MICROBIOLOGY | NGS for Microbiology

Enhancing and Improving Diagnostic Microbiology with qPCR + NGS

Expand clinical impact through enhanced diagnostic power

At Dr. James Snyder’s mycology laboratory at the University of Louisville Hospital, a mold would not sporulate using conventional methods despite repeated attempts. A sample was submitted to MicroGenDX for qPCR+NGS, which identified the isolate as a rare fungus in just four days, and the patient’s symptoms resolved with an appropriate antifungal therapy. View Dr. Snyder video

In another example of NGS diagnostic efficacy, a landmark 15-center study demonstrated 68.6% of revision failures following two-stage exchange were due to untreated or undertreated pathogens missed by culture, but detected by NGS at the time of initial resection. Only 8.6% of failures had negative NGS results at the time of revision surgery. Read about study

13 years of 16S/ITS NGS testing quality and reliability

With over 700,000 qPCR+NGS samples processed, referencing against a validated microbial database of 57,000+ microbes, MicroGenDX ensures representation of a comprehensive and growing library of infective organisms. This precise and expanding identification has been consistently confirmed by a 99% CAP proficiency average from more than 1,000 blinded specimens. MicroGenDX has also been a trusted research partnership lab for the FDA, NASA, and the CDC. View CAP data

Species panel	Test frequency	Number of blinded samples per test	Total number of blinded specimens	Proficiency score
IDR	3 times per year for 7 years	5	105	98.1%
MRS-5M	3 times per year for 12 years	5	180	100%
F1	3 times per year for 12 years	5	180	98.30%
D8	3 times per year for 12 years	5	180	99.40%
VRE	2 times per year for 12 years	2	50	100%
GIP	3 times per year for 4 years	5	60	100%
HC-6	3 times per year for 3 years	5	45	100%
TVAG	2-3 times per year for 2 years	3	21	90.50%
Alternate	3 times per year for 12 years	6	216	100%
See species panel key	Blinded specimen total: 1,037	Mean accuracy rate: 99.2%

Attentive customer service

MicroGenDX accepts the largest selection of media and specimen types, offers a 3.5 day turnaround time for NGS reports and a 24-48 hour turnaround for identification of antimicrobial resistance genes, and provides custom capabilities and validation of lab developed tests in collaboration with customers.

Antimicrobial Resistance Gene Detection using qPCR

Resistance factors are run on a qualitative assay, which will report them out as present or absent.

Quinolone Level 1:
Level 1 test for Quinolone resistance is a real-time qualitative PCR assay utilizing the Roche LightCycler 480. This taqman assay uses dye labeled hydrolysis probes to detect the presence of a unique sequence in the qnr and gyrA gene specific to quinolone resistance. Quinolone antibiotics inhibit bacterial replication by stabilizing the complex between DNA and DNA gyrase or topoisomerase and blocking the polymerase from binding during replication. Quinolone resistance occurs when the DNA gyrase or topoisomerase is protected against the cleaving caused by Quinolone antibiotics. Qnr is a common quinolone resistance gene that blocks the quinolone antibiotics from inhibiting the DNA gyrase activity. A mutation in the DNA gyrase subunit A, gyrA, causes the quinolone antibiotics to become ineffective at stopping the DNA gyrase replication. The accuracy, sensitivity, and specificity of the qPCR assay are 100%. The limit of detection is 0.001ng/uL.

Methicillin Level 1:
Level 1 test for methicillin resistance is a real-time qualitative PCR assay utilizing the Roche LightCycler 480. This taqman assay uses dye labeled hydrolysis probes to detect the presence of a unique sequence in the mecA genes specific to methicillin resistance. The mecA gene is the most common gene used to detect methicillin resistance among Staphylococcus species. The accuracy, sensitivity, and specificity of the qPCR assay are 98.9%, 100%, and 95.2%. The limit of detection is 0.0147ng/uL.

Vancomycin Level 1:
Level 1 test for vancomycin resistance is a real-time qualitative PCR assay utilizing the Roche LightCycler 480. This taqman assay uses dye labeled hydrolysis probes to detect the presence of a unique sequence in the vanA genes specific to vancomycin resistance. The vanA gene is the most common mutated gene used to detect vancomycin resistance. The accuracy, sensitivity, and specificity of the qPCR assay are 100%. The limit of detection is 0.0005ng/uL.

Beta-lactam Level 1:
Level 1 test for beta-lactam resistance is a real-time qualitative PCR assay utilizing the Roche LightCycler 480. This taqman assay uses dye labeled hydrolysis probes to detect the presence of a unique sequence in the TEM and SHV genes specific to beta-lactam resistance. TEM-1 is the most common gene used to detect beta-lactamase resistance due to the majority of beta lactamase resistant bacteria targets the TEM group to cause resistance. The second most common group targeted is the SHV group. The accuracy, sensitivity, and specificity of the qPCR assay are 100%. The limit of detection is 0.0034ng/uL.

Carbapenem Level 1:
Level 1 test for Carbapenem resistance is a real-time qualitative PCR assay utilizing the Roche LightCycler 480. This taqman assay uses dye labeled hydrolysis probes to detect the presence of a unique sequence in the blaKPC, blaNDM, and blaOXA48 genes specific to carbapenem resistance. Carbapenem resistance can be divided into several groups including NDM, KPC, OXA48, IMP, and VIM; however, the most common groups found in the USA are NDM, KPC and OXA48. NDM resistance is typically found in Esherichia coli and Klebsiella pneumoniae, while KPC is common to Klebsiella species and Pseudomonas aeruginosa. OXA 48 resistance is found primarily in Acinetobacter species. The accuracy, sensitivity, and specificity of the qPCR assay are 100%. The limit of detection 3.36ng/uL.

Macrolide Level 1:
Level 1 test for macrolide resistance is a real-time qualitative PCR assay utilizing the Roche LightCycler 480. This taqman assay uses dye labeled hydrolysis probes to detect the presence of a unique sequence in the ermB gene specific to macrolide resistance and is the most common route to macrolide resistance. The accuracy, sensitivity, and specificity of the qPCR assay are 100%. The limit of detection is 0.014ng/uL.

Aminoglycoside Level 1:
Level 1 test for Aminoglycoside resistance is a real-time qualitative PCR assay utilizing the Roche LightCycler 480. This taqman assay uses dye labeled hydrolysis probes to detect the presence of a unique sequence in the aacC6-aph3 and ant-la-aph2 genes specific to aminoglycoside resistance. The most common enzymes causing aminoglycoside resistance target the amino groups using N-acetyltransferases (AAC), O-nucleotidyltransferases (ANT), and O-phosphotransferases (APH). The accuracy, sensitivity, and specificity of the qPCR assay are 100%. The limit of detection is 0.0034ng/uL.

Tetracycline Level 1:
Level 1 test for Tetracycline resistance is a real-time qualitative PCR assay utilizing the Roche LightCycler 480. This taqman assay uses dye labeled hydrolysis probes to detect the presence of a unique sequence in the tetM and tetB gene specific to tetracycline resistance. Tetracycline resistance occurs when proteins stop binding the tetracycline to the bacterial ribosome. There are 14 proteins that can block the binding site, but four cause the majority of blockage. The most common protein found is tetM (at 80%). The proteins tetA and tetB were identified first, but their mechanism is utilized in many of the same bacteria (10%). The protein tetO is the newest discovered at less than 5% and many identified outside the USA. The other 10 proteins make up the remaining 5%. The accuracy, sensitivity, and specificity of the qPCR assay are 100%. The limit of detection is 0.014ng/uL.

Bactrim Level 1:
Level 1 test for Bactrim is a real-time quantitative PCR assay utilizing the Roche LightCycler 480. This taqman assay uses dye labelled hydrolysis probes to detect the presence of a unique sequence in the sul I and sul II genes specific to Bactrim resistance. Sulfonamides and TMP target two enzymes, dihydropteroate synthetase or DHPS and dihydrofolate reductase or DHFR, to disrupt steps in the folic acid production cycle by inhibiting the production of dihydrofolic acid and tetrahydrofolic acid. Transferable resistance to bactrim is mediated by two drug-resistant DHPS enzymes, which are encoded in sul I and sul II genes. The accuracy, sensitivity, and specificity of the qPCR assay are 100%. The limit of detection is 0.004ng/uL.

Extended Spectrum Beta Lactamase CTX-M Level 1:
Level 1 test for Extended Spectrum Beta Lactamase or ESBLs is a real-time quantitative PCR assay utilizing the Roche LightCycler 480. This taqman assay uses dye labelled hydrolysis probes to detect the presence of a unique sequence in the CTX-M gene specific to ESBL resistance. Extended spectrum Beta lactamases or ESBLs have resistance to penicillins, broad spectrum cephalosporins with an oxyimino side chain, such as cefotaxime, ceftriaxone, and ceftazidime, and oxyimino-monobactam aztreonam. There are 10 genes that have been documented to be associated with ESBLs, including CTX-M, SHV, TEM, PER, VEB, BES, GES, TLA, SFO and OXA; however, CTX-M makes up the largest growing group of ESBLs with the highest clinical impact. They can be found in at least 26 bacterial species and the most common are Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis. The accuracy, sensitivity, and specificity of the qPCR assay are 100%. The limit of detection is 0.0006ng/uL.

Industry leader in reference laboratory affordability

$100 for direct colony identification
Competitively priced specimen identification (please contact MicroGenDX or your representative for pricing)

NGS supports physicians with culture-negative infection management

Originally described in 1982, it is now believed that any environmental stressor could send a community into this non-culturable but still viable state (VNBC). Known human pathogens such as E. coli, .E faecalis, P. aeruginosa and S. aureus are all capable of entering and resuscitating from a VBNC state without losing their virulence. During the VBNC state, the microbes maintain their cellular structure and biology (allowing detection with molecular testing) along with significant gene expression, but they are non-culturable by ‘standard’ laboratory methods.

NGS supports direct colony identification from culture

MicroGenDX offers direct colony identification from agar and broth media for bacteria, mycobacteria and fungi using a curated database of 50,000+ microbes. MicroGenDX NGS results have demonstrated 96% concordance with traditional culture based on a study by Dr. Javad Parvizi at Thomas Jefferson University and published in the Bone and Joint Journal, 2018.

References

Tarabichi M, Shohat N, Goswami K, et al. Diagnosis of Periprosthetic Joint Infection: The Potential of Next-Generation Sequencing. The Journal of Bone and Joint Surgery. DOI: 10.2106/JBJS.17.00434 , 2018.
https://www.odtmag.com/contents/view_breaking-news/2020-09-16/next-generation-sequencing-predicts-failure-in-total-joint-arthroplasty/
2009 - 2019 College of American Pathology proficiency results

Show References

qPCR + 16S/ITS NGS testing

When laboratory methods fail to identify fastidious or slow growth organisms, NGS direct identification is the gold standard. This includes:

Direct colony identification from agar and broth media (including positive blood cultures) for bacteria, mycobacteria, and fungi
Direct identification of bacteria, mycobacteria and fungi from specimens
Identification of up to 17 clinically significant antimicrobial resistance genes in Gram-positive and Gram-negative organisms

Widest selection of specimen types

MicroGenDX accepts the following:

Synovial fluid
Hardware/heart valve
Blood
Urine
Cerebrospinal fluid (CSF)
Sputum and saliva
Respiratory swabs
Bronchoalveolar lavage (BAL)
Tissue drainage
FFPE

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About MicroGenDX NGS process