Development of the iCubate Molecular Diagnostic Platform Utilizing Amplicon Rescue Multiplex Polymerase Chain Reaction.

We utilized Amplicon-Rescue Multiplex PCR (ARM-PCR) and microarray hybridization to develop and validate the iC-GPC Assay, a multiplexed, in vitro diagnostic test that identifies five of the most common gram positive bacteria and three clinically relevant resistance markers associated with bloodstream infections (BSI). The iC-GPC Assay is designed for use with the iC-System™, which automates sample preparation, ARM-PCR, and microarray detection within a closed cassette. Herein, we determined the limit of detection for each of the iC-GPC Assay targets to be between 3.0 × 105-1.7 × 107 CFU/mL, well below clinically relevant bacterial levels for positive blood cultures. Additionally, we tested 106 strains for assay inclusivity and observed a target performance of 99.4%. 95 of 96 non-target organisms tested negative for cross-reactivity, thereby assuring a high level of assay specificity. Overall performance above 99% was observed for iC-GPC Assay reproducibility studies across multiple sites, operators and cassette lots. In conclusion, the iC-GPC Assay is capable of accurately and rapidly identifying bacterial species and resistance determinants present in blood cultures containing gram positive bacteria. Utilizing molecular diagnostics like the iC-GPC Assay will decrease time to treatment, healthcare costs, and BSI-related mortality.

Seasonal variation of respiratory pathogen colonization in asymptomatic health care professionals: A single-center, cross-sectional, 2-season observational study.

BACKGROUND: The purpose of this study was to determine the seasonal variance of potentially pathogenic bacterial and viral organisms in nasopharyngeal specimens obtained from asymptomatic health care professionals (HCPs) during the 2014 winter and summer months. METHODS: Nasopharyngeal specimens from 100 HCPs were collected from Huntsville Hospital (Huntsville, AL) during the winter and from 100 HCPs during the summer. All subjects were tested for 22 viruses and 19 bacteria using Target Enriched Multiplex Polymerase Chain Reaction. Both seasonal cohorts were composed of students, nurses, physicians, and residents. RESULTS: Of the 100 HCPs tested during the winter, 34 subjects were colonized with at least 1 bacterium, and 11 tested positive for at least 1 virus. Methicillin-resistant Staphylococcus aureus (MRSA), Moraxella catarrhalis, and coronavirus were the most frequently detected potentially infectious agents. Of the 100 HCPs tested during the summer, 37 tested positive for at least 1 bacterium, and 4 tested positive for a viral agent. The most prevalent bacteria were MRSA and Klebsiella pneumonia. CONCLUSION: Nasopharyngeal carriage among asymptomatic HCPs was common, but the frequency and presence of potential pathogens varied with each season. Understanding the colonization and infection potential of upper respiratory organisms is important, particularly for viruses. Although asymptomatic HCPs certainly harbor a number of different potentially infectious agents, future studies are needed to determine whether colonized pathogens are transmitted or initiate infection in at-risk patient populations.

The frequency of tetracycline resistance genes co-detected with respiratory pathogens: a database mining study uncovering descriptive trends throughout the United States.

BACKGROUND: The Center for Disease Control and Prevention (CDC) indicates that one of the largest problems threatening healthcare includes antibiotic resistance. Tetracycline, an effective antibiotic that has been in use for many years, is becoming less successful in treating certain pathogens. To better understand the temporal patterns in the growth of antibiotic resistance, patient diagnostic test records can be analyzed. METHODS: Data mining methods including frequent item set mining and association rules via the Apriori algorithm were used to analyze results from 80,241 Target Enriched Multiplex-PCR (TEM-PCR) reference laboratory tests. From the data mining results, five common respiratory pathogens and their co-detection rates with tetracycline resistance genes (TRG) were further analyzed and organized according to year, patient age, and geography. RESULTS: From 2010, all five pathogens were associated with at least a 24% rise in co-detection rate for TRGs. Patients from 0-2 years old exhibited the lowest rate of TRG co-detection, while patients between 13-50 years old displayed the highest frequency of TRG co-detection. The Northeastern region of the United States recorded the highest rate of patients co-detected with a TRG and a respiratory pathogen. Along the East-west gradient, the relative frequency of co-detection between TRGs and respiratory pathogens decreased dramatically. CONCLUSIONS: Significant trends were uncovered regarding the co-detection frequencies of TRGs and respiratory pathogens over time. It is valuable for the field of public health to monitor trends regarding the spread of resistant infectious disease, especially since tetracycline continues to be utilized a treatment for various microbial infections. Analyzing large datasets containing TEM-PCR results for co-detections provides valuable insights into trends of antibiotic resistance gene expression so that the effectiveness of first-line treatments can be continuously monitored.

DNA polymerase preference determines PCR priming efficiency.

BACKGROUND: Polymerase chain reaction (PCR) is one of the most important developments in modern biotechnology. However, PCR is known to introduce biases, especially during multiplex reactions. Recent studies have implicated the DNA polymerase as the primary source of bias, particularly initiation of polymerization on the template strand. In our study, amplification from a synthetic library containing a 12 nucleotide random portion was used to provide an in-depth characterization of DNA polymerase priming bias. The synthetic library was amplified with three commercially available DNA polymerases using an anchored primer with a random 3' hexamer end. After normalization, the next generation sequencing (NGS) results of the amplified libraries were directly compared to the unamplified synthetic library. RESULTS: Here, high throughput sequencing was used to systematically demonstrate and characterize DNA polymerase priming bias. We demonstrate that certain sequence motifs are preferred over others as primers where the six nucleotide sequences at the 3' end of the primer, as well as the sequences four base pairs downstream of the priming site, may influence priming efficiencies. DNA polymerases in the same family from two different commercial vendors prefer similar motifs, while another commercially available enzyme from a different DNA polymerase family prefers different motifs. Furthermore, the preferred priming motifs are GC-rich. The DNA polymerase preference for certain sequence motifs was verified by amplification from single-primer templates. We incorporated the observed DNA polymerase preference into a primer-design program that guides the placement of the primer to an optimal location on the template. CONCLUSIONS: DNA polymerase priming bias was characterized using a synthetic library amplification system and NGS. The characterization of DNA polymerase priming bias was then utilized to guide the primer-design process and demonstrate varying amplification efficiencies among three commercially available DNA polymerases. The results suggest that the interaction of the DNA polymerase with the primer:template junction during the initiation of DNA polymerization is very important in terms of overall amplification bias and has broader implications for both the primer design process and multiplex PCR.