Keywords
β-hemolytic streptococci - group A streptococci - throat carriage - surveillance
Introduction
Group A Streptococcus (GAS) is an important cause of infectious disease mortality,
accounting for up to half a million deaths annually.[1]
[2]
[3] It is the most commonly isolated bacterial agent in acute pharyngitis and accounts
for 15 to 30% of all throat infections.[3]
[4]
[5]
[6] However, β hemolytic streptococci (BHS) have also been isolated from asymptomatic
individuals, who serve as reservoirs of infection in the community.[7]
[8] Active surveillance of BHS disease may thus be achieved by periodic evaluation of
the prevalence of streptococcal throat carriage in a community.[8]
[9]
[10]
Streptococcal throat infection may be complicated by early suppurative as well as
medium and long-term nonsuppurative sequelae such as acute rheumatic fever and post-streptococcal
glomerulonephritis, with high morbidity and mortality.[11]
[12] Rheumatic heart disease affects over 15 million persons worldwide, causing about
a quarter of a million deaths annually, with highest prevalence recorded in Sub-Saharan
Africa.[13]
[14] While rheumatic fever is described in carriers, recurrent rheumatic fever episodes,
with higher risk of rheumatic heart disease, have also been reported in some throat
carriers.[15]
[16] Some authors have also stated an immune response in association with asymptomatic
infections.[17]
[18]
Worldwide estimates for asymptomatic streptococcal throat infections show wide variation.
In general, when well children attending outpatient clinics are considered, the rate
of BHS carriage is between 2 and 5%.[4] However, school studies have been known to show a much wider range with BHS carriage
rates between 8 and 40%.[4] A meta-analysis of literature published over the past 70 years by Oliver et al puts
the GAS throat carriage prevalence rate at 5.9% (4.3–8.1%) in low- and middle-income
countries, whereas a higher rate of 10.5% (8.4–12.9%) has been documented in the developed
world.[19]
Studies from Sub-Saharan Africa on streptococcal throat carriage remain relatively
rare. In Nigeria, in the last decade, three studies reported rates varying between
4 and 28.9% for streptococcal throat carriage in children.[9]
[18]
[20] Furthermore, these Nigerian studies have all demonstrated an absence of GAS in asymptomatic
carriers despite purportedly high rates of rheumatic fever/rheumatic heart disease
in the region. This increase in non-GAS BHS throat carriage has also been reflected
in another African study, as well as studies from other parts of the world.[7]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
Streptococcal throat infections are usually treated with a 10-day course of oral penicillin
V or amoxicillin. Erythromycin or azithromycin may be used for penicillin-allergic
individuals.[29]
[30]
[31] Though macrolide resistance has been widely reported especially with erythromycin
and bacteriologic failure occasionally with penicillin, no large community or hospital-based
studies have demonstrated in vitro resistance of GAS throat isolates to penicillin.[30]
[31]
[32]
[33]
[34]
[35]
The current study sets out to establish the prevalence of asymptomatic streptococcal
throat infection and antimicrobial susceptibility patterns in school-aged children
in Uyo, Southern Nigeria.
Methods
The current study was performed in Uyo, a city with six political wards located in
the Nigerian rainforest at an altitude of 70 m above sea level and with average annual
rainfall of 2,509 mm.[36]
Written permission to carry out the study was obtained from the Akwa Ibom State Ministry
of Education. Ethical clearance for the study was obtained from the Institutional
Health Research and Ethics Committee of University of Uyo Teaching Hospital. Written/informed
consent was obtained from all parent(s)/guardian(s) before recruiting their children
into the study.
The study was performed between September 2017 and February 2018, which would correspond
to the late rainy season and dry season in the study area.
Sample size was determined by using Fisher's formula applied to study populations
greater than 10,000.[37] The total number of enrolled pupils in primary schools in Uyo was 71,659 making
the formula appropriate.
Where:
N = minimum sample size
Z = 1.96
D = total width of the expected confidence interval (set at 0.05)
P = prevalence from similar study at a nearby location[18] = 20.6%
Q = 1-P
Therefore:
An attrition rate of 10% was assumed, and thus, the total number of subjects enrolled
in the study was 276.
Primary school students aged 6 to 12 years, whose parents gave consent, were enrolled
in the study. Children with symptoms of acute pharyngitis or those who had used antibiotics
in 2 weeks preceding the study were excluded.
Sampling Method
A multistage sampling method was used to recruit for the study. Overall, 12 schools,
that is, two per political ward were selected from the 55 schools (15 public and 40
private) in the Uyo metropolis. Four public and eight private schools were selected
to achieve proportionate representation from the schools. These schools were selected
by simple random sampling using the table of random numbers.
The number of pupils selected from each of the 12 schools was determined by proportionate
sampling in the second stage. The school sample size was determined by using the formula[9]:
Where:
N2 = number of pupils recruited from a given primary school
a = population of index school
n = total number of pupils in all the selected primary schools
N1 = total sample size
In each school, one arm of each class was selected randomly for the study by using
the ballot method.[38] The number of pupils obtained from the chosen arm in a given school was calculated
by using the class population (all arms), the school population, and the school sample
size as calculated. The calculated sample size of each school (N2) was then distributed
proportionately among the classes by using the formula[9]:
Where:
N3 = class sample size
a = class population (all arms)
b = population of all classes in the school
N2 = school sample size
The first pupil was randomly selected from the list of pupils in that class by using
the ballot method.[38] All subsequent pupils recruited were then selected by using a systematic sampling
method. The list of pupils in the class was arranged alphabetically, and this was
used as the sampling frame. The selected pupils were then given consent forms for
their parents and were only recruited into the study after the filled and signed consent
forms had been retrieved from their parents/guardians.
For each recruited pupil, a proforma containing relevant biodata, household number,
and physical examination findings was filled out. The family socioeconomic status
was determined by using the method described earlier by Olusanya et al.[39]
Throat swabs were taken by using strict aseptic technique. Subjects were asked to
open their mouths wide and say “ah.” With their mouths open, a throat swab was taken
from each participant by gently rubbing the swab stick on the posterior pharynx and
tonsillar bed, after depressing the back of the tongue with a wooden spatula. The
swab stick was then returned to its sterile container, properly labeled and transported
within an hour of collection to the University of Uyo Teaching Hospital laboratory
for immediate plating on 5% sheep blood agar.[40] Cultures were incubated at 35°C for 24 hours in a CO2 jar to enhance growth. Growth of pinpoint colonies with clear surrounding zones of
hemolysis (β hemolysis) was identified and the presence of Streptococci was confirmed
by a negative catalase test, while a gram stain was processed concomitantly to identify
the characteristic gram-positive cocci in chains under the microscope.
The identified BHS were then subcultured for purity and inoculated with Bacitracin
0.04U discs for the appropriate identification of GAS. The Lancefield group was subsequently
determined from positive cultures by using Oxoid Streptococcal Grouping Latex Agglutination
Kit, UK to identify Groups A, B, C, D, F, and G streptococci. Antimicrobial susceptibility
testing was done by using the disk diffusion method.[38] Isolated organisms were cultured on Mueller-Hinton agar with 5% Sheep Blood with
antibiotic susceptibility discs and incubated for 24 hours at 37°C. Oxoid UK sterile
paper discs containing penicillin V (10 μg), amoxicillin (30 μg), erythromycin (15
μg), cefuroxime (30 μg), co-trimoxazole (1.25/23.75 μg), and clindamycin (2 μg) were
used.
The data was analyzed by using the Statistical Package for Social Science for Windows
(SPSS Inc Chicago Illinois, United States), version 20. The prevalence of BHS carriage
was determined by using simple percentages. Results were reported in text, tables,
and figures. Categorical data were recorded as frequencies and percentages, while
continuous variables were recorded as means ( ± standard deviation). Pearson's Chi-square
statistical test of significance or Fisher's exact test was used as required to determine
the relevant associations between throat carriage and the sociodemographic variables.
The level of significance was set at p <0.05.
Results
Two hundred and seventy-six children aged 6 to 12 years were recruited in the study.
The mean age of subjects was 8.7 ± 1.7 years. Overall, 133 (48%) were males and 143
(52%) were females, with a male to female (M:F) ratio of 1:1.1. [Table 1] shows the sociodemographic characteristics of the study group.
Table 1
Sociodemographic characteristics of study population
|
Characteristic
|
Frequency
(n = 276)
|
Percentage (%)
|
|
Age (y)
|
|
6–7
|
70
|
25.4
|
|
8–9
|
114
|
41.3
|
|
10–12
|
92
|
33.3
|
|
Total
|
276
|
100.0
|
|
Gender
|
|
Male
|
133
|
48.2
|
|
Female
|
143
|
51.8
|
|
Total
|
276
|
100.0
|
|
Household size
|
|
≤3
|
31
|
11.2
|
|
4–6
|
156
|
56.6
|
|
7–9
|
73
|
26.4
|
|
10–11
|
8
|
2.9
|
|
> 11
|
8
|
2.9
|
|
Total
|
276
|
100.0
|
|
Religion
|
|
Christian
|
274
|
99.3
|
|
Muslim
|
2
|
0.7
|
|
Total
|
276
|
100.0
|
|
Social class
|
|
Class 1
|
11
|
4.0
|
|
Class 2
|
75
|
27.2
|
|
Class 3
|
82
|
29.7
|
|
Class 4
|
84
|
30.4
|
|
Class 5
|
24
|
8.7
|
|
Total
|
276
|
100.0
|
β-hemolytic streptococci (BHS) were identified in 9 out of 276 subjects by giving
a prevalence of 3.3%.
[Table 2] shows the Lancefield grouping of the BHS isolates. Eight out of nine isolates (88.9%)
were group C streptococcus and one was group G streptococcus.
Table 2
Lancefield grouping of β-hemolytic streptococcal Isolates
|
Lancefield group
|
Frequency (n)
|
Percentage (%)
|
|
C
|
8
|
88.9
|
|
G
|
1
|
11.1
|
|
Total
|
9
|
100.0
|
[Table 3] demonstrates the association between sociodemographic factors and BHS throat carriage.
The association between age groups and streptococcal throat carriage was statistically
significant (p = 0.04). Despite a noticeably higher throat carriage rate in females (66.7%), the
difference was not statistically significant (p = 0.37). The number of household occupants demonstrated a statistically significant
association with asymptomatic BHS infection. Religion and social class did not show
a significant relationship with streptococcal throat carriage.
Table 3
Association of sociodemographic factors with asymptomatic β- hemolytic streptococcal
throat infection
|
Characteristic
|
β-hemolytic streptococcal throat infection
|
Total (%)
|
Fisher's exact test (p-value)
|
|
Positive (%)
|
Negative (%)
|
|
Age
|
|
6–7 y
|
5 (55.6)
|
65 (24.3)
|
70 (25.4)
|
6.47 (0.04)[a]
|
|
8–9 y
|
4 (44.4)
|
110 (41.2)
|
114 (41.3)
|
|
10–12 y
|
0 (0)
|
92 (34.5)
|
92 (33.3)
|
|
Total
|
9 (100)
|
267 (100)
|
276 (100)
|
|
|
Gender
|
|
Male
|
3 (33.3)
|
130 (48.7)
|
133 (48.2)
|
0.82 (0.37)[a]
|
|
Female
|
6 (66.7)
|
137 (51.3)
|
143 (51.8)
|
|
Total
|
9 (100)
|
267 (100)
|
276 (100)
|
|
|
Religion
|
|
Christian
|
9 (100)
|
265 (99.3)
|
274 (99.3)
|
0.01 (0.99)[a]
|
|
Muslim
|
0 (0)
|
2 (0.7)
|
2 (0.7)
|
|
Total
|
9 (100)
|
267 (100)
|
276 (100)
|
|
|
Socioeconomic class
|
|
Class 1
|
0 (0)
|
11 (4.2)
|
11 (4)
|
4.03 (0.40)[a]
|
|
Class 2
|
1 (11.1)
|
74 (27.7)
|
75 (27.2)
|
|
Class 3
|
3 (33.3)
|
79 (29.6)
|
82 (29.7)
|
|
Class 4
|
5 (55.6)
|
79 (29.6)
|
84 (30.4)
|
|
Class 5
|
0 (0)
|
24 (9)
|
24 (8.7)
|
|
Total
|
9 (100)
|
267 (100)
|
276 (100)
|
|
|
Household size
|
|
≤3
|
1 (11.1)
|
30 (11.2)
|
31 (11.3)
|
13.31 (0.01)[a]
|
|
4–6
|
3 (33.3)
|
153 (57.3)
|
156 (56.6)
|
|
7–9
|
3 (33.3)
|
70 (26.2)
|
73 (26.3)
|
|
10–11
|
2 (22.3)
|
6 (2.3)
|
8 (2.9)
|
|
> 11
|
0 (0)
|
8 (3.0)
|
8 (2.9)
|
|
Total
|
9 (100)
|
267 (100)
|
276 (100)
|
|
a Fisher's exact test was used.
[Table 4] shows the antibiotic susceptibility of BHS isolates. Just over three-quarters of
the isolates were susceptible to penicillin, and only one-third to amoxicillin. Susceptibility
was best with cefuroxime and clindamycin. None of the isolates showed sensitivity
to co-trimoxazole. A significantly higher proportion of the streptococcal isolates
in the children demonstrated susceptibility to cefuroxime and clindamycin.
Table 4
Lancefield group-specific antibiotic susceptibilities of beta-hemolytic streptococcal
isolates
|
Antibiotic
|
Susceptibility pattern
|
Lancefield group
|
Total (%)
|
Fisher's exact (p-value)
|
|
Group C (%)
|
Group G (%)
|
|
Penicillin
|
Sensitive
|
6 (75)
|
1 (100)
|
7 (77.8)
|
2.77 (0.10)[a]
|
|
Resistant
|
2 (25)
|
0 (0)
|
2 (22.2)
|
|
Amoxicillin
|
Sensitive
|
2 (25)
|
1 (100)
|
3 (33.3)
|
1.00 (0.32)[a]
|
|
Resistant
|
6 (75)
|
0 (0)
|
6 (66.7)
|
|
Erythromycin
|
Sensitive
|
4 (50)
|
1 (100)
|
5 (56.6)
|
0.11 (0.74)[a]
|
|
Resistant
|
4 (50)
|
0 (0)
|
4 (44.4)
|
|
Cefuroxime
|
Sensitive
|
7 (87.5)
|
1 (100)
|
8 (88.9)
|
5.44 (0.02)[a]
|
|
Resistant
|
1 (12.5)
|
0 (0)
|
1 (11.1)
|
|
Cotrimoxazole
|
Sensitive
|
0 (0)
|
0 (0)
|
0 (0)
|
Nil[b]
|
|
Resistant
|
8 (100)
|
1 (100)
|
9 (100)
|
|
Clindamycin
|
Sensitive
|
7 (87.5)
|
1 (100)
|
8 (88.9)
|
5.44 (0.02)[a]
|
|
Resistant
|
1 (12.5)
|
0 (0)
|
1 (11.1)
|
a Fisher's exact test was used.
b Null values not appropriate for statistical testing.
Discussion
The BHS carriage rate in our study, though slightly lower than the mean rate of 5.6%
reported in low and middle-income countries worldwide, compares favorably with the
prevalence of 4% reported by Baki et al in Calabar, Nigeria.[19]
[20] This can be probably explained by the fact that both cities share similar sociocultural
characteristics. An earlier study in Calabar, Nigeria[18] had recorded a much higher prevalence rate of 20.9%. This could be justified by
their sample collection over a 1-year period as compared with the current study, which
was done toward the end of the rainy season and in the dry season. The former study
would also have been able to consider seasonal variations over an entire year, thus
resulting in a higher prevalence. Our finding also compares favorably with the 4.6%
reported by Singh et al in Northern India.[22] It was however significantly lower than rates (20.9–28.9%) reported from earlier
Nigerian studies.[9]
[18] The difference may be due to improved measures for control of respiratory infections,
such as better hand hygiene and increased rates of exclusive breastfeeding in the
country over the years.[41] Moreover, several authors have acknowledged wide variations in the prevalence rate
of BHS carriage, even within localities in the same regional boundaries.[4]
[9]
[19] This variation may reflect differences in the burden of streptococcal diseases in
various localities.
The pattern of BHS isolates from the present study is consistent with that of recent
Nigerian studies, which also reported GCS as the commonest BHS isolate in asymptomatic
children.[9]
[18]
[20] These earlier reports had also noted a comparable absence of GAS in studies of BHS
carriage. In contrast, an earlier study performed in Lagos in 1972 documented GGS
as the commonest Lancefield group isolate.[42] GAS was isolated in 22% of the children sampled. Changes in the burden of streptococcal
diseases in the country over the years may explain the change in pattern of BHS carriage.
The current study found BHS throat carriage rate to be highest in 6 to 7 years age
group. This is consistent with the well-documented knowledge about BHS carriage that
peaks in the early school age and falls toward adolescence.[11] It also compares well with a study in Northern India, which showed the highest prevalence
in the 5 to 7 years age group.[7] However, the latter study reported the lowest carriage rates among the 7 to 9 years
age group, which had the second highest carriage rate in our study.[7] Furthermore, while no BHS isolates were found in children aged 10 to 12 years in
our study, Baki et al (Calabar) reported the highest carriage rate (36.8%) in the
same age group.[20] A possible change in the epidemiologic pattern of BHS diseases within the region
may account for this difference.
More females than males in the study were streptococcal throat carriers. This is similar
to many other studies showing a slight female preponderance in BHS throat carriage,
though this difference has usually not been proven to be statistically significant.[9]
[21]
[30]
[33] This gender disparity may be attributed to a genetic predisposition of females in
the study to the dominant “emm” type, which has been also reported in a recent Norwegian
study.[43]
Children from households with four to nine occupants were most likely to be BHS throat
carriers in the current study. This association could be explained by the fact that
children from larger households are more likely to live in overcrowded homes with
poor ventilation. Both overcrowding and poor ventilation are associated with streptococcal
diseases.[44] This finding is similar to earlier studies by Owobu et al[9] in Benin, and Nayiga et al[21] in Uganda who found that BHS carriage rates were higher in children from households
with a larger number of occupants.
Overall, 78% of BHS isolates in the current study were susceptible to penicillin.
This finding was lower than the 100% susceptibility to penicillin earlier reported
for BHS.[5]
[26]
[30]
[32] This may be explained by the fact that no GAS was isolated in the current study.
Previous studies with GAS have not demonstrated in vitro resistance to penicillin,
although an Egyptian study had reported evolving resistance of GAS isolates to penicillin.[30] The finding that all the BHS isolates in the current study were resistant to co-trimoxazole
compared well with findings from an earlier study.[7] The 100% resistance of BHS isolates to co-trimoxazole may be explained by the fact
that it is a common over-the-counter remedy for sore throat in the country. The routine
use of the drug in Pneumocystis carinii pneumonia prophylaxis for all patients with
HIV infection may also be a factor in the development of high-grade resistance to
the drug as has been suggested by other studies.[45] The ease of access to antibiotics generally, in the study area as well as entire
country, may also account for the high resistance of isolates to co-trimoxazole, amoxicillin,
and erythromycin.
The 3.3% prevalence of asymptomatic streptococcal throat carriage recorded in school-aged
children in the study is relatively low. The absence of any GAS isolates suggests
a low burden of GAS disease in the study area. It is recommended that larger, multicenter
studies be conducted to elucidate the changing epidemiology of BHS diseases in the
region.
Limitations of the Study
-
This study was only conducted in school-aged children in Uyo. Studies across all ages
of children may have an added advantage.
-
It was often a challenge to confirm the use of antibiotics in the two weeks preceding
the study, in some of the younger children.
-
The low numbers obtained from the study made statistical analysis challenging.
