We conducted a prospective observational study at a leading tertiary care hospital in Turkey. The cohort included 100 infants aged 0-3 months diagnosed with IBIs alongside 100 control infants presenting for non-infectious conditions. Ethical approval was secured from the institutional review board in compliance with the Declaration of Helsinki. Informed consent was obtained in written form from the parents or legal guardians of all participants prior to enrollment.
Invasive bacterial infections categorized in this study included meningitis, pneumonia, sepsis, bacteremia, urinary tract infections, and invasive bacterial gastroenteritis. Diagnostic criteria were stringent: meningitis was confirmed via signs of bacterial infection in cerebrospinal fluid; pneumonia through auscultatory findings and radiographic evidence of pulmonary infiltrates; bacteremia by positive blood cultures; urinary tract infections by significant bacterial cultures from sterile catheterization; and gastroenteritis by the identification of pathogenic organisms in stool cultures.
DNA was extracted from whole blood samples using the QIAamp DNA Mini Kit (Qiagen), according to the manufacturer's instructions. The purity and concentration of the DNA were quantified using a NanoDrop ND-1000 Spectrophotometer. Only DNA samples that met the quality and concentration criteria proceeded to sequencing. The sequence analysis targeted specific regions pertinent to the polymorphisms identified in the study's objectives.
DNA samples were processed using a sequence of analytical techniques to identify the polymorphisms TLR4 rs2149356 (c.261-468T\>G), LTA rs2229094 (c.37T\>C), and RFP175 rs1585110 (c.246+8853G\>A), as detailed in our previous work \[18\]:
Primers targeting the regions encompassing TLR4 rs2149356 (c.261-468T\>G), LTA rs2229094 (c.37T\>C), and RFP175 rs1585110 (c.246+8853G\>A) were designed using the Primer3 web tool. Criteria for primer design included a length of 18-24 base pairs, GC content between 30-50%, specificity to the target DNA region, and a melting temperature (Tm) ranging from 58-62 °C to ensure optimal amplification of a 300-500 base pair region.
Polymerase Chain Reaction (PCR) was employed for the amplification of the target regions. Each reaction mixture, prepared in a 25 µl volume, contained 0.4 µM of each primer, 2 ng of genomic DNA, 4 mM MgCl2, 0.05 µl Taq DNA polymerase, and 0.4 mM of each dNTP. Twelve such mixtures were assembled in PCR tubes. Amplification was performed under optimized conditions in a thermal cycler.
Post-amplification, the PCR products were subjected to agarose gel electrophoresis for verification. A 2% agarose gel was prepared by dissolving agarose in TBE buffer, followed by addition of ethidium bromide post-cooling. For electrophoresis, PCR products were mixed with loading dye and introduced into the gel alongside a DNA ladder. The electrophoresis was run at 120 volts, after which the gel was visualized under UV light using a gel documentation system.
After verification of the target amplicons by agarose gel electrophoresis, the PCR products were prepared for sequencing. The sequencing was performed using the Illumina MiSeq platform. This high-throughput sequencing process generated FastQ files, which were then assembled into BAM files for detailed examination. Sequence alignment and variant identification were conducted using the Integrative Genomics Viewer. Allelic variants within the sequences of the TLR4 rs2149356, LTA rs2229094, and RFP175 rs1585110 genes were meticulously identified and annotated for each subject in the study cohort.
Allelic and genotypic frequencies of the polymorphisms TLR4 rs2149356, LTA rs2229094, and RFP175 rs1585110 were assessed in patients with invasive bacterial infections compared to control subjects. Genotypic frequencies in the dominant model were evaluated by comparing the combined homozygous and heterozygous variants against the homozygous wild-type. Conversely, the recessive model analysis involved comparing homozygous variants to the combined heterozygous and homozygous wild-type genotypes.
Statistical computations were performed using IBM SPSS Statistics version 25. Categorical variables were analyzed using the chi-square test, and the association risks were quantified as odds ratios with 95% confidence intervals. A p-value of less than 0.05 was set as the threshold for statistical significance.
