Study design
We performed a descriptive and analytical cross-sectional study and we involved laboratory investigations to isolate the bacterial pathogens and antibacterial susceptibility pattern. The outcome variables were post-cesarean sepsis. We also assed predictors such as socio-demographic and clinical factors for the study participants.
Study site and setting
The study was conducted in the postnatal ward at Hoima Regional Referral Hospital (HRRH) between the months of July to September 2018. HRRH is a public hospital located in Hoima District with in coordinates; 01 24N, 31 18E and is approximately 230 km by road from Kampala (Capital city of Uganda). The major tribe is Banyoro and the main occupation is animal husbandry and crop farming.
Hoima Regional Referral Hospital is a well-established hospital and it offers both in-patient and out-patient services with a range of departments and clinics, including General Surgery, Orthopedics, Obstetrics and Gynecology and Internal Medicine. The hospital is well equipped with a bed capacity of 400. The Obstetrics and Gynecology Department of Hoima Regional Referral Hospital has four specialists, one Resident doctor, five intern doctors and 13 midwives. The obstetrics and gynecology department has 110 beds. The post-natal ward for post-cesarean mothers is located in the new block. The new block has about 60 beds which are always fully occupied with some patients lying on the floor.
According to the hospital records (Maternity Theatre register), Hoima Regional Referral Hospital performs approximately 8-10 cesarean sections per day. These are done in two theatres that are shared by other surgical teams. The hospital had a range of 10-20 vaginal deliveries per day and also offers antenatal and postnatal services. The main laboratory of Hoima Regional Referral Hospital consists of the following sections: hematology and blood bank, chemistry, parasitology and microbiology. It was composed of 20 staff members and these include three specialists, two laboratory technologists, eight laboratory technicians, six laboratory assistants, and one laboratory attendant. The microbiology laboratory was operated by one laboratory technologist, one laboratory technician, one laboratory assistant and one laboratory attendant. It was well equipped to carry out culture and sensitivity and other microbiological tests. Some of the equipment found in this laboratory were; autoclave, incubator, microscope, hot air oven, refrigerator, safety cabinet and gas cylinder.
Study population
The study population were mothers who have delivered by cesarean section at Hoima Regional Referral Hospital during the period of the study.
Selection criteria
Inclusion Criteria
All adult and emancipated mothers (on ward or re-admitted) who delivered by cesarean section at Hoima Regional Referral Hospital during the study period.
Exclusion Criteria
Cesarean section mothers from other health units that were referred to Hoima regional referral hospital were excluded due to limited access to their medical records. Mothers who would have had a re-exploration due to cesarean section complications other than suspected sepsis, as well as those who were in their early puerperium and those who reported after six weeks, were also excluded.
Sample size
The sample size for this study was 303 and was computed using modified Kish Leslie formula (Daniel, 1999).
Sampling technique
Consecutive enrollment of participants who consent to participate in the study. This was carried out on a daily basis until required sample size.
Data collection instruments
Structured investigator-administered pre-tested questionnaire was used for each participant to collect information on socio-demographic and known factors that may be related to the development of post-cesarean wound sepsis in each patient including obstetric factors, hospital factors and health factors.
A detailed history was elicited (English), translated where necessary for women who did not understand English; and physical examination was performed. Presence or absence of post-cesarean wound discharge (exudate) was noted. Swabs for mothers with discharge were taken and cultured in the laboratory according to standard clinical laboratory guidelines. Susceptibility testing was carried out according to Kirby Baur diffusion methods.
Sample collection and transportation
Patients with post-cesarean wound sepsis who met the inclusion criteria of the study and were, educated, counseled about the study and then consented to participate. They were requested to allow history taking and physical examination and when a patient had post-cesarean wound discharge (Exudate), a sample was taken for microbiological analysis. Using sterile swab sticks, two samples from each participant were collected by gently rubbing the sterile swab sticks in the infected site (wound depth) using aseptic technique and immediately replaced inside the swab sticks case. The sterile swab stick was labeled with each participant’s study number and transported to Microbiology laboratory immediately for processing, and in case of any delay, the sample was stored aerobically in the refrigerator at 4-8⁰C.
Sample processing and Laboratory analysis
Isolation
The collected samples were inoculated on blood agar, chocolate agar, MacConkey agar and mannitol salt agar. They were then incubated both aerobically and anaerobically at 37ᴼC for 24-48hrs. Colony morphology was observed according to shape, size, elevation and margin and surface characteristics.
Direct Gram Microscopy
A direct smear was made for Gram stain; a drop of sterile normal saline was added at the center of a clean dried glass slide and the swab containing the sample rolled in the drop of normal saline spreading it on the glass slide in a circular motion to make a thin smear of the size of a fifty shilling coin. The smear was allowed to air-dry and then heat-fixed by passing it at least three times over a Bunsen flame. The slide was placed on the staining rack and flooded with crystal violet solution for 60 seconds, washed with clean water and covered with Lugol’s Iodine (a mordant) and then allowed to act for a minute.
The slide was again washed in clean water and then decolorized with 50% acetone- alcohol under slow running tap water until a faint pink color was observed or no more color tend to flow from the smear. The process of decolorizing did not exceed 30 seconds. After decolorizing, the slide was washed in clean water and counterstained with neutral red solution. The slide was then washed in clean water; air-dried and observed under the microscope with x100 objective lens (oil immersion lens). Gram-positive bacteria was observed as blue or purple color and Gram-negative as red or pink color. Also, the morphology and shape of the bacteria was used to identify whether they are cocci, diplococcic, cocci in chains, clusters, and whether they are rods in appearance. Pus cells were also observed in the direct Gram-stained slide.
Identification of bacterial isolates
Cultural characteristics
The colony morphological characteristics of the bacterial isolates was observed as follows; color, margin, mucoid, texture, and hemolysis on blood agar medium, among others. This helped in determining the characteristics of the colonies of the bacteria on culture media such as Lactose or non-lactose fermenters on MacConkey agar and type of hemolysis (alpha, beta, and gamma hemolysis) on blood agar.
Biochemical tests
The isolates were identified using the conventional biochemical tests such as API (Analytical Profile Index) which depends on the Gram’s stain reaction of the isolates (Gram-positive or Gram-negative bacteria). Other biochemical tests included catalase, optochin, bacitracin, coagulase, indole, citrate utilization, urea utilization, triple sugar iron agar fermentation, MR-VP test and oxidase as described below:
Catalase test
The Catalase Test was carried out to differentiate between Streptococcus and Staphylococcus species and this was done according to standard methods [22] . A drop of 3% hydrogen peroxide was added to a loop full of the test organisms. Presence of bubbles indicated catalase activity. Streptococcus species was catalase positive while Staphylococcus species was catalase negative.
Indole test
The Indole Test was carried out according to the method described by Cheesbrough [22] to determine the ability of the isolate to degrade amino acid tryptophan and produce tryptophanase enzyme. A 1% tryptophan broth in a test tube was inoculated with 7 days isolate and incubated at 37°C for 48 hours. After 48 hours, 1 ml of chloroform was added to the broth. The test tube was shaken gently, and 2.1 ml of Kovac’s reagent was added and again shaken gently. This was allowed to stand for 20 minutes. The formation of red coloration at the top layer, indicated a positive test, while a yellow coloration indicated negative result. Escherichia coli and Proteus are indole-positive.
Urease test
The Urease test was carried out according to the method described by Cheesebrough [22] to determine the ability of the bacteria to hydrolyse urea and produce ammonia and carbon dioxide. The test organism were inoculated into urease broth and incubated at 30°C for 72 hours. Purplish pink coloration of the medium indicated a positive reaction for Proteus and negative for other enterobacteria like Klebsiella and E. coli.
Citrate utilization
This was carried out by inoculating the test organism in test tube containing Simon’s citrate medium and incubated for 24 to 72 hours. The development of deep-blue color after incubation was indicate a positive result. Klebsiella species are citrate- positive.
Triple sugar- iron test
Triple sugar iron test was carried out according to the method described by Cheesbrough [22]; the test determined the ability of the organism to ferment the three sugar component of the medium: glucose, lactose and sucrose. The medium contains a pH indicator (phenol red) and a detection system (thiosulphate and ferrous sulphate) for hydrogen sulphide (H2S). The medium was prepared as an agar slant. The test organism was inoculated by stabbing the medium using sterilized straight wire loop and the surface of the slope was also streaked with the test organism. The test was incubated at 37°C for 3 days. After incubation, gas production was determined by observing the cracking of the medium, and production of H2S was observed by the blackening of the butt (bottom) of the medium. The triple-sugar iron-agar aided in identification of Escherichia coli which ferments all three sugars and produce acid, turning the media into yellow color. Proteus species produces H2S which is indicated by black coloration of the media and fermentation at the butt of the tube.
Methyl red – Voges – Proskauer test (MR-VP)
Methyl red – Voges – Proskauer test (MR-VP) was carried out according to the method described by Cheesbrough [22]. It was used to determine the ability of the organisms to ferment glucose with production of acid. Five milliliters (5 ml) of MR-VP broth were inoculated with the test organism and incubated for 48 to 72 hours at 37°C. After incubation, 2 to 3 drops of methyl red test were added to 1ml of the broth. A red color signified a positive methyl red test, while yellow color signified a negative test. To what remained, five drops of 4% potassium hydroxide (KOH) were added followed by fifteen drops of 5% α –naphthol in ethanol. The development of red color within 1 hour indicates VP positive test while no color change indicated VP negative test. Escherichia coli is methyl red positive and voges-proskauer negative.
Coagulase test
It was used to identify Staphylococcus aureus which produces the enzyme coagulase. The rapid slide test was done by placing a drop of distilled water on each end of slide. Then a colony of the test organism (previously checked by Gram-staining) was emulsified in each of the drops to make two thick suspensions. A loopful of plasma was added to one of the suspensions (no plasma was added to the second suspension), and mixed gently. Formation of clumps of the organisms within 10 seconds was indicative of a positive test while absence of these clumps was indicative of negative results.
For suspected Staphylococcus aureus isolates which turn negative for the rapid slide test, the test was done by emulsifying several isolated colonies of test organism in 1 ml of diluted rabbit plasma (1:5) dilution to give a milky suspension. The tubes were then incubated at 35°C in water bath for 4 hours. These were then examined at intervals of 1, 2 and 4 hours for clot formation by tilting the tube through 90°. If the test was still negative, the tube was left at room temperature overnight and examined again for Staphylococcus aureus that produced a delayed clot.
Oxidase test
The test was used in identification of organisms which produce the enzyme
cytochrome oxidase. A filter paper soaked with the substrate tetramethyl-p-phenylenediamine dihydrochloride was moistened with sterile distilled water. Using a glass rod, a colony of the test organism was smeared on the filter paper. The development of a blue-purple color within a 10 seconds was indicative of positive test while absence or formation of a blue-purple color after 10 seconds was considered negative. Pseudomonas species and Neisseria species are oxidase positive.
Susceptibility Pattern Determination (Kirby-Bauer disc diffusion technique)
The susceptibility pattern of the identified pathogens was determined by agar disc diffusion technique by Kirby-Bauer using Muller Hinton agar. The Muller Hinton agar was prepared according to the manufacturer’s instructions under sterile conditions to avoid any contamination that may result. About 4-5 colonies of the organism were diluted in sterile peptone water and mixed and incubated for 4-5 minutes and its turbidity compared to a McFarland standard 0.5%.
A prepared Muller Hinton agar was dried in an incubator for at least 15 minutes and using a sterile glass rod or swab stick, the surface were smeared with the diluted organism of the peptone water, and using sterile forceps, ten different antibiotic containing discs were placed on the dried surface of the Muller Hinton agar containing the organism and then placed in the incubator at 37⁰C for 24 hours.
The diameter of a clear zone surrounding the antibiotic disc was measured in millimeters and compared to a standard antibiotic chart for measuring zones of inhibition. Zones of inhibition measured were recorded as susceptible (S), Intermediate (I), and Resistant I according to the standard chart.
Statistical analysis
Data on questionnaires was entered in Microsoft Excel version 2010, and then data from Excel was exported to IBM SPSS statistics version 23 as well as STATA 14.1 (Statacorp, USA Texas). Socio-demographic and clinical factors were summarized as means and medians, standard deviations and interquartile range (for continuous variables). Proportions, percentages and frequencies were used for categorical variables using STATA 14.1.
Prevalence of post caesarian sepsis was summarized as frequencies and percentages. The factors associated with post-cesarean wound sepsis were assessed using binary logistic regression. For factors with odds ratio ≤ 0.2 at bivariate and those with biological plausibility were transferred for multivariate analysis. Bar charts were used to present susceptibility profile.
Quality control
The questionnaires was checked for completeness before collection to ensure valid data is obtained. Two independent microbiologists were involved in identification bacterial isolates. Additionally, standard operating procedures were adhered to.
Ethical considerations
Permission to conduct this study was obtained from the department of obstetrics and gynecology and post graduate directorate. Afterwards approval was obtained from Kampala International University Research Ethics Committee (KIU-REC).