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Background: In Corynebacterium diphtheriae, the dtxR gene plays a role in regulating diphtheria toxin synthesis. The dtxR gene is often used as a marker for identifying C. diphtheriae by the polymerase chain reaction (PCR) method because it is present in all strains of this bacterium. Mutations in the dtxR gene can cause the over-synthesis of diphtheria toxin and reduce PCR assays' sensitivity. Objectives: This study aimed to describe the polymorphisms in the dtxR gene of C. diphtheriae isolated from a diphtheria outbreak in Indonesia. Methods: Forty-eight isolates of C. diphtheriae were obtained from clinical samples (throat/nasopharyngeal swabs) of diphtheria cases and close contacts. The isolates were revived on a blood agar plate (BAP), bacterial colonies were harvested, and deoxyribonucleic acid (DNA) was extracted. The DNA sequencing was carried out using a Whole-genome Sequencing (WGS) approach. The data were converted and analyzed with U-gene software. The dtxR gene analysis was performed with C. diphtheriae PW8 as references. Results: There were 59-point mutation locations in 48 isolates examined. None of these single nucleotide polymorphisms (SNPs) coded for amino acid changes. Based on the mutation pattern, seven clades/groups of the dtxR gene of 48 C. diphtheriae isolates were examined. Conclusions: At least seven types of DNA sequences and more than 50 SNPs of the dtxR gene were identified in 48 C. diphtheriae isolates from a diphtheria outbreak in Indonesia. Although all of them are silent mutations, they must be considered in the design of PCR examination in diphtheria laboratories.

Background: Corynebacterium diphtheriae, C. ulcerans, and C. pseudotuberculosis are known as diphtheria-causing bacteria. Although diphtheria therapy is administered based on the clinical manifestations, some cases are mild and atypical. The immediate and accurate identification of diphtheria-causing bacteria is of paramount importance to prevent the spread of the disease and provide case management as early as possible. Unfortunately, conventional methods as the gold standard are time-consuming. Objectives: This study aimed to develop and implement a multiplex real-time PCR with the dtxR and tox genes as the target to identify three species of diphtheria-causing bacteria and screen their toxigenicity quickly and accurately. Methods: The research sample encompassed seven reference strains, one synthetic DNA, 30 archived isolates, and 924 clinical specimens isolated from 311 diphtheria cases and 613 close contacts. The conventional methods as the gold standard and the established PCR assay were used to verify the results of multiplex real-time PCR developed in this study. Results: The multiplex real-time PCR could identify seven reference strains, one synthetic DNA, and 30 archived isolates as accurately as the conventional methods and the established PCR. Similar to established PCR, the multiplex real-time PCR identified diphtheria-causing bacteria in 120 (38.6%) out of 311 and 12 (2%) out of 613 clinical specimens from diphtheria cases and close contacts, respectively. Meanwhile, the conventional methods identified diphtheria-causing bacteria in 79 (25.4%) out of 311 and three (0.5%) out of 613 clinical specimens. Conclusions: The multiplex real-time PCR developed in this study can be used to identify three species of diphtheria-causing bacteria and screen their toxigenicity quickly and accurately. However, in this study, no diphtheria-causing bacteria other than C. diphtheriae was found in the clinical samples using the PCR or conventional methods. PCR is more sensitive than the conventional methods and can be used as an additional test in diphtheria laboratories.

Background: The World Health Organization reported Indonesia as one of the countries with the most prevalent cases of diphtheria worldwide. The microbiological aspects of diphtheria-inducing bacteria are of great significance in tracing disease transmission and case management. However, clinical aspects are critical for updating clinical features and case management in the field, which may sometimes differ from theoretical foundations. Objectives: This study aimed to identify the microbiological and clinical aspects, including molecular typing and case fatality rates, in diphtheria-confirmed cases from the capital city of Indonesia, Jakarta, and surrounding areas in 2017. Methods: The microbiological aspect of 40 diphtheria-confirmed cases were obtained by re-identify diphtheria-inducing bacteria isolated from the samples, while the clinical aspects of the cases were obtained from the medical records and epidemiological data. The chi-square test was used to examine the correlation between fatal cases and myocarditis and diphtheria antitoxin administration delay. In this study, P ≤ 0.05 was set as the significance level. Results: All 40 diphtheria confirmed cases were induced by toxigenic Corynebacterium diphtheriae with two biotypes, namely intermedius (60.0%) and mitis (40.0%). There are six sequence types of bacteria with two main sequence types, ie, ST534 (46.4%) and ST377 (35.7%). The proportions of cases that had a fever and sore throat were 72.5% and 77.5%, respectively; however, the prevalence rates of the cases with pseudomembrane and bull neck were 100% and 47%, respectively. Most cases were administered a combination of penicillin or erythromycin with other antibiotics (40%), and 22.5% of the cases only received penicillin. Myocarditis was noticed in three fatal cases, and their relationship was statistically significant (P = 0.000). All five fatal cases (12.5% of cases) received diphtheria antitoxin (DAT) lately or had not received it yet. Conclusions: Toxigenic C. diphtheriae with two biotypes (namely mitis and intermedius) and two main sequence types (ie, ST534 and ST377) was the causative agent of diphtheria-confirmed cases from Jakarta and surrounding areas in 2017. It was also concluded that those fatal cases were correlated with myocarditis complications.

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.

In diphtheria laboratory examinations, the PCR test can be applied to isolates and clinical specimens. This study aimed to develop a PCR assay to identify the species and toxigenicity of diphtheria-causing bacteria, including the prediction of some NTTB types. Seven reference isolates, four synthetic DNA samples, 36 stored isolates, and 487 clinical samples used for PCR optimization. The PCR results was confirmed by DNA sequence analysis. The results of the PCR examination of the 7 reference isolates and 36 stored isolates were similar to the results obtained using conventional methods as gold standard, both for diphtheria-causing and non-diphtheria-causing bacteria. The validation of the PCR results using DNA sequence analysis showed that there was no mispriming or misamplification. The multiplex PCR assay developed in this study could correctly identify the species and toxigenicity of diphtheria-causing bacteria, including the prediction of some NTTB types not yet covered by established PCR methods.