Genetic characterization of diphtheria tox B to evaluate vaccine efficacy in Indonesia

Abstract

Background and Objectives: Blocking the attachment of diphtheria toxins to host cells through the intact receptor binding site (tox B) was the initial mechanism of action of the diphtheria vaccine. Diphtheria outbreaks in populations with good vaccination coverage can be caused by mutations or changes in the genetic structure of the tox B protein. The aim of this study was to characterize the Tox B protein produced by Corynebacterium diphtheriae isolated from 2018 to 2019 in patients in Jakarta who had already received the diphtheria vaccine.
 Materials and Methods: Of the 89 throat swab specimens of patients with a clinical diagnosis of diphtheria, 10 were posi- tive for diphtheria and toxin. PCR was used to amplify the tox B DNA fragment in the 10 positive isolates. DNA sequencing was conducted with overlapping primers and the DNA sequences were analysed by using SeqScape V2.7.
 Results: Of the 10 isolates, nine isolate showed a DNA mutation (G30A), but the mutation did not change the amino acid encoding arginin (silent mutation). Our findings indicate that the efficacy of the diphtheria vaccine used in Indonesia has not decreased because of mutations in the tox B genes not change the amino acid.
 Conclusion: Overall, there are no amino acid changes in the tox B protein, indicating that the outbreaks are not affected by mutation in tox B. Another possible mechanism – overexpression of the toxin – is likely responsible for causing diphtheria in patients who have a complete history of immunization in Indonesia.

Challenges of Diphtheria Toxin Detection

“…Much more recent research performed in Indonesia confirms these observations [99]. Among 89 throat swabs from patients with a clinical diagnosis of diphtheria, 10 toxinproducing C. diphtheriae isolates were detected, for which the toxB sequence was analysed.…”

“…The right side of the prophage genomes is characterised by the greatest nucleotide sequence similarity. This region harbours the tox gene and is specified by a decreased GC content [ 98 ]; hence, there is practically no variability observed within the DT gene [ 99 ]. Even though tox is extrinsic, it is regulated by a chromosomally encoded protein DtxR (DT repressor), which is a transcriptional regulator that uses Fe 2+ as a corepressor [ 100 ].…”

“…Nine isolates showed an SNP mutation (G30A), but the mutation did not change the amino acid sequence of the toxin. In consequence, the authors point to other factors that could have reduced the efficacy of the diphtheria vaccine used in Indonesia, for example, mutations in the toxin promoter/operator region ( tox PO) [ 99 ]. The results of the research by Kolodkina et al showed that the increase in the level of DT production is determined by the mutation located in the 9-bp palindrome, which overlaps the –10 sequence of the promoter and the operator region [ 104 ].…”

First report of amphotericin B resistant Candida haemulonii isolated from the ICU of a referral hospital in Indonesia

“…DNA sequencing was performed using overlapping primers. Overlapping editing using SeqScape v.7.0 (Applied Biosystem) and BioEdit was used to analyzed the DNA sequencing result ( 20 ). The primers used for DNA sequencing are the same as those used for PCR amplification.…”

“…The analysis was performed on the six sample sequences and one resistant reference sequence (accession number NW_020289902.1 for the ERG2 gene and MZ711435.1 for the ERG11 gene) using BioEdit to determine any amino acid changes in these sequences ( 20 ).…”