Literature review > Issue_5 > Review on Wade et al. 

For many decades, specimens for the isolation, identification and antimicrobial susceptibility testing of Neisseria gonorrhoeae have been transported in Amies semi-solid medium. Assessments of the efficacy of the Amies transport medium for transporting bacteria including N. gonorrhoeae have shown variable results which have not been attributed to any specific characteristic of the organism.

This study examined whether the auxotype of a gonococcal strain correlated with its survival in Amies transport medium. The authors measured the survival of five isolates belonging to each of six auxotypes (prototrophic [non-requiring], and isolates requiring proline, arginine, hypoxanthine, proline and arginine, arginine and hypoxanthine, and hypoxanthine) after storage in Amies transport medium with, and without, activated charcoal for 24 and 48 h. The organisms were suspended in PBS to an optical density equivalent to a MacFarland no. 1 standard and then diluted 1:1 in PBS or nutrient broth, respectively. Two sets of swabs were each inoculated with 100 µl of the respective suspensions and introduced into sets of Amies transport media with and without activated charcoal. Survival of the organisms after storage at room temperature for 0-, 24-, and 48-h, was measured by vortexing the swab tips in 1 ml of PBS for 30 sec and determining viable counts, in triplicate, on chocolate agar. The median of the three counts for each of the eight inoculum-medium-time combinations for each strain in each transport medium was then recorded. The median viable count for each set of results for the 5 strains representing each auxotype is presented.

Several questions must be raised about the methodology used and presentation of results obtained in this study. The authors serotyped the isolates to select isolates that were distinctly different but did not present these data. One obvious question is whether both IA and IB serovars were represented among some, if not all, of the auxotypes. Based on the fact that IB strains are more resistant to antimicrobial agents than are IA strains, it would have been valuable to determine if IB strains survived storage in Amies medium better than IA strains. It is unfortunate that these data were not presented. Secondly, conventionally, viable counts would be performed on triplicate samples for each inoculum-medium-time combination; triplicate counts of a single test may not sufficiently represent the survival of an isolate and would not substitute for triplicate samples. Thirdly, by presenting only the median result for the inoculum-medium-time combination results for the five strains representing each auxotype, it is not possible to appreciate the range of survival rates for each auxotype. It would have been helpful to know whether these survival rates were tightly clustered or diverse. Further, results for only five of the six auxotypes are presented clearly although it appears that the data indicating the survival of the hypoxanthine-requiring strains may be superimposed on the data indicating the survival of the arginine-requiring strains.

Given the technical limitations of the methodology and data presentation, it is difficult to interpret some aspects of the data. If we assume that the median viable counts are representative of each strain (highly reproducible) and auxotype (tightly clustered), it is not surprising that the isolates survived better in charcoal-containing Amies medium than in the plain medium. It is somewhat surprising that neither the arginine-requiring or hypoxanthine-requiring isolates survived better in the charcoal-containing medium than in the plain medium. Nevertheless, it is apparent that, although many isolates would survive for the 48-h storage interval in the Amies medium with charcoal, a considerable proportion of isolates would not. The authors conclude, justifiably, that clinical specimens could only be stored for 24 h before being inoculated onto an isolation medium for N. gonorrhoeae.

It must be remembered, also, that the measurements made in this study were performed under ideal conditions with inocula of ca. 10⁷ CFU of pure cultures of gonococcal isolates. It might be expected that specimens from symptomatic infections might contain sufficient CFU to survive for 24 h [1] although contaminants (yeast, commensal bacterial species) and other factors such fatty acids and nucleases may reduce the number of viable CFU in such specimens [2]. However, many clinical specimens may contain <= 102 CFU and, based on the survival rates depicted, none of the organisms from such specimens would have survived for 24 h.

To insure the maximum recovery of gonococcal isolates, immediately after collection, specimens should be inoculated onto growth media - selective or nonselective depending upon the anatomic site from which they are collected -and immediately placed in a CO₂-enriched atmosphere (a sealed container with a CO₂-generating system or a candle-extinction jar) and transported to the laboratory for incubation at 35^oC to 36.5^oC within 5 to 6 hours of inoculation [3]. Inoculated media may be maintained at room temperature during transport - a CO₂-enriched atmosphere is more important than incubation at 35^oC to 36.5^oC - but should be protected from extremes of temperature by transporting them in an insulated container [3].

The variable survival rate of gonococci does not mean that there is no role for semisolid transport media. These media may provide, and have provided, a more-than-adequate method for transporting pure cultures for periods of two to three days. Pure cultures may be inoculated at a much higher density than under the controlled conditions for this study and may be expected to survive for longer periods of time.

When it is essential to obtain a representative collection of viable organisms for study, it may be necessary to provide laboratory capabilities at the collection site or a reliable courier service to transport specimens and supplies between a clinic and a central laboratory in a timely manner.

References:

1. Perry J. Assessment of swab transport systems for aerobic and anaerobic organism recovery. J Clin Microbiol 1997;35:1269-1271.

2. Olsen CC, Schwebke JJ, Benjamin Jr WH, Beverly A, Waites KB. Comparison of direct inoculation and Copan Transport Systems for isolation of Neisseria gonorrhoeae from endocervical specimens.

3. Knapp JS, Koumans EH. Neisseria and Branhamella. Pp 587-603 In. Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH. (ed.). Manual of Clinical Microbiology. 7th ed. American Society for Microbiology, Washington D. C, 1999.

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