SERO (FINALS-1)

• Methodologies of Diagnosing Bacterial Disease


  • Culture
  • Nucleic Acid Amplification Technique (NAAT)
  • Serology
• Culture


  • Gold standard; reference methodology
  • Through culture (microbiology section), you are going to identify the specific genus and species
  • You will grow and culture microorganisms and you'll be able to observe and identify them
  • You will grow it on the culture media
  • Disadvantage: some species do not grow under artificial culture medium, usually those that don't grow easily such as Mycoplasma, Intracellular, Spirochetes, Mycobacterium leprae
  • Majority of bacteria can still grow through culture - that's why it's still the gold standard
• Nucleic Acid Amplification Technique (NAAT)


  • Definitively identify the microorganism
  • Confirms genus and species based on gene sequences (DNA or RNA)
  • Sensitive and specific
• Serology


  • To identify antigen or antibody, usually the antibody detected in the patient's serum
  • 2 Methods: Slide method (screening test) and Tube method (confirmatory, quantitative)
• Bacterial Diseases


  • Syphilis
  • Anti-Streptolysin O
  • Febrile Agglutinins
  • Lyme Disease
  • Leptospirosis
  • Primary Atypical Pneumonia
  • C-Reactive Protein
  • Legionellosis
• Syphilis


  • Caused by Treponema pallidum, subspp. Pallidum
  • Acquired through sexual contact, transplacental, blood transfusion
  • One property of T. pallidum is once exposed in ref temp or 4°C for >72 hours, it is killed
• Stages of Syphilis


  • Primary
  • Secondary
  • Latent
  • Tertiary
• Primary Stage


  • Appearance of HARD CHANCRE
  • Located in the genital area
  • 1 - 5 weeks after acquiring the bacteria
  • The chancre is filled with T. pallidum
  • Diagnosed by examining under dark field microscope for motility
  • Serological test NOT RECOMMENDED YET because antibody is not yet produced
• Secondary Stage


  • Organism already spreads in the body and not just in the genital area
  • Characterized by generalized RASH and LESIONS (CONDYLOMATA LATA)
  • Patient is at the peak of antibody production
  • BEST STAGE TO DO SEROLOGY
  • MOST INFECTIOUS stage
• Latent Stage


  • Weeks/ years later, signs and symptoms/ infection might disappear
  • Appears after 2 years of infection
  • Patient is still HIGHLY CONTAGIOUS
  • Patient is ASYMPTOMATIC
  • Only method of diagnosis is through serology
• Tertiary Stage


  • Appears 3-10 years after primary infection
  • Characterized by destructive lesions called GUMMA/GUMMATAS
  • Later complications: Cardiovascular syphilis, Neurosyphilis
  • CSF can be used for diagnosis as T. pallidum has passed through the blood-brain barrier
• Specimen Sample


  • Secondary & Latent: Serum
  • Tertiary: CSF or Serum
• Stages of Syphilis


  • Primary: Hard chancre, painless, punched out appearance, indurated, raised border, red smooth base, 1-5 weeks, Lab test: Darkfield microscope, Early infection: No antibody
  • Secondary: Characterized by generalized rash, lesion (Condylomata lata), Lab test: Darkfield test and Serological
  • Late Latent: Contagious, no clinical signs and symptoms, only indication: Positive serologic test
  • Tertiary: "Gummata", 3 – 10 years after primary, Specimen of choice: CSF, Neurosyphilis
• Specific Serological Tests


  • Non-Specific/Non-treponemal: Wassermann, RPR, VDRL
  • Specific/Treponemal: Reiter Protein Group Ab, TPI, FTA-ABS, MHA-TP, TPHA
• Non-Specific/Non-treponemal Tests


  Ab produced (reagin) is not a true Ab because it also reacts or is produced for other diseases
• Specific/Treponemal Tests


  Ab produced is really specific for T. pallidum subsp. pallidum
• VDRL


  • Uses heated serum as a specimen; subjected to 56°C for 30 minutes
  • Principle: FLOCCULATION is observed due to different colloidal particles
  • Preparation of Specimen: aspirate 0.05mL along with a drop of reagent to produce serum:antigen ratio of 3:1
  • Causes of False (+) Result: SLE, Rheumatic Fever, Infectious Mononucleosis, Malaria, Pregnant Women, Rheumatic arthritis, Hepatitis, Aging, Leprosy, Pneumococcal pneumonia
• Rapid Plasma Reagin (RPR)


  • Uses unheated serum as the sample
  • Principle: FLOCCULATION
  • Causes of False (+) Result: SLE, Infectious Mononucleosis, Malaria, Leprosy, Rheumatoid Arthritis, Lyme Disease, Bejel, Yaws, and Pinta
• Fluorescent Treponemal Antibody Absorption (FTA-ABS)

  • Principle: INDIRECT IMMUNOFLUORESCENT ASSAY
  • For the absorbent, Reiter's treponemes is added to absorb the group Ab and is where the serum is being pre-diluted
  • Lyme Disease: only disease that produces a false (+) result since it causes BFP or biological false positive
• Hemagglutination Treponemal Test for Syphilis (HATTS)


  • Principle: HEMAGGLUTINATION
• Microhemagglutination T. Pallidum Test (MHA-TP)


  • Principle: HEMAGGLUTINATION
• Treponemal Pallidum Inhibition or Immobilization Test (TPI)


  • Considered to be the most specific among all treponemal testing for syphilis, and is the reference test for syphilis
  • Principle: Ab produced against T. pallidum plus the complement system will immobilize the live treponemes
  • Guinea Pig's Complement: added to remove the motility of treponeme
• Serological Test Results


  • FTA-ABS: With Fluorescence / No Fluorescence
  • HATTS: With Hemagglutination / No Hemagglutination
  • MHA-TP: Immobilized Treponeme (>50%)
• Anti-Streptolysin O


  • Ab produced because of a recent infection against S. pyogenes or the Group A Streptococcus
  • Diseases associated: Pharyngitis, Erysipelas, Scarlet fever, Nephritis, Acute rheumatic fever, Acute glomerulonephritis
• ASO Titration (Macrotechnique of Rantz and Randall)

  • Principle = NEUTRALIZATION
  • The ASO Ab actually neutralizes the streptolysin O (SLO) reagent
  • The test estimates the amount of ASO Ab that, in the presence of a constant dose of SLO, can completely inhibit the hemolysis of a control red cell suspension
• MHA-TP


  Tanned formalin sheep RBC coated with treponemal antigen
• TPI


  Live, actively, and motile T. pallidum
• Results can be Positive or Negative
• FTA-ABS


  With Fluorescence or No Fluorescence
• HATTS


  With Hemagglutination or No Hemagglutination
• MHA-TP


  Immobilized Treponeme (>50%)
• ANTISTREPTOLYSIN O (ASO) Ab is produced because of a recent infection against S. pyogenes or the Group A Streptococcus
• Diseases associated with ASO

  • Pharyngitis
  • Erysipelas
  • Scarlet fever
  • Nephritis
  • Acute rheumatic fever
  • Acute glomerulonephritis
• Controls


  • Red Cell Control (Tube 13, no hemolysis)
  • SLO Control (Tube 14, complete hemolysis)
• Normal Values: Children < 125 Todd Units, Adults < 166 Todd Units
• Rapid Latex Agglutination Test

  • Principle: Passive Agglutination
  • Positive: Agglutination of more than 200 U/mL ASO
  • Negative: No agglutination of less than 200 U/mL ASO
• Anti-DNase B Test (ABD)


  • Principle: Enzyme neutralization assay
  • Positive: Green
  • Negative: Colorless
  • Significant Titer: 1:240
• Anti-Hyaluronidase Test (AHT)


  • Principle: Serum + Streptococcal Hyaluronidase + Potassium Hyaluronidase + 2N Acetic Acid
  • Positive: Clot Formation
  • Significant Titer: 1:512
• Febrile Agglutinins


  • Classified with different bacterial diseases
  • Produces high fever when infected
  • The common factor is the antibody causes high fever
• Interpretations of Bacterial Agglutination Tests

  • A rapid slide test is only for screening, a positive test should be confirmed by tube test
  • A single negative test does not necessarily mean no infection
  • A single positive test is of no significance unless the titer is sufficiently high
  • Peak titers may occur in convalescence