Improved Rate of Isolation of Neisseria meningitidis by Direct Plating of Pharyngeal Swabs

Abstract

Culturing of pharyngeal swabs for Neisseria meningitidis is an important clinical and epidemiological tool. Routine methods include direct plating onto solid medium or later plating in the laboratory. A comparison of these methods used with 490 high school students found a significantly higher carriage rate with direct plating (11.8 versus 6.1%; P < 0.001).

Pharyngeal carriage of Neisseria meningitidis is common in the general population, with rates of 20 to 30% in young adults (2, 3). Immediate plating of cotton swabs onto solid culture medium at the site of swabbing (direct plating) is commonly used in epidemiological studies to measure carriage rates (2). An accepted alternative method is to place the swabs in transport medium and send them to the laboratory for plating (3). In clinical practice in the United Kingdom, laboratory plating is usual, as reflected in the Public Health Laboratory Service standard operating procedure for culture of pharyngeal swabs. The assumption is that prompt (i.e., often overnight) transport under appropriate conditions will not significantly affect the rate of isolation of N. meningitidis.

Nasopharyngeal swabs were collected in autumn 1999 from sixth-form students in the Plymouth area of southwest England as part of a United Kingdom multicenter study of meningococcal carriage. Other study centers were in Manchester, Oxford, Nottingham, London, Glasgow, Cardiff, and Bangor. The study protocol allowed either direct plating or transport in Amies medium for laboratory plating, with the latter method being used in Plymouth. The estimated interval between specimen collection and inoculation of plates ranged between 5 and 7 h. An interim analysis, after swabs were taken from 906 students, showed an unexpectedly low meningococcal carriage rate of 6.1% (55 of 906). This contrasted sharply with results from other centers, where direct plating was mostly used and where carriage rates ranged from 10 to 23%. The culture medium used was GCVCAT. This is a modified New York City medium, containing vancomycin, trimethoprim, and amphotericin B. Quality control of the medium revealed no inhibition of meningococcal growth, and the swab collection technique was satisfactory.

We set out to compare meningococcal isolation rates obtained with direct and laboratory plating. Pharyngeal swabs from 490 students were plated on-site and then placed in transport medium for plating at the laboratory. The carriage rate from direct plating was 11.8% (58 of 490), whereas the rate obtained from laboratory plating was 6.1% (30 of 490) (Table 1). This difference was highly statistically significant using McNemar's test (P < 0.001). The carriage rate from laboratory plating during the comparative study (6.1%) was the same as the rate before the study (6.1%), suggesting that the difference was unlikely to be due to loss of organisms from the plate. Swabs were taken from students in many of the same schools for direct plating a year later. For schools in which swabs were laboratory plated in the first year and directly plated in the second year, carriage rates increased from 50 of 761 (6.6%) to 161 of 1,076 (14.9%) (P < 0.0001). For schools in which swabs were directly plated in both years, carriage rates were unchanged, i.e., 42 of 342 (12.2%) versus 61 of 504 (12.1%). These data provide further evidence of higher yields from direct plating.

TABLE 1.

Comparison of meningococcal isolation rates obtained using direct and laboratory plating^a

Result of culture from laboratory plating	No. with the following result of culture from direct plating:
	Positive	Negative	Total
Positive	30	0	30
Negative	28	432	460
Total	58	432	490

^aMcNemar's test statistic = 5.1; P < 0.001. 

Detection of meningococcal carriage is important in epidemiological studies of prevalence, acquisition, and transmission (2, 3, 5) and possibly in outbreak management. Swabbing is also important in individual patient management, as the yield of positive cultures from pharyngeal swabs, as opposed to blood, may remain high for some time after administration of antibiotics (1). The sensitivity of a single swab may be low (5, 6), so any means of maximizing yield is important. The sensitivity of any bacterial culture is greatest when the specimen is inoculated directly onto culture medium immediately after collection. This effect may be particularly important when attempting culture of Neisseria species, as has been reported previously with Neisseria gonorrhoeae (4, 7). The benefits of immediate inoculation must be weighed against the practical difficulties of providing sterile culture medium to the site of specimen collection. A study with military personnel found no clear difference between carriage rates from direct plating and laboratory plating after 5 h in transport medium, but the numbers in that study were too low to exclude such an effect (5). The experience at the Manchester Public Health Laboratory in two separate carriage studies with teenagers gave similar rates (21 to 23%) despite direct plating in one study and laboratory plating (mean delay, 3.5 h) in the other. Direct comparison as performed in Plymouth, however, has not been undertaken (E. Kaczmarski, personal communication).

The reasons for the higher rates of overall carriage found in the Manchester studies are unexplained and deserve further investigation. Nonetheless, our findings indicate that putting swabs in transport medium for more than 5 h as an alternative to direct plating may halve the culture positivity rate. We recommend direct plating of pharyngeal swabs whenever practical, both for studies of meningococcal carriage and for investigation of cases of suspected meningococcal disease.