Pneumococcal Carriage and Antibiotic Susceptibility Patterns in Mother-Baby Pairs in a rural community in Eastern Uganda

Objective: This study aimed to estimate pneumococcal carriage and determine antibiotic susceptibility patterns of the pneumococci isolated in the mother-baby pairs in Ngora district after the roll out of the pneumococcal vaccine. We hypothesized that high carriage of S. pneumoniae in mothers leads to carriage in their babies and hence a greater chance of contacting pneumoniae. Results: Consecutive sampling technique was used to select 152 mother-baby pairs from the community visits and those seeking care at the health facility. We collected nasal swabs from both baby and mother for culture and sensitivity using the Kirby-Bauer’s agar disc diffusion method. This study found that there was a low prevalence of pneumococcal carriage in the mother-baby pair in Ngora district. We also observed high rates microbial resistance to Penicillin which is the rst-line management of pneumonia in Uganda. The relationship between pneumococcal carriage and immunization status suggest that Pneumococcal vaccine is protective against pneumococcal carriage. Resistance of S. pneumoniae to the commonly used antibiotics was high. Key words: Pneumococcal carriage, mother-baby pair, antibiotic susceptibility pattern, immunization with PCV 10, Eastern Uganda.

Pneumococcal vaccine is protective against pneumococcal carriage. Resistance of S. pneumoniae to the commonly used antibiotics was high. Key words: Pneumococcal carriage, mother-baby pair, antibiotic susceptibility pattern, immunization with PCV 10, Eastern Uganda.

Research design and setting
A cross-sectional study was carried out in Ngora district Health Centre IV which serves approximately a population of 142,487. Ngora district is one of the districts in Teso sub region curved out of Kumi district in the year 2010 by the Uganda parliamentary act. Ngora district covers an area of approximately 715.9 square kilometers and predominantly inhabited by the Iteso and Kumam ethnicities. According to the Uganda health care hierarchy of organization, a Health Center IV (HCIV) is expected to serve a population of up to 100, 000 people meaning at its current capacity, this health facility operates above the level of a Health center IV [10].

Specimen collection and transport
Nasal pharyngeal specimens were collected from the posterior nasopharynx of the mother and the baby using sterile cotton swab sticks moistened with 0.9% physiological saline. Separate swabs were used to collect samples from a mother and a baby in a mother-baby pair. To control sample contamination, the swab was placed in a casing containing Amies transport medium and immediately placed into the cool box containing ice packs for transportation to Busitema University Microbiology laboratory for culture and susceptibility testing within 12 hours. For this study, we de ned a baby as any person under the age of 5 years and a mother was considered as either biological or any other female in direct care of the baby.

Laboratory Procedures
Samples were cultured on sheep Blood agar and chocolate agar followed by 24 hours of incubation at 37°C anaerobically. The isolates were identi ed morphologically by colonal appearance and gram staining. Optochin sensitivity and bile solubility testing were conducted on colonies that were potentially identi able as S. pneumoniae by alpha-haemolytic appearance on the culture media and lancet shaped gram positive cocci appearing in pairs. A 0.5 McFaland standard of S. pneumoniae was made from a 24-hour subculture by suspending colonies in sterile normal saline and inoculated by swabbing onto a plate of Mueller Hinton Agar supplemented with 7% sheep blood for susceptibility testing. Antibiotic susceptibility to Penicillin G (1U), Chloramphenicol (30µg), Tetracycline (10µg), Clindamycin (2µg), Erythromycin (30µg) and Ceftriaxone (30µg) was determined using modi ed Kirby-Bauer's agar disc diffusion methods and the disc zone diameters were interpreted using the Clinical and Laboratory Standards Institute Guidelines.

Data analysis
Collected data was entered in Microsoft excel, cleaned, coded and imported to SPSS Version 16.0 statistical package for analysis. Statistical frequency distribution tables and graphs were used for data presentation in terms of proportions, absolute values, percentages and con dence intervals for point approximations at 95% level of con dence with a P<0.05 considered as statistically important.

Demographic characteristics of the study participants
The study participants comprised of 152 mothers and 152 babies. Of the 152 babies, 74 were male and 78 were female with the age range of 0-70 months. The youngest mother was 16 years whereas the oldest was 44 years. None of the mothers who participated in the study reported having formal employment.

Prevalence of Pneumococci in Mother-Baby Pairs.
During the study, 304 samples were collected, 152 from the mothers and 152 from the children making 152 mother-baby pairs. All samples were cultured and antibiotic susceptibility was carried out on the isolated pneumococci. Out of 152 samples from the mothers only ve (5/152) isolates of pneumococci were obtained whereas seven (7/152) isolated from the babies. Only one mother-baby pair (1/152) was found to be colonized with pneumococci in both mother and baby and the rest of S. pneumoniae colonized either the mother or baby.

Immunization coverage
During data collection, immunization status of the baby was categorized in the following divisions; fully immunized, not immunized and partially immunized in the age-groups (Fig. 1). There was high immunization coverage among the children above 12 months old but lower in the 3.5-<12 age group.

Antibiogram
The antibiotic susceptibility testing was done on both positive isolates for mother and baby (Table 1). Generally, high trend of anti-microbial resistance was observed among the S. pneumoniae isolated. The highest resistance patterns were recorded with Chloramphenicol (50%) and Tetracycline (50%) whereas the lowest resistance was recorded in Clindamycin (17%).
Factors associated with pneumococcal carriage Babies that were fully immunized had a less likelihood to be colonized by S. pneumoniae than their nonimmunized counterparts P<0.05. Other factors examined by this study were not signi cantly associated with colonization with S. pneumoniae among the babies ( Table 2).

Discussion
We determined the prevalence of pneumococcal carriage and factors associated with colonization of pneumococci in a mother-baby pair in our study. Out of the 304 nasal swabs cultured, only 12(3.95%) were positive for pneumococci, 7(4.61%) in children below 5 years and 5(3.29%) in mothers. We report a low carriage of pneumococci among mothers and babies that were included in this study. In contrast, a previous study in Iganga/Mayuge reported high carriage rates of over 50% in children aged less than 5 years [4]. In the Iganga/Mayuge study participants were selected on the basis of presentation with pneumonia symptoms as de ned by WHO guidelines as opposed to our study which included all children that ful lled our selection criteria which did not include signs and symptoms of pneumonia. In a similar study carried out in Kenya, 90.0% of children were colonized with pneumococci. Both the Iganga/Mayuge and Kenyan studies were carried out prior to the introduction of PCV10 accounting for the difference in the carriage observed in our study. Different studies have shown varied carriage rates of pneumococci among children below ve years in Uganda and elsewhere [4,11,12] with most of them reporting a higher carriage rate than reported in our study. A systematic review reported a carriage rate in Africa to range between 21-94% [13] with more studies done among children than in adult population. The high immunization coverage for PCV10 in Ngora district could further explain the low carriage rate of pneumococci in our study as opposed to the Iganga/Mayuge study that indicated a high carriage rate of 56% at a lower immunization coverage of 42% PCV10 and 54% PCV13. There was a statistically signi cant relationship between the pneumococcal carriage and immunization status of the babies in our study (Table 2). Pneumococcal carriage is a prerequisite to disease, therefore our ndings suggest that full immunization with PCV10 is protective against pneumococcal carriage and hence pneumonia caused by streptococcus pneumoniae. Several studies have reported decrease in the burden of invasive pneumococcal disease and serotype distribution since the introduction of PCV vaccine [14][15][16][17]. The immunization coverage for the rst dose of PCV10 (PCV1) in Ngora district was 97.78% (133/136) and 2.22% (3/136) of children above six weeks had not received PCV1. Of the 152 participants 10.53% [16] were children below six weeks and were therefore not eligible for immunization with PCV10. The immunization coverage for PCV3 was 90.99% (101/111). Children below fourteen weeks who had not received PCV3 were excluded from the denominator because they were not eligible.
We also report a low carriage rate of pneumococci among the mothers. A similar study in coastal Kenya indicates that pneumococcal carriage was more associated with children below 5 years than the adults [12]. The low carriage rate of pneumococci among adults has been attributed by other studies to development of natural immunity [18]. The upper respiratory tract apparently appears a disadvantageous niche for streptococcus pneumoniae due to development of mucosal host defenses such as sIgA [19,20]. Also vaccination with PCV has resulted into the development of herd immunity in the adult population against S. pneumoniae [12]. Cases of pneumococcal colonization are however reported to rise in the elderly population due to immune senescence [21] with many countries not considering the importance of immunization this group of people.
From our analysis, there was no statistically signi cant association in risk of carriage of pneumococci with sex/gender of the child. This nding is similar to the results of a systematic review in Africa which noted that there was no association between pneumococcal carriage and gender [13] though one study associated pneumococcal carriage with males and the other that reported association of carriage with females.
A high trend of anti-microbial resistance was observed in Chloramphenicol, Tetracycline and Erythromycin. Other studies have similarly reported high resistance of pneumococci to the commonly used antibiotics [22,23]. In a study of erythromycin resistant S. pneumoniae 81% of the isolates were resistant to tetracycline, 76% were multi-drug resistant whereas 12% were resistant to clindamycin, tetracycline, chloramphenicol and kanamycin combined [22]. As opposed to our study, low resistance rates against tetracycline, erythromycin, chloramphenicol and Ceftriaxone were reported in Tanzania [23].
Again, contrary to our ndings, an earlier study in Uganda reported no resistance against erythromycin and ceftriaxone [24], indicating emergency of antimicrobial resistance against those drugs which may be attributed to the irrational use of antibiotics in Uganda and also due to the fact that such drugs are given empirically since there is no laboratory capacity to carry out culture and sensitivity studies.

Conclusions and recommendations
We report low pneumococcal carriage in the mother-baby pair in Ngora district. There was no signi cant relationship between pneumococcal carriage in the mother and prevalence in the baby. The relationship between pneumococcal carriage and immunization status suggest that PCV10 is protective against pneumococcal carriage. Resistance of S. pneumoniae to the commonly used antibiotics was high.

Limitations
We were not able to serotype the pneumococci isolated to determine the circulating serotypes.
Abbreviations PCV: Pneumococcal conjugate vaccine, MRRHREC: Mbale Regional Referral Hospital Research and Ethics Committee.
Declarations Figure 1 PCV 10 Immunization coverage among children below ve years

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. suplimentarymaterials28112019.xlsx