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From the Femeris Women's Health Research Center, Medical Diagnostic Laboratories, L.L.C., a Member of the Genesis Biotechnology Group, Hamilton,. New Jersey ... tions calls for urgent countermeasures.5,13,14 Increases in national and ...
The Journal of Molecular Diagnostics, Vol. 15, No. 1, January 2013

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Multiplex Bead Suspension Array for Screening Neisseria gonorrhoeae Antibiotic Resistance Genetic Determinants in Noncultured Clinical Samples Sergey Balashov, Eli Mordechai, Martin E. Adelson, and Scott E. Gygax From the Femeris Women’s Health Research Center, Medical Diagnostic Laboratories, L.L.C., a Member of the Genesis Biotechnology Group, Hamilton, New Jersey Accepted for publication August 24, 2012. Address correspondence to Sergey Balashov, Ph.D., Femeris Women’s Health Research Center, Medical Diagnostic Laboratories, L.L.C., 2439 Kuser Rd., Hamilton, NJ 08690. E-mail: [email protected].

The increasing threat of antibiotic-resistant Neisseria gonorrhoeae highlights the need for new diagnostic options. A high-throughput multiplex bead suspension array assay was developed for profiling 29 N. gonorrhoeae genomic mutations and 2 plasmid genes conferring resistance to 6 antimicrobial agents: penicillin, ciprofloxacin, cefixime, tetracycline, azithromycin, and spectinomycin. The three steps of this assay include amplification of 12 N. gonorrhoeae chromosomal and plasmid loci, multiplex allele-specific primer extension reaction, and multiplex bead suspension array detection. Antibiotic resistance genetic determinants were identified successfully in 239 cervicovaginal N. gonorrhoeaeepositive noncultured swab samples. This molecular assay can be used for detection of gonococci in clinical specimens, molecular typing, mutation profiling, and predictive assessment of N. gonorrhoeae susceptibility to antibiotics without the need for culture. (J Mol Diagn 2013, 15: 116e129; http://dx.doi.org/10.1016/j.jmoldx.2012.08.005)

Gonorrhea is the second most common reported sexually transmitted disease in the United States with an estimated 700,000 new infections each year.1 The increasing prevalence of antimicrobial-resistant Neisseria gonorrhoeae isolates is a cause for concern. Circulating N. gonorrhoeae strains have acquired resistance to many types of antibiotics used successfully in the past.2,3 These include sulfanilamides, penicillins, tetracyclines, and fluoroquinolones, which currently are not recommended for gonorrhea therapy because of the emerging resistance that has surpassed 20% of the clinical isolates tested in certain urban health care centers.4 The current antibiotic regimen recommended by the CDC for gonorrhea includes dual therapy with a ceftriaxone in combination with either azithromycin or doxycycline owing to frequent co-infections with Chlamydia trachomatis and to slow the development of N. gonorrhoeae antibiotic resistance that occurs more rapidly with monotherapy.1 Extended-spectrum cephalosporins such as the injectable ceftriaxone and oral cefixime have retained efficacy against the pathogen.1e3 Recently, this last line of defense has been breached with the emergence of cefixime Copyright ª 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmoldx.2012.08.005

and ceftriaxone nonsusceptible N. gonorrhoeae isolates observed in Japan, Europe, and the United States.5e11 These N. gonorrhoeae “superbugs” have developed resistance to a spectrum of antimicrobials including most frequently used cephalosporins and fluoroquinolones.6,7,9,11 Alternative therapies are available including spectinomycin, which the CDC recommends be used in patients who cannot tolerate cephalosporins; however, it is expensive, is injectable only, and is not available in the United States.1 Gonococcal resistance to spectinomycin has been described as well.12 The threat of potentially untreatable gonococcal infections calls for urgent countermeasures.5,13,14 Increases in national and international awareness and establishment of coordinated antimicrobial resistance (AMR) surveillance programs play a key role in monitoring the prevalence of multidrug-resistant N. gonorrhoeae.14,15 Improved laboratory diagnostics of gonorrhea infection as well as new Disclosures: All authors of this manuscript are employees of, and E.M. is the owner of, Femeris Women’s Health Research Center, Medical Diagnostic Laboratories, L.L.C., a Member of Genesis Biotechnology Group.

N. gonorrhoeae Resistance Bead Array approaches in N. gonorrhoeae molecular typing and profiling antibiotic susceptibility can facilitate efforts on controlling the dissemination of multidrug-resistant isolates and ensuring pathogen eradication. In a time of very limited antimicrobial treatment options, determination of the AMR profiles is crucial for appropriate antibiotic therapy and prevention of treatment failures. The aim of the present work was the development of a new molecular diagnostic tool for monitoring AMR genetic determinants in N. gonorrhoeae. The assay screens for 29 AMR mutations in 8 chromosomal genes, 2 AMR plasmids, and 1 N. gonorrhoeae species-specific marker. Genetic targets included in the assay are associated with nonsusceptibility to 6 antimicrobial agents: penicillin (PEN), ciprofloxacin (CIP), extended-spectrum cephalosporins (ESC), tetracycline (TET), azithromycin (AZM), and spectinomycin (SPT). It can be applied to DNA extracted from Table 1

N. gonorrhoeae cultures as well as to total DNA isolated from noncultured clinical specimens. The validation and evaluation of the assay are described using cervicovaginal swab samples and N. gonorrhoeae cultures.

Materials and Methods Molecular AMR Targets, Bacterial Strains, Bacterial DNAs, and Patient Samples Twenty-nine N. gonorrhoeae AMR chromosomal mutations, two AMR plasmid-borne genes, one species-specific marker targeted by the multiplex bead suspension array assay, and corresponding resistance types are listed in Table 1. Included N. gonorrhoeae genetic targets were as follows: porA pseudogene, an N. gonorrhoeae speciesspecific marker16; penA encodes penicillin-binding protein

N. gonorrhoeae Chromosomal and Plasmid Genes and Mutations Targeted by the Assay

Gene mutant in (%)

Genetic marker

Marker performance

AMR mutation/gene

Positive in (%)

Associated AMR

porA penA 222 (93%)

porA penA 345A

239 (100%) 224 (94%) 238 (100%)

Mosaic ins345A G545S S91F S91P S91Y D95A D95G D95N D95Y D86N S87R S87N S88P E91G E91K L421P

114 206 0 16 0 0 2 14 0 0 0 15 1 0 0 0 74

(48%) (86%) (0%) (7%) (0%) (0%) (1%) (6%) (0%) (0%) (0%) (6%) (0%) (0%) (0%) (0%) (31%)

PEN ESC

gyrA 16 (7%)

S91 D95

239 (100%) 239 (100%)

parC 16 (7%)

D86 S87 S88 E91

239 239 239 239

ponA 74 (31%) mtrR 69 (29%)

L421

239 (100%)

35 G45 10

217 (91%) 154 (64) 212 (89%)

238 (100%)

-35delA G45D G45S -10insTT G120K G120D A121D A121G A121S V57M

27 54 0 10 22 12 28 2 49 127

(11%) (23%) (0%) (4%) (9%) (5%) (12) (1%) (21%) (53%)

porB 85 (36%)

G120 A121

224 (94%) 212 (89%)

rpsJ 127 (53%) 16s rRNA 0 (0%) bla (TEM) plasmid 2 (1%) tetM plasmid 10 (4%)

V57 G1064 C1192 bla

ND* ND* ND*

G1064C C1192U bla

tetM

ND*

tetM

(100%) (100%) (100%) (100%)

Reference 16 10,11,17e22

CIP

21,23e26

CIP

21,25e27

PEN

21,28

PEN TET AZM

21,25,29e35

PEN TET

21,32e37

TET

21,34,35

0 (0%) 0 (0%) 2 (1%)

SPT

12

PEN

21,38e42

10 (4%)

TET

21,34,43,44

Marker performances and AMR mutation frequencies in 239 N. gonorrhoeae-positive cervicovaginal specimens as determined by the assay. *ND, not determined.

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Balashov et al 2 in which mutations can confer resistance to PEN and ESC10,11,17e22; gyrA encodes subunit A of DNA gyrase in which mutations confer resistance to CIP21,23e26; parC encodes a ParC subunit of topoisomerase IV in which mutations can confer greater resistance to CIP in conjunction with gyrA mutations21,25e27; ponA encodes a penicillinbinding protein 1 in which mutations confer resistance to PEN21,28; mtrR encodes a negative regulator of the MtrCDE multidrug efflux pump MtrR in which mutations can confer resistance to PEN, TET, and AZM21,25,29e35; porB encodes a membrane porin PI in which mutations can confer resistance to PEN and TET in conjunction with mtrR alterations21,32e44; rpsL encodes ribosomal protein S10 in which mutations can confer resistance to TET in conjunction with mutations in mtrR and porB21,34,35; 16S ribosomal RNA (rRNA) in which mutations can confer resistance to SPT12; b-lactamaseeproducing bla (TEM) plasmid, which confers high-level resistance to PEN21,38e42; and plasmid-encoded tetM, which confers resistance to TET.21,34,43,44 Six N. gonorrhoeae strains (Table 2), 7 strains of nongonococcal Neisseria species (Table 3), and 33 bacterial, fungal, and protozoan species (Table 4) were purchased from ATCC (Manassas, VA). N. gonorrhoeae tetM plasmids kindly were provided by Dr. Paola Stefanelli.34 Cervicovaginal swab specimens were collected by obstetrics and gynecology practitioners and shipped to our Clinical Laboratory Improvement Amendments (CLIA) certified laboratory in UTM-RT transport medium (Copan Italia SpA, Brecia, Italy) from July to October of 2010. Specimens were defined as positive or negative for N. gonorrhoeae by an in-house developed real-time PCR assay that has been developed, validated, and used in our laboratory following CLIA requirements and standards (data not shown).

DNA Extraction DNA from bacterial and fungal cultures was extracted using the QIAamp Mini Kit (Qiagen, Valencia, CA). DNA from Table 2

ATCC lyophilized cultures was extracted by the same method after their rehydration in distilled water. DNA from clinical samples was extracted using the automated X-tractor Gene system (Corbett Robotics, Brisbane, Australia) with X-tractor Pack reagents (Sigma-Aldrich, St. Louis, MO). Plasmid DNA from bacterial cultures was extracted using the Plasmid Mini Kit (Qiagen).

Assay Development The assay was based on procedures previously described.45e47 Briefly, 12 N. gonorrhoeae chromosomal and plasmid DNA fragments were amplified in four multiplex PCRs. Amplification products were combined together, treated with ExoSAP-IT (Affymetrix, Santa Clara, CA) and used as templates in the multiplex allele-specific primer extension (ASPE) reaction. The biotin-labeled ASPE primers subsequently were hybridized to MicroPlex-xTAG microspheres (Luminex Corporation, Austin, TX) and labeled with streptavidin-R-phycoerythrin conjugate (Life Technologies Corporation, Carlsbad, CA). MicroPlex-xTAG microspheres with hybridized and streptavidin-R-phycoerythrinelabeled ASPE primers were sorted and counted on the Bio-Plex 200 instrument (Bio-Rad Laboratories, Hercules, CA). The sequences of DNA oligonucleotides used as primers in PCR and ASPE reactions are indicated in Tables 5 and 6, respectively. Multiplex and uniplex PCRs were performed in a 25-mL reaction containing 1 VeriQuest TaqPCR buffer (Affymetrix), 2.5 mmol/L MgCl2, 250 mmol/L each of dNTPs, 2.5 U of Hotstart VeriQuest TaqDNA polymerase (Affymetrix), DNA primers, and 2.5 mL of DNA. Reactions with ExoSAP-IT (Affymetrix) were performed in a 7-mL volume following the manufacturer’s guidelines. Multiplex ASPE reactions were performed in a 20-mL volume containing 1 PCR buffer (Life Technologies Corporation); 1.25 mmol/L MgCl2; 5 mmol/L each of dATP, dTTP, and dGTP; 25 mmol/L of biotin-16-dCTP (ChemCyte, San Diego, CA); 2 U of Platinum GenoTYPE Tsp DNA Polymerase (Life Technologies

N. gonorrhoeae ATCC Isolates Used in the Study and Their Characterization by the Assay

N. gonorrhoeae ATCC strain

ATCC characteristics

Positive markers and AMR mutations as determined by the assay

19424

Type strain

27628

None

31426

Beta-lactamase positive

31953 49226

Resistant to nalidixic acid and streptomycin Quality control strain

49981

Quality control strain

porA, penA, 345A, S91, D95, D86, S87, S88, E91, L421, G120, A121, 35, G45, 10, V57, G1064, C1192. porA, penA, 345A, S91, D95, D86, S87, S88, E91, L421, G120, A121, 35, G45, 10, V57, G1064, C1192. porA, penA, ins345A, S91, D95, D86, S87, S88, E91, L421P, G120D, A121, -35delA, G45D, 10, V57M, G1064, C1192, bla. porA, penA, 345A, S91, D95G, D86, S87, S88, E91, L421, G120, A121, 35, G45, 10, V57, G1064, C1192. porA, Mosaic,* ins345A, S91, D95, D86, S87, S88, E91, L421, G120, A121, 35, G45, 10, V57M, G1064, C1192. porA, Mosaic,* ins345A, S91, D95, D86, S87, S88, E91, L421, G120, A121, 35, G45S, 10, V57M, G1064, C1192, bla.

Positive AMR mutations are shown in bold. *Partial penA sequence corresponding to amino acid residues 537e545 in penicillin-binding protein 2 was identical to mosaic penA GenBank accession number P08149 by sequencing.19

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N. gonorrhoeae Resistance Bead Array Table 3

Neisseria ATCC Isolates Used in the Study and Their Characterization by the Assay

Microbial species

ATCC strain

Positive markers and AMR mutations as determined by the assay

Neisseria Neisseria Neisseria Neisseria Neisseria Neisseria Neisseria

14685 13120 23970 13102 25996 10555 29256

Mosaic,* D95, S87, E91, A121D, G45, V57, G1064, C1192. S87, E91, V57, G1064, C1192. penA, S91, D95, S87, E91, A121D, 35, G45, 10, V57, G1064, C1192. penA, 345A, S91, D95, S87, E91, L421, A121D, G45, 10, V57, G1064, C1192. Mosaic,* S87, V57, G1064, C1192. penA, D95, S87, G45, V57, G1064, C1192. Mosaic,* S87, V57, G1064, C1192.

cinera flavescens lactamica meningitidis mucosa perflava sicca

Positive AMR mutations are shown in bold. *Sequences of penA genes of commensal Neisseria species were determined in this study.

Corporation); 25 nmol/L each of ASPE primers; and 5 mL of ExoSAP-ITetreated PCR reaction. Hybridization reactions were performed in a 75 mL volume of 1 hybridization buffer containing 0.2 mol/L NaCl, 0.1 mol/L Tris pH 8.0, 0.08% Triton X-100, enriched with about 100 of each MicroPlex-xTAG microsphere types, and 5 mL of ASPE reactions. Streptavidin-R-phycoerythrin Table 4

labeling reactions were performed in 1 hybridization buffer with the addition of 2 mg/mL of streptavidin-R-phycoerythrin and hybridized MicroPlex-xTAG microspheres. The cycling parameters for the multiplex PCRs were 95 C for 10 minutes, 35 cycles of denaturation at 95 C for 30 seconds, annealing at 55 C for 30 seconds, and extension at 72 C for 30 seconds, with a final extension at 72 C for

Microbial ATCC Strains Used in the Study and Their Characterization by the Assay

Microbial species

ATCC strain

Positive markers and AMR mutations as determined by the assay

Atopobium vaginae Bacteroides ureolyticus Candida albicans Chlamydia trachomatis Corynebacterium genitalium Cryptococcus neoformans Enterobacter aerogenes Enterococcus faecalis Enterococcus faecium Escherichia coli Gardnerella vaginalis Klebsiella oxytoca Lactobacillus crispatus Lactobacillus gasseri Lactobacillus iners Lactobacillus jensenii Leptotrichia buccalis Listeria monocytogenes Mobiluncus curtisii Moraxella catarrhalis Mycoplasma hominis Peptococcus niger Peptostreptococcus anaerobius Prevotella bivia Proteus mirabilis Pseudomonas aeruginosa Salmonella typhimurium Staphylococcus aureus Staphylococcus epidermidis Streptococcus agalactiae Streptococcus pyogenes Trichomonas vaginalis Ureaplasma urealyticum

BAA-55 33387 90028 VR-901B 33030 32045 13048 700221 19434 11303 49145 13182 33197 19992 55195 25258 14201 7644 35242 25238 15488 27731 27337 29303 29906 BAA427 49416 25923 12228 A909 BAA595 30246 27618

G120K, G1064, C1192 G1064, C1192 G1064, C1192 G1064, C1192 S91, S87, E91, G1064, C1192 G1064, C1192 G1064, C1192 G1064 E91G, G1064, C1192 G1064, C1192 G1064, C1192 G1064, C1192 G1064, C1192 G1064, C1192 G1064, C1192 G1064, C1192 G1064, C1192 E91G, G1064, C1192 G1064, C1192 G1064, C1192 G1064, C1192 G1064, C1192 G1064 G120K, G1064 V57, G1064, C1192 G1064, C1192 G1064, C1192 G1064, C1192 G1064, C1192 G1064, C1192 G1064, C1192 G1064, C1192 G1064, C1192

Positive AMR mutations are shown in bold.

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Balashov et al Table 5 PCR PCR1

PCR2

PCR3

PCR4

PCR Primers Primer Ngon_penA-S Ngon_penA-AS1 Ngon_ponA-S Ngon_ponA-AS Ngon_porB-S2 Ngon_porB-AS2 Ngon_porA-S Ngon_porA-AS Ngon_penA-ins345A-S Ngon_penA-ins345A-AS Ngon_rpsJ-S Ngon_rpsJ-AS Ngon_gyrA-S Ngon_gyrA-AS Ngon_parC-S Ngon_parC-AS1 Ngon_16s_rRNA-S1 Ngon_16s_rRNA-AS1 Ngon_mtrR-S Ngon_mtrR-AS Ngon_bla-S5 Ngon_bla-AS5 Ngon_tetM-ex-S Ngon_tetM_pl-AS2

Sequence 0

5 -CCGTGTGATTGTGGCGGTAACC-3 50 -TGCCCAAGATGTTCAGGCTGC-30 50 -GAGCGGTCGATAATGAGAAAATGG-30 50 -GCATCCAGCGAAACCAAAGC-30 50 -CAACAAACAATCCTTCGTCGGCTTG-30 50 -GGCAAATTCGGGAGAATCGTAGCG-30 50 -CCGTGCGTTACGATTCCCCC-30 50 -ACAGCCGGAACTGGTTTCATCTG-30 50 -TTCGGCAATCAAACCGTTCGTG-30 50 -TGCTTGTGCCGACGTTGGAC-30 50 -GCGTTTCAACATTTTGCGTTCTCC-30 50 -CATCGGTAGTTTTATCGGTCCAATCC-30 50 -AAAATAACTGGAATGCCGCCTAC-30 50 -GAAGTTGCCCTGTCCGTCTATC-30 50 -CGTGGTCGGCGAGATTTTGG-30 50 -CGAACCGAAGTTGCCGATGC-30 50 -AGCCGTAACACAGGTGCTGC-30 50 -GACCATTGTATGACGTGTGAAGCC-30 50 -GGGTTTCATTATACATACACGATTGC-30 50 -GATGTCGTCGCAGATACGTTGG-30 50 -ATAGACAGATCGCTGAGATAGGTGC-30 50 -AAAAGCGGTTAGAGCGGCTATTG-30 50 -CCAGCCCCGTCGTCCAAATAGTC-30 50 -GCATCAATCATTTGCTCATGTGGC-30

3 minutes. Reaction conditions for PCR ExoSAP-IT clean-up were 37 C for 15 minutes followed by 80 C for 15 minutes. Cycling parameters for the multiplex ASPE reactions were identical to the multiplex PCRs minus the initial step of 95 C for 10 minutes. Hybridization reaction conditions were 95 C for 3 minutes followed by 37 C for 15 minutes. PCRs, PCR clean-up, ASPE, and hybridization reactions were performed in 8-tube PCR strips or 48-well PCR plates (Axygen, Union City, CA) on Biometra T3000 PCR cyclers (Biometra GmbH, Goettingen, Germany). DNA sequencing of PCR products was performed on CEQ8000 automated DNA analyzer using GenomeLab DTCS Quick Start Kit (both from Beckman Coulter, Brea, CA).

Synthetic Controls To monitor PCR and ASPE reaction efficiencies, the control plasmid was constructed by cloning the PCR fragment of N. gonorrhoeae porA pseudogene into pCR2.1 vector using the TOPO TA Cloning Kit (Life Technologies Corporation). ASPE performance was monitored in the separate reaction with synthetic oligonucleotide templates of the following structure: 50 -GGG-XXX-Phos-30 , where GGG are three 50 end guanidine residues, XXX is a sequence complementary to the target-specific portion of the ASPE primer, and Phos is a 30 end phosphate group. Synthetic control oligonucleotides as well as PCR and ASPE primers were purchased from Integrated DNA Technologies (Coralville, IA).

120

0

Concentration in PCR, nM

Amplicon size, bp

200 200 200 200 320 320 200 200 320 320 200 200 240 240 200 200 200 200 280 280 200 200 240 240

116 133 202 136 179 116 161 130 209 296 265 181 or 1069

Data Processing and Interpretation Bio-Plex (Bio-Rad Laboratories) raw data were exported into Microsoft Excel (Microsoft, Redmond, WA) for processing and an automated allelic ratio calculation. For both allelic and nonallelic targets, median fluorescence intensity units for at least one allele were required to be 200 or higher to make a positive allele or target call (positive or negative determination). The allelic ratios for the allelic targets were calculated as N/(NþM), where N is the median fluorescence intensity value for a normal allele and M is the median fluorescence intensity value for a mutant allele. The allelic ratio ranges were set to 0.70 to 1.00 for normal calls, 0.30 to 0.70 for ambiguous calls, and 0.00 to 0.30 for mutant calls. Allele call calculations were made and mutations or targets were reported only when the positivity threshold of 200 median fluorescence intensity was reached by the N. gonorrhoeae species-specific marker porA. The run report was generated only in the absence of false-positive and false-negative signals in the no template control, PCR control, and ASPE control.

Assay Validation The analytical sensitivity and specificity were calculated in InStat software version 3.0 (GraphPad Software, La Jolla, CA) using two-way contingency table analysis and the Fisher exact test with 95% CI. The analysis of mutation association was performed in InStat3 software using the Pearson r correlation test. The genetic marker performance was calculated as follows: (NA þMA)/TS, where NA is

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N. gonorrhoeae Resistance Bead Array Table 6

ASPE Primers

Primer name

Sequence

Ngon_porA_tag2 Ngon_porB-G120K-M_tag4 Ngon_ponA-L421P-M_tag6 Ngon_penA-Normal_tag8 Ngon_mtrR-35-N_tag9 Ngon_parC-S87R-M_tag11 Ngon_parC-S88P-M_tag13 Ngon_porB-G120-N_tag14 Ngon_gyrA-S91-N_tag15 Ngon_mtrR-G45S-M_tag16 Ngon_parC-E91G-M_tag43 Ngon_gyrA-D95A-M_tag19 Ngon_mtrR-10-N_tag20 Ngon_porB-A121-N_tag21 Ngon_parC-S87-N_tag22 Ngon_penA-Mosaic-G545S_tag23 Ngon_mtrR-G45-N_tag24 Ngon_penA-ins345A-M_tag25 Ngon_16s-G1064C-M_tag26 Ngon_gyrA-D95G-M_tag27 Ngon_rpsJ-V57-N_tag29 Ngon_parC-E91K-M_tag31 Ngon_porB-G120D-M_tag32 Ngon_penA-345A-N_tag34 Ngon_parC-S87N-M_tag35 Ngon_16s-C1192-N_tag37 Ngon_gyrA-S91F-M_tag38 Ngon_gyrA-D95N-M_tag40 Ngon_porB-A121G-M_tag42 Ngon_tetM_tag44 Ngon_gyrA-D95-N_tag48 Ngon_ponA-L421-N_tag53 Ngon_16s-G1064-N_tag55 Ngon_rpsJ-V57M/L-M_tag57 Ngon_mtrR-35delA-M_tag60 Ngon_porB-A121D-M_tag64 Ngon_bla_tag68 Ngon_parC-D86N-M_tag70 Ngon_16s-C1192U-M_tag73 Ngon_porB-A121S-M_tag76 Ngon_mtrR-10insT-M_tag80 Ngon_mtrR-G45D-M_tag84 Ngon_gyrA-S91P-M_tag45 Ngon_gyrA-S91Y-M_tag41 Ngon_gyrA-D95Y-M_tag36 Ngon_parC-E91-N_tag39 Ngon_penA-Mosaic_tag87

50 -CTTTATCAATACATACTACAATCACGGCAGCATTCAATTTGTTCCGAG-30 50 -TACATTACCAATAATCTTCAAATCCAGCCCCCTGAAAAACACCA-30 50 -TCAACAATCTTTTACAATCAAATCGGTGGTTCAAGAGCCGTTGCC-30 50 -AATCCTTTTACATTCATTACTTACCCGACTGCCAACGGCTATTACG-30 50 -TAATCTTCTATATCAACATCTTACTTATACATACACGATTGCACGGATAAAAA-30 50 -TACAAATCATCAATCACTTTAATCTACCATCCGCACGGCGACC-30 50 -CAATAAACTATACTTCTTCACTAACATCCGCACGGCGACAGTC-30 50 -CTACTATACATCTTACTATACTTTAGCCCCCTGAAAAACACCGG-30 50 -ATACTTCATTCATTCATCAATTCATACCACCCCCACGGCGATTC-30 50 -AATCAATCTTCATTCAAATCATCATTTGAAATGCCAATAGAGCGCGCT-30 50 -CTTTCAATTACAATACTCATTACACCATGCGCACCATCGCCC-30 50 -TCAATCAATTACTTACTCAAATACCGCCATACGGACGATGGTGG-30 50 -CTTTTACAATACTTCAATACAATCGGTTTGACGAGGGCGGATTATAAAAAAGA-30 50 -AATCCTTTCTTTAATCTCAAATCAGGATTCCCAAGCATTGACGTTGGC-30 50 -AATCCTTTTTACTCAATTCAATCACCATCCGCACGGCGACAG-30 50 -TTCAATCATTCAAATCTCAACTTTGCCGACTGCAAACGGTTACTACA-30 50 -TCAATTACCTTTTCAATACAATACTTGAAATGCCAATAGAGCGCGCC-30 50 -CTTTTCAATTACTTCAAATCTTCAAAGAGGGGTAAACATGGGTATCGT-30 50 -TTACTCAAAATCTACACTTTTTCAAACATCTCACGACACGAGCTGAG-30 50 -CTTTTCAAATCAATACTCAACTTTCGCCATACGGACGATGGTGCC-30 50 -AATCTTACTACAAATCCTTTCTTTAACATTTTGCGTTCTCCGCACG-30 50 -TTCACTTTTCAATCAACTTTAATCCCATGCGCACCATCGCCTT-30 50 -ATTATTCACTTCAAACTAATCTACCAGCCCCCTGAAAAACACCGA-30 50 -TCATTCATATACATACCAATTCATAAGAGGGGTAAACATGGGTATCGC-30 50 -CAATTTCATCATTCATTCATTTCAACCATCCGCACGGCGACAA-30 50 -CTTTTCATCTTTTCATCTTTCAATATAAGGGCCATGAGGACTTGACG-30 50 -TCAATCATTACACTTTTCAACAATTACCACCCCCACGGCGATTT-30 50 -CTTTCTACATTATTCACAACATTACGCCATACGGACGATGGTGTT-30 50 -CTATCTTCATATTTCACTATAAACGGATTCCCAAGCATTGACGTTGCC-30 50 -TCATTTACCAATCTTTCTTTATACGTCGTCCAAATAGTCGGATAGATAAAGTACG-30 50 -AAACAAACTTCACATCTCAATAATCGCCATACGGACGATGGTGTC-30 50 -TAATTATACATCTCATCTTCTACAGGTGGTTCAAGAGCCGTTGCT-30 50 -TATATACACTTCTCAATAACTAACAACATCTCACGACACGAGCTGAC-30 50 -CAATATCATCATCTTTATCATTACAACATTTTGCGTTCTCCGCACA-30 50 -AATCTACAAATCCAATAATCTCATTTATACATACACGATTGCACGGATAAAAG-30 50 -CTACATATTCAAATTACTACTTACGGATTCCCAAGCATTGACGTTGT-30 50 -TCATAATCTCAACAATCTTTCTTTGCTGAGATAGGTGCCTCACTGATTAAGC-30 50 -ATACCAATAATCCAATTCATATCAGTAAATACCATCCGCACGGCA-30 50 -ATCAAATCTCATCAATTCAACAATATAAGGGCCATGAGGACTTGACA-30 50 -AATCTAACAAACTCATCTAAATACGGATTCCCAAGCATTGACGTTGCT-30 50 -CTAACTAACAATAATCTAACTAACGTTTGACGAGGGCGGATTATAAAAAAA-30 50 -TCAACTAACTAATCATCTATCAATTTTGAAATGCCAATAGAGCGCGT-30 50 -TCATTTCACAATTCAATTACTCAATACCACCCCCACGGCGATCC-30 50 -TTACTACACAATATACTCATCAATTACCACCCCCACGGCGATTA-30 50 -CAATTCATTTCATTCACAATCAATGCCATACGGACGATGGTGTA-30 50 -TACACAATCTTTTCATTACATCATCCATGCGCACCATCGCCTC-30 50 -AAACTAACATCAATACTTACATCAGCCGACTGCAAACGGTTACTACG-30

Tag sequences complementary to anti-tag sequences of MicroPlex-xTAG microspheres are shown in bold.

a number of normal allelic variants of the marker, MA is the number of mutant allelic variants of the marker, and TS is the total number of samples in the experiment. The limit of detection (LOD) was determined for each of the markers in the assay using serial dilutions of N. gonorrhoeae chromosomal DNA. The accuracy of the assay was determined by DNA bidirectional sequencing of PCR fragments amplified with primers indicated in Table 5.

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Results Assay Design and Validation N. gonorrhoeaeespecific chromosomal mutations and plasmid genes highly associated with PEN, CIP, ESC, TET, AZM, and SPT resistance and listed in Table 1 were included in the assay.11,12,17e44 PCR primers for 9 chromosomal

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Balashov et al N. gonorrhoeae genes were designed to target conserved sequences, which was especially important for highly variable genes such as penA and porB. PCR primers for 2 plasmid-born antibiotic resistance markers bla (TEM) and tetM were designed so that one primer was complementary to the conserved sequence of the resistance gene and another primer was homologous to the N. gonorrhoeae speciesspecific plasmid backbone. ASPE primers for chromosomal mutations were designed in pairs or groups differing by a tag sequence complementary to an anti-tag sequence of the MicroPlex-xTAG microspheres and a single base on the 30 end of the primers complementary to the specific allelic base changes, which allowed for the detection of both the normal and mutant sequences. ASPE primers for plasmidborn bla (TEM) and tetM genes as well as N. gonorrhoeae species-specific chromosomal marker porA were designed to target conserved sequences of these genes. PCR and ASPE primer design was performed on the basis of N. gonorrhoeae FA 1090 genomic sequence, penA ins345A gene sequence of N. gonorrhoeae PEN resistant strain CMRNG, mosaic penA gene allele X sequence of N. gonorrhoeae ESC resistant strain NG-3, mosaic penA gene sequence of N. gonorrhoeae PEN resistant strain CDC77124615, b-lactamaseeproducing plasmids pJD4 and pEM1 sequences, and tetM plasmids pEP5050 and pEP5289 sequences with corresponding GenBank accession numbers AE004969, X54022, AB071984, P08149, U20374, HM756641, GU479464, and GU479466, respectively.17e19,39,40,44 Sequences of N. gonorrhoeae AMR chromosomal mutations and plasmid-borne genes described elsewhere also were used for oligonucleotide design purposes.20e43 Primers targeting chromosomal genes were analyzed by the National Center for Biotechnology Information Basic Local Alignment Search Tool analysis to confirm specificity to N. gonorrhoeae. The homology of plasmid DNA specific primers to the Asia-type and Johannesburg-type bla (TEM) plasmids and the Dutchtype and American-type tetM plasmids also was confirmed by Basic Local Alignment Search Tool analysis. Efficiencies and specificities of DNA amplification with selected primers were estimated in uniplex and multiplex reactions by visualizing PCR products on 3% agarose gels. Efficiencies and specificities of ASPE primer elongation were analyzed in ASPE reactions using either PCR products or synthetic DNA controls as templates. The LOD of the assay was determined using serial dilutions of N. gonorrhoeae chromosomal DNA of two ATCC isolates: 19424 and 31426. The LODs of different genetic markers varied depending on DNA concentration, the multiplex PCR, and the ASPE reaction efficiencies. The highest LOD was for the mtrR alleles with 150 fg of N. gonorrhoeae chromosomal DNA per initial PCR and the lowest LOD was for the N. gonorrhoeae speciesspecific marker porA and alleles of gyrA, parC, and rpsJ with 25 fg of DNA per PCR. The lowest DNA concentration at which all of the chromosomal and plasmid

(if present) markers produced reliable readouts with 100% performances for both 19424 and 31426 N. gonorrhoeae DNAs in three subsequent experiments was 150 fg per PCR, corresponding to about 62 genomic copies, which was considered the LOD for the assay. No responses were obtained for the porB G120 and A121 markers when N. gonorrhoeae ATCC 19424 strain DNA was tested in the assay at any concentration. Sequencing revealed that this strain possessed the porB1a allele with a DNA sequence identical to the previously published.37 The mutation profile of N. gonorrhoeae ATCC 19424 strain is shown in Figure 1. The analytical sensitivity and specificity were determined by applying the assay to a set of 20 positive and 50 negative clinical cervicovaginal swab samples previously diagnosed for N. gonorrhoeae by real-time PCR (data not shown). There was a 100% agreement between the real-time PCR and the multiplex bead suspension array with no falsepositive and no false-negative results. In this set of experiments, both analytical sensitivity and analytical specificity were determined to be 100% (95% CI, 0.83 to 1.00 and 0.9288 to 1.000, respectively). The accuracy of the assay for N. gonorrhoeae chromosomal genetic markers was confirmed by DNA bidirectional sequencing of PCR products generated in uniplex PCRs with chromosomal DNA of two ATCC isolates: 19424 and 31426. The accuracy of the assay for N. gonorrhoeae plasmid-borne genes bla (TEM) and tetM was confirmed by sequencing PCR products generated in uniplex PCRs using plasmid DNA. b-lactamaseecontaining plasmid from the ATCC 31426 strain and previously described tetM-containing plasmids from strains G27, G45, G66, G69, and G181 of the Dutch and American types were used as templates for the amplification.34 AMR mutations subsequently detected by the assay in different N. gonorrhoeae ATCC strains also were confirmed by DNA sequencing (Table 2). Cross-reactivity was determined by analyzing 50 clinical swab specimens negative for N. gonorrhoeae by real-time PCR as well as DNAs extracted from three sets of microorganisms: 6 N. gonorrhoeae strains (Table 2), 7 closely related commensal Neisseria species (Table 3), and 33 bacterial, fungal, and protozoan species indigenous to vaginal environment (Table 4). No signals for any markers included in the assay except for 16s rRNA markers were detected in most of N. gonorrhoeaeenegative clinical swab samples. Both G1064 and C1192 16s rRNA normal alleles were highly positive in all cervicovaginal specimens, showing the abundance of bacterial DNA in them. Positive signals different from 16s rRNA markers were detected in two samples: one sample was positive for normal alleles of porB markers G120 and A121, another sample was positive for the normal alleles of gyrA markers S91 and D95 and parC markers S87 and E91. All 50 clinical specimens negative for N. gonorrhoeae by real-time PCR generated negative signals for N. gonorrhoeaeespecific porA markers included in the assay.

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N. gonorrhoeae Resistance Bead Array

Figure 1

N. gonorrhoeae genetic markers and determinants of antibiotic resistance as determined by the assay. A: Mutation profile of N. gonorrhoeae strain ATCC 19424. B: CIP resistance-associated CIP-R1 mutation profile of N. gonorrhoeaeepositive cervicovaginal swab specimen.

All six N. gonorrhoeae strains consistently were positive for the porA pseudogene and various combinations of chromosomal mutations and plasmid genes (Table 2 and Figure 1). Nongonococcal Neisseria species and 33 other bacterial, fungal, and protozoan species tested produced various normal and mutant responses for some of the genetic markers included in the assay, but all were negative for porA (Tables 3 and 4, respectively). Positive signals for the 16s rRNA marker normal alleles (G1064, C1192) were

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seen in all bacterial DNA samples except for E. faecalis, P. anaerobius, and P. bivia, which were missing the signal for the C1192 16s rRNA marker because of 16s rRNA sequence variations. There were positive signals of lower intensity for 16s rRNA-associated markers when fungal and protozoan DNAs were used as templates for PCRs as well as in the no template control, which can be explained by the presence of the residual bacterial DNA from the Taq polymerase used in the reactions.48

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Screening N. gonorrhoeaeePositive Clinical Cervicovaginal Samples Two-hundred and thirty cervicovaginal swab specimens that tested positive for N. gonorrhoeae by real-time PCR (data not shown) were subjected to the assay, and an additional 20 samples were tested during the validation process, making the total number of screened pathogen-positive specimens 250. Positive responses for N. gonorrhoeae species-specific marker porA were reported for 239 of the samples, therefore bringing the inter-assay sensitivity to 96% (95% CI, 0.92 to 0.98). Fluorescence signals of allelic AMR markers in all samples in the trial were interpreted as either normal or mutant. A single ambiguous response for the mosaic penA allele was detected in one sample. In 230 of 239 samples, at least one sequence alteration or AMR mutation was identified by the assay. Eight samples generated normal responses for all markers and in one sample all markers were normal except for the porB gene, which did not produce a response. Performances of most of the N. gonorrhoeaeetargeted markers varied between 89% and 100%. N. gonorrhoeae chromosomal and plasmid markers included in the assay, their performances, AMR mutations, and their frequencies are summarized in Table 1. Sixty-three different N. gonorrhoeae mutation combinations have been identified in 239 pathogen-positive specimens, 33 of which were seen in single samples only. Most frequently observed were profiles comprising normal alleles of all markers accompanied by mosaic penA and ins345A (n Z 27), ins345A penA insertion alone (n Z 24), and these two mutations plus rpsJ V57M substitution (n Z 22). Some AMR mutations showed a positive association with others as reflected in Table 7. AMR profiles associated with CIP resistance (n Z 16) are shown in Table 8. The most frequent (n Z 7) CIP-resistant profile, CIP-R1, containing mutant copies of gyrA, parC, mtrR, porB, rpsJ, ponA, and penA, is shown in Figure 1.

Discussion The increasing incidence of gonorrhea infection and high prevalence of resistance to antimicrobial agents are major Table 7

concerns worldwide.2,3 National and international efforts on monitoring N. gonorrhoeae epidemiology frequently are complicated by limitations of existing methodologies of antibiotic susceptibility surveillance and trends in clinical diagnostics. Wide acceptance of the nucleic acid amplification technologies (NAAT), PCR in particular, has resulted in the major replacement of conventional microbiological methods for diagnostics of the disease, therefore undermining capabilities of antibiotic susceptibility testing, which traditionally is performed on live bacterial cultures.1,49,50 Meanwhile, enormous progress has been made in understanding the genetic mechanisms underlying AMR in N. gonorrhoeae during the past decade.51 Multiple genetic markers have been associated with nonsusceptibility to certain types of antimicrobials, making it feasible to profile clinical isolates by molecular biology means.21 Although culture-based resistance determination presently cannot be replaced by such methods, they potentially might facilitate epidemiologic surveillance of N. gonorrhoeae and contribute to the future development of predictive NAAT AMR diagnostics. Because of natural constitutive competence and enhanced horizontal gene transfer, circulating N. gonorrhoeae strains undergo constant genetic rearrangements.52,53 Along with de novo mutations, N. gonorrhoeae genome plasticity contributes to the adaptation of the host immune response and development of antibiotic nonsusceptibility.2,49 The ever-changing genetic landscape of the pathogen presents both certain challenges and advantages for the NAAT-based diagnostic approaches. It might result in reduced specificity and sensitivity of NAAT-based assays as a result of both false-negative and false-positive results originating from the loss of assay-specific genetic targets or their acquisition by commensal Neisseria strains.50,54 However, all N. gonorrhoeae genetic typing techniques are based on the detection of genomic rearrangements.33,49,55e57 To date, multiple NAAT-based tests for N. gonorrhoeae identification, typing, and screening antibiotic resistance markers have been described.21,26,33,41,42,49,50,54e58 The molecular assay presented in this study serves all three purposes: it can be a primary or reflex confirmatory assay for N. gonorrhoeae diagnostics in noncultured clinical samples, it can screen for

Association Between N. gonorrhoeae AMR Mutations and Genetic Markers as Determined by the Assay

Mutant gene variant/marker*

gyrA

parC

ponA

porB

mtrR

rpsJ

bla (TEM)

tetM

penA gyrA parC ponA porB mtrR rpsJ bla (TEM)

0.083

0.083 1.000

0.175 0.400 0.400

0.048 0.151 0.151 0.241

0.219 0.272 0.272 0.541 0.197

0.269 0.252 0.252 0.359 0.175 0.348

0.028 0.159 0.159 0.038 0.028 0.065 0.086

0.065 0.056 0.056 0.139 0.024 0.206 0.197 0.019

r values greater than 0.25 indicative of positive association are shown in bold. *Correlation coefficient r values were calculated for pairs of genetic markers in order to measure association between mutant forms of genes and resistance determinants.

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N. gonorrhoeae Resistance Bead Array Table 8

N. gonorrhoeae Ciprofloxacin AMR Mutation Profiles as Determined by the Assay in 239 Pathogen-Positive Specimens

Mutation profile

In number of samples

Positive markers and AMR mutations as determined by the assay

CIP-R1

7

CIP-R2

3

CIP-R3

2

CIP-R4

1

CIP-R5

1

CIP-R6

1

CIP-R7

1

porA, penA, ins345A, S91F, D95G, D86, S87R, S88, E91, L421P, G120K, A121D, L35delA, G45, 10, V57M, G1064, C1192 porA, penA, ins345A, S91F, D95G, D86, S87R, S88, E91, L421P, G120, A121, L35delA, G45, 10, V57M, G1064, C1192 porA, penA, ins345A, S91F, D95G, D86, S87R, S88, E91, L421P, G120, A121S, L35delA, G45, 10, V57M, G1064, C1192 porA, penA, ins345A, S91F, D95G, D86, S87R, S88, E91, L421P, G120, A121, -35A, G45, 10, V57M, G1064, C1192 porA, penA, ins345A, S91F, D95G, D86, S87R, S88, E91, L421P, G120, A121, -35A, G45, L10insTT, V57M, G1064, C1192 porA, Mosaic, ins345A, S91F, D95A, D86, S87R, S88, E91, L421P, G120, A121, -35A, G45, 10, V57M, G1064, C1192 porA, Mosaic, ins345A, S91F, D95A, D86, S87N, S88, E91K, L421P, G120K, A121G, L35delA, G45, 10, V57M, G1064, C1192, bla

Positive AMR mutations are shown in bold.

markers of antibiotic resistance generating predictive profiles of nonsusceptibility, and, because of the large number of targeted genetic markers including chromosomal regions of high diversity, it can be applied as a highthroughput N. gonorrhoeae typing tool similar to the multilocus sequence typing. The assay presented here consists of four initial multiplex PCRs, followed by a PCR clean-up, ASPE reaction, and final processing on the Bio-Plex 200 instrument. In the reported format, the assay LOD was determined as 150 fg or about 62 genomic copies of N. gonorrhoeae DNA per reaction. Four initial PCRs can be combined into one multiplex PCR amplifying all 12 targeted loci of N. gonorrhoeae when DNA is applied in sufficient amounts. A 96% intra-assay sensitivity compared with a real-time PCR test for N. gonorrhoeae most probably was owing to the lower LOD of the real-time PCR, which was reported to be as low as a single genomic copy per reaction. The multiplex bead array assay has been shown to be specific to N. gonorrhoeae, although some nongonococcal bacterial species generated signals for certain AMR markers because of sequence homology (Tables 3 and 4). The assay performed well when testing total DNA samples extracted from cervicovaginal swab specimens collected by obstetrics and gynecology practitioners and shipped to our laboratory. The procedure did not involve isolation and culture of N. gonorrhoeae bacterial cells, resulting in a quick turnaround time of about 6 to 8 hours, most of which was machine time. The assay showed good intra-assay sensitivity and specificity in the screening study of 50 N. gonorrhoeaee negative and 250 N. gonorrhoeaeepositive clinical cervicovaginal swab samples with no false-positive results, and reliable detection of the pathogen in 239 of the 250 specimens that tested positive by real-time PCR. Performances of individual AMR markers associated with genes gyrA, parC, and rpsJ were 100% (Table 1). The lower performance of 64% of the mtrR G45 marker probably was caused by unknown alterations in mtrR affecting PCR or ASPE primer

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binding or novel mutations in the G45 codon. The lower performance of normal/mosaic penA markers (94%) can be explained by high sequence variability of the penA gene.17e22 The lower performance of the porB-associated markers G120 (94%) and A121 (89%) can be associated with either novel mutations affecting PCR or ASPE primer binding sites, G120 and A121 coding sequences, or the presence of the porB1a allele.37,44 Performances of 16s rRNA-associated markers and plasmid-associated markers bla and tetM were not determined. Because PCR efficiencies and LODs varied for all markers, differences in their performances also can be owing to poor amplification in samples with low DNA content. Low performance of certain markers in the assay could pose a problem by generating incomplete AMR profiles. In such situations the assay report should reflect undetermined AMR markers. The choice of the genetic targets included in the assay was based on their reported association with N. gonorrhoeae AMR.11,12,17e44 The only exception was an N. gonorrhoeae species-specific marker porA with utility for pathogen identification shown in PCR already.16 In our study, the porA pseudogene has proven to be a reliable N. gonorrhoeae diagnostic marker, which showed high intra-assay sensitivity and specificity compared with the inhouse developed, real-time PCR assay targeting a different species-specific chromosomal gene. PEN resistance markers incorporated into the assay included mutations in the penA, ponA, porB, and mtrR chromosomal genes as well as detection of plasmid-born bla (TEM) b-lactamase gene (Table 1). Insertion of an additional aspartate codon in penA at position 345 is associated with PEN AMR and was the most frequent genetic alteration, observed in 86% of 239 N. gonorrhoeaeepositive samples. The penA mosaic sequence with nucleotide substitutions corresponding to amino acid residues 538 to 544 in penicillin-binding protein 2 was detected by the assay in 48% of the N. gonorrhoeaeepositive clinical samples. An identical mosaic penA sequence was reported for the

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Balashov et al PEN-resistant isolate CDC77-124615 more than a decade ago.19 A single base change in the targeted locus of mosaic penicillin-binding protein 2 allele X, resulting in G545S substitution, confers ESC resistance in N. gonorrhoeae.17,20 This mutation was not detected in pathogen-positive samples subjected to the assay. Because penA normal and mosaic ASPE primers in our assay were designed to target a sequence of 22 bases directly upstream of G545S mutation, the assay results reflect mosaic sequence rearrangements only at this defined locus. We did not determine the entire penA sequence in any of the N. gonorrhoeae strains or clinical samples. Alterations in ponA, porB, and mtrR genes contributed to PEN resistance and were found in 31%, 36%, and 29% of all samples, respectively. The N. gonorrhoeae plasmid carrying bla (TEM) b-lactamase gene was detected in 1% of all samples. There was a positive association between two markers of PEN resistance: ponA and mtrR, especially between deletion of a single A in the 35 mtrR promoter region and L421P mutation in ponA and between G45D substitution in the mtrR coding sequence and the ponA L421P mutation. There was weak or no intergenic and intragenic associations between other PEN AMR determinants (Table 7) including mutations in porB and mtrR, the simultaneous presence of which is required for antibiotic nonsusceptibility.32 TET nonsusceptibility in N. gonorrhoeae is mediated through both chromosomal and extrachromosomal genetic determinants. To address this kind of antibiotic resistance mutations in mtrR and rpsJ genes and tetM plasmid-borne gene were targeted by the assay (Table 1). Mutations in the promoter region and coding sequence of mtrR were found in 29% of all samples. The overexpression of the mtrCDE multiple transferable resistance complex or multidrug efflux system owing to mutations in mtrR is responsible for TET, PEN, and AZM resistance in N. gonorrhoeae.30,31 V57M substitution in rpsJ was detected in more than half (53%) of all N. gonorrhoeaeepositive samples. The mutant form of rpsJ was associated positively with mutations in mtrR and most of the markers included in the assay (Table 7). As previously reported, a point substitution in rpsJ in combination with the mtrR and porB AMR mutations confers a high level of chromosomally mediated TET resistance in N. gonorrhoeae.35 There was no correlation between the presence of the tetM plasmid in 4% of all specimens and other chromosomal AMR determinants. SPT resistance mutations G1064C and C1192U in 16S rRNA genes of N. meningitidis and N. gonorrhoeae have not been identified in any of the N. gonorrhoeae strains or positive clinical specimens (Table 1). Normal 16S rRNA sequences were detected by the assay not only in N. gonorrhoeaeepositive samples, but also in pathogen-negative swabs and no template control samples owing to low specificity of PCR and ASPE primers targeting conserved loci of bacterial 16S rRNA (Tables 2e4). Positive signals of lower intensity in the no template control samples most probably originated from residual bacterial DNA

contamination of the TaqDNA polymerase.48 Low specificity of 16s rRNA PCR was addressed by adjusting the assay data interpretation algorithm for 16S rRNA-associated targets. We found it a convenient option for monitoring the presence of bacterial DNA in clinical specimens and PCR performance in addition to the plasmid porA synthetic amplification control. Because no positive samples were detected, the assay utility for N. gonorrhoeae SPT resistance mutations in noncultured cervicovaginal samples remains uncertain. However, low specificity of 16s rRNA amplification should not pose a problem for G1064C and C1192U identification in DNA extracted from N. gonorrhoeae cultures. CIP resistance in N. gonorrhoeae originates primarily from point mutations in gyrA and parC. Mutant variants of these genes were found in 7% of all N. gonorrhoeaee positive clinical specimens (Table 1). There was 100% association between gyrA and parC mutations: in all cases gyrA mutations S91F, D95A, and D95G were accompanied by mutations in parC: either S87R or S87N. parC mutations were never found without gyrA mutations in the same sample. Mutations in the parC gene are believed to be complementary to gyrA substitutions and facilitate an increased level of resistance to CIP.25,26 Similarly to other studies, a high co-occurrence of CIP AMR mutations in gyrA and parC with mtrR variants was observed in our sample set.25 An increased association of gyrA and parC mutations with the ponA L421P substitution also was identified (Table 7). The 16 samples positive for gyrA and parC substitutions formed 7 distinct mutation profiles presumably corresponding to N. gonorrhoeae strain genotypes (Table 8). The most frequent among them was CIP-R1 (Figure 1) observed in seven samples: ins345A, S91F, D95G, S87R, L421P, G120K, A121D, -35delA, and V57M, suggesting that seven corresponding N. gonorrhoeae isolates might be clonal and show multidrug AMR properties with decreased susceptibility to PEN, CIP, AZM, and TET. The study and multiplex bead array assay had a number of limitations. The major technologic drawback of the assay was its higher LOD compared with real-time PCR, resulting in a 96% interassay sensitivity. Multiplex PCRs frequently are less efficient than uniplex reactions and require extensive optimization.59,60 Samples with a very low level of infection or degraded DNA therefore could generate falsenegative results. As with any NAAT-based diagnostic test, the assay is susceptible to N. gonorrhoeae sequence variations, which was another assay limitation.50,54 Pathogen identification in the assay depends on the detection of the conserved species-specific sequence of the N. gonorrhoeae porA pseudogene.16 In questionable situations, analysis of AMR marker responses can provide additional insight for reliable N. gonorrhoeae identification. When the assay is applied directly to clinical specimens without N. gonorrhoeae culture, the specificity of AMR mutation profiling might be compromised by mixed infections containing different N. gonorrhoeae strains, commensal Neisseria, or

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N. gonorrhoeae Resistance Bead Array other bacterial species sharing DNA homology with N. gonorrhoeae in targeted loci.55,61 In rare situations of mixed infections of multiple N. gonorrhoeae strains or other nongonococcal Neisseria species, fluorescence signals would be interpreted as ambiguous based on allelic ratio calculations if allelic variants of homologous genes within them were different. This kind of response can be easily detected and resolved by the automated assay data interpretation module. In fact, only 1 of the 239 N. gonorrhoeaeepositive and 50 N. gonorrhoeaeenegative cervicovaginal samples tested produced an ambiguous result. The single instance of an ambiguous allelic ratio was with the penA mosaic variant, which most likely indicated penA sequence variability rather than a nonspecific signal owing to a mixed bacterial population. The assay has not been tested on extragenital specimens such as pharyngeal samples, in which nongonococcal Neisseria species might be prevalent. Given the observed cross-reactions of certain N. gonorrhoeae AMR markers with such species, the assay might not be applicable for clinical samples other than cervicovaginal. This assay limitation will be explored in the future. Plasmid and transposon-based markers tetM and bla (TEM) have a very broad spectrum of microbial hosts, which also poses a problem for their association with N. gonorrhoeae in the analysis of cervicovaginal noncultured DNA samples.38e44 Specificity of tetM and bla (TEM) determination in the assay was ensured by a proper design of corresponding PCRs amplifying these genes only if they were carried by N. gonorrhoeae plasmids. The limited number of AMR markers incorporated into the assay, which obviously does not cover every mutation and gene conferring resistance in N. gonorrhoeae, was also one of the assay limitations. The flexibility of the MicroPlexxTAG bead suspension array platform allows its future modification and expansion for up to 100 individual targets, once new prominent AMR mutations and genes are identified. Because ESC resistance is presently a major concern, newly discovered ESC mutations in penA such as G542S, P551S, P551L, A501P, A501V, and N512Y can be added to the set of targeted markers in the future.7,22,62 The major study limitation was the absence of characterized N. gonorrhoeae strains with a known spectrum of antibiotic susceptibility breakpoints. Although the association of all the genetic markers included in the assay with nonsusceptibility to different antimicrobials is well established in the literature, the correlation of the assay results with conventional phenotypic characterization has not been performed. This will be addressed in a future study, which would establish its predictive values for resistance to different antibiotics. The advantages of the assay include its simplicity, relatively low price, flexibility, and robustness. Without the need to isolate live bacterial cells, the assay allows N. gonorrhoeae identification and typing based on a set of genetic variants conferring resistance to six antibiotics. It can be applied as a primary or secondary diagnostic test for

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gonorrhea infection. Because the test-associated turnaround time, cost, and labor are higher compared with uniplex realtime PCR tests, the most appropriate use of this assay may be reflex or confirmatory testing for the purpose of strain typing and predictive assessment of pathogen antibiotic susceptibility, complementing culture-based diagnostics and AMR surveillance. In situations in which culture is not possible, it may be the only option for pathogen AMR profiling. As a typing assay, it might enrich the current menu of N. gonorrhoeae typing technologies facilitating the monitoring of the clonal spread of multidrug-resistant isolates. In conclusion, we have created and validated a highthroughput multiplex bead suspension array assay for identification of N. gonorrhoeae DNA in cervicovaginal swab samples capable of screening for 31 mutations and genes conferring resistance to six antibiotics used currently and in the past for gonorrhea treatment. The assay validation involved LOD, intra-assay specificity, sensitivity, accuracy, and cross-reactivity determination. We have shown the assay performance in the medium-scale screening of 239 N. gonorrhoeaeepositive clinical specimens showing AMR mutation frequencies, associations, and profiles. Screening of multiple AMR markers by PCR in N. gonorrhoeae isolates has been shown already in a number of studies.21,26,33,42 Up-to-date NAAT-based assays in noncultured clinical samples have been used only for N. gonorrhoeae identification, multiantigen sequence typing, and detection of unique AMR targets.41,50,54,55,63 To our knowledge, the presented work is a first example of profiling tens of N. gonorrhoeae AMR genetic determinants in clinical specimens, omitting the pathogen culture. As proof of principle, it shows feasibility of the approach, paving the road for further assay development aimed at molecular AMR surveillance of N. gonorrhoeae.

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