# Presentation: 056806

## Bioterrorism Agents – PlagueLesson 4

- Laboratory

## Objectives

- List appropriate specimens
- List tests available for diagnosis
- List laboratory criteria for diagnosis
- Describe safety procedures

**Notes:**
Introduction and Objectives
This section covers laboratory issues that clinicians need to understand, including laboratory testing and specimen handling. At the end of this section, you should be able to: 
List the appropriate specimens to obtain in order to diagnose plague. 
List the various laboratory tests available for the diagnosis of plague. 
List the criteria for the diagnosis of plague. 
Describe safety procedures for obtaining and transporting plague specimens. 

## Overview

- Critical for first cases
- Collect immediately and before antibiotics
- Store appropriately
- Presumptive diagnoses

**Notes:**
Overview
Laboratory confirmation is especially important for the first cases in a geographic area, as they will lead to treatment and isolation of additional suspected cases, post- or pre-exposure prophylaxis of those exposed, and other control measures. 

Clinical diagnostic specimens should be collected immediately when you suspect plague and before antibiotics are administered. 

You should make every effort to collect and preserve these specimens so that live bacteria can be observed for species confirmation and characterization.  Identification of a live isolate confirms the diagnosis and permits determination of the agent’s antimicrobial susceptibility and molecular signature. 

PCR, antigen detection, or other non-viable test methods only provide presumptive identification. 

## SpecimensRespiratory

- Pharyngeal swabs
- Tracheal washes or aspirates
- Sputum specimens
- Trans-thoracic lung aspirates
- Pleural fluid collection
- Testing – Staining, culture, DNA amplification

**Notes:**
Specimens - Respiratory
  Respiratory secretions 
  Specimens 
  Pharyngeal swabs
  Tracheal washes or aspirates
  Sputum specimens
  Trans-thoracic lung aspirates
  Pleural fluid collections
  Testing
  Staining
  Culture
  Possible DNA amplification 

## SpecimensTissues

- Lung tissue
- Lymph node tissue
- Spleen
- Liver
- Testing – Culture, Gram stain

**Notes:**
Specimens - Tissue
  Tissues (biopsy) 
  Specimens   
  Lung tissue
  Lymph node tissue
  Spleen
  Liver
  Testing  
  Culture
  Gram stain

## SpecimensTissues (Autopsy)

- Lung
- Liver
- Spleen
- Lymph nodes
- Skin lesions
- Kidney
- Testing – PCR (Specimens kept fresh frozen, unpreserved)

**Notes:**
Specimens - Tissue
  Tissues (autopsy)
  Specimens
  Lung
  Liver
  Spleen 
  Lymph nodes
  Skin lesions
  Kidney
Tissue specimens should be fresh frozen and unpreserved.  These would be examined by culture for recovery of live organism and for evidence of DNA by PCR. 
Whenever possible, specimens should be split into non-glass containers. One set should be stored at 4 degrees Celsius for live recovery.  The other set should be stored frozen at minus 20 degrees Celsius or at minus 70 degrees Celsius for rapid molecular testing. 

## SpecimensBlood

- Baseline before antibiotics
- Convalescent
- Minimum 14 days apart
- 3 to 4 weeks after symptom onset
- Two or more sequentially collected samples preferred

**Notes:**
Specimens - Blood 
Blood cultures should be obtained for Y. pestis isolation before administration of antibiotics. 
Convalescent serum 
At least 14 days apart. 
Preferably 3 to 4 weeks after onset of symptoms. 
May not be practical for all patients in a mass casualty setting. Work with your health department on their needs during this time.
Plasma and whole blood dried on filter paper are acceptable alternatives to serum. 
Two or more sequentially collected samples of blood for Y. pestis are preferred. It is usually low in numbers and multiple sampling provides opportunity for a successful recovery of the live organism. 

## Specimen HandlingRespiratory, Blood

- Respiratory/Sputum
- Sterile screw-capped containers
- Room temperature (store 2C-8C if 2-24 hrs)
- Blood
- Direct to lab at room temperature
- Hold at ambient until incubator
- Do not refrigerate

**Notes:**
Specimen Handling
  Before submitting specimens for testing, you should contact your public health laboratory for the appropriate shipping and transport guidelines and to inform the agency regarding the case or incident.
  Respiratory or sputum specimens
  Sterile, screw-capped containers
  Room temperature
  If it is known that the materials will be transported within 2 to 24 hours after collection, then store the specimen between 2oC-8oC.
  Blood specimens 
  Directly to the laboratory at room temperature
  Hold them at ambient temperature until they are placed onto the blood culture instrument or incubator.  
  Do not refrigerate

## Specimen HandlingTissue

- Sterile container
- 1 to 2 drops normal saline
- Transport at room temperature
- Chill if delay
- Contact public health for detailed instructions

**Notes:**
Specimen Handling
  Tissue or aspirate 
  Sterile container
  For small samples, insert 1 to 2 drops of normal saline to keep the specimen moist. 
  Transport the sample at room temperature for immediate processing.  
  Keep the specimen chilled if processing will be delayed.
  As always, your local health department should be informed as quickly as possible.  Your local health agency is also available for consultation, or assistance with laboratory testing for plague.

## SpecimensTissue Handling

- Split into non-glass containers
- 4C for live recovery
- -20C or -70C for rapid molecular testing
- Formalin-fixed
- Tests – Histopathology, HC, PCR
- NOT frozen
- Packaged separately from unpreserved

**Notes:**
Specimens - Handling
  Whenever possible, specimens should be split into non-glass containers. 
  One set should be stored at 4 degrees Celsius for live recovery. The other set should be stored frozen at minus 20 degrees Celsius or at minus 70 degrees Celsius for rapid molecular testing. 
  Formalin-fixed tissue is suitable for histopathology, immunohistochemistry, and PCR.  It should NOT be frozen.
  Formalin-fixed specimens must be packaged separately from unpreserved autopsy specimens for virus or bacteria isolation.

## Laboratory TestingPneumonic Plague

- Collect before initiating therapy
- Sputum, blood, lymph node aspirate
- Gram Stain and culture
- Gram-negative coccobacillus
- Bipolar (safety pin) staining
- Acute and convalescent bloods for public health

**Notes:**
Specimens should be collected before initiating any therapy. 
Sputum, blood, and lymph node aspirate should be submitted for Gram stain and culture. 
Microscopic Findings: 
gram-negative coccobacillus 
Wright, Giemsa, or Wayson stains may show bipolar (safety pin) staining. 
Sera for acute and convalescent antibody detection should be obtained to assist public health authorities in their management of the case, but are not usually helpful in diagnosis due to the length of time for testing. 

## Laboratory TestingPneumonic Plague

| Leukocyte Count | Elevated |
| --- | --- |
| Differential | Neutrophil predominance, including immature forms |
| Platelets | May be normal or low |
| WBCs | 15,000-25,000/μl, left shift |
| AST, ALT, bilirubin | Elevated |

**Notes:**
Consistent with the systemic inflammatory response syndrome. 
Leukocyte count - Elevated. 
Differential - neutrophil predominance, including immature forms. 
Platelets - may be normal or low. 
WBCs 15,000-25,000/μl, left shift. 
AST, ALT, and bilirubin elevated. 

## Laboratory TestingPneumonic Plague

- Coagulation abnormalities
- Fibrin split products
- DIC possible
- Increased fibrin degradation products
- Hypofibrinogenemia
- Prolongation of PT and PTT

**Notes:**
Coagulation abnormalities 
fibrin split products. 
DIC possible. 
increased fibrin degradation products. 
hypofibrinogenemia. 
prolongation of the prothrombin time (PT) and partial thromboplastin time (PTT). 
DIC possible. 

## Laboratory Case ClassificationSuspect

- Definition
- Clinical sx and exposure compatible AND
- Small, Gram-negative and/or bipolar-staining, plump rods in singles, short chains, catalase (+) oxidase and urease (-)
- Lab Tests – Gram stain, differential stain, culture, catalase, oxidase urease

**Notes:**
Laboratory Results for Case Classification - Suspect
Definition 
Clinical symptoms and exposure history are compatible with plague infection. 
Smear or isolate from affected tissues like the lymph node, blood, or tracheal/lung aspirate that are small, gram-negative and/or bipolar-staining, plump rods in singles, short chains and that are catalase positive, and oxidase and urease negative.
Lab Tests for Suspect 
Gram stain 
Differential stain 
Culture 
Catalase 
Oxidase 
Urease

## Laboratory Case ClassificationProbable

- Definition
- Clinical sx and exposure compatible AND
- Specimen/Isolate positive by one of:  DFA, PCR, IHC
- OR single PHA/HI titer of 1:10 or positive ELISA
- Lab Tests – DFA, DNA, IHC,
- 	Serology

**Notes:**
Laboratory Results for Case Classification - Probable
Definition 
Clinical symptoms or exposure compatible with plague. 
The specimen or recovered isolate is positive by any one of the following tests: 
Direct fluorescent antibody (DFA) staining. 
DNA detection by PCR or plasmids. 
Immunohistochemical staining (IHC). 
OR
Single serum specimen that has a minimum passive hemagglutination/inhibition (PHA/HI) titer of greater than or equal to 1:10 or a positive enzyme-linked immunoassay (ELISA) titer. Known negative and positive serum samples must be run together with test samples on each test. 

Laboratory Results for Case Classification - Probable 
Lab Tests for Probable 
DFA 
DNA detection 
Immunohistochemistry 
Serology assays
These tests should be run by your public health department in Biosafety Level 2 laboratories.

## Laboratory Case ClassificationConfirmed

- Definition
- Positive DFA or DNA detection and bacteriophage lysis of recovered isolate
- OR Paired sera 4-fold rise or fall by PHA/HI or ELISA
- Tests – Bacteriophage lysis, Serology, IHC

**Notes:**
Laboratory Results for Case Classification - Confirmed 
Confirmation of Y. pestis at a Reference Laboratory level can be accomplished by one of the following sets of procedures: 
Positive DFA or DNA detection and bacteriophage lysis of a recovered isolate. 
Appropriate titer change in paired serum sample testing. 
Without an isolated culture (or 4-fold increase in titer), confirmation cannot be verified and can only be considered as presumptive.
Testing 
Biochemical tests and the Antimicrobial Susceptibility Test (E test) are also used at CDC to characterize the bacterium, but are not considered to be confirmatory. 
Bacteriophage lysis 
Serology assays 
Immunohistochemistry 

## Review Questions - Plague

- Lesson 4

## Plague Review QuestionLesson 4, Question 1

- Suspect pneumonic plague
- Sending for labs before antibiotics
- Which of the following is best for organism identification?

**Notes:**
1) Your patient is suspected of having pneumonia caused by a Y. pestis infection. You are sending them to have labs done before prescribing an antibiotic. Which of the following is the best for identifying the organism?

## Plague Review QuestionLesson 4, Question 1

- Which of the following is best for organism identification?
- Obtain initial blood specimen
- Obtain more than one blood specimen
- Get plasma or whole blood specimen on filter paper
- A smear or isolate from affected tissues

**Notes:**
Which of the following is best for organism identification?
Obtain an initial blood specimen
Obtain more than one blood specimen
Get a plasma or whole blood specimen on filter paper
A smear or isolate from affected tissues is sufficient

## Plague Review QuestionLesson 4, Question 1

- Which of the following is best for organism identification?
- D.  A smear or isolate from affected tissues

**Notes:**
D is the best answer.  A smear or isolate from the affected tissues would be sufficient to identify the organism.  However, paired sera, collected 14 days apart, would be critical for public health officials.  A blood specimen should be obtained.

For patient diagnostic purposes, it would be easier to obtain results from a smear or isolate. However, it is critical to obtain one specimen now and another in 14 days for public health management.  

## Plague Review QuestionLesson 4, Question 2

- Suspect pneumococcal plague patient
- Sputum sample
- What tests would your laboratory perform?

**Notes:**
You have obtained a sputum sample from your suspected pneumococcal plague patient.  What tests would you expect your laboratory to perform to confirm infection with Y. pestis?

## Plague Review QuestionLesson 4, Question 2

- What tests would your laboratory perform?
- PCR
- Gram stain and culture
- IHC

**Notes:**
What tests would you expect your laboratory to perform for a suspect pneumonic plague patient?

Polymerase Chain Reaction or PCR
Gram stain and culture
Immunohistochemistry, or iCH

## Plague Review QuestionLesson 4, Question 2

- What tests would your laboratory perform?
- B.  Gram stain and culture

**Notes:**
The best answer is B.
Your laboratory should perform only gram stains, differential stains, culture and biochemical screenings on specimens suspected of containing Y. pestis.  Even if sentinel laboratories have the capability of doing additional testing, the specimens should be sent immediately to public health for confirmation.  

## Plague Review QuestionLesson 4, Question 3

- Lab unable to rule out *Y. pestis*
- Specimens sent to public health
- Patient now suspect case
- At what point would public health rule them a confirmed case?

**Notes:**
Your laboratory has reported that they are unable to rule out Y. pestis and have forwarded the specimens to public health for confirmation.  At this point, your patient is considered a suspect case.  At what point will public health rule them a confirmed case?

## Plague Review QuestionLesson 4, Question 3

- At what point would public health rule them a confirmed case?
- Positive by DFA Staining
- Positive by PCR
- Isolate lysed at 22C-25C and 37C
- Single PHA/HI titer <1:10

**Notes:**
At what point would public health rule your suspect plague patient to be a confirmed case?

Their lab specimen is positive by DFA staining
Their lab specimen is positive by PCR
They have recovered an isolate that is lysed by specific bacteriophage at both ambient temperature (22oC-25oC) and at 37oC 
A single, positive serology titer by PHA/HI of less than 1:10

## Plague Review QuestionLesson 4, Question 3

- At what point would public health rule them a confirmed case?
- C.  Isolate lysed at 22C-25C and 37C

**Notes:**
The best answer is C. A recovered isolate that is lysed by specific bacteriophage at both ambient temperature and 37C would be considered confirmatory.

DFA Staining and a positive PCR is only sufficient for a probable case classification and that is only if the case is clinically compatible.  A single, positive serology titer by PHA/HI would not be sufficient for confirmation.  The titer would need to be more than 1:10 to classify this patient as a probable case and would require a convalescent serum for confirmation.

## Plague Review QuestionLesson 4, Question 4

- About to submit blood specimens
- Possible *Y. pestis*
- What should you do first?

**Notes:**
You are about to submit blood specimens for testing for possible Y. pestis.  What should you do first?

## Plague Review QuestionLesson 4, Question 4

- What should you do first?
- Treat it as you would others
- Do not refrigerate
- Contact public health epidemiology
- Contact public health laboratory

**Notes:**
Treat this specimen as you do other specimens
Do not refrigerate the specimens
C.  Contact public health epidemiology for guidance.
D.  Contact your public health laboratory for the appropriate shipping and transport guidelines.

## Plague Review QuestionLesson 4, Question 4

- What should you do first?
- D.  Contact public health laboratory

**Notes:**
The best answer would be D.  Shipping and transportation guidelines vary according to the pathogen to be shipped. Public health laboratories maintain the most up-to-date protocols regarding shipping and transport of specimens.  It important that the public health laboratory knows that plague is suspected so that they can plan appropriately and provide you with the necessary guidance.

If you suspect you’re dealing with plague, extra precaution should be taken with your specimens for the utmost safety.  Blood specimens should be kept at room temperature, unless there will be a delay in testing. By the time you’re ready to submit your specimens, you should have already talked with public health epidemiology to advise them of your suspicions, so this step should already have been completed.