Abstract
BackgroundDiphtheria outbreaks occurred in endemic areas and imported and indigenous cases are reported in UE/EEA. Because of the high infectiveness and severity of the disease, early and accurate diagnosis of each suspected case is essential for the treatment and management of the case and close contacts.The aim of the study was to establish simple and rapid testing methods based on Loop-Mediated Isothermal Amplification (LAMP) assay for the detection of Corynebacterium diphtheriae and differentiation between toxigenic and non-toxigenic strains.MethodsCorynebacterium diphtheriae and Corynebacterium ulcerans isolates from the National Institute of Public Health-National Institute of Hygiene collection were used for the development of LAMP assay for the diagnosis of diphtheria and nontoxigenic C. diphtheriae infections. Various colorimetric methods for visualization of results were investigated. Sensitivity and specificity of the assay were examined using a collection of DNA samples from various gram-positive and gram-negative bacteria.ResultsThe LAMP assay for tox and dtxR genes was developed. The sensitivity and specificity of the assay were calculated as 100%. The detection limit was estimated as 1.42 pg/μl concentration of DNA template when the reaction was conducted for 60 min. However, the detection limit was lowered 10 times for every 10 min of reduction in the time of incubation during the reaction. Positive results were successfully detected colorimetrically using hydroxynaphthol blue, calcein, QuantiFluor, and lateral flow Milenia HybriDetect dipsticks.ConclusionThe assay developed in the study might be applied for point-of-care testing of diphtheria and other C. diphtheriae infections as well as for other infections caused by diphtheria-toxin producing Corynebacterium species. It is highly sensitive, specific, inexpensive, easy to use, and suitable for low-resource settings.
Highlights
Diphtheria outbreaks occurred in endemic areas and imported and indigenous cases are reported in UE/EEA
The species-specific dtxR gene present in all C. diphtheriae strains and the tox gene present only in potentially toxigenic C. diphtheriae, C. ulcerans, and C. pseudotuberculosis strains were selected as target genes for designing the Loop-Mediated Isothermal Amplification (LAMP) primers
Diphtheria outbreaks occur in endemic countries, and diphtheria cases are reported every year in European Union/European Economic Area (EU/EEA)
Summary
Diphtheria outbreaks occurred in endemic areas and imported and indigenous cases are reported in UE/EEA. Because of the high infectiveness and severity of the disease, early and accurate diagnosis of each suspected case is essential for the treatment and management of the case and close contacts. Diphtheria is an acute, highly infectious and potentially lethal infectious disease of humans. The disease is caused by diphtheria toxin-producing strains of Corynebacterium diphtheriae, Corynebacterium ulcerans, and Corynebacterium pseudotuberculosis. The disease could be presented as respiratory or cutaneous diphtheria, depending on the anatomic site that is affected by the toxigenic corynebacteria. Diphtheria toxin absorbed from the mucosal or cutaneous lesions causes toxic damage to the nervous system, myocardium and kidneys. In respiratory diphtheria cases, formed pseudomembranes can cause obstruction in the airways [1]
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