Characterization of Bacterial Isolates Cultured from the Nasopharynx of Children with Sickle Cell Disease ( SCD )

Method: Sterile cotton-tipped initially dipped into sterile saline thereafter was introduced into nasopharynx of each patient and thereafter applied onto sterile thioglycolate medium and incubated at 370 C for 24 hr, When growth was noticed, samples were inoculated onto enriched, selective and differential bacteriologic media. Bacterial colonies that grew on such media were picked and characterized by Grams’ reaction, cultural, morphologic and biochemical methods. Antibiotic sensitivity tests were determined by the disc diffusion method. Demographic data relating to severity of SCD were provided.


Introduction
A recent report shows about one hundred and fifty thousand infants are born annually with sickle cell disorder in Nigeria [1].The country also ranks highest in the incidence of sickle cell trait in Africa [1].According to a recent release by the Sickle Cell Foundation of Nigeria, one out of four Nigerians carries the sickle cell gene compared with the United States of America where one in twelve of African Americans carry the sickle cell gene [1].It has been shown nasopharyngeal bacterial colonization is common in the young infants which often precedes development of invasive diseases [2].Knowledge of nasopharyngeal bacteria colonization in children is desirable because the nasopharynx harbors potential pathogens.Studies from the eastern and southern parts of Nigeria reported different types of bacterial agents cultured from the nasopharynx of children with sickle cell disease [3].The types and number of nasopharyngeal bacteria recovered SCD patients depend on number of factors relating to the subject and administration of vaccines and antibiotics that suppress resident microflora but increase emergence of resistant strains [4].Some investigators have shown the use of vaccines mitigate the incidence of bacterial agents such as S. pneumoniae, Haemophilus influenzae type b and Neisseria meningitidis which influence the carrier rate of these bacteria often implicated in acute bacterial meningitis [5].For example in the United States, prior to the introduction of Haemophilus influenzae and pneumococcal vaccines, young children below the age of five years with SCD had a 13% risk of developing bacterial sepsis with mortality rate of 30 and 10 % in patients with sepsis and meningitis [6].A recent study of the nasopharyngeal of S. pneumoniae carriage among rural Gambian in West Africa sub-region, showed the nasopharynx carriage of Gambia infants by S. pneumoniae is rapid and highly dynamic, rising from prevalence of 1.5% at birth to 77% at 2 months, 5 months 86% and to 78% at 12 months [7].A Brazilian study reported the nasopharynx may constitute an ecological reservoir or source of dissemination of S. pneumoniae, H. influenzae, S. aureus and other Gram-negative bacilli (GNB) [2].Infants and children are the most vulnerable to opportunistic infections mainly because of their relatively immature immune system besides the elderly and the immunocompromised.We have characterized the bacterial isolates from the nasopharynx of sickle cell disease children attending the infant welfare clinic (IWC) at the Wesley Guild Hospital, Ilesa, southwestern Nigeria because the pattern of colonization, types and number of bacterial isolates from nasopharynx may differ from those reported from other regions.In addition, the study also evaluated the antibiotic resistant profile of the potential pathogenic bacterial isolates since this is an antibiotic pressurized community.We also evaluated the severity or otherwise of SCD in these patients in relation to frequency of blood transfusion and hospitalization.We believe the results obtained from our study will assist clinicians to better manage bacterial infections in children with SCD.

Study Center
Ilesa where the study took place is a semi-urban town with a population of 277,904 inhabitants in Osun State in southwestern Nigeria and the Wesley Guild Hospital is one of the outpost hospitals employed by the Obafemi Awolowo University Hospitals Complex (OAUTHC) to train medical students and other allied healthcare professionals.The hospital is located about 20 minutes' drive from Ile-Ile where Obafemi Awolowo University, the parent institution is situated.

Criteria inclusion
All participants were sickle cell disease patients that attended the infant welfare clinics of the hospital either because they were sick or had a routine checkup appointment with the attending physician.Each participant was recruited after we adequately explained the purpose of the study using each participant's native dialect.Furthermore, demographic information relating to the each participant was obtained from interviews, questionnaire responses from guardian/ parent and case files managed by the attending physicians.Ethical clearance for approval to undertake the study was given by the Ethics committee of the hospital.The age of the participants ranged from 4 months to 15 years comprising of 45 males and 39 females.

Collection of samples
Each sample was collected from each subject's pharynx by the attending physician using a sterile cotton-tipped applicator that was initially dipped into sterile normal saline and introduced into sterile thioglycolate broth.All such samples were incubated at 37 o C for 24 hours for growth and further studied.Duplicate samples were prepared for anaerobes and incubated in AnaeroPack Jar 2.5 Liter, Order No.50-25, product of Mitsubishi Gas Chemical Company Co., Inc.Japan.All samples were analyzed within 24 hours of collection.

Statistical analysis
The resulting data was analysed by the descriptive analysis and t-test using SPSS version 16.0 software.Significant difference was taken as p < 0.05.

Results
Data relating to patients' were obtained from their records and personal files and analyzed.Table 1 shows the profile of the genotype, the frequency of blood transfusion and hospitalization of the SCD patients.Out of the 84 SCD patients screened, 78 (92.85%) were HbSS of which 41 (52.5%) male and 37 (47.4%)female compared to 6 with HbSC of which 4 (66.7%) was male and 2 (33.3%) female.The number of blood transfusion in the 12 months prior the study was analyzed.Of the 81 patients whose records were available, 1 patient received blood transfusion 4 times; another patient had 3 blood transfusions within the period.5 patients also received blood transfusion twice while 23 patients had only one transfusion within the period.In contrast, 51 patients did not receive blood transfusion.Regarding hospitalization in this same group was also based on the number of admis-sion in 12 months prior to study.One patient was hospitalized 5 times, two 4 times, 5 patients were hospitalized 3 times.Furthermore, eight patients were hospitalized 2 times while 33 patients in the group were hospitalized only once.In contrast, 32 patients among the group were not hospitalized (Table 1).
Similarly, the number of significant pain episodes in 12 months preceding the study period was assessed.SCD severity was calculated from the number of hospitalization, blood transfusion and significant pain (significant pain referred to pains necessitating hospital visits (either as outpatient or inpatient and requiring analgesics) (Fig 1).Socioeconomic class (SEC) of parents was derived using the occupations and highest educational qualifications of both parents.24 (30%) each of the SCD patients' parents were categorized in the upper and middle socioeconomic classes while 32 (40%) were classified in the low socioeconomic class.

Discussion and conclusion
The sickle gene confers an increased susceptibility to infection, especially to certain bacterial pathogens, and at the same time infection provokes a cascade of SCD-specific pathophysiological changes.Africa bears the highest burden of sickle cell disease on the globe.(2.52%) S. pneumoniae underscoring a low rate of nasopharyngeal colonization with these organisms in this center which may be attributable with the prophylactic use of penicillin in this center.Despite the high nasopharyngeal carriage which often is a determinant for invasive pneumococci in some regions [9], it is puzzling to note high carriage rates with low rates of invasive pneumococcal disease in sub-Saharan Africa [10,11].According to Kizito et al., [11] a significantly low rate of pneumococ-cal bacteremia in Ugandan children with sickle cell disease (6%, 3/47) [12], and other studies have indicated low pneumococcal bacteremia in Nigerian children [13,14].However, in Zambia low rate of pneumococci frequency was attributable to strict antibiotic policy of the government that reduced access to sale of over the counter drugs.Our data showed widespread multiple antibiotic resistance among the four pathogens we tested.Our study shows all the 5 H. influenzae isolates tested were resistant to tetracycline, chloramphenicol and nalidixic acid.4 isolates were resistant to ampicillin, oxacillin and third generation cephalosporin-ceftriaxone while 3 isolates were resistant to augumentin, erythromycin, ciprofloxacin and nitrofurantoin.In addition, two of H. influenzae isolates were also resistant to streptomycin but none was resistant to gentamycin and kanamycin.Similarly, of the four S. aureus isolates cultured, all were resistant to ampicillin, erythromycin and nalidixic acid and 1 isolate was resistant to augumentin, tetracycline and gentamycin but none was resistant to oxacillin, ceftriaxone, streptomycin, kanamycin, chloramphenicol, ciprofloxacin and nitrofurantoin.Furthermore, our results reveal among the three S. pneumoniae isolates cultured from the nasopharynx, all resistant to the beta lactams antibiotics, augumentin, ampicillin, oxacillin and ceftriaxone.Two of the S. pneumoniae isolates were resistant to streptomycin and one isolate to tetracycline, chloramphenicol and nalidixic acid.
In contrast, none of the isolates was resistant to erythromycin, gentamycin, kanamycin, ciprofloxacin and nitrofurantoin.Finally, among the Moraxella catarrhalis isolates tested, all were resistant to ampicillin, oxacillin, ceftriaxone, chloramphenicol, nalidixic acid.However, 1 isolate was resistant to augumentin, erythromycin but all were sensitive to the other antibiotic used (Table 3).Because of susceptibility to severe pneumococcal infection, children with sickle cell disease (SCD) routinely receive penicillin prophylaxis which unintentionally increased the rates of penicillin resistance reported throughout the world, this practice has led to emergence of penicillin resistant S. pneumoniae (PRSA) untamable to treatment with β-lactams in particular and also a variety of different antibiotics.
The predominance of Corynebacterium spp seen in our study among these SCD patients may also be related to indigenous microflora which represents commensals of the nasopharynx that limit the growth of pathogenic organisms by phenomenon of microbial antagonism.Furthermore, Gram negative organisms accounted for 34.46% of the nasopharyngeal isolates comprising of 11 (26.82%)lactose fermenters and 30 (73.17%) non lactose fermenters.Colonization with Gram negative organisms specifically with lactose fermenters-E.coli, Klebsiella, Citrobacter and Enterobacter spp is of concern but may be attributable to patients' personal hygiene.It is interesting to note that 40% of the population studied comes from low socioeconomic group (15) who live in overcrowded living conditions and lack portable drinking water are therefore at higher risk of parasitic infections such as malaria and diarrhoea.The low rate of nasopharyngeal colonization in children at this center with Haemophilus influenzae 5 (4.2%) and S. pneumoniae, may be attributed to administration of vaccines early in life in this environment.
The multiple antibiotic resistance (MAR) seen in our study was widespread among the four predominant pathogens.The MAR indices for the beta-lactams for example, comprising of ampicillin, augumentin, oxacillin and ceftriaxone were 0.92, 0.57, 0.64 and 0.64 respectively.For tetracycline, the MAR value was 0.50; and for erythromycin 0.57.Regarding aminoglycosides comprising of streptomycin, gentamycin and kanamycin their MAR indices were 0.28, 0.07 and 0.00 respectively.For chloramphenicol the MAR index was 0.71, while for nalidixic acid it was 1.00.When we analyzed ciprofloxacin and nitrofurantoin, MAR value was 0.21 for each antibiotic.These results suggest widespread multiple antibiotic resistance among organisms cultured from SCD patients.Our study showed an unexpected high frequency of nasopharyngeal colonization rarely seen with indigenous microflora specifically Corynebacterium spp at this center.This finding suggests routine use of prophylactic penicillin may have suppressed S. pneumoniae nasopharyngeal colonization among the subjects.It also suggests routine vaccination of infants and children in the center may be assisting in reducing the frequency of colonization by S. pneumoniae and Haemophilus influenzae among subjects.Our data show beta-lactam antibiotics are virtually ineffective against the organisms cultured from these SCD patients compared with aminoglycosides, quinolones and nitrofurantoin.This observation is of epidemiological significance in the event of an epidemic in this center.The widespread resistance recorded for the majority of nasopharyngeal isolates recorded in our study requires urgent and necessity of instituting effective antibiotic policy in this center to reduce the frequency of drug-resistant in this population.
Studies have shown SCD not only originates from abnormality of RBC but also is a multisystem disorder, affecting virtually every organ of the human body.These conditions include haemolysis, many haemotological complications, vasoocclusion, infection and organ dysfunction (8).Our study reveals 49 (60.49%) of the SCD patients were hospitalized at least once.Thirty (61.22%) of these patients also received blood transfusion at least once.SCD patient suffers chronic haemolytic anaemia which allows the patient to carry on normal activities at steady state hemoglobin with narrow reserve capacity to accommodate strenuous physical activities [16,17,18,19].The role of the blood transfusion in the SCD patient is meant at increasing the level of hemoglobin, improve oxygen delivery and reduce proportion of sickle RBCs in circulation [8].In addition, infection caused by H. influenzae and S. pneumoniae is a major cause of concern for children with SCD with haemoglobulin SS; HbSS and HbSC as these children are at risk of bacteremia and chronic anemia that lead to early loss of splenic function [6].Our results, however suggest despite their genotypes, these children had low rate of colonization with H. influenzae and S. pneumoniae at this center which is interesting and may lead to further reduction in infection with these organisms.Furthermore, the preponderance of indigenous microflora in nasopharynx of these children by organisms such as Corynebacterium spp most of which are commensals, are in competition with pathogens because of microbial antagonism, an aspect of innate immunity will reduce the colonization with pathogens.Besides, administration of both pneumococci and influenzae vaccines in booster doses may also lower the rate of nasopharyngeal colonization [20].In conclusion, Gram positive bacterial isolates predominated in this study (65.45%) compared to (34.55%) of Gram negative bacteria.However Corynebacterium spp predominated.Low rate of nasopharyngeal colonization with Haemophilus influenzae, Streptococcus pneumoniae and Staphylococcus aureus strains was recorded.The study also recorded widespread multiple drug resistance among most isolates colonizing the nasopharynx of SCD in this center which is worrisome suggesting for urgent institution of effective antibiotic policy to stem the tide of multidrug resistant organisms in this center.
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Figure 1 :
Figure 1: Severity of sickle cell disease among patients.

Table 1 .
Profile of frequency of blood transfusion, number of hospitalization and Genotypes of the sickle cell patients.

Table 2 .
Distribution of bacterial isolates cultured from the nasopharynx of SCD subjects.

Table 3 .
Antibiotic resistant pattern of potential pathogenic bacterial isolates cultured from the nasopharynx of SCD subjects at the Wesley Guild Hospital, Ilesha

Table 4 .
Multiple Antibiotic Resistance Index of the predominant pathogenic bacterial isolates cultured from the nasopharynx of SCD subjects at Wesley Guild Hospital, Ilesha.