72-Hour transport recovery of antimicrobial resistant Neisseria gonorrhoeae isolates using the InTray® GC method.

Recovery of Neisseria gonorrhoeae isolates exposed to a range of transport times and temperatures was quantitatively assessed for two transport devices, BioMed Diagnostics' InTray GC® and Copan Diagnostics' Liquid Amies Elution Swab (ESwab®) Collection and Transport System. Respective devices were inoculated with N. gonorrhoeae, exposed to simulated transport conditions and spread-plated from serial dilutions in duplicates onto chocolate agar in order to count CFU (colony-forming units) in the range of 25-250. Baseline CFU/mL averages of time-zero transport for each device was compared to either 24 hour (Eswab) or 72 hour (InTray GC) CFU/mL average to assess recovery of six clinical isolates of N. gonorrhoeae, and differences showing no greater than a 3 log10 (± 10%) decline between comparative time points qualified as acceptable. Our findings suggest that the InTray GC system has the potential to transport clinical isolates for ≤72 hours with acceptable N. gonorrhoeae recovery.

Introduction

Neisseria gonorrhoeae is a gram-negative obligate human pathogen and the etiological agent of gonorrhea. Gonorrhea is not typically fatal, but if left untreated, can lead to various symptoms and significant complications, such as pelvic inflammatory disease, sterility, ectopic pregnancy, or, rarely, sepsis or death [1]. After chlamydia, gonorrhea is the second most commonly reported notifiable infection in the U.S. and has experienced a 63% increase in cases since 2014, including a 5.0% increase in incidence in 2018 alone [2]. As of 2016, the global incidence of infection with N. gonorrhoeae was 87 million cases per year [3].

Some N. gonorrhoeae strains have quickly developed resistance to many available antibiotics, often limiting the available treatment options. A single 500 mg dose of intramuscular ceftriaxone is the CDC-recommended first-line drug therapy for uncomplicated anogenital and pharyngeal gonorrhea in adults [4]. In the United States, antibiotic resistance trends are monitored by the CDC’s Gonococcal Isolate Surveillance Program (GISP), which has used resistance surveillance data since the 1980s to stop recommending penicillin, tetracycline, fluoroquinolones, cefixime, and other oral cephalosporins to treat gonorrhea [5]. In recent years, strains with high resistance to ceftriaxone have arisen in several countries, severely limiting treatment options for patients infected with these particularly resistant strains [6].

N. gonorrhoeae testing and detection often involves nucleic acid amplification testing (NAAT) which, although highly sensitive and specific, does not allow for antibiotic susceptibility testing. The benefits of NAAT, particularly increased sensitivity when screening urine, urogenital and pharyngeal swabs, have led to increased popularity and the subsequent decrease in bacterial culturing in clinical settings [7]. However, culturing is necessary not only to test susceptibility to antibiotics, but also to monitor treatment failures and identify outbreaks of antimicrobial resistant N. gonorrhoeae strains. N. gonorrhoeae cultures require specific conditions to optimize their survival, including being inoculated onto nutritive and selective chocolate agar and incubated from 35–37° in a CO2 enriched environment [5]. The difficulty of culturing N. gonorrhoeae makes it difficult to perform antibiotic susceptibility panels, which can further decrease how often they are used.

With ceftriaxone resistance and gonorrhea incidence both on the rise, antibiotic resistance monitoring is more important than ever. Public health efforts must focus on developing new, more advanced technologies to monitor resistance of N. gonorrhoeae, particularly to ceftriaxone and other higher generation cephalosporins. Due to the difficulty of growing and recovering N. gonorrhoeae, growth media and transport systems need to be evaluated for efficacy and must be optimized for maximum recovery and quality control. Prior studies have examined the difference in viability between different commercially available transport systems and generally found that the InTray GC®, a CO2 generating packaging medium manufactured by BioMed Diagnostics, had a high rate of recovery after extended transport times [7]. Additionally, InTray GC plates have a shelf life of approximately one year, so are well-suited for use in settings where standard chocolate agar plates may not be widely available or viable after storage [8]. In an effort to build upon these previous studies, we assessed bacterial recovery of clinical isolates of N. gonorrhoeae in transport conditions in the InTray GC 72 hours after an initial period of incubation. The purpose of this study was also to receive approval from the U.S. Food and Drug Administration (FDA) to modify the intended use to extend the allowable times that the InTray GC device could be used to transport specimens; the methods were chosen to demonstrate viability based on FDA standards.

Materials and methods

We used N. gonorrhoeae AR Bank isolates #0165, #0181, #0197, #0202, and #0175 from the CDC and FDA Antibiotic Resistance Isolate bank [9]. We also used isolate ATCC 43069 from the nonprofit company American Type Culture Collection, located in Manassas, VA, USA. The minimum inhibitory concentrations to several antibiotics, as well as the results to a β-lactamase test, are recorded for each isolate in S1 Table. Each strain was visually confirmed to be N. gonorrhoeae following initial recovery from the suppliers. These six strains were chosen to represent a diverse set of MIC profiles.

The experimental groups consisted of N. gonorrhoeae inoculated onto the InTray GC transport devices (BioMed Diagnostics, Inc., OR, USA), and onto the Liquid Amies Elution Swab (ESwab) Collection and Transport System (Copan Diagnostics, CA, USA). Inoculum verification was also performed on standard chocolate agar plates to determine the viability of the experimental strains.

Colony forming units (CFUs) were calculated using spectrophotometers at 625nm adjusted to a 0.5 McFarland standard in 0.85% physiological saline, as per CSLI guidelines [10]. We calculated the mean log10 CFU/mL for each time point by taking the average of the duplicates of each dilution (dilutions were plated twice prior to calculating CFUs for that specific dilution), then averaging all the dilutions within each triplicate (three plates [A, B, and C] for each time). From there, we averaged the log10 CFU/mL of all three triplicates to create a single value for the time point. Percent decrease was calculated for each strain in both experimental groups by subtracting the final log10 CFU/mL value from the initial log10 CFU/mL value, dividing this value by the initial log10 CFU/mL quantity, then multiplying by 100%. Strain failure was determined by a 100% decrease in viable colonies after 72 hours in the InTray GC group and 24 hours in the ESwab group. If the strain failed, the procedure was repeated. Different time periods were chosen for each experimental group based on the intended use for each device; the ESwab is approved for use in transporting N. gonorrhoeae for up to 24 hours, while the InTray GC is currently approved for up to 48 hours, and in this study we assess the InTray GC for viability in transporting specimen for up to 72 hours [11,12]. Acceptance criteria for quantitative recovery, as well as experimental procedures for inoculation, strain viability verification, length of transport time and plating were based on CLSI M40-A2 Quality Control of Microbiological Transport Systems; Approved Standard 2nd edition [10].

First, we created source tubes for each respective isolate (AR 0165, ATCC 43069, AR 0181, AR 0197, AR 0202, AR 0175) by separately inoculating standard chocolate agar plates with each isolate, then incubating each at 37°C in a 5–7% CO2 environment for 24 hours (Fig 1). We then picked colonies from individual plates with a 1μL loop and mixed each into separate vials of 1.2mL of a 0.85% saline solution, before measuring the mean average absorption with a blanked spectrophotometer at 625nm adjusted to a 0.5 McFarland standard (S2 Table). This process was completed twice for each isolate to create two source tubes, which we then used to make 10-fold and 2-fold serial dilutions from each to achieve concentrations of 107, 106, 105, 104, 103, 5 x 102, 2.5 x 102, and 102 CFU/mL.

Fig 1

Methods flowchart for the InTray GC and ESwab transport recovery of Neisseria gonorrhoeae isolates.

For each individual isolate, we then inoculated 9 InTray GC plates (triplicates from 3 manufacturing lots; A, B, and C) with 20μL from the 103 dilution according to manufacturer instructions, before placing them into a 37°C incubator (Fig 1). 24 hours later, we removed the plates, counted and recorded the colonies on each, then chose the six most homogenous plates (2 from each lot A, B, and C). The two most similar counts from each lot were picked for downstream analysis and the higher count was tagged as plate time-zero InTray GC to avoid over-estimation viability bias for InTray GC time-72 hours. We plated the 3 InTray GC time-zero plates by scraping and removing all colonies from each plate (starting with A) with a 1μL loop and swirling into 1mL of 0.85% saline, then made serial dilutions in the range of 104 to 100 CFU/mL (S2 Table). After, we plated 100μL from each dilution in duplicates onto standard chocolate agar plates and placed them into the CO2 enriched incubator. At the same time, we placed the three InTray GC time-72 hours plates into a therapak-biohazard-specimen transport bag under controlled room temperatures. Temperature was monitored and documented over the 72-hour time period for all strains. For strain AR 0197 and AR 0202, we placed the specimen transport bag in the trunk of a car and monitored temperature changes with a USB data logger to simulate real-world transport conditions. AR 0202 failed in the uncontrolled temperature environment of the car, thus it was repeated at controlled room temperature. We then removed these plates after 72 hours and repeated the plating and dilution process that we used for time-zero InTray GC, then placed these plates into the CO2 enriched incubator. After incubating both the time-zero InTray GC and the time-72 hour InTray GC, we removed them from the incubators and recorded the plate counts in the range of 25–250 CFUs.

In addition to the InTray GC, we also tested Copan ESwabs’ viability over time. For each isolate, we began by inoculating 3 sets of triplicate ESwabs (time-zero A, B, C; time-24 hours A, B, C) by placing each swab into a separate test tube with 100μL of the 107 CFU/mL dilution for 10–15 seconds, before replacing each swab into their respective container of Liquid Amies (S2 Table). The triplicates of ESwab time-24 were then placed into specimen transport bags, under similar conditions to InTray GC time-72 hour. We gave the triplicates of ESwab time-zero 5–15 minutes to rest before we vortexed each and expressed the 100μL of liquid from each swab into separate 5mL test tubes. Then, we made serial dilutions (104, 103 and 102 CFU/mL) for each set (A, B, and C), plated 100μL onto standard chocolate agar plates in duplicates, and placed these into the CO2 incubator. 24 hours later, the plates were removed from the incubator and the CFU per plate was recorded. The same day, we removed the time-24 hours plates from the specimen transport bag and repeated the plating, dilution, and incubation process from ESwab time-zero.

In order to demonstrate strain viability, we also performed inoculum verification for the InTray GC and ESwab. For the InTray GC, we spread 100μL from the 104, 103, 5 x 102, 2.5 x 102, and 102 CFU/mL dilutions onto duplicate standard chocolate agar plates (10 plates total) and incubated them in the CO2 enriched incubator for 24 hours. After 24 hours, we removed the inoculum verification plates and recorded each duplicate’s plate count in the range of 25–250 CFU per plate.

Results

At 72 hours, out of the 6 strains tested on the InTray GC, 5 strains passed and 1 strain initially failed before being repeated at controlled room temperatures (AR 0202) (Fig 2). AR 0165 experienced a 12.9% decrease in log10 CFU/mL (0.06 log10 reduction) from 24 to 72 hours, and ATCC 43069 had a larger percent decrease of 96.37% (1.44 log10 reduction). The other four strains actually had increases in their log10 CFU/mL over the same time period, with AR 0181 having a 18.72% increase (0.09 log10 increase); AR 0197 increased by 90.23% (1.01 log10 increase); AR 0202 experienced a slightly smaller percent increase of 80.5% (0.71 log10 increase); isolate AR 0175 increased by 35.43% (0.19 log10 increase) over the 72 hours. When averaged, there was a 19.27 percent increase (0.08 log10 increase) across successful strains after 72 hours on the InTray GC, which demonstrated acceptable recovery (Table 1).

Fig 2

Time in hours versus Log10 CFU/mL for the InTray GC for all six strains.

CFU; colony forming units.

Table 1

Quantitative recovery results of the subject InTray GC transport device for six Neisseria gonorrhoeae type culture strains.

Neisseria gonorrhoeae type culturestrain	InTray GC replicate	CFU/mL Recovery: time-zero control group		CFU/mL Recovery: 72-hour transport group		Results
		*N	Mean ± SD CFU/mL	*N	Mean ± SDCFU/mL	Log10 Difference	Interpretation
CDC/FDA AR#O165	ABC	644	3.1 x 108 ± 6.7 x 107	423	2.7 x 108 ± 8.4 x 107	-0.06	AcceptableRecovery
ATCC® 43069	ABC	288	2.6 x109 ± 2.9 x109	406	9.3 x 107 ± 1.6 x 107	-1.44	AcceptableRecovery
CDC/FDA AR#O181	ABC	844	1.7 x 108 ± 1.3 x 108	444	2.1 x 108 ± 1.2 x 108	0.09	Acceptable Recovery
CDC/FDA AR#O197	ABC	462	7.3 x 107 ± 6.1 x 107	624	7.4 x 108 ± 4.7 x 108	1.01	Acceptable Recovery
CDC/FDA AR#O202	ABC	224	4.5 x 107 ± 4.5 x 107	444	2.3 x 108 ± 1.3 x 108	0.71	Acceptable Recovery
CDC/FDA AR#O175	ABC	464	9.4 x 107 ± 1.3 x 107	464	1.5 x 108 ± 7.4 x 107	0.19	Acceptable Recovery

Differences in mean average CFU/mL concentrations were analyzed by subtracting the Log10 CFU/mL average of the 72-hour groups from the Log10 CFU/mL average of the time-zero control groups. Each group consisted of three InTray GC devices labeled A, B & C, as listed. N is equal to the number of recovery quantitation plates in the accepted countable 25–250 CFU range.

For the 6 strains tested on the ESwab, after 24 hours, 5 strains passed and the same strain that failed for the InTray GC also initially failed for the ESwab before being repeated at controlled room temperatures (AR 0202) (Fig 3). After 24 hours on the ESwab, AR 0165 decreased in log10 CFU/ml by 94.5% (1.26 log10 reduction), ATCC 43069 decreased by 98.30% (1.77 log10 reduction), AR 0181 decreased by 99.22% (2.11 log10 reduction), AR 0197 decreased by 87.70% (0.91 log10 reduction), AR 0202 decreased by 99.69% (2.51 log10 reduction), and AR 0175 decreased by 98.42% (1.80 log10 reduction). When averaged, the percent decrease across successful strains after 24 hours on the ESwab, was 96.31% (1.73 log10 reduction) (Table 2).

Fig 3

Time in hours versus Log10 CFU/mL for the Copan ESwab for all six strains.

CFU; colony forming units.

Table 2

Quantitative recovery results of the ESwab transport device for six Neisseria gonorrhoeae type culture strains.

Neisseria gonorrhoeae type culturestrain	ESwab replicate	CFU/mL Recovery: time-zero control group		CFU/mL Recovery: 24-hour transport group		Results
		*N	Mean ± SD CFU/mL	*N	Mean ± SDCFU/mL	Log10 Difference	Interpretation
CDC/FDA AR#O165	ABC	644	8.8. x 107 ± 1.2 x 107	222	4.9 x 106 ± 7.8 x 105	-1.26	AcceptableRecovery
ATCC® 43069	ABC	866	1.8 x108 ± 3.9 x107	224	3.1 x 106 ± 1.4 x 106	-1.77	AcceptableRecovery
CDC/FDA AR#O181	ABC	466	1.1 x 108 ± 1.1 x 107	222	8.4 x 105 ± 1.6 x 105	-2.11	Acceptable Recovery
CDC/FDA AR#O197	ABC	666	1.2 x 108 ± 1.2 x 107	222	1.5 x 107 ± 2.5 x 106	-0.91	Acceptable Recovery
CDC/FDA AR#O202	ABC	446	1.2 x 108 ± 3.5 x 107	222	3.8 x 105 ± 1.8 x 105	-2.51	Acceptable Recovery
CDC/FDA AR#O175	ABC	664	1.1 x 108 ± 2.2 x 107	222	1.7 x 106 ± 3.4 x 105	-1.80	Acceptable Recovery

Differences in mean average CFU/mL concentrations were analyzed by subtracting the Log10 CFU/mL average of the 24-hour groups from the Log10 CFU/mL average of the time-zero control groups. Each group consisted of three ESwab devices labeled A, B & C, as listed. N is equal to the number of recovery quantitation plates in the accepted countable 25–250 CFU range.

Strain AR 0197, which was stored in the trunk of a car with a USB data logger, experienced a maximum temperature of 33.5° Celsius, a minimum temperature of 2.5° Celsius, and an average of 15.8°C (Std: 8.3°C) (S1 Fig). Strains AR 0165, AR 0181, and ATCC 43069 were stored under ambient conditions, with a maximum temperature of 22.5° Celsius, a minimum of 19° Celsius, and an average of 20.2° Celsius (Std: 0.6° Celsius) (S2 Fig). Strains AR 0202 and AR 0175 were also stored under ambient conditions, with an average temperature of 23.1° Celsius (Std 0.2° C), a maximum temperature of 24° Celsius, and a minimum temperature of 23° Celsius (S3 Fig).

All inoculum verification tests confirmed strain viability of the experimental groups, including for strain AR 0202, which initially failed for all experimental groups before being repeated.

Discussion

After 72 hours in the InTray GC transport systems, there was a small percent increase in viable colonies of N. gonorrhoeae (Table 1), while the loss of colonies after only 24 hours of transport using the Copan ESwab was fairly high. The minimal decrease in bacterial quantities showed the InTray GC’s potential to maintain a transport environment that facilitates the survival of various clinical isolates of N. gonorrhoeae over increased transport times. After 72 hours, the InTray GC plates experienced an average 19.27% increase in colonies, while the ESwab lost 24.82% of colonies after 24 hours in the same conditions. Strain AR 0197, which experienced more drastic temperature changes due to being stored in the trunk of a car, had an increase of 90.23% in log10 CFU/ml for the InTray GC plates, and a decrease of 87.70% for the ESwab plates. We hypothesize that isolate AR 0202 initially failed all experiments due to overheating, as we initially stored the 72-hour experiment in the trunk of a car to simulate transport in uncontrolled temperatures.

These data support a previous study examining four commercial transport systems and comparing recovery rates between the systems, which found that the InTray GC had optimal recovery of specimen in comparison to other systems [7]. Our study builds upon this research by demonstrating that the InTray GC plates could be subjected to extended transport times up to 72 hours without significant loss of specimen viability. This project was subject to limitations, including the limited number of isolates tested. However, the purpose of this study was to demonstrate acceptable recovery on the InTray GC device for an FDA intended use submission for which the 6 isolates are sufficient. The strain growth in terms of percent recovery for four of the strains on the InTray GC (AR 0181, AR 0197, AR0202, AR0175) could also potentially be confounded by the initial bacterial load in each sample. However, based on prior studies examining recovery on the InTray GC, we did not expect to see vast declines between time points, and rather expected to see small log10 increases and decreases [7].

This research is integral to future research and monitoring of antibiotic resistance in N. gonorrhoeae. The potential of the InTray GC to recover bacteria after long periods of transport should ideally lead to its increased use as a transport system for clinical isolates. With an average of 2.84% of colonies lost after 72 hours, the InTray GC could be used to transport longer distances to facilities with advanced antibiotic susceptibility testing capabilities. In order to help manage N. gonorrhoeae infections and associated antibiotic resistance testing, microbiology laboratories should utilize transport systems that maintain viable clinical isolates during extended transport times. Considering the difficulty in maintaining viable N. gonorrhoeae cultures in transport conditions, as well as the rising rates of antibiotic resistance, it is important to recognize the InTray GC as a potentially viable option for a transport system with high recovery rates after extended periods of transport time.

Time in hours stored in the trunk of a car versus temperature in degrees Celsius for strain AR 0197.

Mean temperature: 15.8°C (Std: 8.3°C), Maximum temperature: 33.5°C, Min temperature: 2.5°C.

(TIFF)

Click here for additional data file.

Time in hours versus temperature in degrees Celsius for strains AR 0165, AR 0181, and ATCC 43069.

Mean temperature: 20.2°C (Std: 0.6°C), Maximum temperature: 22.5°C, Min temperature: 19°C.

(TIFF)

Click here for additional data file.

Time in hours versus temperature in degrees Celsius for strains AR 0202 and AR 0175.

Mean temperature: 23.1°C (Std: 0.2°C), Maximum temperature: 24°C, Min temperature: 23°C.

(TIFF)

Click here for additional data file.

Minimum inhibitory concentrations (MIC) (in μg/ml) of experimental isolates to azithromycin, cefixime, cefpodoxime, ceftriaxone, ciprofloxacin, penicillin, and tetracycline, as well as the results to a β -lactamase test.

MIC; minimum inhibitory concentration. ATCC 43069 does not have antibiotic susceptibility MIC results.

(DOCX)

Click here for additional data file.

Inoculation density and procedure adjustments applied to CLSI M40-A2 section 8.11 Swab Elution Method (Quantitative) for the Eswab and InTray transport GC devices.

(DOCX)

Click here for additional data file.

12 Jul 2021

PONE-D-21-16017

72-Hour Transport Recovery of Antimicrobial Resistant Neisseria gonorrhoeae Isolates Using the InTray® GC Method

PLOS ONE

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Reviewer #1: Gonorrhea incidence is on the rise as is the level of antibiotic resistance in the circulating strains. It is important to develop techniques that maintain viability during transport of this organism. In this study, Paris et. al assessed bacterial recovery of clinical isolates of N. gonorrhoeae in transport conditions using InTray GC (the current standard developed by BioMed Diagnostics), 72 hours after an initial period of incubation. The purpose of this study was also to receive approval from the US FDA to modify the intended use to extend the allowable times that the InTray GC device could be used to transport specimens. The study is rigorous and well written, however there are some concerns that must be addressed:

1. The central finding in this paper is that the recovery is good after 72h in InTrayGC. However, the fact that most of the strains seem to “grow” (in terms of % recovered) during the 72 hours in the InTrayGC (the strain that grew the most, 0197, increased by 1 log) could likely confound any quantitation of the initial bacterial load that was present in the sample. While it is evident that the authors are focused on recovery, this confounder should be at least addressed in the discussion.

2. Continuing from above, the different strains grow to different extents. What do the authors make of this finding?

3. L78- It is not clear why the authors chose to test the particular strains #0165, #0181, #0197, #0202, and #0175. While they provide different MIC values for antibiotics in Table S1 to demonstrate the differences in the strains, how do the authors deflect the criticism that the strains were “cherry picked”? Some reasoning for why they chose these strains would be useful information to provide. Additionally, highlighting the unbiased nature of the process that lead to the selection of these 5-6 strains would allay some of the skepticism associated with strain selection (perhaps they are phylogenetically well spaced out on a tree? Or this is part of a standard set of strains used to test any new method in the field?).

4. L 97- To an uninitiated reader, it is not clear why the 72-hour time point was chosen for InTray GC and 24 hours for Eswab. It would help if appropriate literature describing the current standard in the field was cited.

5. L180 – “the same strain failed”. The word “as” is a typo

6. Figure 3 does not need the 48 and 72 hr timepoints on the X axis

Reviewer #2: The manuscript by Paris and colleagues investigates the recovery of Neisseria gonorrhoeae from two transport systems. The BioMed Diagnostics InTray GC system and Copan Diagnostics Liquid Amies Elution Swab (ESwab) were inoculated with a standard inoculum of six different N. gonorrhoeae strains (five clinical isolates from the CDC FDA specimen bank and one ATCC strain) and held at various times and temperatures to simulate transport conditions from a clinic to a laboratory. There was a 19.3% increase in CFU/ml from GC InTray plates and a 24.8% decrease in CFU/ml from the Eswab system at 72 hours. The data confirms previous studies reporting similar findings and will be used by BioMed Diagnostics to seek a modification from the US Food and Drug Administration to extend the allowable times that the GC InTray can be used to transport clinical specimens.

Comments

General comment: Review the manuscript for consistent use of Neisseria gonorrhoeae and N. gonorrhoeae. The full bacterial name should only be spelled out when it is first used in the manuscript and subsequently referred to as N. gonorrhoeae. There is no need to put N in paratheses as written on line 20.

Line 53: Does the sentence refer to culture being necessary to identify outbreaks of antimicrobial resistant (AMR) N. gonorrhoeae or simply gonorrhea outbreaks? Non-culture methods would be more useful for gonorrhea outbreaks and culture being need for AMR outbreaks.

General comment for the Methods section: Were the strains confirmed to be N. gonorrhoeae following initial recovery from the supplier? The methods used to confirm the identity of the strains should be added.

Reviewer #3: REVIEW NOTES FOR MANUSCRIPT NUMBER- PONE-D-21-16017

The manuscript is well and clearly written, with figures and tables that are easy to comprehend. It describes a research study, assessing the recovery of six Neisseria gonorrhoeae clinical isolates that are inoculated into two different transport devices, the BioMed Diagnostics’ InTray GC and the Copan Diagnostics’ Liquid Amies Elution Swab (ESwab) Collection and Transport System.

The approach used is appropriate. Assessment of recovery from both systems was done after 24h for the ESwab and after 72h for the BioMed Diagnostics’ InTray GC. Each system was assigned to a separate experimental group while a third group consisted of a control group made up of the same clinical samples inoculated unto standard chocolate agar plates to confirm viability of the clinical isolates. Both systems were subjected to the same temperature conditions. The ability of each system to maintain viability of the isolates was determined by comparing the CFU/ml at initial time zero and the CFU/ml at the end point time.

The ability of the InTrayGC system to maintain and transport viable gonococcal isolates for recovery after more than 24hrs has been confirmed by earlier studies (Papp et al., 2016; Beverly et al., 2000). However, the work under review was done to obtain approval from the FDA to use the InTrayGC system to transport clinical specimen for an extended period (up to 72hrs) before recovery of isolates of Neisseria gonorrhoeae. The methods used were chosen to demonstrate viability based on FDA standards.

The study results show that the InTrayGC system can maintain N. gonorrhoeae clinical isolates for up to 72h for recovery of viable isolates.

This study is important in that Neisseria gonorrhoeae is a very fastidious organism that is easily lost to temperature fluctuations during transportation of clinical specimens. There is a need to develop and assess appropriate transport systems to overcome this weakness.

ABSTRACT AND INTRODUCTION

The main research question and the key findings of this study are clearly summarized. Other current literature on the topic has been referenced

FIGURES AND TABLES

All the associated text supports the information in the tables and figures. The figures are clear and readable, captions are complete and accurate.

METHODS

The experiments used are appropriate and the data is enough to support the conclusions that were arrived at, although the assessment of more specimen would have given more weight to the data. However, the author may need to explain why the InTray GC system was incubated here. The system is described as a transport system, when in actuality, incubating the system before transportation has a strong positive influence on isolate survival/viability.

Limitations were addressed adequately; data was collected and interpreted correctly. This study can be replicated.

RESULTS, DISCUSSION AND CONCLUSIONS

The results, discussion and conclusion are well described and clearly written

MINOR COMMENTS/EDITS

Line 66. Space between the end of the sentence and the parenthesis ie. (7) is missing

Line 193. Space between the & and C is missing

Line 261. (ref 6.). The reference title was not complete

Line 233. Typo at “used”, it should be “use”

**********

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Reviewer #1: Yes: aditya bandekar

Reviewer #2: No

Reviewer #3: No

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22 Sep 2021

Thank you for reviewing our paper for publication in PLOS ONE. Enclosed is a revision version of our manuscript, ‘72-Hour Transport Recovery of Antimicrobial Resistant Neisseria gonorrhoeae Isolates Using the InTray® GC Method'. We found reviewer comments insightful and constructive and have revised the manuscript according to their feedback. Below is a detailed account of the revisions made in response to reviewer comments:

Reviewer #1: Gonorrhea incidence is on the rise as is the level of antibiotic resistance in the circulating strains. It is important to develop techniques that maintain viability during transport of this organism. In this study, Paris et. al assessed bacterial recovery of clinical isolates of N. gonorrhoeae in transport conditions using InTray GC (the current standard developed by BioMed Diagnostics), 72 hours after an initial period of incubation. The purpose of this study was also to receive approval from the US FDA to modify the intended use to extend the allowable times that the InTray GC device could be used to transport specimens. The study is rigorous and well written, however there are some concerns that must be addressed:

Thank you for your time in reviewing our manuscript. We appreciate the feedback provided.

1. The central finding in this paper is that the recovery is good after 72h in InTrayGC. However, the fact that most of the strains seem to “grow” (in terms of % recovered) during the 72 hours in the InTrayGC (the strain that grew the most, 0197, increased by 1 log) could likely confound any quantitation of the initial bacterial load that was present in the sample. While it is evident that the authors are focused on recovery, this confounder should be at least addressed in the discussion.

This is a great question. Based on previous experiments examining recovery on the InTray GC (Papp, 2016, Fig 1 A&B), we did not expect to see vast declines between time points, and rather expected to see small log10 differences up or down. For this reason, we were not particularly concerned over bacterial growth during the 72-hour time period. We also performed some unpaired T-tests for the averages between the time-zero and 72 hour groups, though we opted not to put this data into the main manuscript as it was somewhat external to what our study was examining. These tests, however, did seem to support our expectations for small increases and decreases in log10 quantities. To enhance the clarity of our manuscript, we added the following sentences to the discussion starting at line 293: “The strain growth in terms of percent recovery for four of the strains on the InTray GC (AR 0181, AR 0197, AR0202, AR0175) could also potentially be confounded by the initial bacterial load in each sample. However, based on prior studies examining recovery on the InTray GC, we did not expect to see vast declines between time points, and rather expected to see small log10 increases and decreases”.

2. Continuing from above, the different strains grow to different extents. What do the authors make of this finding?

Thank you for your question. Strain AR0197 was exposed to increased temperature (as described in the supplemental file) and we hypothesize there was, in reality, cell growth on the nutritive InTray GC (this is reflected in the small P-value of 0.0059 for this strain from the unpaired t-tests mentioned above). While other strains showed slight log10 increases or decreases, the P-values are non-significant (ranging from 0.10 to 0.55). We suspect the largest decline from the ATCC 43069 strain was due to a “laboratory” adaptation effect. This is an old strain in the lab and we hypothesize it has adapted to growing quite well on media at warm temperatures, but when exposed to cooler temperatures it did not survive as well as the other strains. For strain AR 0202, we would hypothesize that this strain had the ability to grow at lower temperatures based on the small P value of 0.0083.

3. L78- It is not clear why the authors chose to test the particular strains #0165, #0181, #0197, #0202, and #0175. While they provide different MIC values for antibiotics in Table S1 to demonstrate the differences in the strains, how do the authors deflect the criticism that the strains were “cherry picked”? Some reasoning for why they chose these strains would be useful information to provide. Additionally, highlighting the unbiased nature of the process that lead to the selection of these 5-6 strains would allay some of the skepticism associated with strain selection (perhaps they are phylogenetically well spaced out on a tree? Or this is part of a standard set of strains used to test any new method in the field?).

Thank you for this question. Five of the 6 strains we used in our study were from the CDC’s AR isolate bank. We chose these strains for several reasons, the primary one being that these strains had antibiotic resistance patterns that were most likely to be encountered in the field. By choosing strains based on the current epidemiology of N. gonorrhoeae, we hoped to demonstrate the InTray GC’s viability in transporting common strains with a variety of MIC profiles. Furthermore, we used strains with different MIC profiles to ensure a diverse sample. Also, by retrieving all but one strain from the CDC’s AR isolate bank, we tried to keep the sample as standardized as possible. At line 108, we added the sentence “These six strains were chosen to represent a diverse set of MIC profiles”.

4. L 97- To an uninitiated reader, it is not clear why the 72-hour time point was chosen for InTray GC and 24 hours for Eswab. It would help if appropriate literature describing the current standard in the field was cited.

Thank you for your comment. The reason 24 hours was chosen for the ESwab is because it’s current intended use states that it can be used to transport specimen for up to 24 hours, while the InTray can be used for up to 48 hours (although the purpose of this manuscript is to extend the allowable transportation time to 72 hours in the intended use). We added the following to line 129: “Different time periods were chosen for each experimental group based on the intended use for each device; the ESwab is approved for use in transporting specimen for up to 24 hours, while the InTray GC is currently approved for up to 48 hours, and in this study we assess the InTray GC for viability in transporting specimen for up to 72 hours.” We also added the intended use for both the InTray GC and the ESwab as new references.

5. L180 – “the same strain failed”. The word “as” is a typo

Thank you for pointing this out. We have made the appropriate changes.

6. Figure 3 does not need the 48 and 72 hr timepoints on the X axis

Thank you for your comment. To keep the figures consistent between the ESwab and the InTray GC, we have opted to keep the 48 and 72 hour timepoints in figure 3. While the ESwab and InTray GC are testing different timepoints for recovery, we believe it would aid in clarity to keep the figures consistent so readers can easily compare recovery rates between the two.

Reviewer #2: The manuscript by Paris and colleagues investigates the recovery of Neisseria gonorrhoeae from two transport systems. The BioMed Diagnostics InTray GC system and Copan Diagnostics Liquid Amies Elution Swab (ESwab) were inoculated with a standard inoculum of six different N. gonorrhoeae strains (five clinical isolates from the CDC FDA specimen bank and one ATCC strain) and held at various times and temperatures to simulate transport conditions from a clinic to a laboratory. There was a 19.3% increase in CFU/ml from GC InTray plates and a 24.8% decrease in CFU/ml from the Eswab system at 72 hours. The data confirms previous studies reporting similar findings and will be used by BioMed Diagnostics to seek a modification from the US Food and Drug Administration to extend the allowable times that the GC InTray can be used to transport clinical specimens.

Thank you for reviewing our manuscript. We appreciate your feedback.

Comments

General comment: Review the manuscript for consistent use of Neisseria gonorrhoeae and N. gonorrhoeae. The full bacterial name should only be spelled out when it is first used in the manuscript and subsequently referred to as N. gonorrhoeae. There is no need to put N in paratheses as written on line 20.

Thank you for your comment. We have updated the manuscript accordingly.

Line 53: Does the sentence refer to culture being necessary to identify outbreaks of antimicrobial resistant (AMR) N. gonorrhoeae or simply gonorrhea outbreaks? Non-culture methods would be more useful for gonorrhea outbreaks and culture being need for AMR outbreaks.

We were referencing outbreaks of AMR N. gonorrhoeae. To improve the clarity of the manuscript, we edited the sentence beginning on line 71 to say “However, culturing is necessary not only to test susceptibility to antibiotics, but also to monitor treatment failures and identify outbreaks of antimicrobial resistant N. gonorrhoeae strains”.

General comment for the Methods section: Were the strains confirmed to be N. gonorrhoeae following initial recovery from the supplier? The methods used to confirm the identity of the strains should be added.

Each strain was visually confirmed to be N. gonorrhoeae (gray/white, small, translucent, convex, shiny). In the materials and methods section, starting at line 108, we added the following sentence: “Each strain was visually confirmed to be N. gonorrhoeae following initial recovery from the suppliers.”

Reviewer #3: REVIEW NOTES FOR MANUSCRIPT NUMBER- PONE-D-21-16017

The manuscript is well and clearly written, with figures and tables that are easy to comprehend. It describes a research study, assessing the recovery of six Neisseria gonorrhoeae clinical isolates that are inoculated into two different transport devices, the BioMed Diagnostics’ InTray GC and the Copan Diagnostics’ Liquid Amies Elution Swab (ESwab) Collection and Transport System.

The approach used is appropriate. Assessment of recovery from both systems was done after 24h for the ESwab and after 72h for the BioMed Diagnostics’ InTray GC. Each system was assigned to a separate experimental group while a third group consisted of a control group made up of the same clinical samples inoculated unto standard chocolate agar plates to confirm viability of the clinical isolates. Both systems were subjected to the same temperature conditions. The ability of each system to maintain viability of the isolates was determined by comparing the CFU/ml at initial time zero and the CFU/ml at the end point time.

The ability of the InTrayGC system to maintain and transport viable gonococcal isolates for recovery after more than 24hrs has been confirmed by earlier studies (Papp et al., 2016; Beverly et al., 2000). However, the work under review was done to obtain approval from the FDA to use the InTrayGC system to transport clinical specimen for an extended period (up to 72hrs) before recovery of isolates of Neisseria gonorrhoeae. The methods used were chosen to demonstrate viability based on FDA standards.

The study results show that the InTrayGC system can maintain N. gonorrhoeae clinical isolates for up to 72h for recovery of viable isolates.

This study is important in that Neisseria gonorrhoeae is a very fastidious organism that is easily lost to temperature fluctuations during transportation of clinical specimens. There is a need to develop and assess appropriate transport systems to overcome this weakness.

Thank you for your time in reading and reviewing our manuscript and for your positive and constructive comments.

ABSTRACT AND INTRODUCTION

The main research question and the key findings of this study are clearly summarized. Other current literature on the topic has been referenced

Thank you.

FIGURES AND TABLES

All the associated text supports the information in the tables and figures. The figures are clear and readable, captions are complete and accurate.

Thank you for your feedback.

METHODS

The experiments used are appropriate and the data is enough to support the conclusions that were arrived at, although the assessment of more specimen would have given more weight to the data. However, the author may need to explain why the InTray GC system was incubated here. The system is described as a transport system, when in actuality, incubating the system before transportation has a strong positive influence on isolate survival/viability.

Limitations were addressed adequately; data was collected and interpreted correctly. This study can be replicated.

Thank you for your comments. As for why the InTray GC was incubated, this has to do with its current intended use, as the InTray GC is meant to be incubated for 24 to 48 hours prior to transportation (as described in the package insert). The intended use for the InTray GC has also been added as a reference.

RESULTS, DISCUSSION AND CONCLUSIONS

The results, discussion and conclusion are well described and clearly written

Thank you for your comments.

MINOR COMMENTS/EDITS

Line 66. Space between the end of the sentence and the parenthesis ie. (7) is missing

Line 193. Space between the & and C is missing

Line 261. (ref 6.). The reference title was not complete

Line 233. Typo at “used”, it should be “use”

Thank you for this feedback. We have made all the suggested minor edits.

Regards,

Keely Paris

Submitted filename: Response to reviewers_Jul19.docx

Click here for additional data file.

25 Oct 2021

72-Hour transport recovery of antimicrobial resistant Neisseria gonorrhoeae isolates using the InTray® GC method

PONE-D-21-16017R1

Dear Dr. Keely S Paris ,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Academic Editor

PLOS ONE

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #1: Yes

Reviewer #2: N/A

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #3: Yes

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Reviewer #2: The revised manuscript properly addresses comments from the reviewers. This reviewer does not have any additional comments for consideration.

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**********

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: aditya bandekar

Reviewer #2: No

Reviewer #3: No

11 Jan 2022

PONE-D-21-16017R1

72-Hour transport recovery of antimicrobial resistant Neisseria gonorrhoeae isolates using the InTray® GC method

Dear Dr. Paris:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

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Academic Editor

PLOS ONE