2.1. Study Design
166 samples of blood and stool were acquired from Al-Najaf Health Directorate, Public Health Department who were clinically suspicious patients. This study was performed between October 2020 to April 2021.
2.2. Sample Collection
The blood and stool samples were collected from those patients sent to the laboratory in the morning session (9 AM to 12 PM) of patient examination. Venous blood sample (2–3 ml) was collected aseptically using 70% alcohol into a sterile test tube and centrifuged at 3000 revolutions per minute for 5 min to separate the serum. Fresh stool specimen provided by the febrile patient was put into a screw-capped container, labeled, and transported to Microbiology Laboratory of Al-Furat Al-Awsat Technical University, Kufa Technical Institute, Community Health Department for culture.
2.3. Widal agglutination test
Widal antigens (O) and flagellar antigens (H) were used to generate the Widal test for Salmonella typhi (U.K). Antibody titers in serum were detected using the Widal test. Through blood was collected, each patient had approximately 3 ml of blood collected and placed into sterile test containers. Centrifuge tubes at 3000 rpm for 5 minutes, A micropipette was used to collect the serum from each sample into a clean container. A sterile micropipette was used to transfer two drops of each serum sample to the microscope slide. The reagents for detecting Salmonella antigens (O) and (H) were also placed on the microscope slide. Applicator sticks were used to combine the two ingredients, and the tile was gently spun for one minute to ensure visible agglutination. Positive (+++)=1/320 antigens were detected between 0 and 15 seconds, (++)=1/160 antigens were detected between 15 and 45 seconds, and (+)=1/80 antigens were detected between 1 minute. while antigen not reacts were classed as negative (-). (more than one minute). Positive titers were defined as those greater than or equal to 1:80, whereas negative titers were defined as those less than 1:80 .
2.4. Samples Culture
2.4.1. Blood culture: Five milliliters of blood were aseptically placed into a sterile bottle containing fifty milliliters of sterilized Brain heart infusion (BHI) broth and incubated at 37oC for five minutes. The turbidity and color change of the blood culture were indicative of microbial growth constantly. Before reporting a negative result, it is suggested that the culture for at least 7 days. After this, the bottle was thrown 14 days later (7). Subcultures were taken out as follows: a loopful of positive blood was transferred to MacConkey, Salmonella-Shigella agar (SS agar), and incubated at 37ºC for 24 hours. Gram staining and light microscopy were used to examined the isolates . Media were prepared according to the instructions of the manufacturers.
2.4.2. Stool culture: By the second week of infection, stool cultures are generally positive for typhoid fever. Most often, the stool is inoculated into fluid enrichment medium such as Tetrathionate or Selenite broth and then plated on Desoxy Cholate-Citrate Agar. It is possible to test for salmonella O antigens directly on culture plates using slide agglutination and sub-cultured to peptone water to determine the structure of the H antigen and do additional biochemical analysis .
2.5. Isolation and Identification of Salmonella typhi
Aseptically collected blood and stool samples in sterile BHIB containers. Subcultures on solidified sterilized XLD agar and MacConkey agar were created to assess growth after overnight incubation at 37°C. Colonies of Salmonella spp are often red with black center on XLD agar, but off white on MacConkey agar. In accordance with "Bergey's Manual of Determinative Bacteriology, 8th ed." taxonomic guidelines, the selected colonies were then identified as Salmonella spp. by microscopic and standard biochemical reactions such as TSI (Triple Sugar Iron), Urease test, MR-VP test, Oxidase test, Citrate utilization test, gas production .
2.6. Api20E system
Enterobacteriaceae and other gram-negative bacilli can be identified using this method. It is composed of twenty microtubes that hold a dehydrated medium (each microtube consists of a tube and cupul section). The Api20E system was installed and operated in accordance with the manufacturer's instructions.
2.7. Diagnosis by GN-ID with VITEK-2 Compact System
The test was perfumed in accordance with the manufacturer's specifications. This kit has recently been utilized for the fast detection of G+ve and G-ve bacteria.
2.8. DA extraction
The Genetic Material (DNA) of the Salmonella typhi strain investigated was purified using a kit provided by the Geneaid company in the United Kingdom.
2.9. PCR for the screening Virulence Genes of Salmonella typhi
PCR was used to examine for virulence genes in the Salmonella typhi isolates used in this investigation. There are two virulence genes that may be detected using primers, amplification products, and references in Table 1.
The detection procedure began with a 5-minute denaturation at 95 °C, followed by 30 cycles of denaturation at 95 °C for 45 s, annealing at 55 °C for 45 s, extension at 72 °C for 1 minute, and a final extension at 72 °C for 10 minutes. The invA gene fragment was amplified at a 63°C annealing temperature and extension for 1 minute. DNA ladders of 100 bp were used as molecular weight markers in the gel documentation system for electrophoresis 1.5 % agarose gels and ethidium bromide staining.
2.10. Salmonella typhi Susceptibility towards Antibiotic Tested
Salmonella typhi demonstration Susceptibility to various antibiotics was using disk diffusion test [13, 14]. To demonstrate the ability to cultivate pure colonies from previously identified bacteria, we added an isolated colony in culture to saline with the same density as (0.5) McFarland turbidity, which is essentially the same as the density of bacterial broth at 1.5x108 cells/ml. A sterile swab was used to inoculate the cells, which were then plated on Mueller - Hinton agar (MHA). Using burning forceps, antibiotic disks were put regularly over the medium surface and incubated for 18 hours at 37°C. The antibiotic inhibition zone was determined with the use of a specialized ruler. The diameter of the zone in comparison to the accepted clinical laboratory standards institute's standard results (CLSI).
2.11. Statistics Analysis
Data were analyzed using the SPSS (Statistical Package for Social Science) version 23 software, and significance was assessed at P ≤ 0.05. using the Chi-squared test.