Diagnosing Chronic Wound Infection: Efficacy and Cost Effective Dr. Luinio S. Tongson, FPCS, CWS, MSPH Wound Care Conference Singapore
Oct 26, 2014
Diagnosing
Chronic Wound Infection:
Efficacy and Cost Effective
Dr. Luinio S. Tongson, FPCS, CWS, MSPH
Wound Care Conference
Singapore
Chronic Wound
• A wound in which the normal process of
healing has been disrupted.
• “Stuck" in the inflammatory or proliferative
phases of wound healing.
Enoch S.Harding K. Wound Bed Preparation: The Science
Behind the Removal of Barriers to Healing. Wounds. 2003;15(7)
Features of Chronic Wound
Clinical
• Presence of necrotic and unhealthy tissue
• Lack of adequate blood supply
• Absence of healthy granulation tissue
• Lack of reepithelization
• Recurrent wound breakdown due to
superficial bridging
Broderick ,N,, Understanding chronic wound healing . The Nurse Practitioner:
The American Journal of Primary Health Care. Oct 2009 Vol 34 Num 10 , pp 16 - 22
Microbiology
• High levels of bacterial content
• Presence of more than one bacterial strain
• Presence of multi-drug resistant
organisms
• Presence of biofilms
Features of Chronic Wound
Broderick ,N,, Understanding chronic wound healing . The Nurse Practitioner:
The American Journal of Primary Health Care. Oct 2009 Vol 34 Num 10 , pp 16 - 22
Introduction
• Poor clinical performance in diagnosing chronic
wound infection
– Soft tissue infection is the leading indication
for outpatient parenteral antibiotic therapy and
the third most common indication for oral
antibiotic use.1,2
• Sweden study - 60% of patients either were
actively receiving antibiotics or had been treated
with antibiotics in the previous 6 months.3
1. Nathwani D, Moitra S, Dunbar J, Crosby G, Peterkin G, Davey P. Skin and soft tissue infections: development of a
collaborative management plan between community and hospital care. Int J Clin Pract. 1998;52(7):456-60.
2. MacDonald T, Collins D, McGilchrist M, Stevens J, McKendrick A, McDevitt D, Davey P. The utilisation and economic
evaluation of antibiotics in primary care. J Antimicrob Chemother. 1995;35(1):191-204.
3. Tammelin A, Lindholm C, Hambraeus A. Chronic ulcers and antibiotic treatment. J Wound Care. 1998;7(9):435:7.
Introduction
• Clinical perplexity in the current North American
clinical practice guideline on management of
pressure ulcers.1
• Guideline recommends obtaining quantitative
bacterial cultures rather than swab cultures,
quantitative culture techniques are rarely utilized
in the management of most chronic wounds.2
1. Bergstrom N, Allman R, Alvarez O, Bennet M, Carlson C, Frantz R, et al. Clinical Practice Guideline Number
15: Treatment of Pressure Ulcers. Rockville, Md: US Department of Health Human Services. Public Health
Service. Agency for Health Care Policy and Research; 1994. AHCPR Publication 95-0652.
2. Thompson P, Smith D. What is infection? Am J Surg. 1994;167(suppl 1A):75-115.
Introduction
• Infection in chronic wounds may be quite
subtle.*
• Covert infections - out of bacterial balance
when the pathogens in question have
overcome the host immune response.
Dow G. Infection in chronic wounds. In: Krasner D, Rodeheaver G, Sibbald G (eds). Chronic
Wound Care: A Clinical Source Book for Healthcare Professionals, Third Edition. 2001:343-356.
Clinical Presentation
• Acute Wound Infection
or
• Severe
Chronic Wound Infection
• Advancing erythema
• Fever
• Warmth
• Edema / swelling
• Pain
• Purulence
“Classic” Signs & Symptoms
Clinical Presentation
• Critically Colonized
Bioburden
• Bacterial Burden
• Local
Wound Infection
• Delayed healing
• Change in color of wound bed
• Friable granulation tissue
• Absent or abnormal granulation tissue
• or abnormal odor
• serous drainage
• pain at wound site
Cutting & Harding (1994)
Gardner, Frantz & Doebbeling (2001)
“Secondary” Signs & Symptoms
Bacterial burden
™
Contaminated
woundColonised
Critical
colonization
Infection
Healing rate
Tissue biopsy culture
• Remove piece of
viable wound tissue
with a scalpel or
punch biopsy
instrument.
• Used in wound
microbiology research
Method of Diagnosing
Chronic Wound Infection
Deep tissue biopsy culture
• Reference standard for the diagnosis of infection
of chronic wounds
• Indicator of infection of chronic wounds:
– Microbial loads of greater than 105 of any
organism per gram of wound tissue
– Presence of any level of β-hemolytic
streptococcus
• Reddy M, et al. Does This Patient Have an Infection of a Chronic Wound? JAMA. 2012;307(6):605-611
• Schraibman IG. The significance of beta-haemolytic streptococci in chronic leg ulcers. Ann R Coll Surg
Engl. 1990;72(2):123-124 PubMed
Method of Diagnosing
Chronic Wound Infection
• Tissue biopsy culture
– Assist with the recognition of
increased bacterial burden
– Not regularly undertaken
– Wounds can heal despite high bacterial
counts*.
Steer JA, Papini RP, Wilson AP, McGrouther DA, Parkhouse N. Quantitative microbiology in the management of burn
patients. II. Relationship between bacterial counts obtained by burn wound biopsy culture and surface alginate swab
culture, with clinical outcome following burn surgery and change of dressings. Burns 1996; 22(3): 177-81.
Method of Diagnosing
Chronic Wound Infection
• Tissue biopsy culture
• Limitations:
– Skills
– Invasive
– Availability of laboratories
– Expenses
– Further tissue damage and delay of healing
when biopsies are taken
Bamberg R. Sullivan K. Conner-Kerr T. Diagnosis of Wound Infections: Current
Culturing Practices of US Wound Care Professionals. Wounds. 2002;14(9)
Method of Diagnosing
Chronic Wound Infection
Needle-aspiration
technique
• Sensitivity, specificity,
and accuracy of
quantitative needle-
aspiration unclear
• For focal collections
of tissue fluid or
abscess formations
Method of Diagnosing
Chronic Wound Infection
• Wound swabbing is the most common
sampling method used throughout the UK
• Clinical value has been questioned
• Routine swabbing, such as at weekly
intervals or at the time of frequent dressing
changes, is neither helpful nor cost
effective *
Gilchrist B. Taking a wound swab. Nurs Times 2000; 96(4 Suppl): 2.
Method of Diagnosing
Chronic Wound Infection
Wound Swabbing
• Routine swabbing in
the absence of
clinical indicators of
infection is neither
helpful nor cost-
effective.
• Unnecessary cost to
both the NHS and
the patient.
Collier M (2004) Recognition and Management of Wound Infections.
Method of Diagnosing
Chronic Wound Infection
C-reactive protein (CRP)
• Serum investigation
• Identify elevated white cell counts and
protein in serum
• Present in many acute inflammatory
conditions and with necrosis
• Not diagnostic of a chronic wound infection*
Krasner (Ed) D. Chronic Wound Care: a clinical source book for professionals.
Pennsylvania: Health Management Publications, 1990.
Method of Diagnosing
Chronic Wound Infection
Clinical manifestation + Culture
• Wound infection is initially identified by
the recognition of clinical signs + culture.
• Wound swabbing is not a tool that can be
used to diagnose in isolation.
• Assist in the appropriate antibiotic or
treatment options.
Pattern H. Identifying wound infection: Taking a swab. Wound Essential Vo 5. 2010
Method of Diagnosing
Chronic Wound Infection
When Should a Chronic Wound
Be Cultured?
• A wound should be cultured after wound
infection has been clinically diagnosed.
• Clinical diagnosis of infection is essential before
culturing because 100% of wounds are
contaminated at the time of wounding.*
• Soon after the inoculation event, 100% of
wounds become colonized
Bowler P, Duerden B, Armstrong D. Wound microbiology and associated
approaches to wound management. Clin Microbiol Rev. 2001;14(2):244-269.
A six-page, 34-item questionnaire titled
"Wound Culture Survey"
• types of wounds seen in
clinical practice;
• whether wounds are
cultured
• types of wound
specimens collected and
average number per
wound
• factors that prompt a
culture
• specific swab culture and
biopsy techniques used
• specific lab tests
• most common organisms
isolated from wounds
• routine approaches used
in wound diagnosis
• most common treatment
• demographic and
professional information
Bamberg R. etc. Diagnosis of Wound Infections: Current Culturing
Practices of US Wound Care Professionals; Wounds. 2002;14(9)
Type of wounds cared for by
respondents
No. (%)* of Respondents by Frequency Category
Wound Type Frequently or Very Frequently Occasionally Never or Almost Never
Venous insufficiency 277 (80.3) 50 (14.5) 15 (4.3)
Pressure 273 (79.1) 63 (18.3) 7 (2.0)
Arterial insufficiency 181 (52.5) 135 (39.1) 27 (7.8)
Surgical 176 (51.0) 128 (37.1) 37 (10.7)
Neuropathic (diabetic) 166 (48.1) 65 (18.8) 12 (3.5)
Inflammatory 110 (31.9) 159 (46.1) 62 (18.0)
Abscess 108 (31.3) 168 (48.7) 58 (16.8)
Trauma 102 (29.6) 157 (45.5) 76 (22.0)
Neuropathic (nondiabetic) 73 (21.2) 166 (48.1) 87 (25.2)
Burn 39 (11.3) 139 (40.3) 156 (45.2)
Sickle 5 (1.4) 75 (21.7) 253 (73.3)
Fasciitis and/or necrotizing** 4 (1.2) 7 (2.0) - - - -
Pyoderma** 2 (0.6) 7 (2.0) - - - -
Melanoma** 2 (0.6) - - - - - - - -
Cancer** 1 (0.3) 8 (2.3) - - - -
Frost bite** 1 (0.3) - - - - - - - -
Osteomyelitis (refractory)** 1 (0.3) - - - - - - - -
Skin or wound graft** 1 (0.3) 3 (0.9) - - - -
Calciphylaxis** - - - - 1 (0.3) - - - -
Lymphatic** - - - - 1 (0.3) - - - -
Bamberg R. etc. Diagnosis of Wound Infections: Current Culturing
Practices of US Wound Care Professionals; Wounds. 2002;14(9)
Specimen collection:
When, why, and by whomWhen wounds are routinely cultured %*
Before treatment is begun 12.4
After treatment failure 20.2
Varies depending on wound situation 64.7
Only when 3 clinical signs of infection are present 1.0
Did not answer 1.7
Why wounds are routinely cultured %*
To plan a treatment regimen 48.0
For diagnosis of the wound 9.5
For both diagnosis and design of a treatment plan 41.2
Did not answer 1.3
Who makes the final decision to culture %*
Physician or Podiatrist 80.7
Nurse Practitioner or Physician Assistant 7.5
Registered Nurse (RN) 3.6
Wound Care Specialist 2.9
Enterostomal RN 2.6
Physical Therapist 1.6
Did not answer 1.1
Bamberg R. etc. Diagnosis of Wound Infections: Current Culturing
Practices of US Wound Care Professionals; Wounds. 2002;14(9)
Specimen collection:
Type, number, and wound siteType of wound specimens routinely collected %*
Swab only 53.9
Swab and/or biopsy depending on nature of the wound 41.8
Biopsy only 4.3
Number of specimens routinely collected per wound Mean
Swabs 1.8
Biopsies 1.7
Site preparation before wound specimen is collected %*
Saline cleansing 81.7
No preparation 10.1
Aseptic cleansing 8.8
Debridement 4.6
Water and mild soap cleansing 3.9
Irrigation 0.6
Bamberg R. etc. Diagnosis of Wound Infections: Current Culturing
Practices of US Wound Care Professionals; Wounds. 2002;14(9)
Specimen collection:
Techniques and lab tests orderedSwab Specimens* % Biopsy Specimens* %
Collection technique
routinely used:
10-point diagonal 56.7 Punch 54.2
1-point rotation 38.2 Scalpel 33.8
Swab of whole surface 1.4 Surgical scissors 8.5
Levine technique 1.0 Varies 3.5
2-3 point touch 0.3
Varies 2.4
Area of wound from
which
specimens are
routinely collected:**
(Not asked relative to swabs) Most clinically
suspicious area 61.3
Margins 40.8
Center 30.3
Deepest area 2.8
Lab tests ordered:*** Aerobic culture 100.0 Aerobic culture 100.0
Anaerobic culture 54.6 Anaerobic culture 74.6
Gram stain 47.8 Gram stain 58.5
Fungal culture 22.9 Fungal culture 40.8
Viral culture 0.3 Viral culture 11.3
Mycobacteria culture
(i.e., acid fast bacillus) 0.3
Mycobacteria culture
(i.e., acid fast bacillus) 4.2
Bamberg R. etc. Diagnosis of Wound Infections: Current Culturing
Practices of US Wound Care Professionals; Wounds. 2002;14(9)
Significant differences in wound care
practices between practitioners
Wound Care Practices
Work in an OWC*
(n = 132)
Do Not Work in an
OWC (n = 214)
Work time spent in wound care
(mean)**
73.5% 58.0%
Wounds treated without ever
culturing (mean)**
65.7% 72.5%
Culture at least some
wounds***
94.7% 82.5%
Reported non-physician makes
final decision to culture
wound***
18.2% 15.2%
Collect both swab and biopsy
specimens depending on
nature
of wound***
46.2% 31.5%
Use lab culture reports as a
factor in wound diagnosis***
74.2% 68.1%
Bamberg R. etc. Diagnosis of Wound Infections: Current Culturing
Practices of US Wound Care Professionals; Wounds. 2002;14(9)
Results
• Indicate that wound care clinicians are
relying heavily on clinical characteristics
for the diagnosis of wound infection.
– Clinical characteristics (98 %)
– Patient-reported symptoms (88%)
– Wound culturing (70%)
Bamberg R. etc. Diagnosis of Wound Infections: Current Culturing
Practices of US Wound Care Professionals; Wounds. 2002;14(9)
Results
• 79 % of the wounds with positive clinical
signs for infection have positive cultures.
• Supports the practice of using physical
signs as a quick screen to determine
which wounds should be cultured.
Bamberg R. etc. Diagnosis of Wound Infections: Current Culturing
Practices of US Wound Care Professionals; Wounds. 2002;14(9)
Results
• 21% of wound patients with potential
wound infections may go undiagnosed if
clinical signs and symptoms alone are
utilized in diagnosis.
– Immunocompromised patients may not exhibit
typical signs and symptoms
Bamberg R. etc. Diagnosis of Wound Infections: Current Culturing
Practices of US Wound Care Professionals; Wounds. 2002;14(9)
Results
• Situations with False positive wound
infection
• Unrelieved pressure
• Allergies to dressing components
• Have chronic inflammation*
Bamberg R. etc. Diagnosis of Wound Infections: Current Culturing Practices of
US Wound Care Professionals; Wounds. 2002;14(9)
* van Rijswijk L. Wound assessment and documentation. In: Krasner DL, Rodeheaver
GT, Sibbald RG (eds). Chronic Wound Care: A Clinical Source Book for Healthcare
Professionals, Third Edition. Wayne, PA: HMP Communications, 2001
Cultures of wound exudates or swabs
performed with the Z-technique
• Neither predicted nor excluded wound
infection. (CI including 1.0 for positive or negative
results.)
From: Does This Patient Have an Infection of a
Chronic Wound? JAMA. 2012;307(6):605-611.
doi:10.1001/jama.2012.98
Gardner SE, Hillis SL, Frantz RA. Clinical signs of infection in diabetic foot
ulcers with high microbial load. Biol Res Nurs. 2009;11(2):119-128
• Inflammatory marker IL-6 in wound fluid
did not predict the presence or absence of
wound infection.
From: Does This Patient Have an Infection of a
Chronic Wound? JAMA. 2012;307(6):605-611.
doi:10.1001/jama.2012.98
Ambrosch A, Lobmann R, Pott A, Preissler J. Interleukin-6 concentrations in
wound fluids rather than serological markers are useful in assessing bacterial
triggers of ulcer inflammation. Int Wound J. 2008;5(1):99-106
From: Does This Patient Have an Infection of a
Chronic Wound? JAMA. 2012;307(6):605-611.
doi:10.1001/jama.2012.98
Comparisons With Non-reference Standards
• Semiquantitative swab cultures were as
predictive as quantitative swab cultures
• Superficial drainage fluid swab cultures were as
predictive as deep wound swab cultures
obtained after debridement,1
• ↑ white blood cell count or ↑ ESR - as predictive
as clinical features.2
1. Ratliff CR, Rodeheaver GT. Correlation of semi-quantitative swab cultures to quantitative
swab cultures from chronic wounds. Wounds. 2002;14(9):329-333
2. Uzun G, Solmazgul E, Curuksulu H, et al. Procalcitonin as a diagnostic aid in diabetic
foot infections. Tohoku J Exp Med. 2007;213(4):305-312 Pubmed
From: Does This Patient Have an Infection of a
Chronic Wound? JAMA. 2012;307(6):605-611.
doi:10.1001/jama.2012.98
• Comparisons With Non-reference Standards
• Absence of the laboratory marker C-reactive
protein was as predictive as clinical features in
ruling out infection.*
Uzun G, Solmazgul E, Curuksulu H, et al. Procalcitonin as a diagnostic aid in
diabetic foot infections. Tohoku J Exp Med. 2007;213(4):305-312 Pubmed
From: Does This Patient Have an Infection of a
Chronic Wound? JAMA. 2012;307(6):605-611.
doi:10.1001/jama.2012.98
• Comparisons With Non-reference Standards
• Handheld infrared thermometer to assess
periwound skin temperature may be of use in
diagnosing infection of chronic wounds but
needs further study.
Quantitative swab conducted with Levine
technique
• Helpful in predicting wound infection (positive LR, 6.3; 95% CI, 2.5-15).
• Negative swab culture result makes
wound infection less likely
(LR, 0.47; 95% CI, 0.31-0.73).
From: Does This Patient Have an Infection of a
Chronic Wound? JAMA. 2012;307(6):605-611.
doi:10.1001/jama.2012.98
Gardner SE, Hillis SL, Frantz RA. Clinical signs of infection in diabetic foot
ulcers with high microbial load. Biol Res Nurs. 2009;11(2):119-128
• Increasing pain and wound breakdown
have been shown to be particularly good
predictors of infection in the chronic
wound.
Gardner SE, Frantz RA, Troia C, et al. A tool to assess clinical signs and symptoms of localized
infection in chronic wounds: Development and reliability. Ost/Wound Manag 2001;47:40–7
Clinical Manifestation of
Chronic Wound Infection
• Several intrinsic limitations to diagnosing a
wound infection and establishing a
treatment paradigm via clinical signs and
symptoms alone.
• Constantly evolving number of
microorganisms with antibiotic resistance.
Bamberg R. Sullivan K. Conner-Kerr T. Diagnosis of Wound Infections: Current
Culturing Practices of US Wound Care Professionals. Wounds. 2002;14(9)
Clinical Manifestation of
Chronic Wound Infection
• Clinical signs and symptoms may be a
cost-effective method
– Does not inform the appropriate
chemotherapeutic approach to treatment
broad-spectrum chemotherapeutic agents
may be initiated that facilitate the
development of antibiotic resistance.
Bamberg R. Sullivan K. Conner-Kerr T. Diagnosis of Wound Infections: Current
Culturing Practices of US Wound Care Professionals. Wounds. 2002;14(9)
Clinical Manifestation of
Chronic Wound Infection
Best Practice Recommendations for
Preparing the Wound Bed: Update 2006
• Diagnosis of infection is based on clinical
criteria, with bacterial swabs or deep
cultures, laboratory and radiological tests
• All wounds contain bacteria
• Increased bacterial burden in pressure
ulcers delay healing in patients with
chronic ulceration *.
Sibbald. Volume 4, Number 1, 2 0 0 6 Wound Care Canada 15
Volume 4, Number 1, 2 0 0 6 Wound Care Canada 15
* Heggers JP. Defining infection in chronic wounds: Does it
matter? J Wound Care. 1998;7:389-392.
Best Practice Recommendations for
Preparing the Wound Bed: Update 2006
Sibbald. Volume 4, Number 1, 2 0 0 6 Wound Care Canada 15
Volume 4, Number 1, 2 0 0 6 Wound Care Canada 15
Clinical Signs and Symptoms of Wound Infection
Critically Colonized Deep Wound Infection Systemic Infection
Non-healing Pain Fever
Bright red granulation Swelling, induration Rigors
Friable and exuberant
granulation
Erythema Chills
New areas of
breakdown or necrosis
of the wound surface
Increased temperature
Wound breakdown
Hypotension
Multiple organ failure
Increased exudate
Maybe translucent or
clear before becoming
purulent
Increased sized or
satellite areas
Undermining
Foul odor Probing to bone
Clinical Manifestation of
Chronic Wound Infection
• Serous exudate may be increased in a
chronic wound with increasing bacterial
burden before purulence is noted. Chronic
wounds some healing within four weeks to
progress to healing by week 12
• > 12 weeks - ↑ bacterial burden or
infection
Keast DH, Bowering K, Evans W, et al. Measure: A proposed assessment framework for developing
best practice recommendations for wound assessment. Wound Repair Regen. 2004;12:S1-S17
Date of download: 5/21/2012Copyright © 2012 American Medical
Association. All rights reserved.
From: Does This Patient Have an Infection of a Chronic
Wound? JAMA. 2012;307(6):605-611. doi:10.1001/jama.2012.98
Recommendation
• Need to educate professionals
– Use a combination approach
– Screening for wound infection by assessing
for clinical signs and symptoms + culture
Definitive diagnosis of wound infection +
establishing a treatment plan.
Bamberg R. etc. Diagnosis of Wound Infections: Current Culturing
Practices of US Wound Care Professionals; Wounds. 2002;14(9)
Confirming a diagnosis of
wound infection
• After careful assessment,
it is apparent that the wound is infected,
it is important to confirm this and identify the
causative organism(s) and possible
sensitivities to antibiotics.
Diagnosis of wound infection
• Increasing pain
• Friable granulation tissue
• Foul odour
• Wound breakdown
** Gardner SE, Frantz RA, Troia C, et al. A tool to assess clinical signs and symptoms of localized
infection in chronic wounds: Development and reliability. Ostomy/Wound Management. 2001;47:40-47.
Clinical Manifestation of
Chronic Wound Infection
Clinical Manifestation of
Chronic Wound Infection
Symptoms with high positive predictive value:
• Increasing pain (1.0)
• Edema (0.93)
• Wound breakdown (0.89)
• Delayed healing (0.87)
• Friable granulation (0.8)
• Purulent exudate (0.78)
• Serous exudate (0.74)
* Gardner SE, Frantz RA, Doebbeling BN. The validity of the clinical signs and symptoms used
to identify localized chronic wound infection. Wound Repair Regen. 2001;9:178-186.
When to swab?
• Local heat
• Redness/erythema
• Pain or tenderness
• Oedema
• Inflammation
• Increased exudate
• Cellulitis
• Abscess/pus
• Purulent discharge
• Malodour
• Delayed healing– (Beldon, 2001)
• Discoloration of wound bed
• Friable granulation tissue
that bleeds easily
• Pocketing/ bridging at the
base of the wound
• Wound breakdown/
enlargement – (Cutting and Harding, 1994).
Pattern H. Identifying wound infection: Taking a swab. Wound Essential Vo 5. 2010
Other clinical signs:
• Signs of a systemic infection such as pyrexia,
raised white cell count, CRP and/or ESR
• Elderly or immunosuppressed
– Other symptoms: drowsiness, loss of appetite,
nausea, restlessness and confusion
• Screening programme for Methicillin Resistant
Staphylococcus Aureus (MRSA).
Pattern H. Identifying wound infection: Taking a swab. Wound Essential Vo 5. 2010
When to swab?
• Inflammation in isolation is not a reliable
indication for taking a swab or treating a
wound for infection» (Ferguson, 2005)
When to swab?
How to swab?
• Using an effective technique to take a
swab is paramount.
• Incorrect culture swabs technique
– False negative result due to poor technique,
– False positive - only colonising bacteria is
captured but with no clinical indication.
Pattern H. Identifying wound infection: Taking a swab. Wound Essential Vo 5. 2010
How to swab?
Z-stroke swab
• Swab rotated
between the fingers
as the wound is
swabbed from margin
to margin in a 10-
point, zigzag fashion.
• Large portion of the
wound surface is
sampled reflect
surface
Method of Swabbing
Levine technique
• Rotating a swab over
a square centimeter
of tissue for five
seconds with enough
pressure to extract
fluid from the tissue
• Most accurately
reflects wound 'tissue'
bioburden
Method of Swabbing
Gardner S, et al. Diagnostic Validity of Semiquantitative
Swab Culture. Wounds. 2007;19(2):31-38.
Diagnosing Chronic Wound Infection:
Cost Effective and Efficacy:
Clinical manifestation + Culture
Conclusion:
Questions, Comments and Suggestions