HC20: Diagnostics of infectious diseases

Gram-staining

The Gram-stain test was developed by Hans Christian Gram (1853-1938). It works as follows:

1. Fixation of the bacteria on a glass slide
2. Crystal violet staining → slides through the cell wall
3. Iodine treatment → forms crystal complexes
4. Decolorization → the crystal complexes disappear from the gram-negative bacteria
5. Counter stain safranin

Streptococcus pneumoniae:

A 70-year-old woman who smokes has an increased cough with sputum. Gram-stains are made and show Gram-positive duplex cocci. She is infected by the streptococcus pneumoniae bacteria.

Ziehl-Neelsen

Ziehl-Neelsen staining is used to color acid-fast bacteria, for instance mycobacterium tuberculosis. These bacteria have a lipid in their layer which can specifically be colored to identify it as an acid-fast bacterium.

Other colorization methods

Some other colorization methods are:

• Auramine: can also stain acid-fact bacteria
  • Has a higher sensitivity but a lower specificity than Ziehl-Neelsen
• Giemsa stain: used to diagnose plasmodium falciparum in a thin blood smear
  • Plasmodium falciparum causes malaria

Rapid antigen tests

Rapid antigen tests can be useful because they are indeed rapid, however they have a very low sensitivity → the practical value is limited. SARS-CoV-2 immunochromatographic tests, for instance, have a sensitivity of 30-80%. There is a specific antibody on a piece of paper, and it is tested whether an antigen attaches to it. Immunochromatographic tests are based on pregnancy tests.

Culture of microorganisms

There is methodology based on culture of microorganisms. This is usually used for research of bacteria and viruses, but these days hardly for diagnostics. Culturing has certain characteristics:

• Time-dependent
• Specimen quality is important
• Further analysis is required
  • Morphology
  • Metabolism
  • Antigens
• Some bacteria and many viruses cannot be cultured

In contrary to bacteria, viruses can only be cultured on living cells.

Staphylococcus aureus endocarditis:

A 21-year-old man has fever, chills and a heart murmer. He has suffered from intravenous drug abuse. The doctor decides to take a blood culture:

1. The blood culture is taken and put in a bottle
2. The blood culture is left to grow
3. The culture is taken out of the bottle and put on agar
4. Gram staining and determination

This process takes a while.

Afterwards, further analysis is done:

• Rapid simple tests
  1. Catalase test: the bacteria is a gram-positive staphylococcus
  2. Coagulase test: positive → the bacteria is staphylococcus aureus
• Biochemical properties
• MALDI-TOF
• Antibiotic susceptibility testing

MALDI-TOF mass spectrometry

MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time of Flight) is a mass spectrometry test. Several steps are followed:

1. A colony of an unknown organism is selected
2. The organism is prepared onto a MALDI target plate
3. Different organisms spend a different time in the tubes
4. The MALDI-TOF profile spectrum is generated
5. The data is interpreted
6. The species are identified

PCR

PCR is a main diagnostic method these days. Cycles of heating and cooling are used to multiply DNA → PCR is a form of nucleic acid amplification:

1. Target DNA strands are selected
2. Denaturation: DNA is reduced to single strands by boiling
3. Hybridization: primer binding → separate strands receive anneal primers
4. Elongation: the primers are extended by DNA polymerase

This process is repeated multiple times. Each cycle doubles the target DNA copy number.

Molecular methods:

Molecular methods like PCR have many positive, but also negative aspects:

• Advantages
  • Universally applicable
  • Theoretically maximal sensitivity
  • Theoretically maximal specificity
  • Quantification
  • Minimal requirements on specimen conditions
  • Nearly irrelevant processing time (hours)
  • Automation is possible
• Disadvantages
  • Overshoot of specificity
  • Overshoot of sensitivity → irrelevant levels
  • Relative levels are difficult to determine
    • Unlike in blood cultures
  • Cost of tests may be higher

Serology

Serology is a form of indirect detection. It is a way of diagnosing by detecting antibodies. It has both advantages and disadvantages:

• Advantages
  • Blood samples are easy to obtain
  • Rapid
  • Cost-effective
• Disadvantages
  • A host immune response is required
  • Time from onset of disease
  • Interpretation needs to happen with care

Methods:

There are 4 different serology methods:

• Enzyme immuno-assay
• Immunoblot assays
• Immunofluorescence
• Complement fixation

Enzyme Immuno-assay

The goal of enzyme immuno-assay (EIA) is to know whether a viral antigen is present in a clinical sample. EIA can be done directly or indirectly:

• Direct: there is an indicated antibody for the detection of the substance of interest (the antigen)
  • The fluorescent marker is directly linked to the antibody
• Indirect: the fluorescent antibody targets an intermediate, which in turn targets the goal

Complement fixation test

Complement fixation methods aren't used for diagnostics anymore. They are based on the characteristics of the complement system → it is a way to distinguish if a serum has specific antibodies. Complement fixation tests are done with a serum sample:

1. A serum sample is taken that does or does not have antibodies
2. An antigen is added to the serum sample
3. A complement is added to the sample
   • If the sample has antibodies, it binds to the antigen/antibody complex
   • If the sample does not have antibodies, it remains unbound
4. Red blood cells are added → serve as an indicator
   • No red blood cell lysis → test positive
     • The complement binds to the antibodies instead of the red blood cells
   • Lysis of red blood cells → test negative
     • Red blood cells will be lysed by unbound complements

Combining:

Serology and PCR can be combined, for example in case of SARS-CoV2. This is especially used in case the PCR is negative, but the patient has symptoms. By combing PCR with serology after 7-10 days, the change of positive findings is increased.

Interpretation of test results

In the ideal world, a positive test result would mean that the disease is proven and a negative test result would mean that the disease is excluded. However, this is not the case in real life:

• Very reliable tests are often either invasive or expensive
• Most affordable diagnostic tests have limitations
  • Suboptimal sensitivity
  • Suboptimal specificity
  • Suboptimal sensitivity and specificity

Testing HIV in pregnancy:

A 26-year-old woman is 13 weeks pregnant. During her first visit to the midwife, routine blood tests are done, including HIV testing. After several days, the midwife receives the preliminary results. The ELISA for HIV antibodies is positive.

Data from the manufacturer of ELISA shows that:

• The sensitivity is 100%
• The specificity is 99,87%

HIV is present in about 0,05% of all pregnant women in the Netherlands. This is the pre-test risk. This means that the positive predictive value is 27,8%. Thus, the positive test is likely false positive → special confirmation tests are necessary.

After receiving the patient's chart, it shows a history of intravenous drug use. The HIV seroprevalence in this group in the Rotterdam area is about 10%.  This means the new pretest probability is 10% and the positive predicted value needs to be recalculated → it now is 98,8%. Thus, the positive test is now more likely to be true positive, but specific confirmation tests are still necessary.