The Major And Important Branch Of Medicine Biology Essay

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Surgery is one of the major and important branch of medicine that perform a surgical procedure to the human body for diagnostic, treatment, prevention as well as palliative purpose. When a part of the body is operated during surgical procedure, the micro-organism will take this opportunity to get into this wound and multiply in tissues causing a post-operative infection. And such infection occurred post-operatively referred as surgical site infection.

The term 'surgical site infection' (SSI) was introduced in 1992 to replace the previous term 'surgical wound infection' (Horan et al., 1992). Surgical site infection (SSI) is defined as an infection that occurs at an incision site, or any part of the anatomy that was opened or manipulated during the procedure that occurs within 30 days after surgery, or within 1 year in the presence of an implant (Horan et al., 1992).

Since the introduction of antiseptic technique on hand wash by Dr. Ignaz Philipp Semmelweis (Wikipedia), Louis Pasteur's germ theory studies (Wikipedia) and Joseph Lister, 1st Baron Lister on wound cleaning and surgical instruments sterilization by using carbolic acid (Wikipedia), the SSI mortality and morbidity rates was reduced up to 90%. The discovery of antibiotic substance penicillin by Sir Alexander Fleming ( further reduced the incidence of SSI.

However, SSIs remain a significant clinical problem as they are associated with considerable morbidity and mortality and it impose several demands on healthcare resources. SSI account for approximately 20%-25% of total Hospital Acquired Infections (HAI), it can double the length of hospital stay, reduce the wound healing process and increase the cost of health care. In the most unpleasant case, it can lead to re-operation and reduce the patient's quality of life.

Anaerobic bacteria are the major causative agents for the incidence of SSIs because the most common isolated microorganisms from the site of infections are Staphylococcus aureus (included Methicillin-resistant Staphylococcus aureus), Enterococcus spp, and Escherichia coli (Leaper, 2010). The common sources of pathogens is the endogenous flora (FARIDA JAMAL MBBS, 1981) of the patient's skin, mucous membrane or hollow viscera.

There were many studies on prevention of the surgical site infections had provided some new ideas, thoughts, new methods on prevention, and revealing some methods were not necessary as they did not help in preventing the surgical site infection. Therefore another new guideline was published by Centers for Disease Control and Prevention (CDC) on 1999 to replace the guideline version 1985 (Alicia J. Mangram, April 1999).

A study showed that approximately 500,000 SSIs occur in United States (US) each year, and it causes of decreased patient's quality of life, disability, and even death (CG., 2004). An average of 7-12 days extra hospital stays, or readmission due to SSI and subsequent treatments are required for SSI has increased the economic burden of government, from USD $3,000 to USD $5,000 per patient (Kathryn B. Kirkland et al., 1999). Operative patients developed SSI has greater risk of death compared to operative patients without SSI.


2.1 Signs of SSI

The common signs and symptoms presence in surgical site infection:

Fever, from moderate to high grade fever. A low grade fever on the first 2 days is common due to physiological respond following surgery.

Foul smelling drainage or pus from the wound. It can be bloody, greenish, whitish, yellowish or mixed colours. The drainage may be foamy or thick.

Swelling of the wound, sometime can feel hardening as the tissue underneath are inflamed.

Redness of the surrounding skin around the wound, sometime may even feel warm.

Pain around the wound. Normally the pain is steadily and slowly diminished during healing process, but if the pain increases for no reason, probably there is an infection developing in the wound.

The symptoms present on the first 48-72 hours are usually normal physiological respond following surgery due to the healing process, but if they symptoms become severe and worsen, then we should suspect the infection.

2.2 SSI Classification

The SSI can be divided according to the site of incision:

Superficial incisional

Deep incisional

Organ space

C:\Users\JL Wong\Desktop\SSI\ssi.JPG

Figure 2.2 The picture above show the cross section of abdominal wall of the CDC Classifications of SSI (Horan et al., 1992).

Superficial incisional SSI involved the superficial soft tissues which are skin and subcutaneous tissue, Deep incisional SSI involves deep soft tissue, which are fascia and muscle under the subcutaneous tissue, while Organ space SSI involve the organs, for example intestine, spleen, liver, kidney, lungs, heart, bone and other organs.

2.3 Surgical Wound Classification

The surgical wound has been classified into 4 categories:

Table 2.3 Surgical Wound Classification

Class I


An uninfected operative wound in which no inflammation is encountered and the respiratory, alimentary, genital, or uninfected

urinary tract is not entered. In addition, clean wounds are primarily closed and, if necessary, drained with closed drainage. Operative incisional wounds that follow nonpenetrating (blunt) trauma should be included in this category if they meet the criteria.

Class II


An operative wound in which the respiratory, alimentary, genital, or urinary tracts are entered under controlled conditions and without unusual contamination. Specifically, operations involving the biliary tract, appendix, vagina, and oropharynx are included in this category, provided no evidence of infection or major break in technique is encountered.

Class III


Open, fresh, accidental wounds. In addition, operations with major breaks in sterile technique (e.g., open cardiac massage) or gross spillage from the gastrointestinal tract, and incisions in which acute, non-purulent inflammation is encountered are included

in this category.

Class IV


Old traumatic wounds with retained devitalized tissue and those that involve existing clinical infection or perforated viscera. This definition suggests that the organisms causing postoperative infection were present in the operative field before the operation.

(Alicia J. Mangram, April 1999)

2.4 Risk Factors

A number of patient-related and procedure-related factors have been shown in univariate or multivariate analyses to influence the risk of SSIs

Table 2.4 Risk factors that affecting the SSI

Patient related factors

Procedure related factors

Age, e.g. advanced age and extreme age

Nutritional status, e.g. malnutrition and recent weight loss

Uncontrolled blood sugar level, e.g. Diabetes Mellitus



Altered immune status, e.g. HIV/AIDS, chronic steroid use, previous chemo/radio-therapy

Length of pre-operative hospital stay

Co-existent infection at other part of the body


Colonization with micro-organisms (particular Staphylococcus aureus)


Duration of surgical scrub

Skin antisepsis

Preoperative shaving

Preoperative skin preparation

Duration of operation

Antimicrobial prophylaxis

Operating room ventilation

Inadequate sterilization of surgical instruments

Foreign material in the surgical site

Surgical drains

Surgical technique

poor haemostasis

failure to obliterate dead space

tissue trauma

(Alicia J. Mangram, April 1999)

According to the study at 1996, it shows that mild perioperative hypothermia, which is common during major surgery, may promote surgical-wound infection by triggering thermoregulatory vasoconstriction, which decreases subcutaneous oxygen tension (ANDREA KURZ, May 1996). Reduced levels of oxygen in tissue impair oxidative killing by neutrophils and decrease the strength of the healing wound by reducing the deposition of collagen (ANDREA KURZ, May 1996). Hypothermia also directly impairs immune function (ANDREA KURZ, May 1996). However, there are still lacking of clinical data to support that the patient's body temperature will increase the risk of SSI.

Hyperglycemia impairs the immunity system of the patient. High blood sugar lead to non-enzymatic glycation of proteins that can inactivate the Immunoglobulin G (IgG) by decreasing complement fixation and increase collagenase activity (Hennessey et al., 1991). Hyperglycemia also impaired the leukocyte functions which lead to delay of chemotaxis, impair phagocytosis and hinder bacteriocidal activity (Mowat and Baum, 1971; Chang, 1979).

2.5 SSI Prevention Bundle

SSI is a preventable surgical complication. Properly applying the SSI prevention bundle can reduce the risk of SSI. SSI bundle is included the following (Team, 10 March 2008) :

If at all possible avoid hair removal, otherwise use cream or clipper rather than use razors (Institute, 2007; Quality Medical Care Section, 2010). Study shows that removal of the hair around the planned incision site by razor or shaving increase the incidence of wound infection when it compared with no hair removal at all due to the injury of the superficial skin by razor.

Ensure prophylactic antibiotic was prescribed as per local antibiotic policy, for the specific operation category. If antibiotic is required, ensure the antibiotic was been administered within 60 minutes prior to the operation to ensure maximum tissue concentration, because once the incision is made, delivery of the antibiotics to the wound is impaired (Organization, 2009).

Ensure the patient's body temperature was normal throughout the operation (excludes cardiac patients) (ANDREA KURZ, May 1996).

Ensure the patient's blood glucose level was normal throughout the operation (diabetic patients only) (Kazuhiro Hanazaki, 7 September 2009; Mowat and Baum, 1971; Chang, 1979; Hennessey et al., 1991; Quality Medical Care Section, 2010).

Additional prevention can be done in pre-, intra-, and post-operative phases to prevent the incidence of SSI.

2.5.1 Pre-operative Phase

Nasal screening and decolonization

Patient will be screen for Methicillin-resistance Staphylococcus Aureus (MRSA) by using local guidelines and decolonized prior to surgery if found positively.


Patient should have shower, or bath or at least wash, if unable to shower, by using soap to reduce the skin microflora (Quality Medical Care Section, 2010).

2.5.2 Intra-operative Phase

Skin preparation

Use 2% Chlorhexidine gluconate in 70% Isopropyl alcohol and allow to air dry rather than using povidone-iodine, unless patient is hypersensitive to Chlorhexidine gluconate.

Incide drapes

It should be impregnated with antiseptic, if uses.

Supplemented Oxygen

Patient's haemoglobin saturation rate should be maintain above 95% throughout the entire operation as well as post-operative phase.

2.5.3 Post-operative Phase


The wound is covered with an interactive dressing at the end of surgery and it should be kept undisturbed for a minimum 48 hours post-operatively for wound healing, unless there is any leakage. The principle of asepsis (non-touch technique) is used when the wound is being re-dressed.

Hand hygiene

Hand should be decontaminated immediately before and after each episodes of patient contact.

2.6 Problem Statements

There are no new updates on surgical site infection prevention guideline for the past 13 years

There are lack of similar recent studies in Malaysia

Based on the previous studies shows that different hospitals has different rates of surgical site infection, and all these are due to differences in clinical practice, hospital protocol, geolocation of the hospital, populations and other factors.

Due to different risk of bacterial contamination in different surgical procedure, the surgical site infection rates are vary too.

2.7 Justification of the Study

Most of the time, our local data consist of only those who acquired the infection while in the hospital. SSI can develop infection within one month post operatively. Therefore those who get the infection after discharged might not be captured by infection control team.

In this study we would like to determine the prevalence of SSI among clean and clean contaminated surgery in HUSM because we thought that there is under reporting of SSI because to date there is no data available yet regarding SSI. Knowing the incidence is crucial to develop infection control policy for SSI. Therefore we plan to do a prospective cohort study to address this issue.

In this study the patient will be monitor till one month post op to rule out SSI. Furthermore, a cohort prospective study will enable us to do elaborate profiles of the patient who develop SSI and identify the problems with regards to preoperative preparation, during operation, post-operative wound care and co morbid illness associated with SSI.

Determining the causative agents and knowing the antimicrobial profiles are crucial before we can develop a good antibiotic policy for antimicrobial prophylaxis and treatment of SSI.

Antibiogram is unique to every center and we should not adopt other policies without studying our own profiles. To further complicate the matter, most of the SSI diagnosed at outpatient clinic do not have specimen taken for culture. On the other hand, those who have specimens taken for culture were not able to detect the causative organism.

Therefore in this study, we will give emphasize on the specimen collection and processing to determine the best specimen and the causative agents.  

2.8 Research Questions

What is the incidence of SSI in our clean and clean contaminated wound in our hospital?

What are the common organisms and their antibiogram profile of causative agents for SSI in our population?

What is the best method for specimen collection to get the best yield of the causative organisms

What are the preoperative, operative and postoperative practices as well as co morbidities commonly associated with SSI in our population.

What is the antibiogram of the causative agents and are we using the suitable antimicrobial prophylaxis based on our local antibiogram?

How did our population get infected? Did the organisms come from the same clone?

2.9 Objective of Study

To determine the prevalence of SSI among clean and clean contaminated surgery in HUSM.

To identify the risk factors of SSI among clean and clean contaminated surgery in HUSM

To describe the microbiological profile of the causative microorganisms and their antibiotic sensitivity pattern.

To review the antibiotic prophylaxis for clean contaminated surgery based on the result of the antibiogram.

To determine the antimicrobial prophylaxis commonly used in our hospital.

To determine the molecular characterization of MRSA strain causing SSI in clean and clean contaminated surgery.


3.1 Study Design

Prospective cohort study

3.2 Study Area

The study will be done in tertiary-care hospital, Hospital Universiti Sains Malaysia (HUSM), Kubang Kerian, Kelantan. Patients are recruited in Department of Surgery in HUSM. The study will be conducted for a duration of 2 years.

3.3 Study Population

All patients underwent clean and clean-contaminated elective operation.

3.4 Sampling Method

Universal sampling method - take all eligible population in one year.

3.5 Sampling Frame

All patients fulfill the inclusion and exclusion criteria.

3.6 Inclusion and Exclusion Criteria

Table 3.6 Inclusion and Exclusion Criteria of SSI

Inclusion Criteria

Exclusion Criteria

Both sexes

Underwent clean operations

Underwent clean-contaminated operations

Elective operations

Underwent contaminated operation

Underwent dirty-infected operation

Operations that involve implantation

Emergency operations

Sample Size Calculation

Zα = 1.96 (from α = 0.05)

P = Prevalence rate from previous study

∆ = Precision (range 0.01-0.15)

Formula for calculating the prevalence rates (single proportion).

Prevalence of clean surgery (Patel Sachin M, April-June 2012)

Prevalence of clean-contaminated surgery (Patel Sachin M, April-June 2012)

Risk factors are calculated by using PS Power and Sample Size Calculations version 3.0, January 2009, by William D. Dupont and Walton D. Plummer.

Risk factor - Obesity (Arabshahi Ks Fau - Koohpayezade and Koohpayezade)

α = 0.05 power = 0.8 P0 = 0.074 P1 = 0.23 m = 1

experimental subjects = 82 + 82 control subjects

= 164 sample size

Risk factor - Diabetes Mellitus (Arabshahi Ks Fau - Koohpayezade and Koohpayezade)

α = 0.05 power = 0.8 P0 = 0.071 P1 = 0.23 m = 1

experimental subjects = 78 + 78 control subjects

= 156 sample size

Risk factor - Smoking (Arabshahi Ks Fau - Koohpayezade and Koohpayezade)

α = 0.05 power = 0.5 P0 = 0.073 P1 = 0.23 m = 1

experimental subjects = 81 + 81 control subjects

= 162 sample size

Sample size will be taken from clean and clean-contaminated surgery which is 50 and 171, so the total sample size will be 221 subjects.

3.8 Sample Collection (Patient Selection)

Patient will be identifying from elective OT list. Consent will be taken. Nasal swab for MRSA colonization screening will be performed. Patient demographic data will be filled in. Patient will be follow up during pre-, intra- and post-operatively up to 30 days. At any time during 30 days duration, if patient have evidence of infection at his/her surgical site, the specimens (pus aspirates, tissue or swab) will be collected and send for microbiological analysis.

3.9 Sample Collection for Microbiological Analysis

Sample will be collected from patients developed surgical site infection. All wounds will be cleaned by Chlorhexidine. Then the pus, exudate or fluids will be aspirated. Tissue excision from the wound edge by scalpel will be done where the aspiration is not possible.

If aspiration and tissue sample are not possible, swabs from the depths of the wound will be used.

3.10 Sample Transport

All the samples will be placed into an aerobic transport device or a sterile tube, and placed into a sealable plastic bag. Then transport to the microbiological laboratory within 2 hours from the time of collection.

3.11 Study Tools

Blood agar (BA) media plates

MacConkey (MAC) media plates

Chocolate blood agar (CBA) media plates

Gas jar / plastic pouch

Anaerobic gas pack

Selective media plates (Mueller Hinton with 4% NaCl + 6mcg oxacillin)

Antimicrobial sensitivity test disc

Incubator in 35 degree celcius

Incubator with Carbon dioxide in 37 degree celcius

3.12 Laboratory Methods

Bacterial culture method

Antimicrobial susceptibility test

Polymerase Chain Reaction (PCR)

Gram staining

3.12.1 Gram stain

Put 1 small drop of saline on glass slide

Put the sample on the glass slide and fix it

Air dry

Flood the smear with crystal violet solution and wait for 1 minute

Rinse off the Crystal Violet solution with tap water

Flood the smear with Iodine solution and wait for 1 minute

Rinse off the Iodine with tap water

Decolourise the smear by adding Acetone solution and wait for 15 seconds

Rinse off the Acetone with tap water

Counterstain with Safranin solution and wait for 30 seconds

Rinse off the Safranin with tap water

Let it air dry

Examine under light microscope with power 10

Examine with power 100 with a drop of oil on the smear

Grame positive is Blue colour and Gram negative is Red colour

3.12.2 Nasal swab

Streak the sample on BA plate and Mueller Hinton agar + 4% NaCl + Oxacillin plate and put into an enclosed plastic bag with anaerobic gas pack to create an anaerobic environment and place it into incubator.

Both plates will be examined by using gram stain after 24 hours of incubation period.

3.12.3 Pus aspirate

Use a cotton bud to take the pus from the container and place on a small part of 2 blood agar plates, MacConkey plate, chocolate blood agar plate and glass slide for gram stain, then we use sterile loop to streak on the sample on petri plates.

Then we put the CBA plate into incubator with Carbon dioxide, 1 BA plate and the MAC plate into incubator and 1 BA plate put into an enclosed plastic bag with anaerobic gas pack to create an anaerobic environment and place it into incubator.

We stain the smear with gram stain and we will look for the pus cell and epithelial cell under light microscope.

CBA plate will be examined by using gram stain after 48 hours of incubation period while other plates will be examined by using gram stain after 24 hours of incubation period.

3.12.4 Tissue

Use a cotton bud to take the pus from the container and place on a small part of 2 blood agar plates, MacConkey plate and glass slide for gram stain, then we use sterile loop to streak on the sample on petri plates.

Then we put 1 BA plate and the MAC plate into incubator and 1 BA plate put into an enclosed plastic bag with anaerobic gas pack to create an anaerobic environment and place it into incubator.

We stain the smear with gram stain and we will look for the pus cell and epithelial cell under light microscope.

All plates will be examined by using gram stain after 24 hours of incubation period.

3.12.5 Wound swab

Streak the sample on BA plate and MAC plate and let the microorganisms grow in incubator for 24 hours and then examine by using gram stain.

3.12.6 Biochemical Test - Citrate Test

This test is used for screening the ability of bacteria to utilize citrate as its energy and carbon sources. Streak an isolated pure culture onto its surface and incubate at 35 degree Celcius for 18 to 48 hours. Then observe the colour changing.

Citrate "positive" is blue colour and no colour changing in citrate "negative", which is its original green colour. Positive result indicate the presence of gram-negative pathogen.

3.12.7 Biochemical Test - Indole Test

This test is to determine the ability of bacteria to split indole from amino acid tryptophan. Stab an isolated pure culture to the bottom and incubate at 35 degree Celcius for 24 to 48 hours. After that add 5 drops of Kovac's reagent.

Indole "positive" is red-voilet colour and indole "negative" is yellow colour while orange colour is due to tryptophan degradation.

Indole "positive" bacteria includes:

Aeromonas hydrophilia

Aeromonas punctate

Bacillus alvei

most Citrobacter spp.

Edwardsiella spp.

Escherichia coli

Flavobacterium spp.

Haemophilus influenza

Klebsiella oxytoca

Proteus spp. (not P. mirabilis)

Plesiomonas shigelloides

Pasteurella multocida

Pasteurella pneumotropica

Streptococcus faecalis

Vibrio spp.

Indole "negative" bacteria includes:

Actinobacillus spp.

Aeromonas salmonicida

Alcaligenes spp.

most Bacillus spp.

Bordetella spp.

Enterobacter spp.

Lactobacillus spp.

most Haemophilus spp.

most Klebsiella spp.

Neisseria spp.

Pasteurella haemolytica

Pasteurella ureae

Proteus mirabilis

Pseudomonas spp.

Salmonella spp.

Serratia spp.

Yersinia spp.

3.12.8 Biochemical Test - Methyl Red (MR) Test

This test is used to identify bacteria producing stable acids by mechanisms of mixed acid fermentation of glucose. Take an isolated pure culture with a cotton bud and mixed it with Methyl-red solution, then incubate for 48 hours at 37 degree celcius.

Positive result is pH < 4.4 in red colour, negative result is pH > 6.2 in yellow color while in between is orange colour.

Positive result indicates the presence of Escherichia coli.

3.12.9 Biochemical Test - Triple Sugar Iron (TSI) Test

TSI slant is a test tube that contains 1% lactose, 1% sucrose and 0.1% glucose. This test is used in determining the carbohydrate fermentation and H2S production. This test is most frequently used for identifying Enterobacter, as well as other gram negative bacteria. Stab with an isolated pure culture to the bottom and streak on the surface and isolated for 18 to 24 hours at 37 degree celcius.

Expected results:

Table 3.12.9 : Interpretation of Triple Sugar Iron Test






Glucose fermentation only



Glucose and lactose and/or sucrose fermentation



No fermentation


Yellow + bubbles

Glucose and lactose and/or sucrose fermentation, Gas produced


Yellow + bubbles

Glucose fermentation only, Gas produced


Yellow + bubbles

Black precipitate

Glucose fermentation only, Gas produced, H2S produced


Yellow + bubbles

Black precipitate

Glucose and lactose and/or sucrose fermentation, Gas produced, H2S produced



Black precipitate

Glucose fermentation only, H2S produced



Black precipitate

Glucose and lactose and/or sucrose fermentation, H2S produced

3.12.10 Biochemical Test - Urease Test

This test is to identify the micro-organism that are capable of hydrolyzing urea to produce ammonia and carbon dioxide. It's mainly used to distinguish urease-positive Proteeae from other Enterobacteriaceae. Streak an isolated pure culture onto its surface and incubate at 35 degree Celcius for 18 to 24 hours. Then observe the colour changing.

Positive result present with bright pink colour on the slant.

3.12 Data Analysis

Data will be collected, summarized, tabulated, and analyzed using IBM Statistical Package for the Social Sciences (SPSS) software. The results will be presented through histograms, tables, Chi-square test and pie charts.

3.13 Ethical Consideration

Ethical approval will be taken from USM Research Ethics Committee (Human) - (JEPeM) for sample collection. Informed consent will be signed by the participating patients or their relatives.

Get the operation theatre listStudy Flow Chart

Patient discharged from the study

No infection

No evidence of infection

Evidence of infection (fever, purulent discharge, pain)

Follw up the patient on the day 30 post-operatively at OPD

No infection

Continue follow up by phone on third week

No infection

2 weeks follow up during suture to open (STO) at Out-Patient Department (OPD)

Identifying the pathogen (Microscopic. Biochemical, PCR)

Isolation of the pure culture

Take sample from the site of incision

Fill up intra-, post-operation checklist

Fill up patient personal data (name, age, gender, address, contact number etc) and pre-operation checklist

Patient in ward

Patient discharged

Patient underwent operation

Nasal swab for screening MRSA colonization

Get the consent form signed by the patient

Gantt Chart of Research Activities for :


Project Activities




Research Activities


























Patients Recruitment

Data Collection

Identification of microorganisms and their antibiotic sensitivity patterns are performed

Statistical Data Analysis

Report Writing, Presentation and Submission of Report

Submission of Research Papers for Publication

Planned milestones :

December 2013 : Will complete the patients recruitment

March 2014 : Will complete the data collection and identification of microorganisms and their antibiotic sensitivity patterns

July 2014 : Will complete the statistical data analysis

October 2014 : Will complete the report writing, presentation and submission of report

February 2015 : Report will be published