{'fraction_non_alphanumeric': 0.023747079299985693, 'fraction_numerical': 0.02584521482046636, 'mean_word_length': 4.6437029063509145, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Corynebacterium Diphtheriae: A Case Report of Some Isolates in National Ear Care Centre Kaduna

This is a case study of the incidence of diphtheria in National Ear Care Centre Kaduna. Samples included all patients presented with diphtheria symptoms referred to the hospital from October to December; 2018. A total of 8 patients were referred and several samples taken for laboratory investigations. 8 throat swabs were collected for organism isolation by inoculation on some certain agars, incubation, secondary gram and microscopy; colonies appeared to be biconcave, large and whitish. Microscopy revealed characteristic Chinese lettered, pleomorphic bacillus which are features of Corynebacterium diphtheriae. Blood samples were used for urea and creatinine estimation which yielded abnormal high values in all except the creatinine of one of the samples indicating kidney impairment which could be attributed to presence of the tox gene. To further confirm the incidence of the disease; one throat swab was saved at 4C for molecular confirmation and characterisation of the gene. It was then transported to Molecular laboratory and the DNA Case Study Rimi et al.; AJRID, 2(4): 1-15, 2019; Article no.AJRID.52699 2 extracted using the phenol/chloroform extraction method. Both the A and B subunits of the tox gene were amplified from the DNA extract at 248bp and 297bp respectively. Sanger’s Dye Terminator Cycle Sequencing (DTCS) of both the subunits of the tox gene was carried out and the sequences were obtained and converted to the fasta format using a Finch TV for bioinformatics analyses using the NCBI blast tool. The A and B subunits showed a percentage identity of 99.59% and 95.57% to Corynebacterium diphtheriae mitis strain NCTC 3529 genome assembly, chromosome: 1 respectively. The A subunit also showed 99.59% identity to Corynebacterium diphtheriae strain BQ11 chromosome; complete genome while the B subunit also showed 95.57% identity to Corynebacterium diphtheriae mitis strain NCTC 7838 genome assembly chromosome: 1. A mutation was also discovered in the tox genes as indicated by the E-value of 3e-84 and 2e-121 for A and B subunits respectively which are values that are less than 1. This is also confirmed by 5 gaps noticed from the blast results (4 gaps from A and 1 from B) indicating the presence of indels. In conclusion; there are confirmed incidences and sporadic cases of diphtheria in Kaduna State and the organism is said to have mutated.


INTRODUCTION
Diphtheria is a potentially life-threatening upper respiratory infection caused by Corynebacterium diphtheriae which has been largely eradicated in developed nations since the introduction of childhood vaccination programs, but is still reported in the Third World and increasingly in some areas in Eastern Europe. Antibiotics are effective in the early stages, but recovery is generally slow [1].
Corynebacterium diphtheriae; this is the pathogenic bacterium that causes diphtheria. It is also known as the Klebs-Löffler bacillus, because it was discovered in 1884 by German bacteriologists Edwin Klebs (1834Klebs ( -1912 and Friedrich Löffler (1852-1915) [2]. The genome of C. diphtheriae consists of a single circular chromosome of 2,5 Mbp, with no plasmids [3,4]. The genome shows an extreme compositional bias, being noticeably higher in G+C near the origin than at the terminus. The bacterium is sensitive to the majority of antibiotics [5].
DT gene: Diphtheria toxin (DT) is an extracellular protein of Corynebacterium diphtheriae that inhibits protein synthesis and kills susceptible cells [6]. The gene that encodes DT (tox) is present in some corynephages, and DT is only produced by C. diphtheriae isolates that harbor tox + phages. The diphtheria toxin repressor (DtxR) is a global regulatory protein that uses Fe 2+ as co-repressor. Holo-DtxR represses production of DT, corynebacterial siderophore, heme oxygenase, and several other proteins. Diagnostic tests for toxinogenicity of C.
diphtheriae are based either on immunoassays or on bioassays for DT [7]. Molecular analysis of tox and dtxR genes in recent clinical isolates of C. diphtheriae revealed several tox alleles that encode identical DT proteins and multiple dtxR alleles that encode five variants of DtxR protein. Therefore, recent clinical isolates of C. diphtheriae produce a single antigenic type of DT, and diphtheria toxoid continues to be an effective vaccine for immunization against diphtheria [2].

Purpose of Study
The purpose of the study is to report and prove that there are sporadic cases of diphtheria in Kaduna State and to characterise the DTx gene using molecular techniques, bioinformatics and homology searches.

Sample Population
The population included all patients presented with diphtheria symptoms in the hospital from October to December; 2018.

Sample Collection and Transportation
One clinical throat swab was saved after collection from patients with suspected diphtheria in the Centre and was transported to DNA laboratory and Molecular Research Institute in Sterile water at 4ºC.

Bacterial Strains and Processing of Clinical Samples
Control: water was used as the negative control during PCR and the molecular mark of 248bp and 297 bp band appearance served as the positive control Specimen Collection and Processing: A total of 8 samples for throat swabs were collected from patients with suspected diphtheria from October to December 2018; cotton wool swab sticks were used for the collection from the throat and were then used for isolation of organism in microbiology unit by primary gram, inoculation on blood agar, chocolate agar and McConkey agar and incubation at 37ºC for 24 hours; secondary gram and microscopy. Blood samples were collected for urea and creatinine estimation from harvested serum in Chemical Pathology unit through the following protocol: Filled tubes were then incubated for 15minutes and absorbance measured at 600wavelength after blanking with reagent blank

Calculation:
Absorbance of test/Absorbance of standard *concentration of standard (13.5) Absorbance was taking for each after exactly 2 minutes using a stop watch at 340 wavelengths.

Calculation:
Absorbance of test/ Absorbance of Standard *Concentration of standard (100).
*Check appendix III for reagent composition.
Some of the patients underwent surgery to remove a membrane on the pharynx from the theatre that was taken to ABUTH; Shika for histological analyses using the following protocol:  Samples were fixed in formalin and transported to ABUTH; Shika for histological analyses.  They were then grossed using ruler, scalpel blade, pencil for labelling and inserted into cassettes for automatic tissue processing.  They were then inserted and arranged in an automatic tissue processor and passed through the following reagents to confer rigidity for microtomy: 70% alcohol, 90% alcohol, Absolute alcohol (2 changes) and xylene (3 changes) for 2 hours each.  They were then impregnated with liquid paraffin wax and embedded in molten paraffin wax in the embedding chamber.  They were sectioned and stained using the H and E staining technique.  They were mounted with DPX and presented to the consultant pathologist for diagnoses.
Finally; one clinical throat swab was packed in sterile water package for transport to DNA lab Kaduna for further investigations according to following explained methodology.

Isolation and Purification of DNA from the Swabs
This was achieved using the phenol/ chloroform extraction method described by Kirby K. S as follows:  Vials or Eppendorf tubes were first labelled to avoid mix up.  Exactly 200 ul of sterile water sample (containing cells from the swab) was dispensed in a 1.5 ml tube.  Exactly 400 ul of lysis buffer and 10 ul of protenase K was added.  Tubes were then vortexed and placed on heat block at 65ºC for 1 hour for incubation  A total 400 ul of phenol/ chloroform of ratio 1:1 was added and vortexed; it was then spun at 14000 rpm for 10 minutes.  Supernatant was then pippetted out and introduced to new 1.5 ml tubes.  Exactly 400 ul of chloroform was added to the recovered layer and vortexed; it was then spun for 5 minutes and supernatant carefully removed and introduced to new tube.  A total 1000 ul of absolute ethanol was added and 40 ul of 3 M sodium acetate was added and stored at -20ºC in freezer for overnight incubation.  It was then centrifuged in cold centrifuged at 14,000 rpm for 10 minutes and supernatant decanted.  Exactly 400 ul of 70% ethanol was added to the debris and re-centrifuged for 5 minutes in the cool/freezing centrifuge at 4ºC.  Supernatant was then decanted for sole purpose of drying out the ethanol; it was re-centrifuged at 14,000 rpm and removed using a micro-pippette.  The extracted DNA was then dried out at room temperature for 20 minutes and stored at -20ºC for PCR.

Primer Reconstitution Stage
Primers were designed as stated in Table 2 but procured by Bioneer Company.
The primers were reconstituted by first of all spinning for 1minute in a safety cabinet and the following procedure followed: Subunit A-109.2 ul of distilled water was added to tox 1 and 106.7 ul of distilled water was added to tox 2 using micropipette and both were then vortexed.
Subunit B-150.2 ul of distilled water was added to Diphth 6 F and 149 ul of distilled water was added to Diphth6R using micropipette and were then vortexed.
Note: the amount of distilled water added was indicated on the primer containers.
90 ul of water was added to 4 Eppendorf tubes to which 5 ul of each tox 1, tox 2, diphth6F and diphth6R was added and vortexed.
Thus the primers were ready for use in the PCR.

Amplification of the Tox gene (DT gene)
The polymerase chain reaction (PCR) amplification of the DT gene was conducted according to the method of Nakao and Popovic [9,10].

First PCR Reaction
This was performed in a programmable DNA Thermal Cycler PTC-100 with a total of 2 ul of DNA solution and the following master mix of Accupower hot start PCR premix by Bioneer Company:  1 ul of reconstituted tox1 and tox2 primers  1.5 mM MgCl 2 ,  250 ul of deoxynucleoside triphosphate mixture (1 mM each of total dNTP), and  1 U of Taq DNA polymerase  1X PCR buffer  Sterile water was then added to make up to 20 ul.  A negative control was set alongside the samples; just that in the case of negative control, a DNA template was not added, primers and distilled water were added to make up a volume of 20 ul and the lyophilized blue pellet was dissolved by vortexing for 1 minute.  The thermal cycler was set for pre denaturation at 94ºC for 5 minutes, denaturation at 94ºC for 30 seconds, annealing at 45ºC for 30 seconds, extension at 72ºC for 5 minutes and final extension at 72ºC for 5 minutes and it went on for 35 cycles.

Second PCR reaction
 The above procedure in the first PCR was applied to the sample DNA but the only difference was that the 2 nd set of primers Diphth6F and Diphth6R for amplification of the beta subunit of the tox gene were used and annealing temperature of 48ºC.

Visualisation of Amplicon
 A total of 1.5 g of QDLE Agarose was dissolved in 100 ml of distilled water and heated at 30 seconds interval using the highest heat in the microwave until it was completely dissolved and 8 ul of ethidium bromide was added.  It was then cooled down to a temperature of 65ºC.  2 combs were then inserted properly into a cast and the cooled dissolved agarose poured while still molten and allowed to solidify to create wells where the PCR products will be carefully pippetted.  The cast was then inserted into the electrophoretic tank containing buffer after removal of the combs.  Exactly 0.8 ul of the PCR products of each sample was mixed with bromophenol blue stain included in the master mix and carefully pipetted into the wells and made to run for 45 minutes for the amplicons to migrate alongside a ladder (DNA marker).  The amplicon was then finally optimized by repeating some reaction conditions like the annealing temperature to 60ºC from 45ºC to obtain a clearer band ready for sequencing.

Dye Terminator Cycle Sequencing With Quick Start Kit
All reagents were kept on ice during the sequencing reactions; the reaction was carried out in a 2.0 ml tube and reagents added according to the following order:  of sample loading solution. 5. 10 ul of SLS was added to any blank wells in arrow. 6. 250 ul of separation buffer to buffer plate was added. 7. The CEQ machine was then started. 8. "CEQ System" was then clicked on desktop. 9. Direct Control was then clicked on run and then plate accessed: the sample and buffer plates were then put and wetting tray filled. 10. Gel Cattridge was released and new on placed on replenish. 11. Samples were then named and blank written in blank wells. 12. Running method was set for LFR-C-500bp.
(Seq.test-General). 13. Wells were then highlighted with the samples and analysis method clicked and file saved. 14. Run was pressed and reviewed. 15. Finally "OK" was clicked to begin the sequencing.

Microbiology Unit Results
Colonies appeared after the incubation which were large; biconcave and whitish on all agars. Microscopy revealed pleomorphic bacilli resembling a characteristic Chinese letterappearance thus Corynebacterium diphtheriae isolated for all samples of the 8 patients presented with pharyngitis.

Chemical Pathology Unit Results
The estimated values of urea and creatinine were very high of all patients except one creatinine value that fell within normal range. The urea values include 20.

Histopathology Results
The histopathology report of Oropharyngeal masses showed a grey, brown flat tissue measuring 3x1x0.5cm and solid in nature for the macroscopy while microscopy showed tissue fragments composed of necrotic debris admixed with autolysed tissue exhibiting haemorrhage from ABUTH; Shika.

PCR Results
The PCR results presented in Plate 1 Shows 2 positive PCR results of the DTx.

Plate 1. M-1kbp molecular weight marker, lane 1-positive PCR amplicon at 297bp for the B subunit of the DTgene, lanes 2 and 4negative controls, lane 3-positive PCR amplicon at 248bp for A subunit of DTx gene
*the PCR result is that of the 52year old patient

Sequencing Results
The sequence was generally good at about base 18 and was represented by tall distinct peaks that had little overlap; the sequence was of good and high quality accompanied by little stretches of AAAAs as illustrated in the image in appendix I. The sequence belonged to only one product and as such qualified to proceed for bioinformatics analyses (Appendix I).

Sequence for A Subunit of DTx gene
After the sequencing reaction; viewing and editing; the following sequence was obtained for the A-subunit in the fasta format:

Sequence for the B Subunit of the DTx Gene
The string of letters below represents sequence for the B-subunit in the fasta format:

Blast results of A subunit
The A subunit of the gene isolated is a nucleotide with Query ID lcl/Query_61465 and RID 85E16CKE014. It had a distribution of 99 blast hits on 95 subject sequences from the query sequence and it showed 95.57% identity to both Corynebacterium diphtheriae strain NCTC 3529 genome assembly chromosome: 1 (accession number; LR134538.1) and Corynebacterium diphtheriae strain NCTC 7838 genome assembly (accession number; LR134537.1). It had a maximum and total score of 322 after covering 95% of the query sequence. The E-value is 3e-84. There were 4 gaps out of the 174bp in the alignment to both strains signifying "indels" which are a type of mutation. The result is summerised in Table 3.

Blast results for the B subunit
The blast result for the B subunit is presented in Table 3. The B subunit is more or less similar to the A subunit in its homology searches. It has a query ID of lcl/Query_162819 and RID 85DC45V4014 with a distribution of 77 blast hits on the subject sequences from the query sequence. It also showed 99.59% identity to both Corynebacterium diphtheriae strain NCTC 3529 genome assembly chromosome: 1 (accession number; LR134538.1) and Corynebacterium diphtheriae strain BQ11 chromosome complete genome (accession number CP029644.1). It has a total and maximum score of 446 and E-value of 2e-121 out of the 241bp of the B subunit query sequence; 1 gap was noticed signifying indel which is a type of mutation.

DISCUSSION OF RESULTS
High urea and creatinine values indicate kidney imparement; the causative organism of diphtheria release a potent endotoxin that affects the liver, kidney and other organs. As a results diphtheria patients usually have their urea and creatinine values escalated as the kidneys cannot excrete them due to the action of DTx on them [11]. Out of the 8 throat swabs collected; only one was taken for molecular analysis as a representative of others as they share similar symptoms. It was positive for both the A and B subunits of diphtheria tox (DTx) gene. In the United Kingdom, 3 toxigenic strains of Corynebacterium diphtheriae were identified and PCR results showed both A and B subunits of the tox gene in the year 2015 [12]. In a similar study conducted in Atlanta, Georgia; 34 of 36 specimens were positive for one or both subunits of the tox gene using real time PCR assay [13]. In a different report; 170 strains were positive for the A subunit of the DTx gene out of 250 throat swabs obtained from clinical cases and carriers in Moscow, Russia by PCR directed at A subunit of the gene [14]. Similarly; PCR detection of tox gene was positive for the 248bp of the fragment A and the complete tox gene from a specimen obtained from a 38 year old male in a city located in western Canadian province [15,16]. In agreement with the above findings; a study was conducted in Kimba village of Borno State, a cluster of deaths in children following an illness characterized by a swollen neck was reported at Biu General Hospital in Borno State, Nigeria. The village has a population of 1553 people, about 50 km south of the city of Biu. PCR results of pharyngeal swabs were positive for the tox genes confirming the clinical syndrome of diphtheria [17].
The positive sample from the patient was also treated using antibiotics but the symptoms persisted even after administration and infusion with fluids. This is strongly correlated to the   [20]. Within the NIS; biotype var. Mitis isolates were detected during different stages of the epidermis using molecular analyses in the years 1984 through 1995 [21]. Lastly; C. diphtheriae var. Mitis was also isolated in a cutaneous infection in Indonesia [22].
A total of 5 gaps were noted which signify mutation; 4gaps in the A subunit and 1 in the B subunit. A similar study carried out in Colorado; USA agreed with this finding which presented one silent point mutation in the region of tox encoding the A domain of DT, and three silent mutations were detected in the region of tox encoding the B domain of DT, but the amino acid sequences of all the DT proteins encoded by these tox alleles were identical [23].
The A subunit is more significant than the B subunit because of its higher E value and nearness to zero. Because the E value of 1 assigned to a hit can be interpreted as meaning that in a database of the current size one might expect to see one match with a similar score simply by chance; and the E-value of the A and B subunits are 3e-84 and 2e-121 respectively which are less than 1; it can be deduced that the tox gene isolated has mutated. This is also confirmed by the total sum of 5 gaps noticed from the blast results (4 gaps from the A and 1 gap from the B subunit) indicating the presence of "indels".

CONCLUSION AND RECOMMENDA-TION
It can therefore be concluded that there are sporadic cases of diphtheria in Kaduna State and molecular techniques reveal that the DTx gene isolated is homologous to the C. diphtheriae NCTC strain 3529, C. diphtheriae strain NCTC 7838 and C. diphtheriae strain BQ11. Finally; molecular analyses and consequent homology searches show that the organism has mutated because of the E-value and presence of indels. It is highly recommended that Diphtheria antitoxin be purchased in all hospitals to complete treatment, vaccines and their boosters should be taken serious. More researches should be carried out to know the point of mutation and possible production of indigenous vaccines.

CONSENT
As per international standard, patient's written consent has been collected and preserved by the author(s).

ETHICAL APPROVAL
The request for the ethical clearance was made to the The Medical Director through. The Acting Head of Clinical Services; Research and Training; National Ear Care Centre; Kaduna. Since the research was timely, worthy and has direct relation with some of the problems in Kaduna and Nigeria as whole, it was approved.

ACKNOWLEDGEMENT
Authors appreciate the assistance of all the laboratory staff and the management of National Ear Care Centre as well as DNA labs Kaduna.