Comparative Performance of Commercial Amies Transport Media With and Without Charcoal for Neisseria gonorrhoeae Culture for Gonococcal Isolation and Antimicrobial Resistance Monitoring in Ukraine

Abstract

Introduction: Culture is the only laboratory method that provides live gonococcal isolates for monitoring antimicrobial resistance. Many clinical settings do not have direct access to laboratories for the immediate processing of biological samples. Validated and quality-assured transport media are recommended to maintain the viability of Neisseria gonorrhoeae. Methods: In total, 103 clinical samples were divided into two groups: 51 samples were stored in Amies agar gel medium containing charcoal and 52 samples were stored in agar gel medium without charcoal. All samples were stored at 4 °C for 0.5–6 h and then transported in a thermo-protected box within 0.17–0.25 h to the laboratory of Ternopil Regional STI Clinic (Ukraine). Results: The recovery rate was significantly higher for charcoal-containing Amies medium than for charcoal-free Amies medium (86.27%, 44/51 vs. 59.62%, 31/52). A higher rate of specimens transported in charcoal-containing Amies medium was isolated within 24 h (84.31%, 43/51), whereas most isolates from charcoal-free Amies medium grew within 24 h (42.31%, 22/52) or 48 h (17.31%, 9/52). Growth beyond the first quadrant of the agar plate was registered for 59.09% (26/44) of charcoal-containing Amies samples, compared with only 19.35% (6/31) of charcoal-free Amies samples. Conclusions: A high rate of N. gonorrhoeae recovery after transport has considerable public health implications for establishing national antimicrobial susceptibility programs. Charcoal-containing Amies medium had higher viability rate, and it could be used for diagnostic and isolation purposes in future antimicrobial susceptibility studies. Continuous validation studies of transport medium for N. gonorrhoeae culture are needed.

Introduction

Antimicrobial resistance is a major threat to public health globally. The pipeline of effective antimicrobials will become extremely limited if appropriate actions are not taken nationally and internationally [1]. Gonococcal infection caused by Neisseria gonorrhoeae ranks as the second most common sexually transmitted infection (STI) worldwide [2]. Gonococci have a unique ability to acquire resistance to almost all groups of antimicrobials used to treat infected patients [1]. Furthermore, self-treatment is widely performed in countries where antimicrobials are freely available without a prescription [3,4]. This inappropriate treatment could promote the development of antimicrobial resistance in N. gonorrhoeae [1]. Because no vaccine has been developed to prevent gonococcal infections, the management of N. gonorrhoeae relies on highly sensitive and specific diagnostics, notifications of sexual partners, effective treatment based on updated evidence-based guidelines, and the monitoring of the antimicrobial resistance profiles of circulating gonococci [1,5]. Effective and quality-assured antimicrobial susceptibility programs were implemented in many countries following a call for emergency actions [1]. However, substantial numbers of countries have not studied the antimicrobial resistance profile of their N. gonorrhoeae isolates [5,6].

To date, culture is the only laboratory method for obtaining live gonococcal isolates [7] for investigating antimicrobial resistance phenotypes as well as molecular and genomic epidemiological studies [1,3,8]. As N. gonorrhoeae is an extremely fastidious bacterium, point-of-care culture involving the direct inoculation of biological samples onto agar plates using nutritive culture medium and immediate incubation is recommended [9]. The absence of these criteria results in the loss of gonococcal viability and false-negative results [10,11,12]. The risk of obtaining false-negative results hampers the efficacy of treatment and increases infection transmission [1].

Many clinical settings do not have direct access to laboratories for the immediate processing of biological samples [11]. Validated and quality-assured transport media are recommended for these situations [12,13,14,15]. Non- nutritive transport media such as Amies and Stuart media supplemented with charcoal were identified as quality tools for the temporary storage and transport of fastidious microorganisms, including N. gonorrhoeae, based on their ability to maintain the viability of gonococci for 24–48 h at 2–8 °C [9,13]. Nutritive transport media can be used to store and transport biological samples for more than 48 h with a maximum survival rate after preincubation in transport medium at 36 ± 1 °C overnight before transportation to the laboratory [9]. However, it is difficult to ensure an uninterrupted supply of laboratory reagents, kits, and media in financially constrained settings in low- and middle-income countries, including Ukraine [11,12]. Moreover, nutritive transport media were not available for purchase in Ukraine between 2013 and 2018, and charcoal-supplemented transport media were not available in January–May 2018. Under these conditions, it is essential to validate non-nutritive transport media for N. gonorrhoeae culture in Ukraine to ensure the isolation of N. gonorrhoeae samples for diagnostic and surveillance purposes and guarantee the highest possible quality of culture performance.

To improve the isolation of N. gonorrhoeae for antimicrobial resistance research in Ukraine, we evaluated two non-nutritive transport swabs for N. gonorrhoeae culture that were available for commercial purchase during the study period: Amies agar gel medium containing charcoal and Amies agar gel medium without charcoal.

Methods

Study Design

The study design met the guidelines for investigations of diagnostic accuracy [16]. Comparison of the viability of N. gonorrhoeae clinical isolates using non-nutritive Amies transport media with and without charcoal was made to the composite reference standard. Combining two methods was defined as a composite reference standard: positive point-of- care culture using selective agar medium Chocolate agar™ PolyViteX VCAT3 (BioMérieux Ltd., France) and the detection of Gram-negative intracellular diplococci along with leukocytosis in the male urethral smears by microscopy. Preliminary diagnosis of N. gonorrhoeae infection in females was made by detecting intracellular Gram-negative diplococci in the cervical discharge. The final diagnosis of gonorrhea in females was made based on a positive point-of- care culture [3,12].

Patient Population

The inclusion criteria for patients were urogenital discharges as clinical symptoms, detection of Gram-negative intracellular diplococci in urethral smears from males, or cervical smears from females along with leukocytosis. All females were enrolled as contacts to infected partners. Exclusion criteria were treatment with antimicrobials either systematically or using local medications one month before the visit, refusing to sign informed consent, females who had used a vaginal douche in the past seven days, and menstruation or pregnancy in females. We enrolled 103 symptomatic patients, including 88 (85.44%) men and 15 (14.56%) women, aged 26.62 ± 6.83 years (age range: 18–80 years; median age 23 years) who visited the Ternopil Regional STI Clinic (Ukraine) from December 2015 to October 2018 and met all inclusion criteria. The most prevalent clinical symptoms in males were urethral discharge (100%, 88/88), discomfort during urination (13.64%, 12/88), and burning sensation (5.68%, 5/88). In females, the most common clinical symptoms were vaginal discharge (100%, 15/15) and lower abdominal pain (6.67%, 1/15).

All patients received treatment for gonorrhea and concomitant STIs according to the guidelines of the Ministry of Health of Ukraine issued in 2004 and 2009 [3,4].

Biological Specimens

Two clinical urogenital specimens for point- of-care culture and Amies transport medium were simultaneously collected from the urethra in men and cervix in women. Biological samples were randomly divided into two groups according to the non-nutritive transport medium used for temporary storage: odd samples (n = 51) were stored in charcoal-containing Amies medium (Copan Diagnostics Inc., Brescia, Italia), and even samples (n = 52) were stored in charcoal-free Amies medium (Jiangsu Suyun Medical Materials Co., Ltd., China). All samples were held at 4 °C for 1.29 ± 0.78 h (range time: 0.5–6 h; median time: 1 h; interquartile range (IQR) 0.5–1.5 h), depending on how close the patients’ visits were to the time of transportation of samples to the laboratory (one scheduled transportation per clinician’s working hours). Samples were transported in a thermo-protected box to the laboratory of the Ternopil Regional STI Clinic within 0.2 ± 0.03 h (time range: 0.17–0.25 h; median time: 0.2 h; IQR: 0.18–0.23 h). Temporary storage time in the laboratory before processing was with range 0.5–1 h (median time: 0.6 h; IQR: 0.52–0.74 h) for all samples. Thus, the total time between sampling and culturing was within 1.2–6.9 h (median time: 1.8 h; IQR: 1.4–2.3 h) for all samples and, notably, it did not differ between the two studied groups.

N. gonorrhoeae Culture

Laboratory specialists inoculated swabs from the transport media onto 90-mm agar plates containing Chocolate agar™ PolyViteX VCAT3 medium with a combination of antimicrobial and antifungal agents added by the manufacturer to enhance the selectivity (vancomycin, colistin, amphotericin B, and trimethoprim). All samples were incubated at 36 ± 1 °C in a humid candle jar for 24–48 h and, if negative, for up to 7 days according to the national laboratory guidelines. Isolates were confirmed as N. gonorrhoeae by the identification of Gram-negative diplococci on microscopy, rapid oxidase test (OXItest diagnostic strips, Microlatest, Czech Republic) positivity, and rapid sugar utilization test (Neisseria-test, PLIVA—Lachema Diagnostika s.r.o., Czech Republic) positivity. The results were considered positive when at least one typical colony was present and confirmed as N. gonorrhoeae.

Criteria for Transport Medium Evaluation

The cultures of N. gonorrhoeae were compared for differences in recovery (isolation) rates, the time required for isolation, and growth grades. The criteria for growth from culture swabs were previously described [17]. In our study, we categorized all samples into two groups: samples with growth within the first quadrant of the Petri dish (grades 1 and 2) and samples with growth beyond the first quadrant (grades 3–5). The criteria for evaluating N. gonorrhoeae growth from cultured swabs are presented in

Table 1. Criteria for evaluating Neisseria gonorrhoeae growth from cultured swabs.

Group	Grade a	Description of Growth on the Agar Plate
Growth within the first quadrant	1	1–50 colonies in the first quadrant
	2	>50 colonies in the first quadrant
Growth beyond the first quadrant	3	Growth in the first and second quadrants
	4	Growth in the first, second, and third quadrants
	5	Growth in all four quadrants

^a Criteria for growth from culture swabs were as previously described [17].

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Statistical Analysis

Descriptive data are presented as frequencies and percentages. The 95% confidence intervals (CIs) were calculated using the exact binomial distribution method. p < 0.05 denoted statistical significance. Statistical analysis was performed using MedCalc Statistical Software version 19.3.1 (MedCalc Software bvba, Belgium).

Ethical Approval

The Bioethics Commission of I. Horbachevsky Ternopil State Medical University (Ukraine) approved the study (Excerpts from Minutes No. 29, dated 20 May 2015) and publication of data (Experts from Minutes No. 63, dated 15 March 2021).

Results

The proportions of recovered and dead N. gonorrhoeae clinical isolates depending on using transport media are displayed in

Table 2. Viability of gonococcal isolates in Amies transport medium.

Viability of Neisseria gonorrhoeae Isolates		Types of Amies Transport Medium		p Value
		With Charcoal, n = 51	Without Charcoal, n = 51
Recovered isolates	n	44	31	0.003
	%	86.27	59.62
	95% CI	73.74–94.30	45.11–73.00
Dead isolates	n	7	21	0.003
	%	13.73	40.38
	95% CI	5.70–26.26	27.00–54.89

CI—confidence interval.

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Briefly, the viability of gonococcal isolates was significantly higher for charcoal-containing Amies medium than for charcoal-free Amies medium (86.27%, 44/51 vs. 59.62%, 31/52, respectively). The rate of dead isolates was lower in the charcoal-containing Amies medium (13.73%, 7/51) compared with the charcoal-free Amies medium (40.38%, 21/52, p = 0.003).

The results of N. gonorrhoeae cultures from transport swabs based on the duration of incubation are provided in

Table 3. Recovery of Neisseria gonorrhoeae isolates from two types of Amies transport swabs depending on the incubation time.

Incubation Time		Types of Amies Transport Medium		p Value
		With Charcoal, n = 51	Without Charcoal, n = 52
24 h	n	43	22	<0.0001
	%	84.31	42.31
	95% CI	71.41–92.97	28.73–56.80
48 h	n	1	9	0.009
	%	1.96	17.31
	95% CI	0.05–10.45	8.23–30.33
72 h a	n	0	0	NA
	%	0	0
	95% CI	0–6.98	0–6.85

^a If negative, up to 7 days. CI—confidence interval; NA—not applicable.

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The vast majority of specimens were isolated significantly faster (within 24 h) using the charcoal-containing Amies medium (84.31%, 43/51). In contrast, growth within 24 h (42.31%, 22/52) and 48 h (17.31%, 9/52) was observed for isolates from the charcoal-free medium. No isolates grew later than 48 h.

The results of N. gonorrhoeae growth for isolates obtained from the two types of culture swabs are presented in

Table 4. Evaluation of the growth of Neisseria gonorrhoeae isolates obtained from two types of Amies transport swabs.

Grades of N. gonorrhoeae Growth		Types of Amies Transport Medium		p Value
		With Charcoal, n = 44	Without Charcoal, n = 31
1–2	n	18	25	0.0007
	%	40.91	80.65
	95% CI	26.34–56.75	62.53–92.55
3–5	n	26	6	0.0007
	%	59.09	19.35
	95% CI	43.25–73.66	7.45–37.47

CI—confidence interval.

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Bacterial growth was significantly more enriched for isolates obtained from charcoal- containing Amies medium than medium lacking charcoal. Specifically, growth beyond the first quadrant of the agar plate was registered for 59.09% (26/44) of isolates from charcoal-containing Amies medium, versus 19.35% (6/31) of isolates from charcoal-free Amies medium.

Discussion

The validation of alternative transport media options is urgently required in Ukraine and other low- or middle-income countries due to restrictions on the availability of laboratory supplies. On the other hand, not many reports on the efficacy of N. gonorrhoeae recovery were retrieved. Thus, we performed this comparative study of Amies transport media with and without charcoal using clinical urogenital samples from adult patients with urogenital discharge and a confirmed diagnosis of gonorrhea to improve the isolation of N. gonorrhoeae under resource- constricted conditions. Our results comply with recently published studies and support recommendations to validate commercially available culture transport media for N. gonorrhoeae isolation before using them in laboratory practice [19,20,21,22,23,24].

The minimum recommended sensitivity of culture for isolation N. gonorrhoeae is 85% [20]. Arbique JC et al. found that various non-nutritive transport media with and without charcoal could maintain 94% and 82% of N. gonorrhoeae viability for 24 h, respectively [21]. We obtained an acceptable viability rate (86.27%) of N. gonorrhoeae culture using Amies transport medium with charcoal for a temporary storage time of 1.2–6.9 h. A suboptimal recovery rate (59.62%) was obtained for Amies transport medium without charcoal. Previous studies suggested that the viability of N. gonorrhoeae isolates has a negative correlation with the duration of temporary storage in the transport media [15,17,21,22,23,24]. The laboratory guidelines recommend to perform pre-analytical processing as fast as possible, even using transport media [9,13,20]. Results from our study agree with earlier published reports that the risk of lost viability of N. gonorrhoeae is higher in the cases of using Amies transport media without charcoal [21,22,24]. Worryingly, some laboratories operating under limited resource settings continue to use media with suboptimal sensitivity [7,11,25]. Our study highlights that the recovery rate of gonococcal isolates might vary widely across different commercial media. Therefore, validation of media is essential before using any media in laboratory practice [9,13,19,21].

The duration of sample incubation is an important criterion for N. gonorrhoeae culture [9,21]. For example, issues will arise for samples cultured on Thursday and provide colonies within 48 h or more, i.e., over the weekend. Many laboratories do not work during weekends. Thus, the risk exists to obtain nonviable isolates on Monday. Dead N. gonorrhoeae isolates might be acceptable only for diagnostic purposes, but they cannot be used for antimicrobial susceptibility testing. Only fresh N. gonorrhoeae isolates cultured within 18–24 h should be collected for further antimicrobial resistance study [9]. Our testing showed that N. gonorrhoeae isolates grew significantly faster and within 24 h using Amies transport media with charcoal than without charcoal (84.31% vs 42.31%, respectively; p ˂ 0.0001). Almost one-fifth of N. gonorrhoeae isolates (17.31%) were obtained within 48 h using Amies transport media without charcoal, compared with only 1.96% cases using Amies transport medium with charcoal. Substantially prolonged laboratory processing could complicate the isolation of such fastidious bacteria as N. gonorrhoeae for antimicrobial susceptibility testing [20,23].

Most laboratories cannot perform antimicrobial susceptibility testing in routine practice. Specialized leading reference microbiology laboratories support antimicrobial susceptibility testing in many countries [6]. N. gonorrhoeae antimicrobial susceptibility programs are mainly designed as retrospective studies when viable isolates are preserved and stored under particular conditions [9]. To investigate N. gonorrhoeae antimicrobial resistance, the maximum amount of fresh (18–24 h) pure gonococcal colonies from the agar plate should be collected, preserved, and transported to the laboratories [9]. This is crucial to ensure a high restored rate of gonococcal isolates before antimicrobial susceptibility testing. Our study showed that significantly strong growth was obtained using Amies transport media with charcoal (59.09%, p = 0.0007). Most isolates (80.65%) stored in the Amies transport medium without charcoal showed restricted growth, an equivalent area of only one quadrant of the Petri dish. Insufficient collection of N. gonorrhoeae colonies from Petri dish could decrease bacterial viability during temporary storage before antimicrobial susceptibility testing [9]. In such circumstances, the criteria of growth grades described previously by Farhat SE et al [17]. have practical meaning and could be used for further evaluation testing. Thus, comparing the two types of Amies transport media according to the grades of N. gonorrhoeae growth is one of the present study’s strengths.

Another key strength of our study is the comparison of the ability to maintain the viability of N. gonorrhoeae using commercially available non-nutritive Amies transport media in Ukraine. According to good laboratory practice, validation of the performance characteristics of media for fastidious bacteria like gonococcus is essential [9,13,19]. The main output of our study was the improvement of local laboratory operating procedures and provided data for updating national laboratory guidelines. This primary step could help implement antimicrobial surveillance programs for the antimicrobial resistance of N. gonorrhoeae in resource-constrained countries.

The data of the present study impact the diagnostics of gonorrhea in the Ternopil Regional STI Clinic (Ukraine). We have changed the standard operating procedures for collection, temporary storage, and transportation of biological material with the suggestion of using non-nutrition Amies transport media with charcoal. For the first time, we performed the pilot collection of 150 clinical N. gonorrhoeae isolates that circulated in two regions (Ternopil, n = 136; and Dnipropetrovsk, n = 14) in Ukraine in 2013–2018 [3,8]. This has allowed us to perform for the first time an antimicrobial resistance study of these collected isolates from Ukraine using international quality-assured methods (E-test and whole-genome sequence) as previously described [3,8]. Interestingly, none of the isolates from Ukraine were phenotypically resistant to ceftriaxone, cefixime, azithromycin, spectinomycin, or gentamicin. Resistance to benzylpenicillin was detected in only 0.7% of isolates, and 0.7% of isolates had a borderline resistance to the extended-spectrum cephalosporins and belonged to the internationally spreading multidrug-resistant NG-MAST ST1407, MLST ST1901 clone [3,8].

Resistance to benzylpenicillin and/or decreased susceptibility to β-lactam antimicrobials were associated with the mosaic penA-34.001 allele (2.7%), β-lactamase production (0.7%), mtrR (11.3%), penB (12.7%) and the ponA1 mutation (16.7%) [8]. Moreover, 11.3% of isolates were resistant to ciprofloxacin due to gyrA gene mutations (11.3%) and parC gene mutations (8.7%) [3,8]. Resistance to tetracycline was detected in 6% of isolates and was caused by the rpsJ V57M mutation (16.7%) and/or presence of tetM (4.7%) [3,8]. Our data support the need to study antimicrobial resistance nationally and internationally because geographical diversity of gonococcal strains could be present due to the high genetic plasticity of N. gonorrhoeae [1,5,6].

However, our study has several limitations. First, no reference strains were included because they were not available in Ukraine during the study period. Second, no extragenital specimens were tested. Additionally, the study was performed in only one laboratory, and a comparatively low number of tested clinical specimens were included. We did not split the clinical specimens from the same patients between the two types of Amies transport media, as we could not oversample patients. Future research addressing these limitations would be valuable, including reference and clinical strains with different concentrations to avoid bacterial load and sampling procedure as possible biases.

Furthermore, as molecular tests were not available in our setting, we used a composite reference standard with a combination of microscopy and culture methods [7,16,18]. Previously published analytical studies of the diagnostic characteristics of microscopy and culture compared with the international reference nucleic acid amplification tests (NAATs) established a high specificity of culture (100%) and microscopy of urogenital specimens (99.8–100%) [10,25]. However, the sensitivity of microscopy and culture for gonococcal infection is suboptimal, i.e., 71.4% and 57.1%, respectively [10]. Moreover, the diagnostic sensitivity of microscopy of cervical specimens in females (31.8–50%) is crucially lower than of urethral smears in males (75–80%) [10,25]. Notably, the performance of culture for N. gonorrhoeae could vary widely in different clinical settings due to the quality of media, timely updating of local standard operating procedures and national guidelines, as well as the professional knowledge of laboratory specialists [7,25]. Thus, an extensive amount of gonorrhea cases remain undetected in clinical settings without access to NAATs [2,7,14].

The fact that we enrolled symptomatic patients with positive microscopy in our study, as well as a point-of-care culture of N. gonorrhoeae, could explain that the bacterial load in the tested samples was higher than that in asymptomatic gonorrhea cases and/or cases with positive-only NAATs results along with negative microscopy and/or culture results. The high initial bacterial load in the urogenital samples benefits the recovery of N. gonorrhoeae [22]. We can assume that the logistic choices could differ among asymptomatic patients with preference for using point-of-care culture or nutritive transport media [9]. A future extensive study including highly sensitive and specific, quality-assured NAATs as a reference method among symptomatic and asymptomatic patients would be extremely valuable for ensuring the use of transport media with the best performance characteristics.

Conclusions

Transport media are crucial for maintaining the viability of microorganisms in situations in which sites cannot perform point-of-care cultures of isolated gonococci. The widespread implementation of validated transport media is essential internationally for fastidious bacteria such as N. gonorrhoeae. Increased recovery of N. gonorrhoeae after transport may have important public health implications for the establishment of national antimicrobial susceptibility programs. Our study revealed that charcoal-supplemented Amies medium has optimal sensitivity and gonococcal growth grades, making it potentially useful for diagnostic and isolation purposes in future antimicrobial susceptibility studies. Continuous validation research of transport media for N. gonorrhoeae culture should be implemented in routine laboratory practice nationally and internationally.