Laboratory tests

Objectives

Identify the main causes of outbreak in the

work zone

Know the different types of samples and

diagnostic tests that can be conducted, based
on the infection dynamics

Review the role of the laboratory coordinated

with surveillance to characterize outbreaks
Content

Most frequent etiologies in the

Most commonly used tests for
Americas
diagnosing etiologic agents

– Serology
– Molecular
– Microscopic
Specimen type, collection, and
conservation

– Examples of type of sample

and appropriate conservation
Most common etiologies
in the Americas

• Febrile syndrome:
– Dengue, Chikungunya, Zika, yellow fever, influenza, leptospirosis, malaria
• Icteric febrile syndrome:
– Yellow fever, leptospirosis, malaria, hepatitis
• Hemorrhagic icteric febrile syndrome:
– Yellow fever, leptospirosis
• Hemorrhagic febrile syndrome:
– Dengue, yellow fever, leptospirosis, hantavirus
• Neurological syndromes:
– Bacterial, viral (Zika and viral encephalitis), polio, botulism
• Respiratory syndromes:
– Influenza, hantavirus, diphtheria and other respiratory viruses
• Acute diarrheic disease:
– Cholera, rotavirus, norovirus, enterobacteria
• Exanthematous disease:
– Zika, measles, rubella
Most used tests for diagnosing
etiologic agents

Serological: Molecular: Microscopic:

– ELISA (IgM and IgG) – Polymerase chain – Optical microscopy

– Direct and indirect reaction (PCR)
immunofluorescence – Real time PCR
– Sequencing
Serological tests (Serology)

Detect the presence of

Two types: direct and
antibodies
indirect tests

– Direct tests: based on the antigen

antibody reaction to investigate the
presence of antigen. That is, these
Based on the antigen antibody tests investigate the presence of
reaction and constitute a the etiologic agent or one of its
valuable tool in diagnosing components. Example: direct
immunofluorescence.
infectious disease
– Indirect tests: based on detecting
specific antibodies against the
agent or one of its components,
which indirectly allow us to assume
that the pathogen being
Cheaper than molecular tests investigated was present in the host
at some point
Serological tests
(Serology)

Important concepts for interpreting serological

results:

• Positive IgM is related to a recent infection

• Increased antibody titer in paired samples

indicates a recent infection
– First sample: negative
– Second sample: antibodies present
– Antibody titer in the second sample > 4x the antibody titer in
the first sample

• Positive IgG indicates (generally) a past

infection
Types of samples and appropriate
collection
Viral Charge

1 3 5 7 9

Days of symptoms
Antibodies

Primary Secondary
infection infection

5 15 5 15
Serological tests
(Serology)

• Immunofluorescence
– Immunofluorescence techniques may be applied as techniques to
investigate the presence of viral, parasitic, bacterial, or mycotic
antigens in clinical samples or antibodies produced against them in
the patient’s serum.

• ELISA (enzyme-linked immunosorbent assay)

– Immunoenzymatic techniques are tests based on antibody antigen
reaction, making it possible to detect the presence of antibodies
produced against viral, parasitic, bacterial, or mycotic antigens in
clinical specimens in the patient’s serum.
– The ELISA technique may be used to detect IgM or IgG.
Molecular tests

• Are based on detecting the etiologic agent’s genome.

• Make it possible to detect the etiologic agent’s RNA
or DNA with high sensitivity and specificity.
• Are more costly than serology.
• Make it possible to confirm the presence of the
etiologic agent in clinical samples.
• Make it possible to detect RNA or DNA even in very
small sample volumes.
Molecular tests

• Polymerase chain reaction (end point PCR)

– The end point PCR is based on amplifying and visualizing the
complementary DNA or ADNA (cDNA). This technique consists of
amplifying and visualizing a specific DNA fragment from the
etiologic agent.

• Real time PCR

– Also known as quantitative PCR, this test is a variant of the
polymerase chain reaction (PCR).
– Real time PCR makes it possible to amplify and at the same time
absolutely quantify specific DNA or cDNA molecules
Microscopic

• Consists of using special instruments such as

magnifying glasses and microscopes to magnify the
size of structures that are invisible to the naked
human eye, such as viruses, bacteria, and parasites.
• Microscopic observation is necessary in the field of
parasitology.
• Modes of observation vary based on strategies
employed and range from micrometric to nanometric
observation.
• Therefore, various types of microscopes are required,
such as photonic and electronic (scanning,
transmission, and atomic energy).
Microscopic

Optical microscopy

• Optical microscopy technique allows us to visualize

bacteria and parasites simply by examining the
sample with a microscope.

• For bacteria, a Gram stain (a purple stain) is often

performed first. Bacteria is classified as follows:
– Gram positive (appear blue because they retain the Gram stain)
– Gram negative (appear red because they do not capture the stain)

• A blood swab is performed to detect parasites found

in the blood, such as filariasis, malaria, or babesiosis.
For this test, a drop of blood is placed on a
microscope slide, stained, and examined under the
microscope to visualize the parasite.
Sample type, collection, and
conservation

Successful laboratory diagnosis depends on two factors prior to the

laboratory phase:

Selecting the type of sample: Sample quality:

• It is important to select the correct type • There are three factors that have a
of sample, based on clinical material direct influence on the quality of the
and days since the onset of symptoms, sample to be received by the
whenever it is possible to obtain a laboratory:
positive result (serologically or
molecularly) – Correct collection
– Conservation of the cold chain
during transport
– Conservation at the appropriate
temperature based on the
processing time
Examples of sample type and
appropriate conservation

Dengue, Chikungunya,
yellow fever, hantavirus, Zika, measles, and rubella
leptospirosis

Influenza and other respiratory

Hepatitis
viruses

Rotavirus, norovirus,
Bacterial and viral encephalitis
enterobacteria

Malaria Meningeal disease

Dengue, Chikungunya,
yellow fever, hantavirus,
leptospirosis

• Type of sample: serum

• Quantity: 3 to 7 mL
• Transport medium: no additives
• Transport conditions: 2 to 8°C
• Conservation: -20°C (up to 1 week) / -70°C (period
greater than 1 week)
• Laboratory diagnosis:
– 1 to 5 days following onset of symptoms: PCR
– 5 to 10 days following the onset of symptoms: PCR +
ELISA IgM
Zika, measles, and
rubella

• Type of sample: serum

• Quantity: 3 to 7 mL
• Transport medium: no additives
• Transport conditions: 2 to 8°C
• Conservation: -20°C (up to 1 week) / -70°C (period
greater than one week)
• Laboratory diagnosis:
– 1 to 5 following the onset of symptoms: PCR
– 5 to 10 days following the onset of symptoms: PCR +
ELISA IgM
Zika, measles, and
rubella

• Type of sample: urine

• Quantity: 3 to 7 mL
• Transport medium: no additives
• Transport conditions: 2 to 8°C
• Conservation: -20°C (up to 1 week) / -70°C (period
greater than 1 week)
• Laboratory diagnosis:
– 1 to 15 days following the onset of symptoms: PCR
Zika – congenital syndrome or
fatal cases

Transport Transport Conservation

Sample Quantity Laboratory test
medium conditions >1 week

Mother’s serum 5–7 mL No additives 4 / 8 °C -20 / -70 °C PCR, ELISA IgM, PRNT, others

Umbilical cord blood 5–7 mL No additives 4 / 8 °C -20 / -70 °C PCR, ELISA IgM, PRNT, others

Placenta 0,5–1 mL Buffered formalin 4 °C – TA* 4 °C – TA* Immunohistochemical

Placenta 5–7 mL Saline solution 4 / 8 °C -20 / -70 °C PCR

Umbilical cord (tissue) Buffered formalin 4 °C – TA* 4 °C – TA* Immunohistochemical

Umbilical cord (tissue) Saline solution 4 / 8 °C -20 / -70 °C PCR

Newborn’s blood 0,5–1 mL No additives 4 / 8 °C -20 / -70 °C PCR, ELISA IgM, PRNT, others

Newborn’s CSF** 0,5 mL No additives 4 / 8 °C -20 / -70 °C PCR, ELISA IgM, PRNT, others

Mother’s total blood 5–7 mL EDTA, others 4 / 8 °C 4 °C Biochemical, others

Newborn’s total blood 2–5 mL EDTA, others 4 / 8 °C 4 °C Biochemical, others

Tissue** 3x3 cm (approx) Buffered formalin 4 °C – TA* 4 °C Immunohistochemical

Tissue** 3x3 cm (approx) Saline solution 4 / 8 °C -20 / -70 °C PCR

* Ambient temperature *** Fatal cases: brain, liver, kidney, others

** Under medical indication for suspected neurological syndrome
Hepatitis

• Type of sample: serum

• Quantity: 3 to 7 mL
• Transport medium: no additives
• Transport conditions: 2 to 8°C
• Conservation: -20°C (up to 1 week) / -70°C (period
greater than 1 week)
• Laboratory diagnosis:
– 1 to 5 days after onset of symptoms: PCR + Elisa
HBsAg
– 5 to 10 after onset of symptoms: PCR + ELISA IgM
Influenza and other
respiratory viruses

• Type of sample: nasopharyngeal swab

• Quantity: 2 nylon swabs
• Transport medium: viral transport medium or saline
solution (3 mL)
• Transport conditions: 2 to 8°C
• Conservation: 4°C until aliquots are prepared; aliquots to
a -20°C (up to 48 hours) and -70°C (period greater than
48 hours)
• Laboratory diagnosis:
– PCR or IF (only typification) followed by PCR
Influenza and other
respiratory viruses

• Type of sample: nasopharyngeal aspiration;

nasopharyngeal wash
• Material: suction device
• Transport medium: saline solution
• Transport conditions: 2 to 8°C
• Conservation: 4°C until aliquots are prepared; aliquots to
a -20°C (up to 48 hours) and -70°C (period greater than
48 hours)
• Laboratory diagnosis:
– PCR or IF (only typification) followed by PCR
Bacterial and viral
encephalitis

• Type of sample: serum

• Quantity: 3 to 7 mL
• Transport medium: no additives
• Transport conditions: 2 to 8°C
• Conservation: -20°C (up to 1 week) / -70°C (period
greater than 1 week)
• Laboratory diagnosis:
– 1 to 5 following onset of symptoms: PCR
– 5 to 10 following onset of symptoms: PCR + ELISA IgM
Bacterial and viral
encephalitis

• Type of sample: blood

• Quantity: 3 to 7 mL
• Transport medium: no additives
• Transport conditions: 2 to 8°C
• Conservation: -20°C (up to 1 week) / -70°C (period
greater than 1 week)
• Laboratory diagnosis:
– 1 to 5 following onset of symptoms: PCR
– 5 to 10 following onset of symptoms: PCR + ELISA IgM
Rotavirus, norovirus,
enterobacteria

• Type of sample: fecal material

• Conservation: 2 to 8°C
• Laboratory diagnosis:
– Generally, molecular methods are used based
on PCR and ELISA kits for antigen detection
Malaria

• Type of sample: total blood

• Conservation: 2 to 4°C
• Laboratory diagnosis:
– Large drop (extended) for microscopy
Meningeal disease

Bacteria culture

Hemoculture

PCR

Viral isolation in cell culture

© Pan American Health Organization, 2023

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