Case study; Microbiology

Case; Leo is 23 years old. He is well-behaved and is a motorcycle enthusiast. During the summer, he works as a plattsetter in Sweden. In the second part of the year he goes motorcycle, preferably in Asia. During a motorcycle holiday in Thailand, Kenny is in a serious traffic accident and arrives at a hospital. He is lucky to survive, but gets serious injuries, with multiple fractures and internal injuries. After five days at the hospital, he gets high fever and suspects a catheter-related sepsis.
For this reason, samples are taken for microbiological analysis; from catheterspets, instillation and blood cultures. In addition, basic blood and electrolyte status tests and CRP are taken. In the microbiological analysis, Staphlococci are isolated, which are further typed using MALDI- ToF to Staphylococcus epidermidis. For this reason, a resistance determination is also made (see results below).
The following results are obtained from the laboratory, among other things:
Weight: 85 kg
Length: 183 cm

Resistance determination Staphylococcus epidermidis

Breakpoints for antibiotics

Tests Resuts Age (y) Referense Range
P-Sodium 140 mmol / L 318 137-145 mmol/L
Β-potassium 4.0 mmol / L 318 3,5-4-6 mmol/L
P-CRP 200 mg / L < 3 mg/L
Antibiotics Zon mm Antibiotics Zon mm
Isoxazolyl-pc 20 Fucidic acid 25
Gentamicin 20 Clindamycin 17
TrimSulfa 20 Rifampicin 28
Linezolid 24
Antibiotics S.aureus KNS Antibiotics S.aureus KNS
Isoxazolyl-pc 22/21 27/21 Fucidic acid 24/24 24/24
Gentamicin 18/18 22/22 Clindamycin 22/19 22/19
TrimSulfa 17/14 17/14 Rifampicin 26/23 26/23
Linezolid 21/21 21/21

Answers of the following questions related to the above case.

1-Is it likely that Kenny has a sepsis?

Answer: Yes, Kenny has sepsis.

2-What does the resistance pattern look like for the Staphylococcus that has been isolated? (SIR)?

Answer: I think this is look like staphylococcus epidermidis with methicillin sensitive pattern. But as this case missing beta-lactamase drugs then it seems to methicillin resistant staphylococcus epidermidis.

3-What can be the cause of the disease?

Answer: The cause of the disease is nosocomial infection of staphylococcus epidermidis which is resulted by catheterization.

The principle, advantages, dis-advantages, pre-analytical. analytical and post-analytical errors of the following methods which help in solving the above case.

MALDI-ToF (Matrix assisted laser desorption ionization-time of flight)

Principle: The sample is mixed with matrix (energy absorbent) and then sample crystallizes together with matrix by drying. When the laser bean is passes through causes the ionization of trapped sample in the matrix and generated singly protonated ions of the analytes in the sample. These ions are accelerated at a fixed potential and separated according to mass to charge ratio (m/z). the charge analytes detected by TOF analyzer.

Advantages:

It is a rapid and have good accuracy at species level, correct and faster method.  It not very expensive and no fragmentation is formed because of low internal energy. It is also very useful in subspecies level identification. It is good in taxonomical and epidemiological studies.

Limitations

This method having low analytical sensitivity. It is not suitable in the identification of low amount bacterial in sterile sample such CSF. For such a sample needed more optimization and standardization.

Preanalytical phase

it is important to perform MALDI-ToF is a very sterile condition. It is important to use biological safety cabinets in pre-analytical phase.  It is important to record the bacterial growth medium. It is important to note the patient record. It is important to use lab coat, gloves and glasses. It is important to select the bacterial colony which has to be test. the preparation of the sample for MALDI-ToF is very simple but it need attention that a proper amount of sample is applied by touching the surface of the selected colony. It the direct identification is perform from blood culture the sample preparation step involved the use of lysis solution or lysis filtration.

Analytical phase

It is important to perform the test with quality practice to ensure accurate result. It is important to check the external and internal quality control. The laboratory must calibrate the MALDI-ToF system with the specific calibration standard (extract of E. coli or specific E. coli calibration strain). It is important to use specific positive and negative control for external control. It is important to maintain spectral quality for accurate result.

Postanalytical phase

The result report is also a consideration of the quality control. Different spectral databases are used for the MALDI-ToF reporting. The reporting of the result is also depended on the type of specimen. If the report is not correlate the conventional identification the procedure should be repeat because increase amount of the sample decreases the quality of the result.  If there is no peak formation its means that MALDI-ToF is insufficient in identification while the second sample correctly identified.

Immunoturbidimetry
Principle:The immunoturbidimetric assay is quantitative determination of protein is done by the formation of agglutination of antigen antibody complex. The agglutination causes the turbidity. The light when passes through the mixture some of the light is absorbed by the mixture and change in the intensity of the transmitted light is measured photometrically.
Advantages

It has high reaction analyzer speed, ease of automation and having good precision and accuracy. It is a sensitive, rapid method and blank interference is reduced by using low concentration of PEG (polyethylene glycol).

Disadvantages
It is an expensive, inflexible reagents and inflexible protocol.

Preanalytical phase

The sample for C-reactive protein is collected by standard procedure and in a sterile tube. The reagents for the test should be stored at 2-10 oC. It is important to rejected contaminated, lipemic and hemolyzed sample for CRP. If the sample is not using on the same collecting day so it is important to store at 2-10 oC for 2 months and at -20 oC for longer than a year. The blood sample collected in EDT or sodium heparin anticoagulant tube for plasma and for serum in a tube without anticoagulant.

Analytical phase

It is important to use appropriate immunoassay and specific antibody or antigen for the assay. It is important to check the function and quality control of the equipment. The correct pipetting is important. It is important to used standard laboratory procedure. It is important to maintained regularly of the equipment. The reagent should be properly handled. To check the turbidimeter for wavelength calibration, accuracy, linearity of detector and light should be performed at regular intervals.


Postanalytical phase

The normal level of CRP is less than 3mg/L. The interpretation of CRP result is very important because it is measure in a number of conditions. It is increases in limit from 10-40mg/l in mild inflammatory condition or in viral infection. The serum CRP level increases up to 200mg/l in bacterial infection and severe inflammation. For the sepsis 50mg/l is consider a cut-off value by Pedro Póvoa. IF the level of the CRP is not correlated to clinical feature or have doubt of interference substance so the test should be repeated. 

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