Professional Documents
Culture Documents
Detection and Identification of Microorganism
Detection and Identification of Microorganism
Example: Salmonella typhi is initially present in peripheral blood but not in urine or stool
until at least 2 weeks after infection.
The quantity of target organisms as well as the clinical implications should be taken into
account when interpreting the significance of positive results, as molecular detection can
reveal infective agents at levels below clinical significance.
Equipment and reagents used for specimen collection are also important for molecular
testing Blood draws should go into the proper anticoagulant, if one is to be used. Although
wooden-shafted swabs may be used for throat cultures, dacron or calcium alginate swabs
with plastic shafts are recommended for collection of bacteria, viruses, and mycoplasma
from mucosal surfaces.The plastics are less adherent to the microorganisms and will not
interfere with PCR reagents as do emanations from wooden shafted swabs.
________________________ tube system-
- designed for maximum recovery of microorganisms from swabs by centrifugation.
Commercial testing kits- supply an optimized collection system for a particular test
organism. Microbiological specimens may require special handling to preserve the viability
of the target organism. Special collection systems have been designed for collection of
strict anaerobes, viruses, and other fastidious organisms. Although viability is not as
critical for molecular testing, the quality of nucleic acids may be compromised if the
specimen is improperly handled. DNA and especially RNA can be damaged in lysed or
nonviable cells. Due to the sensitivity of molecular testing, it is also important to avoid
contamination that could yield false-positive results.
SAMPLE PREPARATION
LESSON 3: VIRUSES
Molecular-based methods have benefited the laboratory diagnosis of viruses probably
more than any other organism!
In general, viruses are diagnosed by testing for antibodies against the virus, by:
1. Measuring the presence or absence of ______________
2. By detecting the growth of a virus in a _________________
Although some of these methods are well-established for certain viruses, they all have
major disadvantages associated with them.
DISADVANTAGES OF VIRAL ANTIGEN:
1. Even though laboratory testing is available for antibodies against most viruses, the
detection of antibodies against a virus is an indirect method of diagnosis. The host
immune response needs to be stimulated by the virus to produce antibodies. If a
patient is immunodeficient and does not make antibodies, the lack of antibodies is
due to host factors and not due to the lack of the virus, although the lack of
antibodies is often interpreted as a lack of the virus. Using antibodies to diagnose
an infection is often a retrospective indication of the infection. To interpret
antibody testing with the most confidence, paired sera should be collected, one
collected during the acute phase of the infection and the other collected as the
patient is convalescing, and the titers of antibodies measured in both samples. A
fourfold or greater rise in titer level from the acute sample to the convalescent
sample indicates the presence of the virus during the acute stage.
2. Detecting ____________ antibodies in particular during an acute infection
is the best evidence for the presence of that virus. But even detecting IgM, the
first isotype of antibody produced in an acute infection, is not without problems.
If the patient is in the very early stages of infection, IgM titers may be below
detection limits and would be interpreted as negative.
3. When the patient is infected with a virus and the antibodies are not detectable,
they are in the “______________” period. During this time, the patient is
infected and infectious, yet antibodies are not detectable. Antigen detection testing
is available in the clinical laboratory for only some viruses.
Assays that measure viral antigens are available more often for Respiratory Syncytial Virus,
Influenza Virus, Rotavirus, Herpes Simplex Virus (HSV), Cytomegalovirus (CMV), and
Hepatitis B Virus (HBV).
Molecular methods are well suited to target the various configurations of nucleic acids
found in human pathogenic viruses Target amplification assays such as:
1. PCR, reverse transcriptase PCR (RTPCR),
2. Quantitative (or real-time) PCR (qPCR), and
3. Transcription-mediated amplification (TMA)
As well as signal amplification assays such as branched DNA (bDNA) amplification and
hybrid capture are used in the clinical virology laboratory to diagnose or monitor viral
infections. Molecular-based tests for HIV and HCV are used more extensively in clinical
laboratories; they are discussed in more detail below.
(Image taken from Molecular Diagnostic Fundamentals, Methods and Clinical Applications
Parasites
Parasites are typically detected and identified by morphology directly in clinical specimens.
This method of diagnosis is subject to false negatives because of low organism
concentrations and depends greatly on appropriately trained personnel. Molecular-based
testing has been limited for the parasites mainly because parasites are not a major cause
of disease in developed countries. Recognition that travelers from parasite-endemic
countries bring the parasite to developed countries and can serve as a reservoir for
transmitting the parasite and that expertise in identifying parasites by morphology is
declining have made the development of molecular-based assays for parasite detection
and identification more of a need than a luxury. Recently, PCR assays have been
developed for the following parasites:
• __________________________ in patients with chronic Chagas’ disease
• __________________________
• __________________________ in water
The development of ____________ PCR assays to detect multiple parasites in stool samples
would be extremely useful. First, multiple parasites can cause diarrhea, and morphology is the
only way to differentiate between causative agents. Second, patients can have multiple
intestinal parasites at the same time, and laboratory detection of the presence of all parasites is
important. Finally, multiple parasites are transmitted in the same water supply; thus, detection
of all parasites and appropriate water treatment will reduce large-scale outbreaks of waterborne
parasites.