Prevalence of malaria resistance-associated mutations in Plasmodium falciparum circulating in 2017-2018, Bo, Sierra Leone.

Introduction: In spite of promising medical, sociological, and engineering strategies and interventions to reduce the burden of disease, malaria remains a source of significant morbidity and mortality, especially among children in sub-Saharan Africa. In particular, progress in the development and administration of chemotherapeutic agents is threatened by evolved resistance to most of the antimalarials currently in use, including artemisinins. Methods: This study analyzed the prevalence of mutations associated with antimalarial resistance in Plasmodium falciparum from 95 clinical samples collected from individuals with clinically confirmed malaria at a hospital in Bo, Sierra Leone between May 2017 and December 2018. The combination of polymerase chain reaction amplification and subsequent high throughput DNA sequencing was used to determine the presence of resistance-associated mutations in five P. falciparum genes - pfcrt, pfmdr1, pfdhfr, pfdhps and pfkelch13. The geographic origin of parasites was assigned using mitochondrial sequences. Results: Relevant mutations were detected in the pfcrt (22%), pfmdr1 (>58%), pfdhfr (100%) and pfdhps (>80%) genes while no resistance-associated mutations were found in the pfkelch13 gene. The mitochondrial barcodes were consistent with a West African parasite origin with one exception indicating an isolate imported from East Africa. Discussion: Detection of the pfmdr1 NFSND haplotype in 50% of the samples indicated the increasing prevalence of strains with elevated tolerance to artemeter + lumefantrine (AL) threatening the combination currently used to treat uncomplicated malaria in Sierra Leone. The frequency of mutations linked to resistance to antifolates suggests widespread resistance to the drug combination used for intermittent preventive treatment during pregnancy.

Comparison of capillary and venous blood for malaria detection using two PCR-based assays in febrile patients in Sierra Leone.

BACKGROUND: Rapid and sensitive diagnostics are critical tools for clinical case management and public health control efforts. Both capillary and venous blood are currently used for malaria detection and while diagnostic technologies may not be equally sensitive with both materials, the published data on this subject are scarce and not conclusive. METHODS: Paired clinical samples of venous and capillary blood from 141 febrile individuals in Bo, Sierra Leone, were obtained between January and May 2019 and tested for the presence of Plasmodium parasites using two multiplexed PCR assays: the FilmArray-based Global Fever Panel (GFP) and the TaqMan-based Malaria Multiplex Sample Ready (MMSR) assay. RESULTS: No significant differences in Plasmodium parasite detection between capillary and venous blood for both assays were observed. The GFP assay was more sensitive than MMSR for all markers that could be compared (Plasmodium spp. and Plasmodium falciparum) in both venous and capillary blood. CONCLUSIONS: No difference was found in malaria detection between venous and capillary blood using two different PCR-based detection assays. This data gives support for use of capillary blood, a material which can be obtained easier by less invasive methods, for PCR-based malaria diagnostics, independent of the platform.

Use of real-time multiplex PCR, malaria rapid diagnostic test and microscopy to investigate the prevalence of Plasmodium species among febrile hospital patients in Sierra Leone.

BACKGROUND: Malaria continues to affect over 200 million individuals every year, especially children in Africa. Rapid and sensitive detection and identification of Plasmodium parasites is crucial for treating patients and monitoring of control efforts. Compared to traditional diagnostic methods such as microscopy and rapid diagnostic tests (RDTs), DNA based methods, such as polymerase chain reaction (PCR) offer significantly higher sensitivity, definitive discrimination of Plasmodium species, and detection of mixed infections. While PCR is not currently optimized for routine diagnostics, its role in epidemiological studies is increasing as the world moves closer toward regional and eventually global malaria elimination. This study demonstrates the field use of a novel, ambient temperature-stabilized, multiplexed PCR assay in a small hospital setting in Sierra Leone. METHODS: Blood samples from 534 febrile individuals reporting to a hospital in Bo, Sierra Leone, were tested using three methods: a commercial RDT, microscopy, and a Multiplex Malaria Sample Ready (MMSR) PCR designed to detect a universal malaria marker and species-specific markers for Plasmodium falciparum and Plasmodium vivax. A separate PCR assay was used to identify species of Plasmodium in samples in which MMSR detected malaria, but was unable to identify the species. RESULTS: MMSR detected the presence of any malaria marker in 50.2% of all tested samples with P. falciparum identified in 48.7% of the samples. Plasmodium vivax was not detected. Testing of MMSR P. falciparum-negative/universal malaria-positive specimens with a panel of species-specific PCRs revealed the presence of Plasmodium malariae (n = 2) and Plasmodium ovale (n = 2). The commercial RDT detected P. falciparum in 24.6% of all samples while microscopy was able to detect malaria in 12.8% of tested specimens. CONCLUSIONS: Wider application of PCR for detection of malaria parasites may help to fill gaps existing as a result of use of microscopy and RDTs. Due to its high sensitivity and specificity, species coverage, room temperature stability and relative low complexity, the MMSR assay may be useful for detection of malaria and epidemiological studies especially in low-resource settings.

Antimicrobial resistance of Klebsiella pneumoniae stool isolates circulating in Kenya.

We sought to determine the genetic and phenotypic antimicrobial resistance (AMR) profiles of commensal Klebsiella spp. circulating in Kenya by testing human stool isolates of 87 K. pneumoniae and three K. oxytoca collected at eight locations. Over one-third of the isolates were resistant to ≥3 categories of antimicrobials and were considered multidrug-resistant (MDR). We then compared the resistance phenotype to the presence/absence of 238 AMR genes determined by a broad-spectrum microarray and PCR. Forty-six genes/gene families were identified conferring resistance to ß-lactams (ampC/blaDHA, blaCMY/LAT, blaLEN-1, blaOKP-A/OKP-B1, blaOXA-1-like family, blaOXY-1, blaSHV, blaTEM, blaCTX-M-1 and blaCTX-M-2 families), aminoglycosides (aac(3)-III, aac(6)-Ib, aad(A1/A2), aad(A4), aph(AI), aph3/str(A), aph6/str(B), and rmtB), macrolides (mac(A), mac(B), mph(A)/mph(K)), tetracyclines (tet(A), tet(B), tet(D), tet(G)), ansamycins (arr), phenicols (catA1/cat4, floR, cmlA, cmr), fluoroquinolones (qnrS), quaternary amines (qacEΔ1), streptothricin (sat2), sulfonamides (sul1, sul2, sul3), and diaminopyrimidines (dfrA1, dfrA5, dfrA7, dfrA8, dfrA12, dfrA13/21/22/23 family, dfrA14, dfrA15, dfrA16, dfrA17). This is the first profile of genes conferring resistance to multiple categories of antimicrobial agents in western and central Kenya. The large number and wide variety of resistance genes detected suggest the presence of significant selective pressure. The presence of five or more resistance determinants in almost two-thirds of the isolates points to the need for more effective, targeted public health policies and infection control/prevention measures.

Effect of Linker Length on Cell Capture by Poly(ethylene glycol)-Immobilized Antimicrobial Peptides.

Development of antimicrobial peptide (AMP)-functionalized materials has renewed interest in using poly(ethylene glycol) (PEG)-mediated linking to minimize unwanted interactions while engendering the peptides with sufficient flexibility and freedom of movement to interact with the targeted cell types. While PEG-based linkers have been used in many AMP-based materials, the role of the tether length has been minimally explored. Here, we assess the impact of varying the length of PEG-based linkers on the binding of bacterial cells by surface-immobilized AMPs. While higher surface densities of immobilized AMPs were observed using shorter PEG linkers, the increased density was insufficient to fully account for the increased binding activity of peptides. Furthermore, effects were specific to both the peptide and cell type tested. These results suggest that simple alterations in linking strategies-such as changing tether length-may result in large differences in the surface properties of the immobilized AMPs that are not easily predictable.

Evanescent wave fluorescence biosensors: Advances of the last decade.

Biosensor development has been a highly dynamic field of research and has progressed rapidly over the past two decades. The advances have accompanied the breakthroughs in molecular biology, nanomaterial sciences, and most importantly computers and electronics. The subfield of evanescent wave fluorescence biosensors has also matured dramatically during this time. Fundamentally, this review builds on our earlier 2005 review. While a brief mention of seminal early work will be included, this current review will focus on new technological developments as well as technology commercialized in just the last decade. Evanescent wave biosensors have found a wide array applications ranging from clinical diagnostics to biodefense to food testing; advances in those applications and more are described herein.

Antimicrobial resistance genotypes and phenotypes from multidrug-resistant bacterial wound infection isolates in Cambodia.

This study aimed to identify the molecular determinants responsible for antibiotic resistance among human wound isolates in Cambodia. Staphylococcus spp. (n=10) and a variety of Gram-negative isolates (n=21) were taken from a larger collection of wound isolates collected during 2011-2013 and were analysed for the presence of >230 resistance determinants using a broad-spectrum DNA microarray. These isolates were chosen to represent the species most commonly found in wound isolates referred during this time and to include some of the most resistant strains. Resistance determinants detected among the staphylococci included blaZ (90%), mecA (100%), erm(B) (70%), erm(C) (20%), tet(38) (90%), tet(K) (40%), tet(Lp) (10%), tet(M) (20%), lnu(A)/lin(A) and lnu(B)/lin(B) (10% each), msr(A)/msr(B)/msr(SA) (10%), norA (80%) and dfrA (10%). Eleven different ß-lactamase genes were detected among the Gram-negative bacteria, including genes encoding the TEM (48%), CTX-M-1 (48%), CTX-M-9 (5%), SHV (5%) and VEB (10%) families of broad-spectrum and extended-spectrum ß-lactamase enzymes, as well as the carbapenemase gene blaOXA-23. Forty additional genes were also detected in the Gram-negative isolates conferring resistance to aminoglycosides (11 genes), phenicols (5 genes), macrolides [4 genes, including mph(A)/mph(K) (10%)], lincosamides [lnu(F)/lin(F), lnu(G)/lin(G)], tetracycline (4 genes), rifampicin [arr (29%)], quaternary amines [qacEΔ1 (43%)], quinolones [qnrS (14%) and qnrB (5%)], sulfonamides [sul1 (29%), sul2 (38%) and sul3 (10%)], streptothricin (sat2) and trimethoprim (6 genes). The results obtained here provide a snapshot of the broad variety of resistance determinants currently circulating within Cambodia.

Antimicrobial resistance determinants in Acinetobacter baumannii isolates taken from military treatment facilities.

Multidrug-resistant (MDR) Acinetobacter baumannii infections are of particular concern within medical treatment facilities, yet the gene assemblages that give rise to this phenotype remain poorly characterized. In this study, we tested 97 clinical A. baumannii isolates collected from military treatment facilities (MTFs) from 2003 to 2009 by using a molecular epidemiological approach that enabled for the simultaneous screening of 236 antimicrobial resistance genes. Overall, 80% of the isolates were found to be MDR, each strain harbored between one and 17 resistant determinants, and a total of 52 unique resistance determinants or gene families were detected which are known to confer resistance to ß-lactam (e.g., blaGES-11, blaTEM, blaOXA-58), aminoglycoside (e.g., aphA1, aacC1, armA), macrolide (msrA, msrB), tetracycline [e.g., tet(A), tet(B), tet(39)], phenicol (e.g., cmlA4, catA1, cat4), quaternary amine (qacE, qacEΔ1), streptothricin (sat2), sulfonamide (sul1, sul2), and diaminopyrimidine (dfrA1, dfrA7, dfrA19) antimicrobial compounds. Importantly, 91% of the isolates harbored blaOXA-51-like carbapenemase genes (including six new variants), 40% harbored the blaOXA-23 carbapenemase gene, and 89% contained a variety of aminoglycoside resistance determinants with up to six unique determinants identified per strain. Many of the resistance determinants were found in potentially mobile gene cassettes; 45% and 7% of the isolates contained class 1 and class 2 integrons, respectively. Combined, the results demonstrate a facile approach that supports a more complete understanding of the genetic underpinnings of antimicrobial resistance to better assess the load, transmission, and evolution of MDR in MTF-associated A. baumannii.

Molecular characterization of multidrug resistant hospital isolates using the antimicrobial resistance determinant microarray.

Molecular methods that enable the detection of antimicrobial resistance determinants are critical surveillance tools that are necessary to aid in curbing the spread of antibiotic resistance. In this study, we describe the use of the Antimicrobial Resistance Determinant Microarray (ARDM) that targets 239 unique genes that confer resistance to 12 classes of antimicrobial compounds, quaternary amines and streptothricin for the determination of multidrug resistance (MDR) gene profiles. Fourteen reference MDR strains, which either were genome, sequenced or possessed well characterized drug resistance profiles were used to optimize detection algorithms and threshold criteria to ensure the microarray's effectiveness for unbiased characterization of antimicrobial resistance determinants in MDR strains. The subsequent testing of Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae hospital isolates revealed the presence of several antibiotic resistance genes [e.g. belonging to TEM, SHV, OXA and CTX-M classes (and OXA and CTX-M subfamilies) of ß-lactamases] and their assemblages which were confirmed by PCR and DNA sequence analysis. When combined with results from the reference strains, ~25% of the ARDM content was confirmed as effective for representing allelic content from both Gram-positive and -negative species. Taken together, the ARDM identified MDR assemblages containing six to 18 unique resistance genes in each strain tested, demonstrating its utility as a powerful tool for molecular epidemiological investigations of antimicrobial resistance in clinically relevant bacterial pathogens.

Multidrug-resistant tet(X)-containing hospital isolates in Sierra Leone.

The tet(X) gene encodes a flavin-dependent monooxygenase that confers resistance to all clinically relevant tetracycline antibiotics including tigecycline. It has only previously been identified in environmental and non-human pathogenic bacteria. To investigate levels of multidrug resistance in Bo, Sierra Leone, a molecular epidemiological study was conducted using an antimicrobial resistance determinant microarray (ARDM), PCR and DNA sequencing. The study found that 21% of isolates from Mercy Hospital (Bo, Sierra Leone) were tet(X)-positive, all of which originated from urinary specimens. Use of molecular epidemiological surveillance tools has provided the first evidence of tet(X)-containing multidrug-resistant Gram-negative hospital isolates in a hospital in Sierra Leone.

Enhancement of deoxyribonucleic acid microarray performance using post-hybridization signal amplification.

Microarray performance depends upon the ability to screen samples against a vast array of probes with the appropriate sensitivity and selectivity. While these factors are significantly influenced by probe design, they are also subject to the particular detection methodology and reagents employed. Herein we describe the incorporation of super avidin-biotin system (SABS) and secondary enzymatic enhancement (SEE) as post-hybridization signal amplification techniques to improve the sensitivity of oligonucleotide microarrays. To these ends, we tested these methods on electrochemically interrogated arrays using both purified influenza A PCR products and randomly amplified genomic Francisella tularensis DNA as targets. While SABS treatment did not improve sensitivity for CombiMatrix ElectraSense(®) arrays using purified influenza A cDNA, chip sensitivity was improved 10-fold for randomly amplified targets. SEE improved performance to a greater degree and was able to lower the detection limits 10-fold for influenza A and 100-fold for F. tularensis DNA. These results indicate the promising capability of post-hybridization amplification techniques for enhancing microarray performance.

Multiplexed electrochemical detection of Yersinia pestis and staphylococcal enterotoxin B using an antibody microarray.

The CombiMatrix antibody microarray is a versatile, sensitive detection platform based on the generation and transduction of electrochemical signals following antigen binding to surface antibodies. The sensor chip described herein is comprised of microelectrodes coupled to an adjacent bio-friendly matrix coated with antibodies to the biological pathogens Yersinia pestis and Bacillus anthracis, and the bacterial toxin staphylococcal enterotoxin B (SEB). Using this system, we were able to detect SEB and inactivated Y. pestis individually as well as in two-plex assays at concentrations as low as 5 pg/mL and 10(6) CFU/mL, respectively. We also introduce super avidin-biotin system (SABS) as a viable and effective means to enhance assay signal responses and lower detection limits. Together these technologies represent substantial advances in point-of-care and point-of-use detection applications.

Comparison of detection and signal amplification methods for DNA microarrays.

One of the factors limiting the use of DNA microarray technology for the detection of pathogenic organisms from clinical and environmental matrices has been inadequate assay sensitivity. To assess the effectiveness of post-hybridization secondary detection steps to enhance the sensitivity of DNA microarray-based pathogen detection, we evaluated a panel of 11 commercial and novel hybridization detection and signal amplification methods (direct labeling, indirect aminoallyl labeling, antibody, DNA dendrimers, viral particles, internally fluorescent nanoparticles, tyramide signal amplification, resonance light scattering nanoparticles and quantum dots) using a multiplex PCR and spotted long oligonucleotide microarray for Vibrio cholerae. Quantitative parameters such as sensitivity, signal intensity, background, assay complexity, time and cost were assessed and provide comparative criteria to be considered for DNA microarray experimental design. While the most important parameter is likely to vary based on the assay, when weighted equally, the findings suggest that recognition element- and dye-functionalized viral particles provide the most attractive option for microarray detection and signal amplification.

Array Biosensor for Toxin Detection: Continued Advances.

The following review focuses on progress made in the last five years with the NRL Array Biosensor, a portable instrument for rapid and simultaneous detection of multiple targets. Since 2003, the Array Biosensor has been automated and miniaturized for operation at the point-of-use. The Array Biosensor has also been used to demonstrate (1) quantitative immunoassays against an expanded number of toxins and toxin indicators in food and clinical fluids, and (2) the efficacy of semi-selective molecules as alternative recognition moieties. Blind trials, with unknown samples in a variety of matrices, have demonstrated the versatility, sensitivity, and reliability of the automated system.

Antimicrobial Peptides: New Recognition Molecules for Detecting Botulinum Toxins.

Many organisms secrete antimicrobial peptides (AMPs) for protection againstharmful microbes. The present study describes detection of botulinum neurotoxoids A, Band E using AMPs as recognition elements in an array biosensor. While AMP affinitieswere similar to those for anti-botulinum antibodies, differences in binding patterns wereobserved and can potentially be used for identification of toxoid serotype. Furthermore,some AMPs also demonstrated superior detection sensitivity compared to antibodies: toxoidA could be detected at 3.5 LD50 of the active toxin in a 75-min assay, whereas toxoids B andE were detected at 14 and 80 LD50 for their respective toxins.

Multiplexed measurement of serum antibodies using an array biosensor.

The array biosensor provides the capability for simultaneously measuring titers of antibody against multiple antigens. Human antibodies against four different targets, tetanus toxin, diphtheria toxin, staphylococcal enterotoxin B (SEB) and hepatitis B, were measured simultaneously in sera from eight different donors in a single assay and titers were determined. The assays could measure amounts of bound antibody as low as approximately 100 fg. Each individual serum exhibited a different pattern of reactivity against the four target antigens. Applications of this biosensor capability include monitoring for exposure to pathogens and for efficacy of vaccination.

Antimicrobial peptides for detection of bacteria in biosensor assays.

Bacteria, plants, and higher and lower animals have evolved an innate immune system as a first line of defense against microbial invasion. Some of these organisms produce antimicrobial peptides (AMPs) as a part of this chemical immune system. AMPs exert their antimicrobial activity by binding to components of the microbe's surface and disrupting the membrane. The overall goal of this study was to apply the AMP magainin I as a recognition element for Escherichia coli O157:H7 and Salmonella typhimurium detection on an array-based biosensor. We immobilized magainin I on silanized glass slides using biotin-avidin chemistry, as well as through direct covalent attachment. Cy5-labeled, heat-killed cells were used to demonstrate that the immobilized magainin I can bind Salmonella with detection limits similar to analogous antibody-based assays. Detection limits for E. coli were higher than in analogous antibody-based assays, but it is expected that other AMPs may possess higher affinities for this target. The results showed that both specific and nonspecific binding strongly depend on the method used for peptide immobilization. Direct attachment of magainin to the substrate surface not only decreased nonspecific cell binding but also resulted in improved detection limits for both Salmonella and E. coli.

Evanescent wave fluorescence biosensors.

Since discovery and first use in the mid-1970s, evanescent wave fluorescence biosensors have developed into a diverse range of instruments, each designed to meet a particular detection need. In this review, we provide a brief synopsis of what evanescent wave fluorescence biosensors are, how they work, and how they are used. In addition, we have summarized the important patents that have impacted the evolution from laboratory curiosities to fully automated commercial products. Finally, we address the critical issues that evanescent wave fluorescence biosensors will face in the coming years.