1312. Evaluation of a Multiplexed PCR Pneumonia Panel in a Tertiary Care Medical Center

Background Syndromic PCR testing for lower respiratory pathogens may give rapid, actionable results to aid in management decisions for suspected pneumonia cases. We sought to evaluate the performance of a multiplexed PCR pneumonia panel compared to routine microbiologic work-up in a tertiary care patient population. Methods Sputum and bronchoalveolar lavage (BAL) samples from Keck Medical Center (Los Angeles, CA) inpatients submitted for clinical microbiology work-up Dec 2019-Jun 2020 were tested by a multiplexed PCR panel (FilmArray Pneumonia Panel, BioFire Diagnostics). We compared panel results for typical bacterial pathogens to those of quantitative culture and susceptibility testing. We retrospectively determined the incidence of non-panel respiratory pathogens as detected by standard of care tests in this patient cohort. Results 68 of 180 samples yielded 80 positive bacterial PCR results: 34 were detected by both PCR panel and culture and 46 by PCR panel only, yielding a sensitivity of 100% (34/34) for pathogens detected and specificity of 73.1% (114/156) among negative cultures (normal flora or no growth). Concordant results had PCR Bin values ≥10^5 copies/mL whereas all 18 targets detected at 10^4 copies/mL were culture-negative. Among resistance gene targets, the panel detected 12 MRSA specimens, of which MRSA grew in only 4 cultures; E. coli and CTX-M in 1 specimen from which grew normal flora; and multiple gram-negative organisms and KPC in 1 specimen from which culture isolated carbapenem-resistant P. aeruginosa. Quantitation from positive BAL cultures (n=25) correlated weakly with PCR Bin values (R-squared=0.17). Non-PCR panel pathogens were detected in 22 of 180 (12.2%) specimens through routine methods (16 molds, 3 AFB, and 3 non-fermenter gram-negative bacteria). Conclusion The pneumonia panel had excellent sensitivity for its target bacterial pathogens, but results were often positive in negative cultures. This could be due to antecedent antibiotic therapy, differences in reporting threshold versus culture, or inability of PCR to discern results from normal flora. Non-panel pathogens were detected in a significant proportion in our population. The pneumonia panel should be implemented and interpreted carefully with consideration of antimicrobial stewardship. Disclosures All Authors: No reported disclosures

Prider – multiplexed primer design using linearly scaling approximation of set coverage

Designing oligonucleotide primers and probes is one of the key steps of various laboratory experiments such as multiplexed PCR or digital multiplexed ligation assays. When designing primers and probes to complex, heterogeneous DNA data sets, an optimization problem arises where the smallest number of oligonucleotides covering the largest diversity of the input dataset needs to be identified. Tools that provide this optimization in an efficient manner for large input data are currently lacking. Here we present Prider, an R package for designing primers and probes with a nearly optimal coverage for complex and large sequence sets. Prider initially prepares a full primer coverage of the input sequences, the complexity of which is subsequently reduced by removing components of high redundancy or narrow coverage. The primers from the resulting near-optimal coverage are easily accessible as data frames and their coverage across the input sequences can be visualised as heatmaps using Prider's plotting function. Prider scales linearly to increasing sequence data and therefore permits efficient design of primers to large and highly diverse DNA datasets. Prider is available on GitHub under the permissive BSD-3-clause license: https://github.com/manutamminen/prider .

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.

Autochthonous Austrian Varieties of Prunus avium L. Represent a Regional Gene Pool, Assessed Using SSR and AFLP Markers

Sweet cherry production faces new challenges that necessitate the exploitation of genetic resources such as varietal collections and landraces in breeding programs. A harmonized approach to characterization is key for an optimal utilization of germplasm in breeding. This study reports the genotyping of 63 sweet cherry accessions using a harmonized set of 11 simple sequence repeat (SSR) markers optimized in two multiplexed PCR reactions. Thirty-eight distinct allelic profiles were identified. The set of SSR markers chosen proved highly informative in these germplasm; an average of 6.3 alleles per locus, a PIC value of 0.59 and above-average expected and observed heterozygosity levels were detected. Additionally, 223 amplified fragment length polymorphism (AFLP) markers derived from eight selective primer combinations were employed to further differentiate 17 closely related accessions, confirming the SSR analysis. Genetic relationships between internationally known old cultivars were revealed: SSR fingerprints of “Schneiders Späte Knorpelkirsche” and “Germersdorfer” were found to be identical to those of the standard cultivar “Noire de Meched”, among others, whereas four accessions known as “Hedelfinger Riesenkirsche” and four known as “Große Schwarze Knorpelkirsche” showed allelic differences at various loci. The genetic diversity of locally-grown cultivars worldwide might be currently underestimated. Several autochthonous Austrian sweet cherry germplasm accessions were genotyped for the first time and their genetic relationships analyzed and discussed. Interestingly, seven Austrian sweet cherry landraces were shown to be clearly genetically separated from international and modern varieties, indicating that Austrian germplasm could include valuable genetic resources for future breeding efforts.

High diversity droplet microfluidic libraries generated with a commercial liquid spotter

Droplet libraries consisting of many reagents encapsulated in separate droplets are necessary for applications of microfluidics, including combinatorial chemical synthesis, DNA-encoded libraries, and massively multiplexed PCR. However, existing approaches for generating them are laborious and impractical. Here, we describe an automated approach using a commercial array spotter. The approach can controllably emulsify hundreds of different reagents in a fraction of the time of manual operation of a microfluidic device, and without any user intervention. We demonstrate that the droplets produced by the spotter are similarly uniform to those produced by microfluidics and automate the generation of a ~ 2 mL emulsion containing 192 different reagents in ~ 4 h. The ease with which it can generate high diversity droplet libraries should make combinatorial applications more feasible in droplet microfluidics. Moreover, the instrument serves as an automated droplet generator, allowing execution of droplet reactions without microfluidic expertise.

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. We observed no significant differences in Plasmodium parasite detection between capillary and venous blood for both assays. The GFP assay was more sensitive than MMSR for all markers that could be compared (Plasmodium spp. and P. 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.

Novel CRISPR-based sequence specific enrichment methods for target loci and single base mutations

The programmable sequence specificity of CRISPR has found uses in gene editing and diagnostics. This manuscript describes an additional application of CRISPR through a family of novel DNA enrichment technologies. CAMP (CRISPR Associated Multiplexed PCR) and cCAMP (chimeric CRISPR Associated Multiplexed PCR) utilize the sequence specificity of the Cas9/sgRNA complex to target loci for the ligation of a universal adapter that is used for subsequent amplification. cTRACE (chimeric Targeting Rare Alleles with CRISPR-based Enrichment) also applies this method to use Cas9/sgRNA to target loci for the addition of universal adapters, however it has an additional selection for specific mutations through the use of an allele-specific primer. These three methods can produce multiplex PCR that significantly reduces the optimization required for every target. The methods are also not specific to any downstream analytical platform. We additionally will present a mutation specific enrichment technology that is non-amplification based and leaves the DNA in its native state: TRACE (Targeting Rare Alleles with CRISPR-based Enrichment). TRACE utilizes the Cas9/sgRNA complex to sterically protect the ends of targeted sequences from exonuclease activity which digests both the normal variant as well as any off-target sequences.

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

Background. Malaria parasites infect over 200 million people and cause more than 400,000 deaths each year. 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. We observed no significant differences in Plasmodium parasite detection between capillary and venous blood for both assays. The GFP assay was more sensitive than MMSR for all markers that could be compared (Plasmodium spp. and P. 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.

NGS-based identification and tracing of microsatellite instability from minute amounts DNA using inter-Alu-PCR

Sensitive detection of microsatellite instability (MSI) in tissue or liquid biopsies using next generation sequencing (NGS) has growing prognostic and predictive applications in cancer. However, the complexities of NGS make it cumbersome as compared to established multiplex-PCR detection of MSI. We present a new approach to detect MSI using inter-Alu-PCR followed by targeted NGS, that combines the practical advantages of multiplexed-PCR with the breadth of information provided by NGS. Inter-Alu-PCR employs poly-adenine repeats of variable length present in every Alu element and provides a massively-parallel, rapid approach to capture poly-A-rich genomic fractions within short 80–150bp amplicons generated from adjacent Alu-sequences. A custom-made software analysis tool, MSI-tracer, enables Alu-associated MSI detection from tissue biopsies or MSI-tracing at low-levels in circulating-DNA. MSI-associated indels at somatic-indel frequencies of 0.05–1.5% can be detected depending on the availability of matching normal tissue and the extent of instability. Due to the high Alu copy-number in human genomes, a single inter-Alu-PCR retrieves enough information for identification of MSI-associated-indels from ∼100 pg circulating-DNA, reducing current limits by ∼2-orders of magnitude and equivalent to circulating-DNA obtained from finger-sticks. The combined practical and informational advantages of inter-Alu-PCR make it a powerful tool for identifying tissue-MSI-status or tracing MSI-associated-indels in liquid biopsies.