Reduction in the Incidence of Acute Bronchitis by an Oral Haemophilus influenzae Vaccine in Patients with Chronic Bronchitis in the Highlands of Papua New Guinea 1- 3


Introduction SUMMARY following the administration of a standardized questionnaire, 62 adult patients with

Chronic lung disease (CLD) is a major chronic bronchitis were enrolled Into a double-blind controlled trial of an oral killed Haemophllus cause of morbidity and mortality in Influ.nza. vaccine In the highlands of Papua New Guinea. A 3-day course of vaccine or placebo Papua New Guinea (PNG). In a recent was given monthly for 3 consecutive months. Participants were monitored weekly over 12 months population-based study in the Asaro Valfor acute exacerbations; early morning sputum specimens were collected monthly and during acute ley, Eastern Highlands Province the overexacerbations. Density of colonization by H. Influenzae and H. para/nfluenzae was determined by standard quantitative and semiquantitative technique., and the latter method (quadrant score) was all prevalence of chronic bronchitis was used to determine the density of growth of pneumococcI. A total of 30 patients received vaccine 250/0, but was as high as 50070 in people and 32 placebo. The Incidence rate of acute bronchitis In the vaccine group (0.011 eplsodes/personaged 65 yr or more, and the prevalence weeks) was significantly lower than that In the placebo group (0.021 eplsodes/person-weeks), but of chronic airflow limitation (CAL) was there was no difference between the two groups In the Incidence rates of more severe disease. 6070 (1). In the PNG highlands, CLD acVaccine efficacy was maximal at times of peak Incidence of disease. There was no evidence of counts for at least 20 to 250/0 of deaths a decline In vaccine efficacy for acute bronchitis over the 12-month follow-up period. The number in subjects aged 45 yr or more; acute of viable H. Influenza. In the sputum declined In both vaccine and placebo groups over the 12lower respiratory tract infections (ALRI) month follow-up period. The average concentration of H. Influenzae In the vaccine group fell below account for a further 25070 in the same that In the placebo group within 1 to 2 months after first Immunization and remained so for 12months, age group, but pulmonary tuberculosis although the difference between the two groups narrowed during the follow-up period. The quadrant score of pneumococci was lower In the vaccine than placebo group within 1 month of first is rare (2). Immunization and remained so for a further 7 months. AM REV RESPIR DIS 1991; 144:324-330 Streptococcus pneumoniae and Haemophilus influenzae are the most important pathogens associated with ALRI in reporters employed by the PNGIMR wereinMethods PNG (3-5). High colonization rates of formed of the purpose of the study and asked Study Area both S. pneumoniae and H. influenzae to identify people suffering from chronic lung (in particular nonserotypable strains) The study took place in sections of Goroka disease. A standard questionnaire was dehave been reported among patients town and nearby peri-urban and rural areas signed and administered in Melanesian pidgsuffering from CLD in developed coun- in the Asaro Valley, Eastern Highlands Prov- in English (although frequently interpreted tries (6-8), and it has been suggested that ince, PNG that were under regular demo- into the local language as well) to all potenthese two bacteria may be implicated in graphic surveillance by staff of the Pneumo- tial participants, as wellas some people who the etiology of ALRI complicating nia Research Program of the Papua New werenot suspected of suffering from chronic Guinea Institute of Medical Research chronic lung disease (7, 9). lung disease to validate the questionnaire. A (PNGIMR). The study area is situated beA prospective placebo-controlled tri- tween 1,500and 1,800 m above sea level;there person was eligible for entry into the trial if al of an oral vaccine containing killed H. is a clear wet and dry season, with 75070 of influenzae in an Australian population rain falling between November and April; the (Received in original form March 5, 1990 and in of subjects suffering from CLD demon- environmental temperature varies daily be- revised form October 15, 1990) strated protection against acute episodes tween 16 and 30° C with minimal seasonal of bronchitis over a 3-month study peri- variation. In the urban areas people live in 1 From the Papua NewGuinea Institute of Medod (10). The aim of the present study was houses of permanent material; in the rural ical Research, Goroka, Eastern Highlands Provto determine whether the same oral vac- areas families live in houses made of wood ince, Papua New Guinea, and the Department of Pathology,Universityof Newcastle, Newcastle, New cine could prevent acute exacerbations and woven canegrass with a thatched roof. South Wales, Australia. Generally in the rural areas the houses have in patients with chronic bronchitis in an 1 Supported by Auspharm International, Ltd., a singlelivingarea, no windows,a singledoorarea with a high prevalence of CLD over way to the outside, and a central unvented Sydney, Australia. 3 Correspondence and requests for reprints a 12-month follow-up period and wheth- fireplace. should be addressed to Deborah Lehmann, Papua er the vaccine had any effect on bacteriNew Guinea Institute of Medical Research, P.O. Trial Population al colonization of the lower respiratory Box 60, Goroka, Eastern Highlands Province, tract. Villagers and town residents as well as lay Papua New Guinea. 324


he or she had either (1) a productive cough for 3 months or more annually for the past 2 yr or more, or (2) a continuously productive cough for 6 months or more in the past year.

Immunization Schedule and Follow-up A prospective double-blind controlled trial of a killed H. influenzae oral vaccine (Broncostats; Auspharm International, Ltd., Australia) was started in October 1987 after ethical clearance wasgiven by the Medical Research Advisory Committee of PNG. Recruitment of the 62 trial participants continued until April 1988.Each enterically coated tablet of the vaccine contains 1011 killed nonencapsulated biotype I H. influenzae. This strain was originally a serotype b but lost its capsule through repeated subculture (11). Placebo tablets were similarly constructed enterically coated tablets without the active ingredient. The courses of Broncostat or placebo were randomized, packaged, and given consecutive case numbers by the Pharmacy Department at the Royal Newcastle Hospital, New South Wales. There was no visible difference between vaccine and placebo tablets. The randomization code was held by Auspharm International, Ltd. until completion of the trial. A clinical examination was performed and lung function assessed by spirometry at the time of entry into the trial after informed consent was givento participate. Twotablets were administered by a nurse or doctor in the morning on 3 consecutive days at monthly intervals for 3 consecutive months. Participants wereasked if they noted any side effects. Each participant was followed for a period of 12 months and was visited monthly by medical staff who collected an early morning sputum sample if the person was able to produce sputum.

Morbidity Surveillance On weeklyvisits,lay reporters recorded whether the participants were seen and if they suffered any illness in the preceding week; if they were ill, then a doctor or nurse visited them. Patients were also encouraged to come to the offices of PNGIMR in the event of any illnessfor medical examination, specimen collection, and treatment. On monthly home visits by medical staff, participants wereagain asked whether they had been sick since the preceding visit. If a participant receivedtreatment through the routine health services at any time, any available information on illness and treatment provided was also recorded. An acute exacerbation was defined as acute bronchitis or pneumonia (12) as follows:

Acute bronchitis: an increase in volume and purulence of sputum with no evidence of respiratory distress, with or without fever or chest pain. Moderatepneumonia: cough, fever,chest pain, respiratory rate 30 to 40 per min, evi-

dence of respiratory distress (intercostal indrawing not previously present or use of accessory muscles of respiration) but no cyanosis or evidence of heart failure of recent onset. Severe pneumonia: cough, fever, chest pain, evidenceof respiratory distress, respiratory rate> 4O/minand/or cyanosisand/or signs of heart failure of recent onset. Since there were only two episodes of severe pneumonia, moderate and severepneumonia wereanalyzed together. Sputum was collected from patients suffering from an acute exacerbation before giving antibiotics.

Bacteriology All sputum samples weredelivered to the laboratory in sterile containers within 4 h of collection. On receipt at the laboratory, all samples were examined macroscopically for consistency and color. A wet preparation was examined microscopically, and only those specimens that contained leukocytes and < 5 squamous epithelial cells/low-power field (x 100)were cultured; they were stained with Gram stain before culture and the presence of leukocytes and bacteria noted. A ZiehlNeelsen stain was also prepared to check for tuberculosis if this was indicated. Dithiothreitol (Sputolysin;Behring, La Jolla, CA) was diluted 1:10 with sterile distilled water giving a final concentration of 50 ug/ml, The sputum sample was weighed and an equal volume of diluted dithiothreitol added (together with 3-mm sterile beads) to digest the mucus. Specimens were vigorously mixed and incubated at 360 C for 15 to 30 min until the sputum was digested. Serial 10-fold dilutions from 10-1 to 10-12 wereprepared in microtiter plates using 180 III brain heart infusion broth (Grand Island Biologicals, Grand Island, NY) and 20 III homogenized sputum for the initial dilution. Each dilution (20 Ill) was seeded onto a quarter-plate of 5070 chocolate agar (Oxoid, Basingstoke, England); defibrinated horse blood (Commonwealth Serum Laboratories, Melbourne, Australia) with and without 300 ug/ml of bacitracin for detection of Haemophilus species. The number of H influenzae or H parainfluenzae per milliliter of sputum was calculated from the number of colonies counted x 2 x dilution. Semiquantitative culturing by quadrant scoring was done for both Haemophilus speciesand Streptococcuspneumoniae, the latter on 5070 horse blood agar (Oxoid); defibrinated horse blood (Commonwealth Serum Laboratories), with and without 5 ug/ml of gentamicin sulfate. All cultures were incubated at 360 C in a humidified incubator, supplemented with 5070 CO 2 , and examined at 24 and 48 h. An antimicrobial assay was set up for each digested sputum using standard methods (13). Respiratory bacteria weresubcultured from single colonies and identified by standard methods (13). Four single colonies each of Haemophilus speciesand S. pneumoniae were

325 subcultured for identification. H. influenzae and H. parainfluenzae were differentiated using X and V factors; H. parainfluenzae was confirmed by the porphyrin test. H. influenzaewereserotyped by slideagglutination using Wellcome antisera (Beckenham,England) and biotypedusing the Minite~ differentialsystem (BBL Microbiology Systems, Cockeysville, MD). S. pneumoniae were serotyped using Statenserum Institut antisera (Copenhagen, Denmark).

Analysis Incidence rates were calculated using a person-time at risk method. Temporary breaks in surveillance (e.g., due to temporary movements away from the area under surveillance) or periods following permanent exit from the study were excluded from the calculation of the period at risk, as weredays when patients weresuffering from acute exacerbations. Periods at risk were classified according to the sex, age group, vaccination status, time since first course of vaccine or placebo, and month of the year. Log-linear models using the logarithm of time at risk as an offset (14) were used to compare incidence rates while allowing for covariates. The term "statistically significant" refers to a 5070 significance level throughout. The relative risks quoted compare incidence rates of placebo to vaccine groups.


Description of Trial Population There were 62 participants in the trial, 30 of whom received vaccine and 32 placebo. A description of the study population is given in table 1. None ofthe participants suffered from tuberculosis. Three people (two vaccine and one placebo) complained of side effects: three had abdominal pain, one had diarrhea, and one person vomited once. Eleven people (180/0) (eight in the vaccine and three in the placebo group) were not followed for a full year. Three people did not complete the 3-month course of vaccine or placebo (two moved out of the area, and one no longer wanted to take part in the study). The remaining eight people (six vaccine and two placebo) were in the study for an average of 22.5 ± 8.3 wk (standard deviation, SD): four people (three vaccine and one placebo) moved out of the area, three were unwilling to continue participating in the study (two vaccine and one placebo), and one woman was excluded when she was diagnosed as having cancer of the cervix. There were four deaths (three vaccine and one placebo): three (two vaccine and one placebo) died of complications of chronic lung disease (within 1,2, and 3 months of entry into the study). The fourth, who died




chitis were calculated for periods 1,2 to 3, 4 to 6, 7 to 9, and 10 to 12 months following the first 3-day course of vaccine or placebo (figure 2). The picture is confounded by the seasonal variation in incidence rates (figure 1). However, analysis using log-linear models showed that vaccine efficacy did not vary significantly over the 12months following the first immunization (likelihood ratio chisquare, 1.33; 3 DF). In particular, there was no evidence of a decline in protection afforded by the vaccine.


Number of participants Person-weeks monitored Mean age ± SO Sex, M/F Never smoked Current smokers Died Lost to follow-up Mean FEV1 on entry Mean FEV1/FVC, % on entry



30 1113.4 52.6 ± 11.4 15/15

32 1398.3 53.7 ± 9.2 15/17

8 10 1

3 8


1.41 ± 0.67 63.3 ± 16.2

1.31 ± 0.59 62.9 ± 13.4

100 25 29

92 21 42

Isolation rate, % on entry: H. influenzae H. parainfluenzae Pneumococcus

within 2 wk of entering the study, had long-standing silent cirrhosis, which was only detected when she developed a toxic hepatitis following the course of doxycycline.

5 13

person-time at risk was low in October and November 1987, while participants were being recruited and also from December 1988 onward, when most of the participants had completed 12 months of follow-up. There wasevidence of a seasonal variation in the incidence rate of acute bronchitis (and the pattern was the same for all acute exacerbations), with peak incidence during the wet season, that is, during the first 4 months and the last 3 months of 1988.Log-linear models were used to determine whether the observed seasonal variation in vaccine efficacy was significant; the study time was divided into 3-month periods (excluding the very small person-time at risk in 1989, during which there was no illness). Vaccine efficacy varied significantly according to the time of year,the maximal effect occurring during the two peak periods of infection (likelihood ratio chi-square, 17.36; 4 DF) (figure 1). To determine whether vaccine efficacy waned over the year following immunization, incidence rates of acute bron-

Effect of Oral Vaccine on Incidence of Acute Exacerbations A total of 63 acute exacerbations (24and 39 in vaccine and placebo groups, respectively) were reported during the trial: 41 episodes of acute bronchitis (12 vaccine and 29 placebo) and 22 episodes of moderate or severe pneumonia (12 vaccine and 10 placebo). The overall incidence rate of acute exacerbations in the trial was 0.025 episodes/person-weeks at risk, equivalent to 1.3 episodes/person/ year. The incidence rates of acute exacerbations were0.022and 0.028/person-week in the vaccineand placebo groups, respectively, a nonsignificant difference (relative risk 1.29, 95070 confidence interval 1.03 to 1.55, likelihood ratio chi-square, 1.005; 1 degree of freedom, DF). Loglinear modeling showed that the incidence rate of acute bronchitis in the vaccinated group (O.Ol1/person-week) was significantly lower than in the placebo group (0.021/person-week) (relative risk 1.92, 95070 confidence interval 1.58 to 2.26, likelihood ratio chi-square, 3.92; 1 DF), but there was no difference in the incidence rate of pneumonia between the two groups (relative risk 0.66,95070 con- ~ fidence interval 0.23 to 1.09, likelihood ratio chi-square, 0.92; 1 DF). The over- :: all efficacy of the vaccine in preventing acute bronchitis was 480/0 with 950/0 confidence intervals of 0 to 740/0; there was .~ a 21% reduction in the total number of acute exacerbations among those who received vaccine. The 3-month running mean incidence rates of acute bronchitis in vaccine and placebo groups are shown in figure 1.The


Carriage of H. influenzae The mean log concentration of H. influenzae isolated/milliliter sputum was 6.19(± 5.59) log cfu/ml (geometric mean



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Effect of Oral Vaccine on Bacterial Carriage A total of 362 sputa from 59 of the 62 trial participants were collected in the course of the study, 313 during the routine monthly visits and 49 during acute exacerbations. A further 53 sputa were collected but subsequently excluded from the study either because they were betel nut stained or they had more than four squamous epithelial cells/low-power field. The average number of sputum specimens per participant collected during monthly visits and subsequently cultured was 5.2 ± 3.8 and 5.4 ± 3.3 in the vaccine and placebo groups, respectively. The overall isolation rates of H. influenzae, H. parainfluenzae and pneumococci were similar in the vaccine and placebo groups (table 2). Quadrant scores on routine specimens collected from the placebo group showed that 57, 37, and 11070 of specimens had heavy growths of H. influenzae, S. pneumoniae, and H. parainfluenzae, respectively.



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Fig. 1. The a-month running mean incidence rate of acute bronchitis in vaccine (solid line) and placebo (dashed line) groups (October 1987-January 1989).




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Fig. 2. Incidence rate of acute bronchitis in vaccine (solid line) and placebo (dashed line) groups at increasing time intervals (in months) from the first course of tablets {time 0) over a 12-monthfollowup period.

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Reduction in the incidence of acute bronchitis by an oral Haemophilus influenzae vaccine in patients with chronic bronchitis in the highlands of Papua New Guinea.

Following the administration of a standardized questionnaire, 62 adult patients with chronic bronchitis were enrolled into a double-blind controlled t...
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