DETECTION AND IDENTIFICATION OF MICROORGANISM

LESSON 1: SPECIMEN COLLECTION AND PREPARATION

SPECIMEN COLLECTION
 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. Collection techniques designed to avoid contamination from the
surrounding environment of adjacent tissues apply to molecular testing, especially by
amplification methods.
 Sampling must include material from the original infection. The time and site of collection
must be optimal for the likely presence of the infectious agent.

Example: Salmonella typhi is initially present in peripheral blood but not in urine or stool
until at least 2 weeks after infection.

Classical methods (include culturing of ___________________ testing

the agent)
Sufficient number of microorganisms Minimum numbers (as few as 50
(103/mL specimen) must be obtained for organisms) can be detected successfully.
agar or liquid culture growth.

 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

PREPARED BY DON PASCUAL

 Isolating nucleic acids from microorganisms is similar to isolating nucleic acids from human
cells with only a few additional considerations.
 Considerations:
1. First, depending on the microorganism, more rigorous lysis procedures may be
required. Mycobacteria and fungi in particular have thick cell walls that are more
difficult to lyse than those of other bacteria and parasites. Gram-positive bacteria
having a thicker cell wall than gram-negative bacteria may require more rigorous
cell lysis conditions. Mycoplasma, on the other hand, lacks a cell wall, and so care
must be taken with the sample to avoid spontaneous lysis of the cells and loss of
nucleic acids.
2. Second, the concentration of organisms within the clinical sample must be
considered. Samples should be centrifuged to concentrate the fluid and the
organisms within the fluid from the milliliters of sample that are often received,
down to microliter volumes that are used in nucleic acid amplification procedures.
3. Third, inhibitors of enzymes used in molecular analysis may be present in clinical
specimens; removal or inactivation of inhibitors must be a part of specimen
preparation.
4. Finally, if RNA is to be analyzed, inactivation or removal of RNases in the sample
and in all reagents and materials that come into contact with the sample must
occur. Any clinical specimen can be used as a source of microorganism nucleic acid
for analysis. Depending on the specimen, however, special preparation procedures
may be necessary to allow for optimal nucleic acid isolation, amplification, and
analysis.

 In ______________, inhibitors of DNA polymerase have been demonstrated;

therefore, careful isolation of nucleic acid from other molecules present in the sample will
more likely result in an amplifiable sample.
 When processing a _________________, it is critical to remove all of the hemoglobin
and other products of metabolized hemoglobin because they have been shown to be
inhibitors of DNA polymerase and thus may prevent the amplification of nucleic acid in the
sample, resulting in a false negative. White blood cells, such as lymphocytes, can be used
as a source of nucleic acid for organisms, primarily viruses that infect these cells. In this
case, the cells are isolated from the red blood cells using ____________ and then lysed.
 ___________________ can also be used as a source of microorganism nucleic acid.
 __________________ is used as a source of nucleic acid from organisms that cause
respiratory tract infections. Acidic polysaccharides present in sputum may inhibit DNA
polymerase and thus must be removed.
 ______________, when sent for nucleic acid isolation and amplification, is treated
similarly to cerebrospinal fluid; the specimen is centrifuged to concentrate the organisms
and then subjected to nucleic acid isolation procedures. Inhibitors of DNA polymerase;
1. ______________
2. ______________
3. ______________
4. ______________
The type of specimen sent for molecular testing may also affect extraction and yield of
nucleic acid.
Example: Viral nucleic acid from plasma is easier to isolate than nucleic acid from
pathogenic Enterococcus in stool specimens.

PREPARED BY DON PASCUAL

 Reagents and devices have been developed to combine collection and extraction of
nucleic acid from difficult specimens; for example, stool transport and recovery (STAR,
Roche Diagnostics) buffer or the FTA paper systems that inactivate infectious agents
and adhere nucleus.

LESSON 2: BACTERIAL TARGETS OF MOLECULAR BASED TEST

 Molecular methods are extremely sensitive and specific, but these qualities are limited by
the choice of target sequences for primer or probe hybridization. The primary nucleotide
sequence of many clinically important microorganisms is now known. The specificity of
molecular methods targeting these sequences depends on the primers or probes that
must hybridize to the chosen point in the genome of the microorganism. Choosing a
sequence target is critical for the specificity of a molecular test. Many microorganisms
share the same sequences in evolutionarily conserved genes. These sequences would not
be used for detection of specific strains as they are likely to cross-react over a range of
organisms. Sequences unique to the target organism are therefore selected.
 Some organisms, such as HIV, have variable sequences within the same species. Such
variations may be informative, for instance, in determining drug resistance or for
epidemiological information; however, not all types would be detected by a single
sequence.
 The variable sequences may be included in the probe or primer areas to differentiate
between types.
 These type-specific probes/primers can be used in a confirmatory test after an initial test
using probes or primers directed to a sequence shared by all types. In addition to their
strain- or species-specificity, the target sequences must meet technical requirements for
hybridization conditions. Primers should have similar annealing temperatures and yield
amplicons of appropriate size. Probes must hybridize specifically under the conditions of
the procedure. Sequence differences can be distinguished using sequence-specific probes
or primers. Probe design includes decisions as to the length of the probe, whether the
probe is DNA, RNA, or protein; how the probe is labeled; and, for nucleic acid probes, the
length of sequences included in the probe. The source of the probe is also important, as
probes must be replenished and perform consistently over long-term use. Probes are
manufactured synthetically or biologically by cloning. Synthetic oligonucleotides
may be preferred for known sequences where high specificity is required. Primer design
includes the length and any modifications of the primers and type of signal generation for
quantitative PCR. Many tests currently used in molecular microbiology are supplied as
commercially designed systems, including prevalidated probes and/or primers. Several of
these methods are FDA-approved.
 Manufacturers of these commercial reagents provide quality assurance requirements
including controls and assay limitations. Each system must be validated on the type
of specimen used for clinical testing, including serum, plasma, cerebrospinal and other
body fluids, tissue, cultured cells, and organisms.
 In addition to the commercial reagent sets, many professionals working in clinical
laboratories have been developing laboratory protocols (home-brew PCR) for most of the
testing that they perform. Primers are designed based on sequence information that has
been published; the reagents are bought separately, and the procedures are developed
and optimized within the individual laboratory cleared.

PREPARED BY DON PASCUAL

Molecular Detection of Bacteria Molecular-based methods that have been used to detect and
identify bacteria include:

1. Nucleic acid sequence–based amplification (NASBA),

2. ____________________, and
3. _____________________
Including the following modifications:
1. ________________or quantitative
2. __________________
3. __________________
4. __________________

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).

PREPARED BY DON PASCUAL

 Viral antigens are detected by ________________ immunoassays or direct
immunofluorescent assays most often.
 The primary method used in clinical virology laboratories to detect and identify viruses in
body fluids is by _________________cultures. Monolayers of host cells are grown in
vitro, the patient’s specimen is inoculated onto the cells, and changes in the cells due to
viral infection, called cytopathic effect, are observed microscopically by the technologist.
The identity of the virus is confirmed using _______________ labeled monoclonal
antibodies.
 Culture is often the gold standard for many viruses, in particular:
1. Adenovirus
2. Enteroviruses
3. CMV,
4. Influenza, and
5. HSV
 Culture is not applicable for other viruses because the viruses do not grow in current in
vitro culture systems, such as the hepatitis viruses.

DISADVANTAGES OF VIRAL CULTURE:

1. It is the amount of time before viral growth is detectable. Although centrifuging

the sample onto the monolayer in the shell vial system has decreased detection
time; days to weeks, depending on the virus, can pass before detection of
cytopathic effect.
2. Some viruses do not produce a cytopathic effect on infecting cells, or the
cytopathic effect that is produced is subtle and easily missed. In these cases,
the cultures will be reported as a false negative.
3. Another disadvantage of using viral cultures to detect and identify viral
infections is that the specimen must be collected in the acute phase of the
disease, i.e., in the first 5 days of the illness. After 5 days, the amount of virus
in body fluids decreases significantly and may result in false-negative cultures.

 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.

PREPARED BY DON PASCUAL

Figure 1.7: Summary of the viruses for which nucleic acid amplification assays are available
(either commercially or performed in-house) along with the type of amplification procedure, the
targeted genes, and clinical utility.

(Image taken from Molecular Diagnostic Fundamentals, Methods and Clinical Applications

PREPARED BY DON PASCUAL

p.101)

LESSON 4: FUNGI AND PARASITES

Fungi
 Applications of molecular-based testing for fungal organisms are less numerous than those
for bacterial or viral organisms.
 Fungi are important causes of human disease, especially in immunocompromised
patients. Fungal infections are most often diagnosed by direct staining methods and
isolation of the causative agent in culture.
 As for other organisms, traditional smears and cultures are affected by sensitivity,
organism viability, and the length of time required for the organism to grow. In addition,
laboratory-acquired infections from fungi are a major risk for laboratory personnel. Despite
these problems, _________________ and _________________ of fungi are still
the major method for detecting fungi in clinical samples.
 To detect fungi in clinical samples, ______________ PCR and subsequent analysis
is most often used. In this assay, primers anneal to DNA sequences that are common to
most of the clinically relevant fungi, such as Candida, Aspergillus, Rhizopus and other
zygomycetes, and Histoplasma and other dimorphic fungi. Once the sequences are
amplified, hybridization to species-specific probes or sequencing is used to identify the
fungus to the genus or genus and species level. Fungi growing in culture are typically
identified by their microscopic and macroscopic morphologies. For some of the fungi,
though, gene probes have been developed to confirm the identity of the organism growing
in culture are available for:
1. ______________________,
2. ______________________,
3. ______________________
4. ________________________
 The use of DNA probes for these organisms is faster and less hazardous than determining
the microscopic morphology.

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

• _________________________ subspecies _______________ and

_________________

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 • ___________________ in blood and speciation

• ___________________ and differentiation to the species level

• ____________________ in suspected congenital and central nervous system

infections

• __________________________

• __________________________ in water

• ________________________ detection in stool and small intestine biopsies.

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.

PREPARED BY DON PASCUAL