Application of Polymerase Chain Reaction in Diphtheria Laboratory Examination: A Field Need

Abstract

Background: Indonesia is one of the five countries with the highest number of diphtheria cases worldwide. Diphtheria is caused by the toxigenic strains Corynebacterium diphtheriae, C. ulcerans, and C. pseudotuberculosis. The diphtheria-causing bacteria can be identified using conventional and molecular methods, including polymerase chain reaction (PCR) assay. We used the PCR assay as additional testing, because in island countries like Indonesia, specimen transport is a serious challenge to maintaining bacterial survival. Objectives: This study aimed to evaluate the PCR assay as additional testing to identify diphtheria-causing bacteria in the diphtheria laboratory. Methods: In this cross-sectional study, a total of 178 pairs of the throat and nasal swabs from diphtheria suspected cases and close contacts were collected from seven provinces in Indonesia in 2016. All samples were directly identified by the conventional method and multiplex PCR assay. Statistical analysis was conducted using the 2 × 2 tables to determine the sensitivity and specificity of both methods, while the χ2 test was used to examine the correlation between specimen examination delay and the differentiation of results. A P-value < 0.05 was considered as statistically significant. Results: Out of 178 examined samples, eight samples were identified as diphtheria-positive by both the conventional method and PCR assay, while nine samples were only detected by the PCR assay. All diphtheria-causing bacteria found in the positive samples were toxigenic C. diphtheriae. The diphtheria-causing bacteria were found in 27.6% of cases and 6.0% of close contacts using the PCR assay versus 13.8% of cases and 2.7% of close contacts using the conventional method. Statistical analysis showed that the PCR assay is about twice more sensitive than the conventional method. There was a significant correlation between the differentiation of results and > 72 hours’ specimen examination delay with a P-value of 0.04 (< 0.05). Conclusions: The PCR assay is more sensitive than the conventional method to identify diphtheria-causing bacteria and may be applied as additional testing to increase the positivity rate of diphtheria-confirmed cases in Indonesia as an archipelago country where geographical factors and specimen transport are real obstacles.

Development and Application of Dtxr and Tox Genes Targeting Real-time PCR to Identify Corynebacterium diphtheriae, C. ulcerans, and C. pseudotuberculosis Simultaneously

“…Unlike our previous study (13), we did not identify the NTTB strains to reduce the cost in this study because of the high cost of the PCR probe. Furthermore, previous studies documented that NTTB strains were never observed in diphtheria samples in Indonesia (4,10,13). The NTTB strain originated in Indonesia was isolated from the non-diphtheria sample (21).…”

“…Although the diagnosis and treatment of diphtheria are based on clinical manifestation, some cases are mild and atypical. Laboratory tests are needed to rule out a differential diagnosis (9,10). The fast and accurate laboratory tests help prevent the spread of diphtheria-causing bacteria and provide case management as early as possible.…”

“…This is a major problem for countries such as Indonesia. In this case, PCR can be an alternative, which is fast and relatively less affected by sample delivery problems and history of antibiotic use (10). Diphtheria laboratory examination using the PCR method has long been introduced as a screening method to examine toxigenicity.…”