Rapid Detection Of Pathogenic Bacteria In Blood Samples With 23S rRna Assays

Every year thousands of patients get diagnosed and hospitalized due to various bloodstream infections caused by different pathogenic bacteria. These microorganisms get into our bodies via air, water, food, and sometimes contamination of open wounds. If not treated on time, these bloodstream infections caused by bacteria can be fatal.

Therefore, studying and diagnosing these bacterial infections at the right time is crucial for maintaining good health. Our human body does wonders by fighting with foreign cells on its own. Sepsis, a common body’s natural response to infection, aids in identifying and fighting foreign bacterial cells that cause bloodstream infections. However, the definition of sepsis is still challenging for clinicians. Therefore, for the evaluation of the intensity of sepsis, a dependable method is needed.

The availability of standard guidance can help clinicians to manage patients and increase patient survival. Let’s look into the basis of sepsis and how we can easily detect pathogenic bacteria in our bloodstream for diagnosis.

Understanding the Need For Reliable Methods

Bacteria are the major cause of bloodstream infections. Gram-positive bacteria have been identified as the most common causes of bloodstream infection (30-50%), while 25- 30% of these infections are due to gram-negative bacteria. However, the most used method for bloodstream infections is the blood culture method through centrifugation. But this method comes with a few limitations.

Therefore, a series of sensitive and rapid molecular methods are now being introduced for the detection of a wide range of pathogenic bacteria in the bloodstream. These molecular methods have the ability to identify infective bacteria even if inactivated by antibiotics. These robust methods have the ability to reduce mortality rates, ICU stays, and duration of hospitalization due to the shortened time of detection. These methods can even identify bacteria that are usually missed in blood cultures.

A survey was conducted to better understand the detection of pathogenic bacteria. The aim of the survey was to speed up the diagnostic trend and identification of the causes of BSIs (bloodstream infections) in the blood samples using 23S rRNa PCR assays and compare it to the standard blood culture sample.

The Streamlined Procedure For Analysis

Patients

The study was performed on 265 patients in the ward of Infectious Diseases at Shahid Beheshti Hospital. The criteria for the selected candidates were fever ( > 38°C ), white blood count (12,000/mm3 or less than 4000/mm3. ), and respiratory rate ( > 20 breaths/min).

Sample Collection

Blood samples were acquired from the catheter blood of patients for blood culture prior to the introduction of antimicrobial therapy. For PCR assays, 4 ml of blood with EDTa was introduced in 250 ml centrifuge tubes and was delivered to the research laboratory within 1-2 hours. The sample was stored at 4°C until further processing. These blood samples were then immunized into anaerobic and aerobic Bactec bottles. These sterilized bottles of samples were then incubated for 7 Days at 35°C. Bottles were evaluated via gram staining and then sun-cultured. Bacterial cells isolated from the bottles were detected by standard bacteriological tests.

PCR Assays

The DNA sample extracted from the EDTA whole blood was done using DNA blood mini kit as per the manufacturer’s protocol. The optical density of the blood sample was measured via spectrometry using NanoDrop. The confirmed extracted DNA was then stored in sterilized 500 ml centrifuge tubes at -20°C until the PCR assay. For further DNA amplification and PCR assay, the reaction mixture was brought to 50 µL by applying a thermal cycler. The thermal program for PCR assays carried initial denaturation at 95°C for 5 min with a total of 35 cycles, including 30 sec at 95°C, 1 min at 58°C, 1 min at 72°C, and finally 10 min at 72°C for the final run.

Statistical Analysis

Complete statistical analysis was executed using SPSS for the 16.0 version of Windows. The statistical evaluation was carried out by Chi-square test, and P< 0.05 was considered a significant result.

Results

The results of the bacterial detection showed that, out of 265 hospitalized patients, only 27 candidates ( 10.2%) were detected with positive blood cultures. Out of the remaining, 117 (44.2%) were females, and 148 ( 55.8%) were males, with their ages ranging from 2 months to 96 years old. The following data shows the ratio of patients and their diseases.

No. Of  Patients	Acquired Diseases
47 ( 17.7%)	Diabetes Mellitus
22 ( 8.3%)	Kidney Failure
12 ( 4.9%)	Respiratory Failure
30 (11.3%)	Infectious Disease
14 (5.3%)	Malignancies
2 (0.8%)	Hospitalized Infants

 

Of 27 patients with positive blood cultures, 22 were aged 60 years or more. Of all the hospitalized patients, only 110 of them received antibiotical treatment. The PCR assays by 23S rRNA genes identified 80 ( 30.2%) of all patients with bloodstream infections.

Discussion

Bloodstream infection is one of the most prominent causes of mortality among hospitalized patients. Quick diagnosis and proper treatment of this infection could be an effective way to reduce the mortality rate associated with BSIs.

The results of these tests show that molecular methods have higher sensitivity and can even detect minimal levels of bacteria in the bloodstream in contrast to the blood culture method.

Molecular methods for 23S rRNA PCR methods are proven to be rapid and sensitive, especially for the diagnosis of sepsis among candidates in whom criteria are difficult to diagnose.

These methods are considered precise as they incorporate the use of standard microbiological lab instruments and apparatus. For instance, the use of NanoDrop technology, Bactec bottles, 250 ml centrifuged tubes, and a thermal program makes the process more precise and error-free.

Final Verdict

All in all, the procedure proves that with a 23S rRNA PCR assay, we can determine and diagnose bloodstream infections more rapidly and effectively. The research validates that the right implementation, streamlined process, and complete statistical analytical data can help us fight bacterial infections in a better way, reducing the rate of bacterial infection mortality.

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