Correlation between chronic periodontitis and rheumatoid arthritis: A periodontist perspective

In this study, we evaluate the relationship between rheumatoid arthritis (RA) and chronic periodontitis on the basis of clinical attachment present and severity of attachment loss in both the cases. First of all Diagnosis of rheumatoid arthritis and chronic periodontitis was performed, thereafter bacterial DNA extraction from blood serum sample and subgingival dental plaque of each group through PCR and later DNA purification through Spin protocol was performed, oligonucleotide primer was used to detect t.forcythia and PCR amplification was done to detect T. Denticola for both the groups .PBDNA was detected in both SGP and serum samples of both the groups. In SGP samples, Tannerella forsythia was more frequently detected as compared to serum samples of both the groups. In result theclinical attachment Level (CAL) was observed to be higher in RA group as compared to CP group. Comparison of CAL according to severity was also observed in both the groups which suggested that RA group has mild periodontitis as compared to CP group in which moderate to severe periodontitis was seen, Detection of periodontal bacterial DNA by PCR assay PBDNA was detected in both SGP and serum samples. In SGP samples, Tannerella forsythia was more frequently detected as compared to serum samples of both the groups. So these are two common chronic inflammatory diseases with a similar host-mediated pathogenesis. Current evidence suggests that an association exists between periodontitis and RA. Well-designed multicenter longitudinal clinical trials and studies with sufficient sample sizes are needed to ascertain the relationship between these two diseases and whether periodontal treatment can reduce the severity of RA or prevent its onset.

A five-year retrospective study on the epidemiology of hand, foot and mouth disease in Sabah, Malaysia

Hand, foot, and mouth disease (HFMD) is endemic in Malaysia, with the number of cases increasing. Sabah has experienced several HFMD outbreaks, but information on the epidemiology and molecular characteristics of responsible viruses is scarce. In this study, data of 17,574 reports of HFMD cases in Sabah from 2015 to 2019 were extracted from a public health disease surveillance system and analyzed. Twenty-one swab samples from 13 children were collected from Beaufort, Sabah, during an outbreak in August 2018 for detection and serotyping of causative viruses by semi-nested reverse transcription-polymerase chain reaction (snRT-PCR) of the VP4–VP2 region and consensus degenerate hybrid oligonucleotide primer PCR of the VP1 region, respectively. Nucleotide sequencing and phylogenetic analysis were conducted by the neighbor-joining method. The average annual incidence of HFMD was 94.3 per 100,000 people, with the greatest yearly increase between 2017 and 2018. Swabs from six children were tested positive for enterovirus, of which five were positive for CVA16 and one for EV71. All CVA16 strains belonged to sub-genotype B1a, and the EV71 strain belonged to sub-genotype B5. Phylogenetic analyses indicate that enterovirus genotype shift might be responsible for the increasing trend of HFMD in Sabah, however, further study is needed.

IN SILICO OLIGONUCLEOTIDE PRIMER DESIGN FOR Campylobacter jejuni cytolethal distending toxin B GENE AMPLIFICATION

<p>Cytolethal distending toxin B (cdtB) is a genotoxinexpressed by <em>Campylobacter jejuni</em>. cdtB is a DNasethatinduces DNA double-strand breaks (DSB) in the nucleus causing cell cycle arrest at the G2/M phase and apoptosis. This study aimed to design and analyze in silico primer pairs to amplify cdtB gene of<em>C. jejuni</em>.The cdtB gene sequence with accession number AY445094.1was retrieved from GenBank NCBI and primer pairs were designed by using Primer-BLAST. Further analysis of primer quality such asself dimer, hairpin, repeats, and runwere done by NetPrimer. The results showed that forward primer pair 3 (5’-AGCAAGTGGAGTGTTAGCGT-3’) and reverse primer pair 3 (5’- TTGGAGTGGCTGTTCTTGGT-3’) met requirements as an ideal primer set to amplify cdtB gene in the term of primer length, Tm and GC% with a product length of 103 bp.In addition, based on NetPrimer analysis results, this primer pair had no self dimer, hairpin, repeats, and run. It can be concluded that a primer set to amplify cdtB gene of <em>C. jejuni</em>has been successfully designed<em>.</em>However, a wet experiment is needed to run this primer set in the laboratory setting.</p><p> </p><p>Keywords: <em>Campylobacter jejuni</em>, cdtB gene, in silico, primer.</p>

The role of infectious agent in development of tooth decay

Aim: to assess the relationship between colonization of the oral cavity with S. mutans and different genotypic characteristics and the degree of tooth decay in children.Materials and methods. 274 children aged 5 to 17 years (153 girls and 121 boys) who received a preventive dental checkup were included in the study. The dental caries experience was assessed by the DMFT index (number of decayed, missing due to caries, and filled teeth), according to WHO recommendations. The plaque was collected with sterile wooden toothpicks from the buccal gingival margin or from fissures of the first molars and placed in 1.5 mL Eppendorf tubes, and then plated on Mitis Salivarius Agar medium (HiMedia, India). 481 strains of S. mutans were selected for further study. DNA was extracted by an express method. Amplification was performed in the CFX-96 thermal cycler (Bio-Rad, USA). Serotyping was performed by multiplex PCR. PCR products were analyzed by gel electrophoresis in 1.5% agarose gel with ethidium bromide (10 mg/mL) manufactured by Helicon, Moscow, and visualized in UV light in transilluminator UVT1 by Biokom. Genotyping was performed according to the methodology (Saarela et al., 1996) with the oligonucleotide primer OPA-02 (5’-TGCCGAGCTG-3’). Strains of S. mutans were studied for the presence of the following genes: gtfB, spaP, cnm, fruA, gtfB, htrA, comE, mutA x(I), mutA (II), mutA (III), nlmAB (IV), adcA, Smu.399, Smu.583, Smu.761, Smu.940c, Smu.1449, Smu.2130.Results. S. mutans was isolated from all the examined children. Dental decay was detected in 82.4% of the children. Among the strains studied, all 4 serotypes were found: in children with a DMFT = 0 only serotypes k and f were detected; the predominant serotype in children with tooth decay was serotype c (74.7%). 19 genotypes of S. mutans were identified. In children without caries (DMFT = 0), S. mutans did not contain the genes spaP, comE, adcA, Smu.2130, Smu.1449, gtfB, htrA. With the increase in the DMFT index, the frequency of their detection increased. 9 genotypes of S. mutans had all 7 virulence factors. In 94.9% of children colonized by these “virulent” genotypes, high DMFT index scores were observed.Conclusion. The data obtained indicate that only a limited number of specific strains have a cariogenic potential. Strains of S. mutans belonging to serotypes e and c with a combination of virulence genes spaP, gtfB, comE, adcA, Smu.2130, Smu.1449, and htrA were isolated from children with tooth decay. Strains without these factors did not cause any damage to the teeth. The degree of tooth decay increases with colonization by several genotypes with the combination of virulence factors described above.

Development of a method for determining the levels of normalized expression of structural and functional genes in patients with knee joint arthropathy

Objective : to develop a method of molecular genetic analysis for determining the normalized expression levels of COL2A1, COL6A1, MMP-2, and MMP-9 genes in the biological material of patients with gonarthrosis. Material and methods . The biopsy samples of knee joint cartilage of 10 patients with gonarthrosis were used as a biological material for the study. Nucleic acids were isolated from the samples of the biological material using TRIZol Reagent. Reverse transcription was performed using the SuperScript III reverse transcriptase kit, dNTP and Ribonuclease inhibitor (Invitrogen, USA). Results . The usage of VectorNTI software has made it possible to select pairs of primers (forward and reverse) and TaqMan probes for COL2A1, COL6A1, MMP-2 , and MMP-9 genes, and to study the possibility of the use of the selected pairs of primers and probes for the purpose of identification of each of the studied genes. In-house test systems in the multiplex format have been designed for reference and target genes. Conclusion . High specificity (100 %) of the selected oligonucleotide primer sets was confirmed using the online application NCBI/Blast. The developed molecular genetic method can be applied to determine the normalized expression levels of COL2A1, COL6A1, MMP-2 , and MMP-9 genes in knee joint arthropathy

Development of real-time RT-PCR for N2 subtype avian influenza RNA-virus detection

Currently, N2 subtype avian influenza (AI) virus actively circulates in domestic and wild bird populations and is regularly detected in China, other Asian countries and Russia, particularly in combination with H9 hemagglutinin. Therefore, a method for rapid detection of the said infectious agent is urgently required. Data on oligonucleotide primer selection and reverse transcription real-time polymerase chain reaction condition optimization for N2 AI virus detection are presented in the paper. Modified primers and probe proposed by B. Hoffmann in 2006 as well as original primers and probes with the viruses available in the Laboratory working collection and selected during testing were assessed for N2 neuraminidase gene fragment amplification. Optimal concentrations of real-time RT-PCR master mix components and temperature-time mode were determined. Various combinations of primers were tested against ten N2 avian influenza virus isolates that genetically differed from each other in N gene. Nine viruses were isolated from birds in the Russian Federation regions and classified to different genetic groups. The real-time RT-PCR assay was tested for its specificity using AI virus isolates of different neuraminidase subtypes (H5N8, H3N6, H4N6, H5N1, H10N7) as well as samples containing other RNA-viruses: Newcastle disease virus, infectious bronchitis virus and infectious bursal disease virus. As a result of the testing, real-time RT-PCR conditions providing high sensitivity and specificity of the assay were selected and optimized.

Validation of a quantitative PCR assay for the detection of 2 Flavobacterium columnare genomovars

Flavobacterium columnare is the causative agent of columnaris disease in a variety of fish hosts. Using modifications to previously established protocols, a quantitative PCR (qPCR) assay was validated for the detection of 2 predominant F. columnare genomovars. The oligonucleotide primer and probe combination was designed to amplify a 203-bp region of the chondroitin AC lyase gene (GenBank AY912281) of F. columnare. There were no significant differences in amplification between genomovars. Comparable quantities of genomic DNA from 10 F. columnare strains, 5 representatives of each genomovar, produced similar results. Serial dilutions of purified PCR product demonstrated the limit of sensitivity for the assay was ~ 10 copies per reaction. The presence of gill and spleen tissue did not significantly affect the sensitivity of the assay. Comparably, bacterial DNA detected from the liver and kidney was less sensitive than pure bacterial DNA. However, detection from these tissues was within one order of magnitude of other tissues, indicating this reduction may have minimal analytic significance. This validated assay was used to approximate the minimum infectious dose for F. columnare isolate 94-081 in channel catfish and assess bacterial loads in gill and kidney tissues 48 h post-infection.

Detection of Aspergillus fumigatus by Polymerase Chain Reaction (PCR)

Aspergillosis refers to fungi infections of the respiratory tract caused by Aspergillus species, especially Aspergillus fumigatus. Infection of A. fumigatus was increased in the last few years due to either resistances to antibiotics or the influence of other factors such as other fungal infections. The present study aimed to review the impact of Aspergillus fumigatus in Aspergillosis cases, and study the role of Singleplex PCR for amplification of ITS1, ITS4 of rRNA gene in the detection of fungal isolate. In this study, One hundred sputum samples were collected from patients admitted to the specialize chest and respiratory diseases center / Baghdad who were suffering from respiratory problems. During these studied, molds were isolation and identification based on Conventional method (Direct microscopy by using 10% KOH, and fungal culture was done on Sabouraud Dextrose agar supplemented with chloramphenicol and on Czapek-Dox agar incubated at 37°C and examined for 3-7 days then macroscopic, microscopic examination of the colony by(lactophenol cotton blue stain )and molecular methods by using Polymerase chain reaction (PCR)technique for identification. The 10% KOH examination was positive for 35 cases, while laboratory culturing was positive for 53 cases. Aspergillus sp were isolated from 44(83%) patients; A. fumigatus was isolated in 23 (42. 4%) patients while A. flavus, A. niger, and A. terreus were isolated from 11 (20. 08%), (13. 2%) and 3 (5. 7%) patients respectively, also isolated Penicillium spp. at percentage 1(1. 9%). In this study. The ages of participants ranged from 10-70years with a mean age of 34years, the males were more susceptible to fungal infection, were recorded 35/53 (66. 3), compared to females were 18/53 (33. 96). The infection of fungi was more prevalent in ages 30-40recorded 26(53. 06%) followed by ages 40-50, 13(26. 5), while the lowest infection recorded in the age group 10- 20 years was 2(2. 04%). DNA isolated from twenty-three A. fumigatus isolates was used as a template, and the specific of oligonucleotide primer sequences were used in conventional PCR to detect the presence of internal transcribed spacer region ( ITS) region of the rRNA gene for Aspergillus fumigates. The results of the PCR amplification of the rRNA gene showed that this gene was present in 19 samples out 23 positive samples which isolation with a PCR product size of approximated 385 bp, while 4 samples out 23 positive samples showed negative results for the presence of this gene as indicated by the absence of the PCR products in their relevant lanes. Statistical analysis revealed that the PCR to have a sensitivity of 95. 1% in the detection of Aspergillus fumigatus in Aspergillosis cases. Polymerase chain reaction (PCR) is a rapid, specific, and sensitive method to detect Aspergillus fumigatus in aspergillosis cases of humans.

1786. An Automated Method to Assess Oligonucleotide Primer and Probe Complementarity to Genomic Targets in Infectious Disease qPCR Assays

Background Success of real-time TaqMan PCR (qPCR) in detecting pathogen targets and quantifying pathogen load is dependent upon frequent assay monitoring. This is due to i) the high degree of complementarity needed between primers / probes and genomic targets for assay accuracy and ii) natural pathogen variation and evolution. Failure to monitor and refine may result in false negativity or under quantification. Here we present a bioinformatics tool to identify potential problems resulting from newly discovered genomic mutations in primer/probe regions. Methods The tool performs an unbiased and automated search of the NCBI database, collects relevant genomic sequences based on user-defined Taxon-ID and executes a Python program to discard synthetic sequences. A profile of primer-probe sequence complementarity to targets is then generated. While the tool can be used for any microbe, here we present results for our laboratory’s cytomegalovirus (CMV) qPCR primer-probe analysis. In addition, our laboratory’s traditional approach utilizing alignment software was performed (download of all CMV sequences (~10,000) followed by iterative alignment building of these against our primers and probes). The amount of time to perform the automated and manual methods was recorded. Results The tool retrieved 8,732 sequences from NCBI and compared these to the CMV qPCR primers and probes. The tool found 2,501 alignments between the primers / probes and the downloaded genomic data (~15 minutes to finish (6 CPUs)). A total of 64% (1,624/2,501) of BLASTn alignments were exact matches between all primers / probes and viral genomic sequences. 17.5% (439/2,501) of alignments had 1 mismatch at either 5’ or 3’ terminus, and 1% (25/2,501) of alignments had two mismatches with the primers / probes. Similar results were found using a primarily manual approach (which took approx. 5 hours computing time and 20 hours of labor). Conclusion This new bioinformatics approach performed indistinguishably vs. a manual approach and did so in minutes rather than days. Both methods led to the conclusion that, by virtue of our design involving overlapping primers and probes, none of the identified mismatches are predicted to lead to false negativity or under quantification in our current CMV qPCR assay. Disclosures All authors: No reported disclosures.