynonymous mutations was predicted using evolutionary conservation scores. The G524R mutation did not have effect on 14α-demethylase functionality, while the Y166S mutation was found to affect the enzyme. This observation suggests a possible link between the mutation and dose-dependent sensitivity to voriconazole in the clinical isolate of C. krusei. Although the presence of the Y166S in phenotype of reduced azole sensitivity observed in isolate C. kruseidemands investigation, it might contribute to the search of new therapeutic agents against resistant Candida isolates.     

Targeting the Cell Stress Response of Plasmodium falciparum to Overcome Artemisinin Resistance

Successful control of falciparum malaria depends greatly on treatment with artemisinin combination therapies. Thus, reports that resistance to artemisinins (ARTs) has emerged, and that the prevalence of this resistance is increasing, are alarming. ART resistance has recently been linked to mutations in the K13 propeller protein. We undertook a detailed kinetic analysis of the drug responses of K13 wild-type and mutant isolates of Plasmodium falciparum sourced from a region in Cambodia (Pailin). We demonstrate that ART treatment induces growth retardation and an accumulation of ubiquitinated proteins, indicative of a cellular stress response that engages the ubiquitin/proteasome system. We show that resistant parasites exhibit lower levels of ubiquitinated proteins and delayed onset of cell death, indicating an enhanced cell stress response. We found that the stress response can be targeted by inhibiting the proteasome. Accordingly, clinically used proteasome inhibitors strongly synergize ART activity against both sensitive and resistant parasites, including isogenic lines expressing mutant or wild-type K13. Synergy is also observed against Plasmodium berghei in vivo. We developed a detailed model of parasite responses that enables us to infer, for the first time, in vivo parasite clearance profiles from in vitro assessments of ART sensitivity. We provide evidence that the clinical marker of resistance (delayed parasite clearance) is an indirect measure of drug efficacy because of the persistence of unviable parasites with unchanged morphology in the circulation, and we suggest alternative approaches for the direct measurement of viability. Our model predicts that extending current three-day ART treatment courses to four days, or splitting the doses, will efficiently clear resistant parasite infections. This work provides a rationale for improving the detection of ART resistance in the field and for treatment strategies that can be employed in areas with ART resistance.