J. Microbiol. Infect. Dis., (2023), Vol. 13(3): 125–127

Original Research

10.5455/JMID.2023.v13.i3.4

Quest for ideal transport conditions and factors affecting revival of Neisseria gonorrhoeae isolates: Experience of a National Reference Laboratory

Madhavi Kirti¹, Sumathi Muralidhar¹*, Abhishek Lachyan¹, Devanshi Sharma¹, Naveen Joshi¹ and Niti Khunger²

¹Apex Regional STD Centre and SRL-HIV, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India

²Department of Dermatology and STD and Apex Regional STD Centre, Safdarjung Hospital, New Delhi, India

*Corresponding Author: Sumathi Muralidhar. Apex Regional STD Centre and SRL-HIV, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India. Email: sumu3579 [at] yahoo.com

Submitted: 15/05/2023 Accepted: 01/09/2023 Published: 30/09/2023

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Abstract

Background: Neisseria gonorrhoeae (Gonococcus-GC) is a fastidious, autolytic Gram-negative diplococcus with stringent growth requirements, and cannot be cultivated in a routine microbiology laboratory, without well-equipped incubators, reagents, and special media. Hence, many clinics and laboratories prefer to ship the specimens or isolates to a dedicated referral laboratory for confirmation of isolates and antimicrobial susceptibility testing. Thus, transportation conditions for gonococcal isolates, become crucial for its recovery and successful isolation in the laboratory. This retrospective study was conducted at a national referral laboratory for gonococcus, in India, over 3 years.

Aim: In this study, an attempt was made to determine the factors affecting the revival of isolates of gonococci, that were despatched, from across India, to the referral laboratory for confirmation of species and antimicrobial susceptibility patterns.

Method: Over 3 years, the culture plates, test tubes, or vials used for transporting gonococcal isolates, and their modes of transport to the referral laboratory, were studied in detail. The isolates were revived (whenever possible), subcultured, and identified by standard methods in the referral laboratory.

Results: A total of 77 samples were processed for revival and 83.12% of isolates were recovered, with failure of recovery in 16.88% of specimens.

Conclusion: Several factors play a role in the successful revival of N. gonorrhoeae from culture isolates transported across the Indian subcontinent. These include purity of growth, culture media used for transport, sending of isolates in duplicates, temperature, time, distance, and season of transport. All these factors must be kept in mind when transporting gonococcal isolates, for successful revival. Finally, the skills of the laboratory technician are of immense importance too.

Keywords: Gonococcus, Neisseria gonorrhoeae, Speciation, Transport media, Transit time.

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Introduction

Neisseria gonorrhoeae, the etiological agent of gonorrhea, is the second most prevalent agent of sexually transmitted infection, after Chlamydia trachomatis. This organism is known to be fastidious, in terms of growth requirements, and is amenable to rapid autolysis (Symington, 1975).

The isolation and accurate laboratory identification of gonococcus is vital, especially in cases with suspected cephalosporin treatment failure, and as test-of-cure if an alternative treatment regimen (e.g., cefixime combination therapy) is used (CDC's sexually transmitted diseases treatment guidelines-2010, 2012). For this, efficient specimen transport and submission, from the clinic to the laboratory, is significant (Gumede et al., 2017). Several factors affect the recovery and final isolation of the organism, such as the method of collection, time of transport, and transport conditions, including temperature, CO₂ content, overgrowth of contaminants, or dilution of the organism in surrounding collection medium (Ng and Martin, 2005).

Recovery rates can be improved by following appropriate guidelines for isolation, including the use of Dacron swabs for specimen collection, and immediate plating onto a suitable medium with minimal delay. The primary specimens should be inoculated onto nonselective routine chocolate agar (CA) containing synthetic hemin and growth factors, as well as selective agar such as Thayer–Martin, Martin Lewis and/or New York city medium containing antimicrobial agents such as vancomycin (to suppress gram-positive organisms), that inhibit the growth of indigenous microflora (Martin and Lester, 1971; Janda and Knapp, 2003; and Unemo and Ison, 2013). The inoculated CA plates should be held at room temperature for no longer than 5 hours in a CO₂-enriched atmosphere while being transported to a local laboratory. If long-distance mailing is required, the specimens should be inoculated onto media contained in a CO₂-generating system, incubated for 18–24 hours before shipping, and should have visible growth (Ng and Martin, 2005).

Neisseria gonorrhoeae cultures have been reported to survive up to 4 days on Thayer Martin media upon mailing (Cross et al., 1970; Jephcott, 1997). According to reports, N. gonorrhoeae isolates can be maintained for 12–18 months on CA slants under sterilized paraffin oil, successfully (Cody, 1978; Tapsall, 1990).

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Materials and Methods

The Apex Regional STD Centre, Safdarjung Hospital, New Delhi, being the national referral laboratory for N. gonorrhoeae, receives isolates of gonococcus from all over India, for confirmation, identification, and stock keeping (lyophilisation), via courier transportation with different transit times.

The culture plates, test tubes, or vials used for transporting gonococcal isolates, and their modes of transport to the referral laboratory were studied in detail. The isolates were revived (whenever possible), subcultured, and identified by standard methods in the referral laboratory.

The isolates received were immediately plated onto CA at room temperature and incubated at 35°C at 5% CO₂ level for 18–24 hours. Plates were examined for colony characteristics and a Gram smear to look for Gram-negative, diplococci as per standard methods. Final identification was made based on biochemical tests–a positive superoxol test and oxidase test followed by a rapid carbohydrate utilization test showing a color change only for glucose. The details of the revived isolates were documented and preserved.

Ethical approval

This study was approved by the Institutional Ethics Committee.

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Results

Of the total of 77 isolates of Gonococcus (GC) received from various laboratories across the country, 64 could be revived at the Apex Referral laboratory (83.12%), giving a 16.88% failure rate (13 isolates).

Most samples received were sent by the laboratories, on routine media, such as CA plates, while some were sent in CA slopes, with or without an overlay of liquid paraffin. Six CA slopes were not overlaid with paraffin oil, and all of them failed to be revived. A few plates were sent in lysed blood agar plates. The longest time taken for samples to reach the Apex laboratory from various parts of the country, was 7 days.

The largest number of samples were received from the state of Maharashtra (Nagpur + Mumbai) (18), followed by Punjab and Telangana with 15 samples each. Other states from which samples were received, included Assam (8), Gujarat (4), Karnataka (13), Tamil Nadu (1), and Uttar Pradesh (3). The revival status of GC isolates from laboratories across various Indian states was as follows-Assam-75%, Gujarat-100%, Karnataka-61.53%, Maharashtra-94.44%, Punjab-86.67%, Tamil Nadu-0, Telangana-93.33%, and Uttar Pradesh-66.66%.

All the samples were shipped at ambient temperatures, wherein no specific temperature conditions were maintained while shipping. Considering the seasonal aspect, 36.3% of samples were received in the monsoon (June–September) season, with other seasons showing smaller numbers. The revival rates were least in the winter season (December–Feburary).

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Discussion

Our study results revealed that CA serves as a simple, cost-effective, and successful method for transportation as well as storage of N. gonorrhoeae isolates. Our results are in concurrence with a study by Sidharath D. Thakur and Jo-Anne R. Dillon (Thakur and Dillon, 2018).

Survival rates of isolates can be enhanced if culture media are coated with sterilized paraffin oil before shipping. Cultures on CA slopes, covered with paraffin oil, were stored for 3 months and were successfully revived in all the cases by recipients (Thakur and Dillon, 2018). As per our results, six slopes that were not overlaid with paraffin oil, failed to be revived, indicating that coating slopes with sterilized paraffin oil might increase the chances of revival.

Although nonrevived specimens were from varying distances, six samples shipped from Bangalore (2,195 km away, in South India), could not be revived, while most samples from Chandigarh (North India) and Nagpur (central India) were revived, clearly denoting the impact of distance, and delay, on successful revival and isolation of organisms.

Eight of the nonrevived samples were obtained between October and January (winter season); however, it is still not clear if climate and season have a role to play in the viability of isolates.

Many factors are responsible for the revival of isolates-the slants should be kept at 35°C–37°C in 5% CO₂ for 18–24 hours before being shipped. Also, a correlation of optimal pH with the viability of N. gonorrhoeae has been studied. A slight change in pH would affect the viability drastically (Finlayson et al., 1975).

In our study, the temperature during transportation did not have too much of a role to play as, all the specimens were transported under similar temperature conditions, which is in sync with the results found by Evans et al. (1986) wherein the number of revivable specimens at room temperature or under refrigeration did not differ dramatically from the number from which organisms were recovered from plates held at a recommended temperature (Evans et al., 1986).

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Conclusion

The revival rates of N. gonorrhoeae, transported across laboratories, at varying distances, depend on several factors. CA slopes with an overlay of sterile liquid paraffin seem to be a satisfactory method if other factors, such as temperature, distance, season, and duplication, are also taken cognizance of. Finally, the skills of the laboratory technician also contribute significantly to the successful isolation and detection of microorganisms.

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Acknowledgment

The authors would like to thank all participants of the study for their participation in the investigation.

Conflict of interest

All other authors declare no competing interests.

Funding

No funding sources

Data availability

All data generated or analyzed during this study are included in this published article.

Authors’ contributions

MK and SM designed and conceptualized the study. MK collected the data. NJ performed the laboratory testing. MK and SM analyzed the data. MK, SM, DS, and AL wrote the entire manuscript together. SM and NK reviewed the manuscript. All authors read and approved the final manuscript.

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