Indonesia, with the world's fourth largest population, has an unknown rate of invasive Haemophilus influenzae type b (Hib) disease, as do many other Asian countries.1 Although studies in Hong Kong,2 Taiwan3 and Singapore4 have reported invasive disease incidence rates more than 10-fold less than pre-vaccine era studies in Europe and North America, it is unclear whether this difference derives from surveillance, diagnostic or laboratory limitations; from more extensive and early use of antibiotics in Asia; or from a true absence of Hib bacteria among Asian populations.
The Program for Appropriate Technology in Health (PATH), the Association Pour l'Aide à la Médecine Préventive and the Indonesian Ministry of Health have proposed a collaborative Hib vaccine intervention trial in Lombok, Indonesia, to determine the effectiveness of vaccine in reducing the incidence of childhood pneumonia and meningitis. Hib vaccine is not currently included in the routine schedule of vaccinations and, although vaccine can be purchased through private providers, immunization records suggest a very small percentage of children receive it. We conducted a preliminary population-based nasopharyngeal carriage study, the first in Indonesia, to determine the prevalence of Hib nasopharyngeal colonization and thus whether one of the conditions for invasive Hib disease is present in Indonesia.
Study population. Lombok Island is located in the center of the Indonesian archipelago just east of Bali. The island has an area of 5435 km2, a volcano rising 3726 m in height and a tropical equatorial climate with a wet season from November to April. The population of 2.6 million is predominantly Muslim and of the Sasak ethnic group. A rural agrarian economy predominates, although tourism is becoming increasingly important. Mataram is the largest city with a population of approximately 250 000.
We conducted a prospective, population-based study. We selected a sample size based on identifying a carriage rate of at least 5%, ± 2%, with a power of 80% and a 95% confidence interval. We stratified sampling by the four administrative regions of Lombok (Mataram, West, Central and East) and then randomly selected 5 hamlets in each region, for a total of 20 hamlets. In each hamlet we attempted to culture bacteria from the nasopharynges of 8 children in each of 3 age groups: 0 to <6 months; 6 to <12 months; and 12 to <25 months. We selected the first 8 children of each age group to come to a well child clinic known locally as the outreach integrated health post or posyandu; children with illness were excluded. Because occasionally extra children arrived for evaluation, we finished with 484 study participants rather than the planned 480. This study was approved by the PATH institutional review board and by the Indonesian Ministry of Health.
Risk factor evaluation. A field-tested questionnaire was administered to the parent or guardian regarding various potential risk factors for nasopharyngeal carriage of Hib or other serotypes. Risk factors evaluated included: gender; current breast-feeding; current use of foods other than breast milk; consumption of premasticated food; use of wood for heat; use of kerosene for heat; the presence of someone who smoked cigarettes in the household; a previous history of at least one child death in the family; a history of sickness, diarrhea or cough during the previous 30 days; the presence of more than one other child in the household; and a household size of five or more.
Specimen collection. After obtaining informed consent, field workers placed calcium alginate-tipped swabs (Calgiswab®; Spectrum Laboratories, Dallas, TX) into the nasopharynx of each subject. The depth of swab insertion was estimated as one-half the distance between the tip of the child's nose and the anterior portion of the ear. The swab was immediately placed in Amies transport medium for shipment to the central laboratory (Biomedical Research Unit, Mataram Hospital, Lombok).
Lombok laboratory methodology. Laboratory methods were derived from surveillance procedures recommended by the World Health Organization5 as modified for the Invasive Bacterial Infections Surveillance Group project.6 Within 24 h of nasopharyngeal swab collection, laboratory personnel at the Biomedical Research Unit in Lombok streaked the swab onto a chocolate agar plate (Becton-Dickinson, Franklin Lakes, NJ) with 300 μg/ml bacitracin. After overnight incubation at 35-37°C in 5% CO2, colonies typical of Hib were identified and plated on chocolate agar. Again after overnight incubation colonies with morphology typical of Hib were identified and plated on trypticase soy agar (Becton-Dickinson) with x- and v-factor discs (Difco TSA/XV; Difco Laboratories, Detroit, MI). All specimens with x and v factordependent growth were frozen at −70°C in trypticase soy broth with 20% glycerol (Becton-Dickinson) while awaiting delivery of serotyping materials.
After ∼2 months frozen specimens were serotyped with the Phadebact® Haemophilus test (Boule Diagnostics AB, Huddinge, Sweden), an agglutination test that identified specimens as Hib, typable H. influenzae but not type b, and nontypable H. influenzae or not H. influenzae. If type b was identified no further serotyping was performed. Specimens that were identified as typable but not type b were tested with antisera (Difco) to determine the specific serotype.7
Antibiotic sensitivity was determined for all Hib isolates by the Kirby-Bauer disc diffusion method8, 9 for ampicillin, cefotaxime, chloramphenicol, sulfamethoxazole and tetracycline and for beta-lactamase production with cefinase discs (BBL Microbiology Systems, Cockeysville, MD). A standard Hib isolate (ATCC 49247) was used for quality control of all media and procedures.
Reference laboratory methodology. A sample of H. influenzae-positive specimens was sent to the National Haemophilus Reference Laboratory, Oxford, UK. Specimens were plated onto both chocolate agar and Hib antiserum agar to maximize the recovery of type b strains. All specimens yielded H. influenzae strains on biochemical identification. Serotyping was then performed by a slide agglutination method and genotyped by PCR. The PCR method identifies all capsular (a through f) and capsular-deficient strains (b−) and confirms strains that are genetically noncapsulated.10 For the few samples for which the Lombok and reference laboratory results differed, we used the reference laboratory results for analysis.
Statistical analysis. All analyses were performed using the CSample function within Epi-Info Version 6.01 computer software. Region was used as the stratification variable and the hamlet was used as the primary sampling unit. We weighted for age and the population of children <2 years of age within each region. Because hamlet populations were not available, we assumed that all hamlets had approximately the same number of people and thus did not weight for this variable. Results were considered statistically significant if the 95% confidence interval (CI) did not include 1.
Prevalence. We collected 484 samples, of which 155 (32%) yielded organisms that were presumptively H. influenzae based on growth on TSA/XV. The laboratory in Lombok determined the serotype of all 155 positive isolates, whereas 36 isolates were sent to the reference laboratory for confirmatory serotyping. For 5 isolates Lombok and the reference laboratory disagreed. For 3 cases the reference laboratory found nonencapsulated strains whereas Lombok found encapsulated strains (types a, b and c). For 2 cases the reference laboratory found encapsulated strains (types b and c) whereas Lombok found nonencapsulated strains. Using the reference laboratory as the standard, we found 34 encapsulated organisms, including 22 type b, 4 type e, 3 each of types d and f and 1 each of types a and c.
Among all 484 children, the age- and population-weighted Hib carriage prevalence, adjusted for design effect, was 4.6% (95% CI 3.7 to 5.5%). We found that children 6 months of age and older had a higher carriage prevalence than younger infants, although this difference was not significant (Table 1). Variations among the age groups were similar for the prevalence of Haemophilus carriage and the prevalence of Hib carriage.
We also found that the Hib carriage prevalence varied among the four administrative regions in Lombok, although again the difference was not significant (Table 2). The prevalence of H. influenzae carriage varied to a lesser extent among regions than did the prevalence of Hib carriage.
Risk factors. We compared the rates of nasopharyngeal carriage of H. influenzae, of encapsulated H. influenzae and of Hib associated with the evaluated risk factors. For all three analyses and for all evaluated risk factors, relative risks varied between 0.5 and 2.0. For only two risk factors did the relative risk achieve statistical significance; the use of food other than breast milk and the consumption of premasticated food both were associated with carriage of any H. influenzae (both relative risks, 1.4; 95% CI 1.1 to 2.3 and 1.1 to 1.9, respectively).
We also evaluated the risk of carriage of any encapsulated H. influenzae and of Hib, compared with children who carried nonencapsulated H. influenzae. Again for both analyses and for all evaluated risk factors, the relative risks varied between 0.5 and 2.0; none achieved statistical significance.
Antibiotic sensitivity. All 22 specimens with Hib identified at the laboratory in Lombok were tested for antibiotic sensitivity. None were resistant to ampicillin, sulfamethoxazole or cefotaxime. Ten isolates (45%) were resistant to tetracycline and 2 (9%) were resistant to chloramphenicol.
This is the first study examining Hib carriage in Indonesia, one of the first to examine carriage among an Asian population and one of the few population-based studies with systematic sampling from any area. We identified a population-based, age-weighted carriage prevalence of 4.6%, thus accomplishing our primary goal of documenting that Hib carriage exists in Lombok, Indonesia. The carriage prevalence from our study compares with prevalences of 1.3% among a Vietnamese population in Hong Kong,11 ∼1% in Japan,12 8% in Papua New Guinea,13 10% in the Gambia14 and 4.7% in Cape Town, South Africa.15
Moreover the carriage prevalence we found is equal to or greater than that reported from developed countries during the prevaccine era or among unvaccinated individuals, including the US,16, 17 Finland18 and England.19 Carriage prevalences of this magnitude were associated with prevaccine era invasive disease annual incidence rates in these same countries of 35 to 130 per 100 000 children younger than 5 years of age.20-22 In Lombok, however, culture-positive invasive Hib disease had not been documented before our survey and, in Asia as a whole, reported rates of invasive disease have been an order of magnitude less than in Western countries,2-4, 23 with the only exception being among the Vietnamese population of Hong Kong.2 Our demonstration that a high carriage prevalence exists in this part of Indonesia suggests that absence of the organism does not explain these low rates. Instead other explanations, such as limited laboratory facilities, diagnostic resources, or surveillance capacity, more widespread and early use of antibiotics, or differences in risk factors for disease, must be sought.
We found variation in the carriage prevalence between different regions. We do not believe that this was related to swabbing technique, because teams were not allocated to specific regions. Unfortunately we do not know whether these differences reflect different population characteristics or simply random variation in the distribution of Hib organisms, because we did not identify any factors, other than older age, that predicted carriage. Although age has been associated with carriage in previous studies, few studies have found risk factors that would explain reported geographic or ethnic variations in carriage prevalence.11, 14, 15, 24, 25 Interestingly the overall H. influenzae prevalence varied less than the Hib prevalence among regions, suggesting that these findings may be specific to type b organisms.
Other than tetracycline we found little antibiotic resistance in Lombok, including a lack of beta-lactamase production. This is in contrast to the situation among more developed areas of Asia such as Japan,26 Singapore4 and Hong Kong27 and also differs from the situation in India.6 Because antibiotic resistance patterns of H. influenzae identified through carriage can predict resistance patterns among invasive isolates,28, 29 this information suggests that ampicillin remains an effective first line therapy against invasive infections in Lombok. Additionally the lack of resistant organisms suggests limited exposure to antibiotics.30 Consequently if subsequent studies find low rates of invasive Hib disease, it is unlikely to be explained by early and widespread antibiotic use.
Our study had several limitations. Because of logistic difficulties with supply delivery, specimens remained frozen for 2 months before we performed serotyping. Prolonged freezing may lead to loss of the capsule in some instances, making the results we report minimum estimates. We collected specimens from children at only one point in time during the dry season and can make no statements regarding temporal or seasonal variation in carriage prevalence; at least one study has suggested that temporal variation may be substantial.14 Finally our inability to adjust our results for differences in hamlet population may have had a small effect on the overall carriage prevalence estimate.
The finding of substantial Hib carriage in Lombok has convinced us to proceed with a study to determine the effectiveness of Hib vaccine in reducing Hib-related disease, including pneumonia and meningitis. A recent report from the Gambia provides evidence that Hib vaccine may prevent 20% of cases of radiologically proven pneumonia and >95% of Hib meningitis cases.31 In Lombok the infant mortality rate is approximately 90 per 1000 live births (Indonesian Statistics Bureau, 1995) with one-third to one-half of deaths attributable to pneumonia (PATH verbal autopsy survey, 1996, unpublished data; Indonesian Ministry of Health, 1992 Family Health Survey). If the Hib carriage we have demonstrated in Lombok is accompanied by high rates of invasive disease, Hib vaccination may be as useful at reducing these figures in Lombok as it was in the Gambia.
This publication was made possible partially through support by the Office of Health and Nutrition, US Agency for International Development, under the terms of Cooperative Agreement HRN-A-00-95-00025-01 with PATH and through support by the Mérieux Foundation, France. The opinions expressed herein are those of the authors and do not necessarily reflect the views of USAID or the Mérieux Foundation.
We acknowledge the support of Philippe Stoeckel, Association pour l'Aide à la Médecine Préventive; James Maynard and I. G. P. Suradana, Program for Appropriate Technology in Health; and Cyrus Simanjuntak, Indonesian Ministry of Health.
1. Funkhouser A, Steinhoff, MC, Ward J. Haemophilus influenzae
disease and immunization in developing countries. Rev Infect Dis 1991;13:S542-54.
2. Lau YL, Oppenheimer SJ, Ho A, et al. Invasive Haemophilus influenzae
type b infections in children hospitalized in Hong Kong, 1986-1990: Hong Kong Hib Study Group. Acta Paediatr 1995;84:173-6.
3. Wang CH, Lin TY. Invasive Haemophilus influenzae
diseases and purulent meningitis in Taiwan. J Formos Med Assoc 1996;95:599-604.
4. Tee NW, Lin RV. Serotypes and antimicrobial resistance in Haemophilus influenzae
in a hospital practice. Ann Acad Med Singapore 1996;25:184-7.
5. Manual for national surveillance of antimicrobial resistance of Streptococcus pneumoniae
and Haemophilus influenzae:
epidemiological and microbiological methods. Geneva: WHO, CDC, 1991.
6. Invasive Bacterial Infections Surveillance Group. Invasive Haemophilus influenzae
disease in India: a preliminary report of prospective multihospital surveillance. Pediatr Infect Dis J 1998;17(Suppl)S191-5.
7. Kilian M. Haemophilus.
In: Lennette E, Balows A, Hausler W, Shadomy JH, eds. Manual of clinical microbiology. 4th ed. chap 33. Washington, DC: American Society for Microbiology, 1985:387-93.
8. Barry, AL, Thornsberry, C. Susceptibility tests: diffusion test procedures. In: Lennette E, Balows A, Hausler W, Shadomy JH, eds. Manual of clinical microbiology. 4th ed. chap 102. Washington, DC: American Society for Microbiology, 1985:978.
9. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disk susceptibility tests. 5th ed. Document M2-A5. Villanova, PA: National Committee for Clinical Laboratory Standards, 1993.
10. Falla TJ, Crook DW, Brophy LN, Maskell D, Kroll JS, Moxon ER. PCR for capsular typing of Haemophilus influenzae.
J Clin Microbiol 1994;32:2382-6.
11. Sung RY, Ling JM, Fung SM, Oppenheimer SJ, Crook DW, Lau JT. Carriage of Haemophilus influenzae
and Streptococcus pneumoniae
in healthy Chinese and Vietnamese children in Hong Kong. Acta Paediatr 1995;84:1262-7.
12. Kuroki H, Niimi H, Sonobe T, Himi K, Uehara S, Ishikawa N. Nasopharyngeal colonization with Haemophilus influenzae
type b among infants and children in Japan. Acta Paediatr Jpn 1997;39:541-5.
13. Montgomery JM, Lehmann D, Smith T, et al. Bacterial colonization of the upper respiratory tract and its association with acute lower respiratory tract infections in Highland children of Papua New Guinea. Rev Infect Dis 1990; 12(Suppl 8):S1006-16.
14. Bijlmer HA, Evans NL, Campbell H, et al. Carriage of Haemophilus influenzae
in healthy Gambian children. Trans R Soc Trop Med Hyg 1989;83:831-5.
15. Hussey GD, Coetzee G, Hitchcock J, van Schalkwyk E, van Wyk H, Kibel M. Carriage of Haemophilus influenzae
in Cape Town children. S Afr Med J 1994;84:135-7.
16. World Health Organization. Technical bases for the WHO recommendations on the management of pneumonia in children at first-level health facilities. Publication No. 91.20. Programme for the Control of Acute Respiratory Infections. Geneva: World Health Organization, 1993.
17. Lerman SJ, Kucera JC, Brunken JM. Nasopharyngeal carriage of antibiotic-resistant Haemophilus influenzae
in healthy children. Pediatrics 1979;64:287-91.
18. Takala AK, Eskola J, Leinonen M, Kayhty H, Pekkanen E, Makela PH. Reduction of oropharyngeal carriage of Haemophilus influenzae
type b (Hib) in children immunized with an Hib conjugate vaccine. J Infect Dis 1991;164:982-6.
19. Barbour ML, Mayon-White RT, Coles C, Crook DW, Moxon ER. The impact of conjugate vaccine on carriage of Haemophilus influenzae
type b. J Infect Dis 1995;171:93-8.
20. Takala AK, Eskola J, Peltola H, Makela PH. Epidemiology of invasive Haemophilus influenzae
type b disease among children in Finland before vaccination with Haemophilus influenzae
type b conjugate vaccine. Pediatr Infect Dis J 1989;8:297-302.
21. Broome CV. Epidemiology of Haemophilus influenzae
type b infections in the United States. Pedatr Infect Dis J 1987;6:779-82.
22. Howard AJ, Dunkin KT, Musser JM Palmer SR. Epidemiology of Haemophilus influenzae
type b invasive disease in Wales. Br Med J 1991;303:441-5.
23. Ishikawa T, Yamaguchi H, Watanabe K, et al. Epidemiology of bacterial meningitis in children: Aichi Prefecture, Japan, 1984-1993. Pediatr Neurol 1996;14:244-50.
24. Hall DB, Lum MK, Knutson LR, Heyward WL, Ward JI. Pharyngeal carriage and acquisition of anticapsular antibody to Haemophilus influenzae
type b in a high-risk population in southwestern Alaska. Am J Epidemiol 1987 Dec;126:1190-7.
25. Stephenson WP, Doern G, Gantz N, Lipworth L, Chapin K. Pharyngeal carriage rates of Haemophilus influenzae,
type b and non-b, and prevalence of ampicillin-resistant Haemophilus influenzae
among healthy day-care children in central Massachusetts. Am J Epidemiol 1985;122:868-75.
26. Nishioka K, Shirato K, Tanno Y, Ohno I, Ogihara H. The incidence of respiratory tract pathogens and antimicrobial susceptibilities of Streptococcus pneumoniae, Haemophilus influenzae
and Moraxella (Branhamella) catarrhalis
isolated between 1990 and 1993. Tohoku J Exp Med 1996;179:111-21.
27. Ho P, Luk W, Sai-yin Wong S, Yam W, Yuen K. Changing patterns of susceptibilities of blood, urinary and respiratory pathogens in Hong Kong. J Hosp Infect 1995;31:305-17.
28. Mastro TD, Schwartz B, Breiman RF, et al. Use of nasopharyngeal isolates of Streptococcus pneumoniae
and Haemophilus influenzae
from children in Pakistan for surveillance for antimicrobial resistance. Pediatr Infect Dis J 1993;12:824-30.
29. Ostroff SM, Harrison LH, Khallaf N, et al. Resistance patterns of Streptococcus pneumoniae
and Haemophilus influenzae
isolates recovered in Egypt from children with pneumonia. Clin Infect Dis 1996;23:1069-74.
30. Weinberg GA, Spitzer ED, Murray PR, et al. Antimicrobial susceptibility patterns of Haemophilus
isolates from children in eleven developing nations. Bull WHO 1990;68:179-84.
31. Mulholland K, Hilton S, Adegbola R, et al. Randomised trial of Haemophilus influenzae
type-b tetanus protein conjugate for prevention of pneumonia and meningitis in Gambian infants. Lancet 1997;349:1191-7.
FIRST INTERNATIONAL CONFERENCE ON HAEMOPHILUS INFLUENZAE TYPE b INFECTION IN ASIA
The Editors thank the Association pur l'Aide à la Médicine Préventive, the Foundation Mérieux, and the World Health Organization for supporting publication of these proceedsings, and Jennifer Wells for her editorial assistance.