BURNOUT Y LA RELACIÓN CON VARIABLES SOCIODEMOGRÁFICAS

The study included all patients with signs suggestive of sepsis. Samples were obtained from 388 patients in order to achieve the acceptable sensitivity (or specificity) of 80% with a confidence interval of 95%, the following formula will be used¹¹⁹:

FP+TN = Z² X (SP x (1-SP) / W²

Z = standard normal deviation which correspond to 95% confidence level set at 1.96 W = desired degree of accuracy set at 0.05.

SP = 80%

N (Sp) = FP + TN / (1- P) = 245.888/1-0.3

= 351

A total of 388 patients participated in the study.

35 3.4 Sample collection and processing

Blood samples were obtained for complete blood count and differential and blood culture.

For procalcitonin determination, blood was collected into the EDTA tube. The plasma samples were used for procalcitonin analysis.

3.4.1 Blood culture: Two samples were taken 1 hour apart from different limbs. Blood obtained was put into BD BACTEC^TM Peds Plus ^TM/F culture vials (Becton Dickson Diagnostic MD, USA) and it was transported to the microbiology laboratory immediately after collection. It was incubated in an automated detection system, BACTEC 9050 (Becton Dickson Diagnostic Instrument System, Sparks, MD, USA). Incubation was at 35-37⁰C for five days. Cultures that did not indicate growth within 5 days of incubation in the BACTEC system were considered negative. Growth within this period in the system was detected as a rise in CO2 and the system flagged it on the computer screen as a positive growth. From the positive samples, a small volume was obtained for gram staining and inoculation on 5% of sheep blood agar, chocolate agar and MacConkey agar. Chocolate agar and Blood agar were incubated in 5% CO2 and MacConkey agar was incubated in ambient air for 18 to 24 hours.

3.4.2 Procalcitonin assay: Procalcitonin concentration was measured in plasma samples by use of an immunochromatographic assay (PCT-Q, B.R.A.H.M.S. Diagnostica GmbH, Berlin, Germany). The test procedure was carried out on a non-haemolysed blood sample that was previously centrifuged, and 200 µL of plasma was pipetted into the round cavity of the test strip. The tracer in the strip bound to the PCT in the sample and a marked antigen antibody complex was formed. This complex moved by means of capillarity through the test system, and in the process, passed through the area containing the test band. Here, the

36 marked antigen antibody complex was bound to the fixed anticalcitonin antibodies and formed a sandwich complex. At a PCT concentration ≥ 0.5 ng/ml, this sandwich complex was seen as a reddish band. The colour intensity of the band was directly proportional to the PCT concentration of the sample and it was related to different PCT concentration ranges (≥

0.5 ng/ml, ≥ 2.0 ng/ml, ≥ 10 ng/ml) with the help of a reference card. Non-bound tracer diffused into the control band zone, where it was fixed and produced an intensely red colour control band. The functional ability of the test system was checked by means of this control band. After an incubation period of 30 minutes, the results of the test was read and the procalcitonin concentration ranges was determined by comparing the colour intensity of the band with colour blocks of the reference card. Lipids and bilirubin have no effect on the measured result.

Table 1: Reference ranges of semi-quantitative procalcitonin value.

PCT < 0.5ng/ml Systemic infection (sepsis) is not likely;

local infection is possible.

PCT ≥ 0.5 - < 2ng/ml Systemic infection (sepsis) is possible with moderate risk for progression to severe systemic infection (severe sepsis).

PCT ≥ 2-10ng/ml Systemic infection (sepsis) is possible with high risk for progression to severe systemic infection (severe sepsis).

PCT ≥10ng/ml Systemic inflammatory response syndrome almost due to severe bacterial sepsis or septic shock.

37 3.4.3 Identification of isolates: This was based on the following: colonial morphology, Gram stain, biochemical tests (API), oxidase test and motility.

3.4.4 Biochemical tests: API 20E and 20NE (bioMerieux, Marcy-L’Etoile, France) kits was used in the identification of gram negative organisms to species level. The organisms was emulsified in normal saline, matched with the recommended McFarland’s standard and added to the micro wells. The color changes were read and fed into the software which gave identification of the organism. The manufacturer’s instructions were strictly adhered to.

Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used to quality control the API 20E and 20NE kits respectively.

Catalase and coagulase tests were used to differentiate gram positive cocci. Optochin sensitivity and bile esculin agars were used in identification of catalase negative gram positive cocci. The tests were quality controlled with Staph aureus ATCC 25923 and Enterococcus faecalis ATCC 29212 organisms.

3.4.5 Antibiotic susceptibility testing: All isolates were tested for antimicrobial susceptibility. The following antibiotics were tested against Gram positive isolates:

clindamycin (2µg), gentamicin (10µg), cefoxitin (30µg), piperacillin-tazobactam (110µg), co-amoxiclav (30µg) levofloxacin (5µg), cefuroxime (30µg) and erythromycin (15µg), chloramphenicol (30µg), rifampicin (5µg). For Gram negative isolates the following were tested: Cefotaxime (30µg), Ceftazidime (30µg), co-amoxiclav (30µg), Cefepime (30µg), levofloxacin (5µg), Amikacin (10µg), piperacillin-tazobactam (110µg), aztreonam (30µg), ertapenem (10µg) and Meropenem (10µg).

38 This was done using the Modified Kirby-Bauer disc diffusion method. Discrete colonies were emulsified in 3-5 mls of normal saline and the turbidity of the resulting suspension matched against a 0.5 Mcfarland Standard.

The suspension was streaked on Mueller Hinton agar using a sterile swab stick. 6 discs were placed on a 90mm plate. The plates were incubated in ambient air at 35-37⁰ C for 24 hours.

The zones of inhibition were read the next day using a ruler or calipers. Zone sizes were interpreted as susceptible, intermediate or resistant using the Clinical Laboratory Standards Institute (CLSI) standards) interpretive tables.¹²⁰

For vancomycin the E-test method was used to detect sensitivity or resistance of Gram positive isolates (Staphylococcus spp, Enterococcus spp). One E-test strip of vancomycin was placed on Mueller Hinton agar inoculated with the test organism. Incubation was for 24 hours and the minimum inhibitory concentration (MIC) was read off directly from the ellipse which was the zone of convergence.

3.4.6 Antimicrobial resistance testing

Gram negative organisms that showed resistance to 3^rd Generation Cephalosporin's were tested for extended spectrum beta lactamase production (ESBL).

Staphylococcal isolates were screened for methicillin resistance using cefoxitin 30µg disc.

3.4.6.1 ESBL production

Principle: Amoxycillin-clavulanic acid a beta lactam - beta lactam inhibitor combination is stable to ESBL's while 3^rd generation cephalosporin's are inhibited by it.

ESBL production was detected by an enlarged or distorted zone of inhibition on the

39 side of the cephalosporin disc facing the amoxycillin-clavulanic acid disc (CLSI guidelines).

Procedure: Amoxycillin-c1avulanic acid disc was placed 30 mm apart centre to centre from a 3^rd generation cephalosporin on Mueller Hinton agar. Distortion or enlargement on the side of the cephalosporin facing amoxycillin-clavulanic acid was observed.

3.4.6.2 Methicillin Resistant Staphylococcus aureus (MRSA)

Principle: Cefoxitin a cephamycin inhibits the growth of Staphylococcus species that do not possess methicillin resistance while resistant one will show no zone of inhibition or faint growth within the zone of inhibition. Procedure: Cefoxitin disc was placed on Mueller Hinton agar that has been streaked with the test organism. The plate was incubated at 33-35⁰ Celsius for a full 24 hours and the zone size measured and interpreted using the CLSI standard. A zone of inhibition less than 22mm was interpreted as resistance.

3.4.6.3 Vancomycin resistance

This was performed on all methicillin resistant Staphylococcus aureus, Coagulase Negative Staphylococci and Enterococcus spp. A suspension of the organism was made in sterile normal saline, and matched to 0.5 McFarland standard. With a sterile swab stick the organism was streaked uniformly on Mueller Hinton agar. Thereafter sterile forceps or an applicator was used to place the E-test strip aseptically on the Mueller Hinton agar plate and incubated at 35-37⁰C for 18-24 hours.

The following day the zone of convergence of the ellipse was measured as the Minimum Inhibitory Concentration (MIC).¹²⁰

40 Vancomycin Sensitive Staphylococcus aureus: 4µg/ml

Vancomycin Intermediate Staphylococcus aureus: 8-16µg/ml Vancomycin Resistant Staphylococcus aureus: >32µg/ml Vancomycin Resistant Enterococci: > 1-4µg/ml