Lesson 13 - Diagnosing Infectious Diseases

Clinical Specimens

• Various types of specimens that are collected from patients and used to diagnose or follow the progress of infectious diseases

  • E.g., blood, urine, feces, and CSF

  • High-quality specimens are crucial for accurate lab results.

  • Healthcare professionals play a vital role in specimen collection and transport.

Role of Healthcare Professionals

• Close collaboration between clinicians and the microbiology lab is essential.

• Healthcare professionals select, collect, and transport specimens, and communicate findings to clinicians.

• Safety precautions (Standard Precautions) must be strictly followed during specimen handling.

Laboratory Professionals

Make laboratory observations and generate test results that are used by clinicians to diagnose infectious diseases and initiate appropriate therapy

Importance of High-Quality Specimens

• Specimen quality directly impacts the accuracy of lab results.

• High-quality specimens require:

  • Proper specimen selection (correct type)

  • Proper specimen collection

  • Proper specimen transport

• High-quality clinical specimens are required to achieve accurate, clinically relevant laboratory results

• Lab P&P Manuals provide guidelines for specimen handling.

Proper Selection, Collection, and Transport of Clinical Specimens

• General Precautions

  • Select the correct specimen type.

  • Collect specimens aseptically to minimize contamination.

  • Collect from the site most likely to contain the pathogen.

  • Collect before antimicrobial therapy begins (if possible).

  • Collect during the acute stage of the disease

  • Collect sufficient specimen quantity.

  • Use sterile containers.

  • Transport specimens promptly to the lab.

  • Protect specimens from heat, cold, and oxygen (for anaerobes).

  • Label specimens correctly and include a completed lab requisition.

  • Handle specimens with care to prevent contamination.

  • Should be collected and delivered to the laboratory as early in the day as possible to give CML professionals sufficient time to process the material

Contamination of Clinical Specimens

• Contamination with indigenous microflora can interfere with pathogen identification.

• Proper collection techniques are essential to minimize contamination.

Type of Clinical Specimen | Blood

• Usually sterile within the body.

• Cultures are typically collected in pairs (aerobic and anaerobic bottles).

• _____ for culture is usually obtained from a vein located at the antecubital fossa

• should be incubated at 37C

• should never be refrigerated

• Aseptic technique is crucial to prevent skin contamination.

Blood | Bacteremia

• Presence of bacteria in the bloodstream.

• May or may not be a sign of disease

Blood | Septicemia

• Serious disease with bacteria or their toxins in the bloodstream.

  • Characterized by chills, fever, prostration, and the presence of bacteria or their toxins in the bloodstream

  • Most severe are caused by Gram-negative bacilli

Serum

Plasma that no longer contains clotting factors

Type of Clinical Specimen - Urine | Urine Collection

• Urine is normally sterile within the bladder.

  • Contamination occurs during urination due to contact with the distal urethra.

  • Clean-Catch, Midstream Urine (CCMS) Collection

    • Cleanse the urethral opening with soap and water.

    • Direct the initial portion of urine into the toilet.

    • Collect the midstream portion of urine in a sterile container.

  • Alternative Collection Methods: Catheterized urine collection.

    • Suprapubic needle aspiration (more invasive).

Type of Clinical Specimen - Urine | Urine Specimen Handling

• Process urine specimens within 30 minutes of collection or refrigerate at 4°C.

• Refrigerated specimens should be cultured within 24 hours.

• Failure to refrigerate can lead to bacterial overgrowth and inaccurate results.

Type of Clinical Specimen - Urine | Urine Culture Analysis, Colony Count

• A way of estimating the number of viable bacteria that are present in the urine specimen

  • Estimates the number of viable bacteria in the urine.

  • Uses a calibrated loop to inoculate a specific volume of urine onto a blood agar plate.

  • Counts colonies after incubation.

  • Calculates colony-forming units (CFU) per milliliter of urine.

Type of Clinical Specimen - Urine | Urine Culture Analysis, Isolation and Identification

Isolates and identifies the specific bacterial species causing the infection.

Type of Clinical Specimen - Urine | Urine Culture Analysis, Antimicrobial Susceptibility Testing

Determines the effectiveness of different antibiotics against the isolated bacteria.

Type of Clinical Specimen - Urine | Urine Culture Analysis, Interpretation of Results

• A colony count of 100,000 CFU/mL or higher is generally indicative of a UTI.

  • High colony counts can also result from specimen contamination or improper handling.

  • The presence of bacteria in urine is not always indicative of a UTI.

  • Two or more bacteria per 1,000 microscopic fields in a Gram-stained urine smear can suggest a UTI with a high CFU count.

Type of Clinical Specimen | Cerebrospinal Fluid (CSF)

• Obtained via lumbar puncture (spinal tap)

• Crucial for diagnosing meningitis, encephalitis, and meningoencephalitis.

• Treated as a STAT specimen due to the severity of CNS infections.

• Gram stain results are often reported immediately (preliminary report).

• All must be cultured for organism identification and antibiotic susceptibility testing

• Important: Do not refrigerate CSF specimens

Type of Clinical Specimen | Sputum

• Collected from patients with respiratory infections like pneumonia and tuberculosis.

  • Pus that accumulates deep within the lungs of a patient

  • Differentiate _______ from saliva, as saliva analysis is not informative.

  • Proper mouth hygiene is important to minimize oral flora contamination.

  • Refrigeration is generally acceptable for short periods.

  • Through bronchial lavage aspiration, a better quality specimen may be obtained

Type of Clinical Specimen | Throat Swabs

• Primarily for diagnosing strep throat (Streptococcus pyogenes).

  • Proper collection technique is crucial: swab inflamed areas or tonsils, avoid touching cheeks, teeth, or gums.

  • Specify any other suspected pathogens on the lab requisition.

Critical Value

Pertains to laboratory reports that are communicated (usually by telephone) to a health care provider and provide information that may be critical to the proper care of patient

Type of Clinical Specimen | Wound Specimens

• Aspirates are preferred over swabs to minimize contamination.

• Indicate the type of wound (e.g., dog bite, surgical site) on the lab requisition.

Type of Clinical Specimen | GC Cultures (Neisseria gonorrhoeae)

• Use a sterile swab to gather a sample from the suspected infection site, which could be the urethra (for men), cervix (for women), rectum, or throat, depending on symptoms and potential exposure;the swab is then placed in a specialized transport medium to preserve the bacteria for laboratory analysis.

  • Use Dacron or calcium alginate swabs, avoid ordinary cotton swabs.

  • Inoculate immediately onto appropriate media (Thayer-Martin, Martin-Lewis)

  • Detection can be accomplished through molecular-based assays

  • Incubate in a CO2 environment.

  • Do not refrigerate GC swabs.

Type of Clinical Specimen | Fecal Specimens

• Collect and process promptly to prevent pH changes that can kill pathogens (Shigella and Salmonella species).

• Use preservatives to maintain pH of 7.0 if immediate processing is not possible.

• Pathogens often overwhelm the normal gut flora in gastrointestinal infections.

Gastrointestinal Infections

In ________, the pathogens frequently overwhelm the indigenous intestinal microbiota, so that they are the predominant organisms seen in smears and cultures

Pathology Department (The Lab)

• Divided into two major divisions

  • Anatomical Pathology

    • Deals with autopsies, tissue examinations, cytology, etc.

  • Clinical Pathology

    • Includes the CML, Clinical Chemistry, Hematology, Blood Bank, and Immunology.

• Employs pathologists, specialized scientists, medical technologists (MTs), and medical laboratory technicians (MLTs).

Pathology Department (The Lab) | Anatomical Pathology

• Deals with autopsies, tissue examinations, cytology, etc.

• Houses the Histopathology Lab, Cytology Lab, and the Cytogenetics Lab

Pathology Department (The Lab) | Clinical Pathology

Includes the CML, Clinical Chemistry, Hematology, Blood Bank, and Immunology.

Clinical Microbiology Laboratory (CML)

• Responsible for diagnosing infectious diseases.

  • Exact steps in the processing vary depending on the specific section of the CML to which the specimen is submitted

  • Sometimes called for environmental sample processing

  • Divided into sections

    • Bacteriology, Mycology, Parasitology, Virology, Mycobacteriology.

CML | Bacteriology

• A CML category that Isolates and identifies bacteria.

  • Uses various techniques: Gram staining, colony morphology, cell shape, biochemical tests.

  • Bacterial pathogens are identified by gathering clues (phenotypic characteristics)

CML | Mycology

• Isolates and identifies fungi (yeasts, molds).

  • Uses KOH preparation, culture on Sabouraud dextrose agar (for fungi), microscopic examination of tease mounts.

  • KOH (Potassium Hydroxide Preparation)

  • Performed on hair clippings, nail clippings, and skin scrapings

  • Normally held for 3 to 4 weeks because of slow growth of many fungi

CML | Parasitology

• Diagnoses parasitic infections.

• Identifies parasites by observing characteristic microscopic features of the various life cycle stages that are seen in clinical specimens

CML | Virology

• Diagnoses viral infections.

• Uses techniques like immunodiagnostic tests, electron microscopy, cell culture, and molecular methods.

CML | Mycobacteriology (TB or AFB)

• Focuses on tuberculosis diagnosis and mycobacterial infections

• Uses acid-fast staining, culture, and biochemical tests, susceptibility testing

• Full identification of mycobacteria is accomplished by molecular probe techniques or by nucleic acid sequencing

Endoparasites

Parasite that live within the body

Parasitic Infections

They are diagnosed by observing and recognizing various parasite life cycle stages

Amplification Assays

Identify individual organisms

Multiplex Assays

Can identify a broad range of pathogens

Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF)

• It measures the mass-to-charge ratio of ionized particles

  • E.g., proteins from an organism

Types of WBC

• Granulocytes

• Monocytes

• Lymphocytes

Clean-catch (For Urinary Specimen)

Refers to the fact that the area around the external opening of the urethra is cleansed by an antiseptic towelette or washing with soap and rinsing with water before urinating

Midstream (For Urinary Specimen)

Refers to the fact that the initial portion of the urine stream is directed into a toilet or bedpan, and then the urine stream is directed into a sterile container

Chromogenic Agars

• Produce different colored colonies

• Used for fungi and yeast identification

Moulds

Identified using a combination of rate of growth and macroscopic and microscopic observations

Enzyme Immunoassays (EIAs)

They are rapid, relatively inexpensive, and easy to perform but lack the sensitivity of molecular-based assays

CML | General Process

• Examining the specimen macroscopically and recording pertinent observations

• Examining the specimen microscopically and recording pertinent observations

• Inoculating the specimen to appropriate culture media in attempt to isolate the pathogen from the specimen and get them growing in pure culture

CML | Key Responsibilities

• Process the various clinical specimens submitted to the lab

• Isolate pathogens from those specimens

• Identify (speciate) pathogens to the species level

• Perform antimicrobial susceptibility testing.

Label

It contains the patient’s name, unique hospital identification number, hospital room number, requesting clinician’s name, culture site, and date and time of collection

Laboratory Text Requisitions

This contains the patient’s name, age, sex, and unique hospital identification number

• name of the requesting clinician

• specific information about the type of specimen and the site from which it was collected

• date and time of collection

• initials of the person who collected the specimen

• information about any antimicrobial agent(s) that the patient is receiving

Plasma

• liquid portion of the blood

• makes up about 55% of the volume of blood

Types of Granulocytes

• neutrophils

• basophils

• eosinophils

-emia

refers to the bloodstream, often the presence of something in the bloodstream

Phlebotomists

these professionals collect, transport, handle, and process blood specimens for analysis

Urine Culture

it is the most common test processed in the CML

Calibrated Loop

Refers to a bacteriologic loop that has been manufactured so that it contains a precise volume of urine

Complete Urine Culture

It consists of a colony count, isolation and identification of the pathogen, and anti-microbial susceptibility testing

Potassium Hydroxide Preparation (KOH Prep)

• Acts as a clearing agent by dissolving keratin in the specimens

  • Performed on hair clippings, nail clippings, and skin scrapings

Antimicrobial Susceptibility Testing | Disk Diffusion

This testing requires a standardized inoculum of the organism to be tested

Antimicrobial Susceptibility Testing | Broth Dilution Method

This testing requires a dilution series of the antibiotic and can provide a minimum inhibitory concentration (MIC) that is defined as the lowest concentration of the antibiotic that results in no visible growth of the organism after incubation

Antimicrobial Susceptibility Testing | Gradient Diffusion Method

This testing combines some of the properties of the disk diffusion and broth dilution