Ethics Approval
This study was approved by the Federal Capital Territory of Nigeria Ethics Committee, Zankli Medical Center Ethics Committee, National Hospital Abuja Ethics Committee, Keffi Medical Center Ethics Committee, The Joint Gambia Government and MRC Ethics Committee, and Michigan State University Institutional Review Board.
Setting
Nigeria is the most populated country in sub-Saharan Africa with a population of almost 150 million [13]. Abuja is the capital city of Nigeria, located in the Federal Capital Territory (FCT) which is the geographical centre of Nigeria. It has a land area of 8,000 square kilometers. It is bounded on the north by Kaduna State, the west by Niger State, the east and southeast by Nassarawa State and the southwest by Kogi State. Abuja is a "planned" city as it was mainly built in the 1980s and officially became Nigeria's capital in 1991, replacing the previous capital in Lagos. In 2006 the population was estimated at 1.7 million but may currently be about 5.7 million [13]. Abuja and the FCT have experienced a huge population growth; it has been reported that some areas around Abuja have been growing at an annual rate of 20 - 30%. The rapid spread of squatter settlements and shanty towns in and around the city limits is believed to be an important contributor to this rapid growth. The rainy season begins in April and ends in October. Within this period there is a brief interlude of harmattan, occasioned by the Northeast Trade Wind, with the main features of dust haze, intensified coldness and dryness. The annual total rainfall is in the range of 1,100 to 1,600 mm. The population is diverse, with increasing representation from the major ethnic groups of Hausa, Yoruba and Igbos following the development of the FCT and relocation of the federal capital.
There is perennial malaria transmission, mostly due to Plasmodium falciparum and HIV prevalence of 5.6% among pregnant women attending antenatal clinics.
National Immunization Program
The national expanded program on immunization calendar in Nigeria includes Bacillus Calmette Guerin vaccination, oral polio vaccine (OPV) and Hepatitis B at birth, OPV, diphtheria-tetanus whole cell pertussis (DPT) and Hepatitis B at 6 weeks, OPV and DPT 10 and 14 weeks, Hepatitis B and Measles at 9 months and Yellow Fever at between 9-12 months. National immunization coverage varies across the country but in the FCT has been about 85% for DTP3 [14]. Additional vaccines such as conjugate pneumococcal and Haemophilus influenzae type b are available at a few private clinics.
In 2008, Michigan State University in collaboration with the National Hospital Abuja established a research project to determine the etiologic agents of community-acquired bacteremic syndromes (sepsis, pneumonia, meningitis and bacteremia) in young Nigerian children aged 2 months -5 years. The enrollment sites for this Community-acquired Bacteremic Syndrome (CABSYNC) study include a number of hospitals within Abuja city and other rural settlements on the outskirt of Abuja, carefully selected to capture a mix of urban and rural population within easy reach of the diagnostic laboratories in Abuja city. Together, these facilities have an annual pediatric outpatient attendance of over 110,000 and 12,000 admissions and include the following;
a) The National Hospital, Abuja
The National Hospital of Abuja was originally designed to cater to the needs of women and children in Nigeria and the West African sub-region, with a view to reducing morbidity and mortality rates, and to carrying out extensive research into the particular causes of diseases in women and children in Africa. Phase 1 of the Hospital presently contains 200 beds, but the centre has facilities for a future expansion to 500 beds. The pediatric department admits about 90-100 patients per month and attends to 1300-1500 patients per month in the out-patient department. The facility provides postgraduate training in Pediatrics and has eight Attending Pediatricians and twenty five resident doctors.
b) University of Abuja Teaching Hospital, Gwagwalada
This is a district general hospital which has just recently been designated the teaching hospital for the University of Abuja. The hospital has a 300 bed capacity and serves a rural population from several villages and settlements on the outskirt of Abuja, within the Federal Capital Territory and also from the adjoining states. There are currently three Attending Pediatricians with 14 residents and with its new status; the university is in the process of employing more attending Pediatricians.
c) Nyanya District Hospital
This is a small district hospital located on the outskirts of Abuja and functions as a primary health care center with limited in-patient capacity. This facility is staffed by five generalist physicians, supported by nurses and midwives. Patients are generally referred to the National Hospital for specialist care. The facility has minimal laboratory support for urine microscopy and malaria blood films.
d) Zankli Medical Center
Zankli Medical Center (ZMC) is a private hospital based in Abuja. It has approximately 150 members on staff, 45 beds for in-patient admission, 17 full time doctors, including 9 consultants. In addition to the above there are four independent specialist clinics: Dental, Dermatology, Ophthalmology and ENT, which operate within the hospital premises. Constant power and water supplies are guaranteed by in-house generators to maintain 24-hour electricity and a borehole as a source of water. Communication is facilitated through broadband internet access and a network system within the hospital maintained by an in-house engineer. The Hospital is approved by the National Postgraduate Medical College as a tutelage centre for senior registrars in general practice and has recently embarked on the development of academic and research activities in collaboration with the Federal Ministry of Health of Nigeria and Liverpool School of Tropical Medicine, UK. The facility provides care for over 30,000 pediatric outpatients annually, with 1,255 admissions. The Research Laboratory has two senior laboratory scientists and ten other laboratory scientists working in the routine laboratory. The lab is equipped with two light microscopes, one fluorescent microscope, a Bactec™ MGIT machine for mycobacterial culture, and a Bactec™ 9050 for bacterial culture.
e) Garki General Hospital
Garki Hospital Abuja (GHA) is a private public hospital established in 1988. The hospital is located within less than 5 miles of the National Hospital and carters for a mix of population consisting of middle class and low socioeconomic class population from the outskirt of Abuja city. The pediatric facility has 25 beds with 784 annual admissions and 13,716 outpatients. It is staffed by one Attending Pediatrician and four medical officers.
f) Maitama Hospital
Maitama Hospital Abuja (MHA) is a government run general hospital within Abuja city. This facility provides care for middle class and low socioeconomic class population and has a large clientele from some of the new periubran settlements on the outskirt of Abuja. It is staffed by one attending pediatrician and four medical officers.
g) Federal Medical Center Keffi
This is a general hospital which is owned by the Federal Government and provides clinical services to the semi-urban population of Keffi and several rural settlements. This facility is staffed by one Attending Pediatrician and five medical officers.
The laboratories for this surveillance are primarily based at ZMC. This site was instrumental in supporting the generation of preliminary data that led to the development of this project. The Center for Disease Control and Prevention, Atlanta provided logistic support for setting up the study laboratories and also provided control bacteria strains.
Prior to the commencement of this study, two senior Microbiology Scientists were identified for special training for study procedures and the operation of the Bactec culture instrument. Training was provided at the Medical Research Council Laboratories in the Gambia and upon return to Nigeria, these Technicians have continued to provide supervision and guidance for other laboratory staff. Since commencement, we have procured an additional Bactec™ 9050 culture instrument through a generous donation from Beckton Dickinson, which is now placed at the National Hospital. All the facilities are within 30-45 minutes drive to either of the laboratories.
Participant enrolment
Following an initial period of sensitization and training for medical officers and residents, blood culture bottles and vacutainer sets were made available to the different sites.
We restricted enrollment initially to just NHA and ZMC and gradually expanded to include the peripheral sites at Gwagwalada, Nyanya, Keffi Medical center and subsequently, GHA and MHA. Not all eligible subjects were approached for enrollment because of insufficient manpower to commit specifically for this study at the different enrollment sites. Enrollment depended very much on the availability of existing medical and nursing staff at the different locations for subject enrollment and also for completion of the questionnaire. Some of the sites are currently understaffed and this clearly retarded our ability to enroll more subjects and to obtain additional specimens such as CSF from clinically suspected cases of meningitis. Such detailed clinical evaluation is currently not routine care at most of these facilities due to lack of adequate diagnostic laboratory services or affordability by caregivers.
Full scale enrolment commenced in November 2008 after formal training was provided to all laboratory personnel. Children who presented with fever or hypothermia were identified by a triage nurse and consent for participation was sought.
Children attending the out-patient clinics or emergency units were triaged and evaluated for eligibility. Informed consent was obtained from the parent or guardian. A physician administered a detailed questionnaire to obtain information on clinical history and physical examination findings. Appropriate clinical specimens were then obtained and these include 1-3 ml of blood for culture using the vacutainer set after cleaning the skin with alcohol swabs. The specimen was collected directly into the Bactec™ culture bottle and promptly delivered by the project driver to ZMC microbiology laboratory within 4 hrs of collection.
Data Acquisition
The Biomedical Research and Informatics Core (BRIC), Michigan State University provided support and supervision for data management of this study. BRIC is a data management unit for MSU that is involved in nearly 100 active clinical research protocols. Primary research informatics support is provided by a locally developed database application known as RIX.
Metadata design and entry
For the CABSYNC study data was collected from several diverse sources. Forms were entered during the medical exam, at the laboratory after tests specimen processing, and at the time of follow-up. In order to streamline data entry medical examinations forms were acquired from the National Hospital in Abuja (NHA) and laboratory forms from Zankli Medical Center. Next, online forms were designed for RIX to closely mimic the actual forms used onsite. This helped ensure that physicians and laboratory technicians would not have to deviate much from their normal workflow to enter study forms. Data collection instruments that are used for online data capture tools are designed differently as compared to paper forms. CABSYNC forms were edited to reduce rates of implicit uncertain responses.
Training
The onsite staff in Abuja had to attend an online seminar conducted by the Human Research Protection Program at Michigan State University. This seminar is required for anyone who is given credentials to the RIX system. Additionally, the onsite staff needed to be trained to properly use the RIX system. We used Skype for communicating across borders.
Training sessions included: RIX training for data entry, training for specimen tracking, barcode printing, and printer maintenance and methods of subject tracking and data integrity checks.
Data Cleaning and analysis
RIX is capable of adding many restrictions to data entry fields but for some variables these fields were left unrestricted. For this pilot stage of data collection it was not possible to predict some of the responses for string fields. During the post pilot data collections phase some of these variables have been coded. Using frequencies of some of the data collected during this pilot phase, it has been possible to provide selectable options for future CABSYNC forms instead of open string fields. Data analysis was performed with Predictive Analytics Software version 19.
Laboratory Methods
Blood culture
In order to determine the etiology of bacteremia, the impact of prior antibiotic exposure on culture yield was minimized by the use of the Bactec™ culture system with culture bottles containing an antibiotic-removing device (antibiotic resins). Bacteria were isolated from blood using an automated blood-culture system (Bactec™ 9050, Becton Dickinson, Temse, Belgium). The study utilized only aerobic blood culture bottles. The work-up for a positive culture vial included a subculture using sheep blood agar or chocolate agar, both of which were supplemented with micronutrients, e.g., iso-vitalex or vitox, for enhanced growth of fastidious bacteria, or McConkey agar plates. Inoculated media was incubated under aerobic and 5% CO2 conditions at 35°C for 18-24 hours. Bacteria were identified by a combination of morphology, Gram stain, and chemical methods; for S. pneumoniae, by susceptibility to ethyldrocupreine hydrochloride (Optochin) and bile solubility; for H. influenzae type b, X and V growth factor dependency detection and slide agglutination with type-specific antisera (bioMerieux, France) and for Enterobacteriacae, we used the API 20 E system (bioMerieux, France). Antibiotic susceptibility assessments were determined by Kirby-Buaer disc diffusion test using standard interpretative criteria and using antibiotic discs for locally available antibiotics for the immediate management of patients.
The blood agar plates for isolating bacteria from blood cultures were either obtained commercially from (Remel) Fisher Scientific USA or when this was not available, sheep blood was obtained from a local abattoir. Sheep blood was collected in to a citrate phosphate donor bag and used for preparation of 5% blood agar plates or chocolate agar plates, using previously described methods for collection and defribrination [15].
Following primary identification of the bacteria isolates at the study site lab, confirmation was sought at reference laboratories. This service was provided by the Medical Research Council Laboratories, The Gambia and Sparrow Hospital Microbiology laboratory, an affiliate of Michigan State University. Michigan State Department of Health Laboratory confirmed the typing of the Salmonella species.
Bacteremia was defined as the isolation of at least one non-contaminant bacteria from the admission blood culture. Coagulase-negative staphylococci, Bacillus, Corynebacterium species or Micrococcoci were classified as contaminants. S. viridians isolates were considered significant when isolated from children with underlying risk factors for S. viridians infection, such as congenital heart defect.
Antimicrobial activity detection in serum
Micrococcus luteus-
ATCC 7468 (provided by the Centers for Disease Control and Prevention, Atlanta) was grown overnight on agar plate. Colonies from fresh culture plates were suspended in normal saline, and calibrated to 0.5 McFarland standard. Using another cotton-tipped-sterile applicator, this was inoculated on to a nutrient agar plate, streaking the entire surface of the plate, rotating the plate 60°between streaks and ultimately rimming the plate to ensure confluent growth to the edges. The inoculum was allowed to dry for 5 minutes before depositing the 6-mm sterile paper filter discs. Using a sterile forceps 2 sterile discs were applied ~3 cm apart from each other, taking care not to remove and replace any disc after it has been applied. Once all of the above processes have been accomplished, add 20 μl of the test serum in one disc and 20 μl of sterile saline to the other disc. A positive control disc of chloramphenicol was used in each experiment. The inoculum was allowed to dry and the plates inverted so that the media is on the upper surface and cannot be contaminated by condensation. Plates were then incubated at 37°C in air atmosphere for 18-24 hours before final reading. A ruler was used to measure the zone of inhibition. Any inhibition zone bigger than the saline disc (6 mm) was interpreted as positive for previous antibiotic use.