Study area: The study was conducted in Tikur Anbesa Specialized Hospital (TASH), the teaching Hospital of health Science College, Addis Ababa University. TASH is the largest specialized Hospital in Ethiopia, with over 700 beds, and serves as a training center for undergraduate and postgraduate medical students, dentists, nurses, midwives, pharmacists, medical laboratory technologists, radiology technologists, and others who shoulder the health problems of the community and the country at large. With more than 70 percent of childhood deaths attributable to communicable diseases and malnutrition, Ethiopia’s healthcare resources have been directed primarily to treat and prevent diseases such as malaria and diarrhea .
Study design and period: A cross-sectional study was conducted from September 2017 to June 2018 to identify the bacterial profiles and antimicrobial susceptibility pattern among septicemia under five patients with acute febrile illness in Tikur Anbesa Specialized Hospital in Addis Ababa.
Source population: All under 5 years pediatric patients who were suspected for septicemia and seek medical care at the study site during the study period
Study population: All under 5 years pediatric patients who were requested for blood culture in the study site during the study period.
Inclusion and Exclusion criteria: Children aged under five years including neonates with fever
Patients who are diagnosed with sepsis, Sever sepsis and septic shock. In addition, all children who gave blood sample and their parents volunteer to give permission to participate on the study. However those participants clinically none febrile patients under five years. Patients who took antibiotics currently within the last 7 days were excluded.
Sample size calculation
The sample size for the study that infers the total population was determined using a single population proportion formula. The study considered the previous study of prevalence and antibiotic resistance of bacterial pathogens isolated from children under five in septicemia patients at Tikur Anbesa Specialized Hospital 27.9% bacterial isolation (20), at 95% level of confidence and 5% margin of error.
n = (Zα)2 (pq)/ d2 Where: n = sample size Zα/2 = level of confidence P = diarrhea prevalence q = 1-p d2 = margin of error (0.05) n= z² * p * q, p=0.279, q=0.721, d=0.05, Zα/2=1.96
Considering 10% non- response rate, the totals of 340 children patients were enrolled in the study. Sampling technique
The study subjects were selected using convenient sampling technique from all patients attending Tikur Anbesa specialized Hospital among under five children with febrile illness clinically diagnosed at pediatric OPD, ICU and impatient pediatric ward admitted during the study period. Sampling technique was employed for those children fulfill the inclusion criteria.
Data collection procedure
Well standardized questionnaire was used to collect socio-demographic characteristics (sex, age, clinical presentation (fever, vomiting and household income). Patients visiting outpatient departments (pediatric and general medicine) and those admitted in the inpatient units were investigated for bloodstream infections by respective unit physicians. At the onset of fever (>37∘C) or in the presence of any clinical symptoms compatible with infection.
Blood sample collection: A venous blood culture specimen was taken with aseptic technique by cleansing of the collection site with 70% alcohol and subsequently followed by 10% povidone-iodine solution by trained laboratory personnel. About 2.5-5ml of blood specimen was collected and inoculated into aerobic 30ml BacT/ALERT PF Plus pediatric bottles at the blood to broth ratio of 1: 10-1:30. At least 2 sets of blood cultures were collected from a patient with suspected bacteremia prior to the initiation of antimicrobial therapy.
Culture Isolation and Identification: Venous blood to BacT/ALERT culture bottles were incubated in automatic BacT/ALERT® 3D at 37oC of 5% CO2 for 5 days for the primary isolation of the microorganism. Two aerobic blood culture bottles were used for each patient and growth in both bottles were considered positive. The microbial growth that could be detected by flag and audible sound of the instrument will subsequently be sub culture on 5% sheep blood Agar, chocolate, and MacConkey Agar plate (Oxoid Ltd, UK) and incubate at 37oC for 18-24 for bacterial isolation. The MacConkey agar plate was incubated aerobically while chocolate and blood agar were incubated in microaerophilic atmosphere (5-10% CO2) candle Jar. A negative result was checked by examining the flag and doing gram stain and a final subculture at the end of 5th day prior to discarding as negative. The significant growth colonies were examined morphologically for size, consistency, shape, hemolytic and ability to ferment lactose .
Blood agar, chocolate and MacConkey agar (Oxoid Ltd, UK) were used for subcultures and gram stain was performed for preliminary result. All positive cultures from blood samples were characterized by colony characteristics, Gram stain and biochemical tests. For gram negative bacteria convectional biochemical test and serological identification was performed for Salmonella and Shigella spp [21-22].
Antibiotic Susceptibility Test: Pure Colony of isolated bacterial organism was mixed with 0.85% normal saline and measured at 0.5 McFarland standards for susceptibility testing. The bacterial isolates were tested against the following drugs commonly used; for gram negative bacteria Tobramycin (10μg), Amoxicilin-Clavulanate (20/10μg), Amikacin (30μg),
Gentamycin(10μg), Ampicilin(10μg), Piperacillin-Tazobactam(100/10μg), Cefotaxime (30μg), Cefepime (30μg), Ceftriaxone (30μg), Ciprofloxacin (5μg), Impenem/ Meropinem (10μg), Trimethoprim- Sulfamethoxazole (1.25/23.75μg), Nalidixic Acid (30μg) were tested. Kirby-Bauer’s Disk Diffusion method was used for susceptibility of the isolates on Muller Hinton agar was referred to the standard interpretative chart reporting the zone sizes of each antimicrobial in the book of Cheesbrough, 2009 and CLIS guidelines .
Detection of Carbapenem Resistance: All the Carbapenem (imipenem or Meropenem) resistant or intermediate isolates were checked for the presence of carbapenemase using modified Hodges test (MHT), also known as the clover leaf test as per the EUCAST and CLSI. Presence of indentation indicates a positive test and the isolate was a carbapenemase producing strain. No growth of the ATCC E. coli 25922 along the organism growth streak indicates a negative test and the isolate is not a carbapenemase producer.
Detection of Extended spectrum beta-lactamase: Initial screening for ESBL was done by the diameters of zones of inhibition produced by Ceftazidime (30µg), Ceftriaxone (30µg) and Cefotaxime (30µg) found to be within the CLSI screening criteria. These breakpoints indicative of thought for ESBL production are: for CAZ≤22mm, CRO, ≤ 25 mm and for CTX≤ 27mm. Phenotypic detection of ESBL production was confirmed by double disk synergy test and combined disk test according to EUCAST(2017) and CLSI(2017) guidelines respectively.
Combined disk (double disk potentiate) Test (CDT): A Ceftazidime (30 µg) disk and Cefotaxime (30µg) dick were used alone and their combination with Clavulanic acid (30 µg/10 µg) for phenotypic confirmation of the presence of ESBLs. A ≥5 mm increase in zone diameter for either of the Cephalosporin disks and their respective Cephalosporin/Clavulanate disk were interpreted as ESBL producer. This method (according to CLSI) is used as reference phenotypic method for comparing double disk synergy method.
Double Disk Synergy Test (DDST): The organism to be tested was spread onto a Mueller–Hinton agar plate. The antibiotic disks used are Ceftriaxone (30 µg), Cefotaxime (30 µg), Ceftazidime (30µg), Aztreonam (30µg) and Amoxicillin/ Clavulanic acid (20/10 µg). The four antibiotics were placed at distances of 20 mm (edge to edge) from the Amoxicillin/Clavulanic acid disk placed in the middle of the plate. After 24-h incubation, if an enhanced zone of inhibition between either of the cephalosporin antibiotics and the Amoxicillin/Clavulanic acid disk occurred, the test was considered positive.
Data Quality assurances: Media preparation as per manufacturer instructions and laboratory Standard Operating Procedures (SOP) was strictly followed. Verify that media meet expiration date and quality control parameters per CLSI. Labeling container, media, filling the forms were carried out.
Visual inspections of cracks in media or plastic petri-dishes, unequal fill, hemolysis, evidence of freezing, bubbles, and contamination were performed. Use ATCC control strain for each isolated bacterium including E. coli 25922, S. aureus 25923, Pseudomonas areuginosa 27853, H.influnzae 10479. Report the results on log sheet and stored for further data. Samples were stored at -80 0c in skim milk.
Data analysis and interpretation : SPSS versions 20.0 was employed to analyze the work and to make inferences on the frequency of occurrence of the bacterial pathogens associated with febrile illness and to show the resistance pattern to antibiotic substances. Descriptive statistics to analysis by using frequency, proportions graphs, crosstabs and odds ratio. Bivariate analysis was performed for each factors associated with enteric pathogens in pediatrics diarrhea. Regression analysis was conducted to identify associated factors and how they are associated with dependent variables .The strength of association was presented by odds ratio and 95% confidence interval and p-value of <0.05 was considered as statistical significant association between risk factors and enteric pathogens causing diarrhea and antimicrobial resistance of bacterial infection.
Ethical Considerations: The study was conducted after ethical clearance was obtained from the research ethical committee of Department of Medical Laboratory sciences. An informed consent was obtained before collection of blood specimens and results were used in the management of patients. Those patients who clinically diagnosed as BSI in pediatric OPD and admitted willing to participate in the study and able to give blood sample during the study period were informed about the purpose of the study and written consent was sought for the study. Any information related with the patient result and clinical history was kept confidential.
Dissemination and Utilization of Results
After the completion of the study the research were disseminated to Department of Medical Laboratory Sciences, School of Allied Health Science, College of Health Science, and Addis Ababa University. It will also be submitted for scientific publication.
Antimicrobial resistance: occurs when microorganisms change in ways that render the medications used to cure the infections they cause ineffective.
Extended spectrum β-lactamase (ESBL): Extended-spectrum beta-lactamases (ESBL) are enzymes that confer resistance to most beta-lactam antibiotics, including penicillins, cephalosporins, and the monobactam aztreonam.
Multidrug resistance (MDR): is antimicrobial resistance shown by a species of microorganism at least to one drug in three different classes of antibiotics.