- Study design, settings and duration
From 28th September to 30th October 2020, a cross-sectional analytical study was carried out in two hospitals within the city of Yaounde, including the Yaounde Gynaecology Obstetrics and Paediatrics hospital – a referral hospital with the capacity of 240 beds and the Animation Social and Sanitary centre of Nkoldongo, a medical center with the capacity of 62 beds. Pregnant women in labor were consecutively enrolled in the study following an informed consent. The neonates born from these women were also included in the study. Clinical specimens were collected from all study participants and transported in the Bacteriology Laboratory of the Yaounde Gynaeco-Obstetrics and Paediatrics hospital for analysis.
- Inclusion and exclusion criteria
Were included from our study, all women in labour who had given their free and informed consent and all new-borns whose mothers had given their free and informed consent. Were excluded from our study, all women who were unable to produce stool and were on antibiotics three months prior to childbirth and all stillbirths.
Our minimum sample size was estimated to 68 couples of mothers and their neonates, following the Lorentz formula.
- Data collection methods and laboratory procedures
A standardized questionnaire was used to collect demographic, epidemiological and clinical data for all study participants.
- Sample collection and transport
A stool sample was collected from each study participant using a labelled sterile stool container and transported in the Bacteriology Laboratory immediately after collection using a refrigerated bag containing ice packs which maintained the temperature between 4-8°C.
Eosine Methylene Blue medium (BIOCHEM Chemopharma, Cosne-cours sur loire, France) supplemented with cefotaxime solution (Bio-Rad Marnes-la-Coquette, France) was used for Enterobacteriaceae selection. Strain identification was performed using Api 20E system (Biomérieux Marcy-l’Etoile France) after an oxidase test (Bio-Rad Marnes-la-Coquette, France). Each identification plate was inoculated with a bacteria suspension prepared with an opacity of 0,5 on the Mc Farland scale.
The presence of Extended Spectrum Beta-Lactamase (ESBLs) in isolates was confirmed using the double-disk synergy test (DDST), which was performed by placing the disk of cefotaxime (30 μg), (BIOCHEM Chemopharma, Cosne-cours sur loire, France) ceftazidime (30 μg) (Bio-Rad Marnes-la-Coquette, France), and combination of amoxicillin/clavulanic acid (20 μg/10 μg) ( Bio-Rad Marnes-la-Coquette, France ) on a lawn culture of bacteria on Muller-Hinton agar (BIOCHEM Chemopharma, Cosne-cours sur loire, France) plate, with a 20 mm distance between each disk from center to center. The expression of Extended Spectrum Beta-Lactamase, a reduced sensitivity (Inhibition diameter <25mm) to the Ertapenem antibiotic disc were the selection criteria for the detection of carbapenemases.
- Detection of carbapenemase production
The detection was done using the Modified Hodge Test (MHT)[10] in which when the test isolate produces the enzyme and allows the growth of the carbapenem-sensitive strain (Escherichia coli ATCC 25922) around a carbapenem disc, the appearance of a characteristic notched clover leaf is indication of the presence of a carbapenemase enzyme.
- Classification of carbapenemases
Classification tests were done by inhibitory synergy tests, using Boronic acid 30mg/ml (BIOCHEM Chemopharma, Cosne-cours sur loire, France) and Chelating agent (EDTA) (BIOCHEM Chemopharma, Cosne-cours sur loire, France) tests for class A (KPC) and class B (NDM) respectively. A negative result to the tests using the two inhibitory agents was considered as a class D. The phenotype interpretation was done according to the Antibiogram Committee of the French Society of Microbiology (AC-FSM) guidelines 2020[13].
The data on socio-demographical and bacteriological characteristics, and potential risk factors were recorded on the software Excel 2013 and analysed using the software statview version 4.0. The results were expressed using the mean and associated to their confidence interval (CI) at 95%. The research of potential risk factors associated to the carriage of carbapenemases producing Enterobacteriaceae in mothers and new-borns, and a potential vertical transmission, was done using the logistic regression method. P value <0.05 was considered as significant.
The ethical approval was obtained from the Institutional Ethical Review Board - University des Montagnes and cities local authority (AUTORISATION N°2020/164/UDM/PR/DE). Permissions to conduct the study were granted by the Director of the the Yaounde Gynaeco-Obstetrics and Paediatrics hospital and at the Social Animation and Sanitary centre of Nkoldongo. All study methods were performed in accordance with the relevant guidelines and regulations.