Use of Silica Desiccant Packets for Specimen Storage and Transport To Evaluate Pneumococcal Nasopharyngeal Carriage among Nepalese Children

It is important to monitor serotype distribution within the principal reservoir for Streptococcus pneumoniae, which is asymptomatic nasopharyngeal (NP) carriage among children. Monitoring changes of carriage serotype distribution over time can provide insights into the epidemiology of pneumococcal disease.

Pneumococci easily autolyze, making carriage studies difficult. NP specimens must be cultured quickly, with pneumococcal cultures immediately frozen at −70°C in freezing medium (5). We tested silica desiccant packages (SDPs) (Grace Davison, Baltimore, MD) containing 1.5 g of silica powder for storage and transport of NP specimens. SDPs are effective for transporting specimens for delayed culture of several bacterial species (2, 4, 7, 9), but their utility for delayed culture of NP swabs for pneumococcal isolation has not been demonstrated.

NP swabs were collected from 302 healthy children ranging from 1 to 15 years of age at a children's homeless shelter in Ranibari, Kathmandu, Nepal. A sterile cotton swab was inserted into the nasopharynx and rolled twice prior to inserting into an SDP and sealing. The length of time between collection of initial and last samples was 8 days. Transportation of NP samples at room or environmental temperature from Kathmandu to the CDC required 72 h. Isolates were stored for up to 13 additional days at room temperature before culture, with a maximal storage duration of 25 days. NP samples were incubated (37°C and 5% CO₂) for 4 h in 5 ml of Todd-Hewitt broth containing 3 drops of defibrinated rabbit blood. Cultures were streaked onto Trypticase soy agar II-5% sheep blood agar and incubated for 18 to 24 h.

Of 302 NP samples, 184 (61%) contained pneumococci according to alpha-hemolysis, optochin sensitivity, and bile solubility analysis results (Table 1). The level of carriage was high (69 to 83%) within the age groups 1 to 2 years, 3 to 4 years, 5 to 6 years, and 7 to 8 years and incrementally decreased among older ages (Table 1). Carriage among 1- to 10-year-old children (71% [156/221[) (Table 1) was comparable to the carriage previously observed among Nepalese children in the same age group in a different region (84 to 88%) (3). Incrementally decreased carriage was evident among groups of older children (Table 1). We observed no differences in pneumococcal recovery between NP specimens processed after 12 days and those processed after 25 days (data not shown).

TABLE 1.

Carrier status by age and gender

Characteristic	No. of subjects	No. (%) of carriers
Age (yr)
1-2	29	24 (83)
3-4	42	30 (71)
5-6	55	38 (69)
7-8	51	38 (75)
9-10	44	26 (59)
11-12	37	18 (49)
13-15	44	10 (23)
Total	302	184 (61)
Gender
Male	175	102 (58)
Female	127	82 (65)

Of isolates from 184 pneumococcus-positive samples, 160 were serotypeable (43 serotypes) by a Quellung assay using CDC antisera, with one mixed-carriage isolate detected (Table 2). Twenty-five (13.5%) isolates were nontypeable. All isolates were tested by the Etest (AB Biodisk, Sweden) and found to be penicillin susceptible (MIC ≤ 068 μg/ml). Four of nine conventionally serotyped 6A isolates were subsequently typed as newly discovered serotype 6C (6) by use of 6C-specific PCR (1) but were identified in the present study as representing serotype 6A. Thirty-seven (23%) of 160 isolates were covered by the 7-valent conjugate vaccine (vaccine serotypes 4, 6B, 9V, 14, 18C, 19F, 23F, and 6A), and 52 of 160 (32%) were covered by a 13-valent vaccine (vaccine serotypes 1, 3, 5, 6A, 7F, and 19A) in development (Wyeth) (8).

TABLE 2.

Serotype distribution among 185 NP isolates from children living in a homeless shelter in Nepal

Serotype(s) (in order of prevalence)	No. (% of total) of isolates per serotype, combined no. of isolates
Nontypeable	25 (13.5)
15B/15Ca	16 (8.6)
34	13 (7.0)
13	11 (5.9)
6Ab	9 (2.7)
23F	8 (4.3)
16F	7 (3.8)
3, 6B	6 (3.2), 12
14, 17F, 33F, 39	5 (2.7), 20
1, 18C, 19F, 33B, 35C, 37	4 (2.2), 24
5, 8, 11A, 15A, 23A	3 (1.6), 15
15F, 18B, 19A, 19B, 31, 35F	2 (1.1), 12
7C, 9L, 9N, 9V, 18A, 21, 22A, 27, 32A, 35B, 36, 45, 48	1 (<1%), 13
Total	185c

^aData for serotypes 15B and 15C are combined, since they are known to interconvert at high frequency.

^bFour of the 6A isolates were predicted to represent the newly discovered 6C serotype.

^cTwo different strains (serotypes 8 and 18C) were obtained from 1 child among the 184 children.

In summary, we found high levels of pneumococcal carriage among Nepalese children. In addition to the small scale of the study, one limitation was that comparisons were not made with analogous NP samples stored at −70°C in freezing medium. However, an important finding is that NP swabs can be maintained in SDPs in room or environmental temperature storage for at least 25 days, with up to 83% carriage evident in samples from members of subgroups consisting of younger children (Table 1).