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Trial registered on ANZCTR


Registration number
ACTRN12621001637831
Ethics application status
Approved
Date submitted
30/10/2021
Date registered
30/11/2021
Date last updated
26/11/2023
Date data sharing statement initially provided
30/11/2021
Date results information initially provided
7/11/2022
Type of registration
Prospectively registered

Titles & IDs
Public title
The sensitivity and specificity of Gram stain on formalin fixed tissue sections in the diagnosis of bacterial skin infection - a study of diagnostic test accuracy
Scientific title
The sensitivity and specificity of Gram stain on formalin fixed tissue sections in the diagnosis of bacterial skin infection - a study of diagnostic test accuracy
Secondary ID [1] 305677 0
Nil known.
Universal Trial Number (UTN)
Trial acronym
Linked study record

Health condition
Health condition(s) or problem(s) studied:
Bacterial skin infection 324148 0
Condition category
Condition code
Skin 321623 321623 0 0
Dermatological conditions
Infection 321858 321858 0 0
Studies of infection and infectious agents

Intervention/exposure
Study type
Observational
Patient registry
False
Target follow-up duration
Target follow-up type
Description of intervention(s) / exposure
This project represents a study of diagnostic test accuracy, aiming to evaluate the test performance (sensitivity, specificity, predictive values, and likelihood ratios) of Gram stain - when performed as part of histological assessment - in the diagnosis of bacterial skin infection.

The work-up of patients with a cutaneous disease (e.g. patients with an ulcer, a pustular eruption, or panniculitis) will sometimes involve punch or incisional biopsies wherein part of the tissue is placed in formalin for histological assessment, and part placed in a sterile transport medium (e.g. saline) for microbiological assessment.

Microbiological assessment of fresh tissue specimens involves microscopy and culture with or without assessment of antibiotic sensitivities.

Histological assessment involves formalin fixation which, in addition to preserving tissue architecture and cell morphology, is bactericidal. After fixation, the tissue is dehydrated, infiltrated with paraffin, and embedded in a rectangular paraffin block. 0.004 mm slices of tissue (so-called tissue sections) are taken from the surface of this paraffin block, stained with haematoxylin and eosin (H&E), and examined at a microscope.

Gram stain has been used in in anatomical pathology laboratories in the assessment of formalin-fixed paraffin-embedded (FFPE) tissue sections since the 1880s, but appears to have been accepted as an ancillary test in anatomical pathology laboratories with a relative lack of objective data on test performance to inform its use and interpretation.

Gram stain can either be performed as on fresh tissue specimens as part of microbiological assessment or on formalin fixed paraffin embedded (FFPE) tissue as part of histological assessment. Under both circumstances, Gram stain involves the use of a primary stain (crystal violet) and an iodine solution which bind to bacterial nucleic acids, followed by a clearing agent (alcohol or acetone) and a counterstain (e.g. safranin). Gram positive bacteria appear purple due to retention of the crystal violet-iodide complex, a result of their cell wall structure particularly in terms of the amount and configuration of peptidoglycan, whereas Gram negative bacteria are decolourised by the clearing agent and appear red due to the counterstain.

Although Gram stain plays an important role in microbiological assessment in terms of visualisation and classification of bacteria, its use on FFPE in histological assessment has multiple limitations which include: the time intensive nature of searching for small numbers of bacteria on stained sections, the lack of visual contrast between bacteria (particularly Gram negative bacteria) and background tissue, and the ambiguity in distinguishing between bacterial colonisation and genuine infection.

The potential advantages of using Gram stain on tissue sections include: the potential value of visualising bacteria in a particular tissue plane (e.g. deep dermis, blood vessel wall) to distinguish genuine infection from colonisation (where bacteria tend to be limited to the surface of the specimen), the identification of bacteria which are slow or difficult to culture (e.g. B. henselae), and in attempting to determine the pathogenic organism when culture reveals a combination of Gram positive and negative bacteria.

Notwithstanding, there is a relative lack of objective data pertaining to the diagnostic test accuracy of using Gram stain on FFPE tissue sections to diagnose infection. Guarner et al (2007) evaluated the test performance of Gram stain and immunohistochemistry in diagnosing S. pneumoniae infection, using tissue culture as the reference standard (n = 46). In this context, Gram stain had sensitivity of 0.61 (61%) and specificity of 0.50 (50%) in diagnosing S. pneumoniae infection. These results suggest that the test performance or diagnostic accuracy of Gram stain on tissue sections is poor, at least in the diagnosis of S. pneumoniae.

This project aims to add to existing literature by using a cohort with a large sample size to evaluate the test performance of Gram stain on FFPE tissue sections (index test) in diagnosing bacterial skin infection. These aims will be addressed by reviewing a reviewing case notes and pathology reports as part of a retrospective and consecutive series of cases (with a time interval spanning September 2017 to the date on which data collection begins) wherein skin tissue was concurrently submitted for microbiological and histological assessment at SA Pathology. Cases will be excluded if the Gram stain result was not described in the anatomical pathology report, or if the patient was diagnosed with bacterial infection due to a pathogen which cannot reasonably be identified using Gram stain (e.g. mycobacteria).

For each case, medical records including case notes available through SA Health, microbiology reports, histopathology (anatomical pathology) reports, and other test results (e.g. white cell count, c-reactive protein) will be reviewed.

This project will not involve active participant participation, as the data to be reviewed (case notes and test results) will be that which was collected as part of the participant’s routine medical care. A waiver of consent has been granted in line with the NHRMC National Statement on Ethical Conduct in Human Research 2007 (Updated 2018).

For each case, the duration of observation will be encompassed by the review of medical records spanning the patient’s first presentation to the hospital system (with the cutaneous condition being investigated) until the point at which a working diagnosis was established and treatment initiated. For each case, relevant medical records are expected to span a one to two-week period.

The performance of the index test will then be evaluated by comparing the Gram stain results as described in anatomical pathology reports with tissue microscopy and culture results and the final diagnosis assigned to each patient’s cutaneous disease. The reference standard will be defined as the final diagnosis assigned to each patient’s cutaneous disease, determined through a process of clinicopathological correlation (i.e. the reconciliation of clinical and pathological information).
Intervention code [1] 322073 0
Diagnosis / Prognosis
Comparator / control treatment
This study will compare the test performance of Gram stain on FFPE tissue sections (index test) against the actual presence or absence of infection (reference standard). The reference standard represents the result of clinicopathological correlation i.e. in each case, the presence or absence of infection will be defined by reviewing the result of tissue microbiological studies (microscopy and culture) along with other key elements of the patient’s case notes including use of antibiotics prior to biopsy, white cell count, c reactive protein, and the documented diagnosis.
Control group
Active

Outcomes
Primary outcome [1] 329385 0
The primary outcome is the test performance (sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio) of Gram stain on FFPE tissue sections from skin biopsy specimens (index test) in diagnosing bacterial skin infection.

In evaluating the test performance of the index test, the reference standard is the final diagnosis assigned to each patient’s cutaneous disease, a diagnosis which will be defined through a process of clinicopathological correlation by reviewing the result of tissue microbiological studies (microscopy and culture) along with other key elements of the patient’s case notes including use of antibiotics prior to biopsy, white cell count, c reactive protein, and the documented diagnosis.
Timepoint [1] 329385 0
The completion of work-up for the patient’s cutaneous disease (typically within one week of performing skin biopsy).
Secondary outcome [1] 402404 0
Severity of disease among cases positive (reference standard) for infection. The nature of the treatment administered (no treatment, topical antibiotics, oral antibiotics, parenteral antibiotics, admission to intensive care unit) for the patient’s cutaneous disease, as documented in the patient's medical records, will be used as a marker of disease severity
Timepoint [1] 402404 0
The initiation and de-escalation of treatment of the patient’s cutaneous disease, typically within one to two weeks of the patient’s first presentation to the hospital system.
Secondary outcome [2] 403203 0
Distribution and frequency of non-infectious cutaneous diseases, as per the diagnosis documented in the case notes for each patient.
Timepoint [2] 403203 0
The completion of work-up for the patient’s cutaneous disease, typically within one week of performing skin biopsy.

Eligibility
Key inclusion criteria
This study will include a retrospective consecutive series of all cases encountered at SA Pathology from 4th September 2017 wherein skin tissue specimens were concurrently submitted for histological and microbiological assessment, provided histological assessment included use of Gram stain. SA Pathology is the public pathology provider for South Australia, serving patients across all age groups, and serving patients in both inpatient and outpatient settings.
Minimum age
No limit
Maximum age
No limit
Sex
Both males and females
Can healthy volunteers participate?
No
Key exclusion criteria
Cases will be excluded is the Gram stain result was not described in the anatomical pathology report, or if the patient was diagnosed with bacterial infection due to a pathogen which cannot reasonably be identified using Gram stain (e.g. mycobacteria).

Study design
Purpose
Natural history
Duration
Selection
Timing
Statistical methods / analysis
In this study, estimated sample size requirements are dependent on sensitivity, as the sample size required to calculate sensitivity is larger than the sample size required to calculate specificity. Setting alpha at 0.05, using an estimated sensitivity of 0.61 (Guarner et al, 2007), and accepting a margin of error of 0.11, the required number of positive cases for calculating sensitivity is 75. With a prevalence of skin and soft tissue infection of 10% among hospitalised patients (Ki and Rotstein, 2008), the estimated total sample size for calculating sensitivity is 750 cases.

Point estimates for sensitivity, specificity, and predictive values will be calculated by constructing a standard 2 x 2 contingency table, tabulating the results of the index test in rows against the results of the reference standard in columns, such that sensitivity is a/(a+c), specificity is d/(b+d), positive predictive value is a/(a+b) and negative predictive value is d/(c+d). The positive likelihood ratio will be calculated as sensitivity/(1-specificity) while the negative likelihood ratio will be calculated as (1-sensitivity)/specificity.
95% confidence intervals for sensitivity and specificity will be calculated using formulae reproduced by Hess et al (2012), while 95% confidence intervals for likelihood ratios will be calculated using formulae reported by Simel at al (1991).

Recruitment
Recruitment status
Completed
Date of first participant enrolment
Anticipated
13/12/2021
Actual
1/02/2022
Date of last participant enrolment
Anticipated
Actual
10/10/2022
Date of last data collection
Anticipated
Actual
10/10/2022
Sample size
Target
750
Accrual to date
Final
163
Recruitment in Australia
Recruitment state(s)
SA

Funding & Sponsors
Funding source category [1] 310037 0
Hospital
Name [1] 310037 0
SA Pathology
Country [1] 310037 0
Australia
Primary sponsor type
Individual
Name
Brendan Stagg
Address
Royal Adelaide Hospital, Port Road, Adelaide, SA 5000
Country
Australia
Secondary sponsor category [1] 311089 0
None
Name [1] 311089 0
Address [1] 311089 0
Country [1] 311089 0

Ethics approval
Ethics application status
Approved
Ethics committee name [1] 309737 0
Central Adelaide Local Health Network Human Research Ethics Committee
Ethics committee address [1] 309737 0
Roma Mitchell Building, 136 North Terrace, Adelaide, SA 5000
Ethics committee country [1] 309737 0
Australia
Date submitted for ethics approval [1] 309737 0
01/09/2021
Approval date [1] 309737 0
17/10/2021
Ethics approval number [1] 309737 0
15388

Summary
Brief summary
This study aims to determine the test performance and diagnostic accuracy of Gram stain on FFPE tissue sections in diagnosing bacterial skin infection.

Measures of test performance and diagnostic accuracy include sensitivity (the “true positive rate” or the ability of a test to identify positive cases correctly), specificity (the “true negative rate” or the ability of a test to identify negative cases correctly), positive predictive value (the probability of having a particular disease given a positive test result), negative predictive value (the probability of not have a particular disease given a negative test result), positive likelihood ratio (LR(+)) (the degree to which a person’s probability of having a particular disease is increased by having a positive test result), and negative likelihood ratio (LR(-)) (the degree to which a person’s probability of having a particular disease is decreased by having a negative test result). Although each of these parameters will be reported, our hypothesis focuses on test performance in terms of likelihood ratios, because likelihood ratios a more patient specific and therefore clinically appropriate measure of test performance.

Our hypothesis is that Gram stain on FFPE tissue sections has poor test performance in diagnosing bacterial skin infection, as defined by LR(+) of less than 2 and LR(-) of more than 0.5. These thresholds for acceptable test performance are consistent with those reported by Schecter and Sheps (1985).

If our hypothesis is confirmed, the findings of this study may encourage a more informed approach to test selection, improvements in healthcare resource utilisation, and enhance the interpretation of histopathology reports.
Trial website
Trial related presentations / publications
Public notes

Contacts
Principal investigator
Name 115214 0
Dr Brendan Stagg
Address 115214 0
Royal Adelaide Hospital, Port Road, Adelaide, SA 5000
Country 115214 0
Australia
Phone 115214 0
+61870740000
Fax 115214 0
Email 115214 0
bmj.stagg@gmail.com
Contact person for public queries
Name 115215 0
Dr Brendan Stagg
Address 115215 0
Royal Adelaide Hospital, Port Road, Adelaide, SA 5000
Country 115215 0
Australia
Phone 115215 0
+61870740000
Fax 115215 0
Email 115215 0
bmj.stagg@gmail.com
Contact person for scientific queries
Name 115216 0
Dr Brendan Stagg
Address 115216 0
Royal Adelaide Hospital, Port Road, Adelaide, SA 5000
Country 115216 0
Australia
Phone 115216 0
+61870740000
Fax 115216 0
Email 115216 0
bmj.stagg@gmail.com

Data sharing statement
Will individual participant data (IPD) for this trial be available (including data dictionaries)?
No
No/undecided IPD sharing reason/comment


What supporting documents are/will be available?

No Supporting Document Provided



Results publications and other study-related documents

Documents added manually
TypeIs Peer Reviewed?DOICitations or Other DetailsAttachment
Study results articleYes Stagg, B, Foster-Smith, E, Tam, PCK. The diagnosti... [More Details]

Documents added automatically
No additional documents have been identified.