1.1 Nucleic acids in human cells
There are about 6 billion bases in the human genome, of which
2-5 % code for genes. The majority of the remaining DNA has no
known function, and is often referred to as “jund” DNA on the assumption that it is indeed functionless.
There are estimated to be in theregion of 35.000 to 70 000 genes in
human DNA. The vast majority of hu man DNA is found in the cell
nucleus, and this is where most of the information on human genetic
disease in this course will be concentrated.
However, there is also an important source of DNA in the
mitochondria, which are found in the cytoplasm. These organelles
have a different arrangement of genes compared to the nucleus and
the mitochondrial genome contains only 37 genes.
Owing to a high mutaion rate in mitochondrial DNA and the fact that the
mitochondrial genome is densely packed with coding sequences, this
DNA is more important than might be expected in human genetic
disease.
Human nuclear DNA is complexed with proteins in the form of
Chromosomes. During most of the cell cycle these structures are
dispersed in the nucleus but during cell division they condense and can
be seen under the microscope with the correct preparative techniques.
Normal human cells contain 22 pairs of chromosomes (called
autosomes), one of each pair inherited from each parent, plus 2 sex
chromosomes which are known as X and Y.
Females are XX and males XY.
Figure 1.3 shows a diagram of the human chromosome complement,
which are numbered in size order from 1 to 22. The chromosomes
represented in Figure 1.3 would have been treated with a stain called
Giemsa, to give the light and dark pattern known as G banding.
Light bands are much more gene rich than the dark bands.
Figure 1.3 also shows that chromosomes have 2 arms, with a
constriction where they meet called the centromere.
The short arm is the p arm (from the French petit), the long one is
the q arm (because q comes after p). The centromere is essential for
segregation of duplicated chromosomes when they have divided at mitosis.
The end of each chromosome arm is called a telomere, and this
contains large numbers of repeated lengths of noncoding DNA.
These are very variable in number between different individuals, and
this variation is one component that is used to produce a “DNA
fingerprint”.
Most genes are interrupted by noncoding regions as summarized in Figure 1.4. RNA is copied from DNA, and initially this RNA contains
both coding and noncoding regions (Figure 1.4). The stretches of RNA
which code for amino acids are called exons, and the intervening sequences introns.
There are 2 bases at the beginning and end of every intron that are
invariant; these are GT at one end and AT at the other.
This intron/exon boundary is known as a splice junction, because during processing to produce the final mRNA the intron sequences are
spliced out at these points (Figure 1.4). The production of a messenger
RNA molecule from DNA is known as transcription (Figure 1.4), and
in part this process is controlled by particular sequences of bases upstream of the coding region. These are indicated as ‘promoter’ regions
in Figure 1.4.
There is an ‘initiation’ codon, AUG, in mRNA, where
translation into protein begins in the cytoplasm. Upstream of this site,
a specially modified nucleotide, 7-methyl guanosine (termed a ‘cap’)
is added immendiately after transcription, and most mRNAs have a series of adenosine molecules added at their 3’ end, the so-called ‘poly(A)
tail’. These are the processes referred to as capping and polyadenylation
in Figure 1.4. RNA is normally single-stranded and there are three types
of RNA molecule in a eukaryotic cell. The most important in terms of
genetic disease is mRNA, which carries the coding sequence that the
cell recognizes to translate into protein. This translation involves the other two RNA species; ribosomal RNA (rRNA) and transfer RNA
(tRNA). The former is a component of ribosomes, where the mRNA is
translated into protein, while the tRNA carries the amino acids to the ribosomes, and ensures that the correct amino acid is added as each triplet
base code.
1.2 Nucleic acid structure in micro organisms
1.2.1 Viruses
Viruses are genetic elements enclosed in a protective coat that allows
them to move from one cell to another. Viruses may be subdivided by
genome type, into those with:
• Double stranded (ds) DNA genomes (including the Adenoviridae),
which are amongst the largest of viral genomes;
• Single stranded (ss) DNA genomes, typically smal, such as parvovirus.
• ds RNA /RNA genomes, (e.g., reovirus);
• ssRNA genomes, which can be subdivided into those that funcion as
mRNA (positive sense),
• and those that are compplementary to the
mRNA produced from them (negative sense).
• Viruses with RNA genomes that use a DNA intermediate (a provirus)
to produce the RNA genome (Retroviridae);
• Viruses with DNA genomes that use an RNA intermediate stage to
produce a DNA genome (Hepadnaviridae).
The replication of RNA viral genomes occurs through the formation of
complementary strands, a process that is catalyzed by RNA
polymerase enzymes known as replicases.
As mentioned above, there are so called negative strand RNA viruses
(e.g., influenza) and positive strand viruses (such as polio).
The difference is that in the former case the infecting viral RNA does not
code for protein; it is only the complementary strand and there must
be a preformed replicase present to produce infectious strands.
In the case of positive strand viruses the viral strand is coding
and can act as a messenger RNA in the cell. Some viruses can replicate
in the cytoplasm, or can incorporate into the host cell DNA. The RNA
viruses that do this are called retroviruses, such as HIV, where part of
the genome codes for an enzyme not present in higher cells;
reverse transcriptase. This produces DNA from RNA, and in this way
the viral RNA genome can be integrated into that of the cell.
1.2.2 Bacteria
Like higher organisms, bacterial genomes are composed of DNA, from
which messenger RNA molecules are produced. There are no mitochondria and hence no mitochondrial DNA. However, there are DNA
molecules within bacterial cells which can self-replicate. They are called plasmids, and have a small circular chromosome. Plasmid DNA does
not usually encode genes with essential functions for the bacteria, they
carry resistance genes for antibiotics.
There are often multiple copies in each bacterium, and their division
occurs independently of the bacterial chromosome. E. coli, one of the
best studied bacteria has a circular chromosome of 4.700.000 bases.
Unlike human cells, bacterial DNA is not enclosed in a nucleus, but it is
centrally located within the organism.
About 90% of the bacterial DNA codes for messenger RNAs,
compared to about 5-10% in humans.
In bacteria, many genes with related functions,
such as members of particular metabolic pathways, are clustered together in what are calles ‘operons’. These clusters are transcribed as
single messenger RNA molecules. There are no introns in bacterial ge
nes, so the process of transcription from DNA to RNA is much
simpler with no splicing necessary to remove noncoding regions.
Almost all E. coli genes occur as single copies, with the exception
of those that code for ribosomal RNA, which in most strains of E.
Coli have seven copies.
Genetic elements called transposons exist in bacterial DNA (as they
do in eukaryotic DNA).
These are DNA sequences that code for enzymes which can cause a new
copy of the transposon to be inserted into another site of the
chromosome.
2.6 Some pitfalls in nucleic acid diagnosis
This is especially true of PCR where the sensitivity of the technique can
be a real problem .Great care has to be taken, as it is the PCR product,
rather than DNA from other individuals, that is the most likely cause of
contamination.
To minimize the problem, solutions for PCR are prepared in one
room, sample is added in another, and the PCR is carried out in
another.
PCR products are never allowed into the other areas. Some
laboratories have different colored lab coats for those people in each
room, and different colored pipettes, so it is immediately obvious if
anyone or anything is in the wrong place.
This may seem excessive, but there are many papers in the literature
where positive viral results have proved to be due to contamination.
Mutations in the primer sites in PCR can produce anomalous
results, especially if they are at the most 3’ position. In this case the
primer will almost certainly not amplify, and this can give the
appearance of a deletion for this region, whereas it is simply a point
mutation.
2.7 Future diagnostic research
The use of robotics is certain to increase in the next few years as DNA
testing becomes more common. The area where technology will have
the greatest impact will be the use of silicon chips .
In this method, short oligonucleotides (about 15 bases long) are attached
to a silicon chip, and this is used as a hybridization target for an
individual’s DNA.
Infectious diseases I- viruses
4.1 Introduction
Molecular probes have the advantages over conventional methods of
speed, specificity and sensitivity.In addition they may be the only applicable techniques as many microorganisms are fastidious and cannot be easily grown . This is particularly pertinent to the study of viruses
which, as obligate intracellular parasites, require mammalian cells for
their culture. Apart from detection, molecular methods are now
becoming standard technologies for establishing viral load,
particularly with regard to monitoring treatment, and resistance to
antiviral therapy.
Table 4.1 shows a comparison of molecular probes with more
conventional methods.
Viruses are obligate intracellular pararites. They possess either RNA or
DNA. From the viewpoint
of probe technologies it is useful to know the type of target nucleic
acid . In theory it should be possible to diagnose virus infections rapidly by electron microscopy and culture. In practice, the
former is insensitive, requiring about 106 virions to be present in a
sample, and the latter slow and not always possible. Molecular probes, and gene amplification, overcome these problems and have become routine procedures in the diagnostic setting.
4.2 Sample collection and preparation
Nucleic acid from DNA viruses can be processed in a manner not
dissimilar to that used for DNA from eukaryotic and bacterial sources.
RNA is, however, more labile and specimens containing viral RNA need
to be handled rigorously. Containers should be both sterile and treated
with diethylpyrocarbonate to minimize RNAse activity. Collection
should be undertaken with gloves as RNAse activity is high in sweat and
other bodily fluids. The use of RNAse inhibitors may also be required if
viral load is low. Extraction of RNA as template requires rigorously
controlled conditions and most often employs a guanidiumisothiocyanate (GI ) extraction step.
4.3 Detection of virus
4.3.1 PCR methods
For DNA viruses this is straightforward, for RNA viruses an additional reverse transcription step is required either by a separate RT enzyme or a DNA polymerase, such as Thermus thermophilus DNA polymerase, which has RT activity.
Human papillomaviruses (HPV) cannot be routinely grown in cell
culture and serological assays, are of poor sensitivity and crossreactivity of antigens. These viruses are recognized by their genotype
of which more than 120 are now recognized.
Certain types of HPV, such as 16 and 18, are strongly associated with
the risk of cervical cancer. Other types may be more common but are
not oncogenic.
PCR studies have shown that up to half of Papanicolaou smear-negative
cervical specimens carry HPV, including type 16,
suggesting that this is a more sensitive test for determining the risk
of subsequent cervical cancer. PCR also allows genotyping with the
use of type-specific primers. Other viruses that cannot be grown
routinely are shown in Table 4.3. Some viruses can be routinely
grown in cell culture but grow so slowly as to be clinically unhelpful. An example of this is the cytomegalovirus (CMV). Standard
cell culture techniques can take up to 3 weeks for positive isolation
although this can be speeded up by immunofluorescent or enzymatic detection of early antigens in 24-48 hours. Both simple probe
and amplification methods have been applied to the detection of
CMV. Positive results may occur because of latent virus which is
common in adults. In one study in which primers were used that
hybridized to regions of the MIE gene (the ‘major immediate
early’ antigen gene, an expressed nonstructural product), all 44
culture-positive neonates were PCR positive in their urine but
none of the 27 culture-negative neonates were.
The most problematic specimen type is blood as the virus is
lymphotropic and latent virus can be detected in white cells.
Unless there is bleeding into the meninges, however, CSF would not
normally be expected to harbor latent virus and its use on CSF of
AIDS patients with neurological disorders has shown PCR
to be able to detect almost all cases with disease even though cell culture was more commonly negative. With viruses expected to be
present in low concentrations, nested PCR is often used and adds to
specifity; the increased sensitivity also, however, makes the risk of false positives due to contamination. With specimens which may
have a number of different viruses or even mixed infections,
multiplex PCR methods have been developed. Specimens such as
CSF and respiratory secretions are ideal samples for the use of
multiplex PCR as the clinical features are unlikely to identify the
causative virus. Another use of multiplex PCR is to type viruses.
There are, for example,
four serotypes of dengue virus which can cause an illness ranging
from simple fever to hemorrhagic fever. It is useful to be able to type
this virus as it is recognized that infection with one serotype, following prior infection with another is a risk factor for the serious hemorrhagic fever manifestation.
Other Amplification Techniques
Following the heels of PCR, a number of alternative in-vitro amplification
techniques have been developed, of which some are now available commercially.
Examples of these alternative techniques include ligase chain reaction (LCR),
nucleic acid sequence based amplification/isothermal amplification (NASBA), and
branched DNA probes. Of these techniques, LCR, NASBA and branched DNA are
now available commercially in an automated or semi-automated format. A NASBA
assay is available for the quantification of HIV-RNA (Organon), and an LCR assay
is available for the detection of chlamydia (Abbott). Branched DNA assays are
available for the detection of quantification of HIV-RNA, HBV-DNA, and HCVRNA (Chiron).
With the exception of the branched DNA probe, all these techniques
involve exponential amplification of either the target nuclei acid or the
probe.
Therefore, they are all as susceptible to contamination as PCR.
The branched DNA system is really an intermediate between classical
hybridization techniques and the newer in-vitro amplification techniques. It is not
as sensitive as those techniques which involve exponential amplification but is
considerably more sensitive than the classical hybridization techniques. Below is a
brief summary of the features of the different amplification methods available
tion method, such as PCR ,LCR can be highly sensitive and is useful for the
detection of point mutations.
So LCR is useful for the detection of mutations which are frequent in HIV . It
depends on the ability of a thermostable DNA ligase to seal nicked double
stranded DNA.
A method of DNA amplification similar to PCR. LCR differs from PCR because it
amplifies the probe molecule rather than producing amplicon through
polymerization of nucleotides. Two probes are used per each DNA strand and are
ligated together to form a single probe. LCR uses both a DNA polymerase enzyme
and a DNA ligase enzyme to drive the reaction. Like PCR, LCR requires a thermal
cycler to drive the rxn and each cycle results in a doubling of the target nucleic
acid molecule. LCR can have greater specificity than PCR.
NASBA amplification pathway. Target ssRNA (in this case,
Noroviral genome) binds to Primer 1. An RNA/DNA hybrid is
formed by the action of reverse transcriptase. RNaseH then
degrades the RNA component of the hybrid and reverse
transcriptase using Primer 2 makes a cDNA of the target region.
Because Primer 1 contains a T7 RNA polymerase promoter, many
copies of the target RNA are made. NASBA reagents are available
from Biomerieux under the product name Nuclisens
http://www.biomerieux-usa.com/clinical/nucleicacid/index.htm
Molecular Beacon probe in the unbound hairpin conformation
(upper figure) and in the bound, fluorescent conformation.
Amplification of the target sequence can be monitored by
fluorescence measurements made every minute.
quence.
bDNA quantitative assays are commercially available for HBV,HCV and HIV.
4.3.3Molecular epidemiology
Viruses evolve through mutation and recombination events at a rate
much faster than living organisms; this is greater in those with an
RNA genome. As genetic variation is a precursor of antigenic variation, subtler differences can best be detected by genome analysis.
Two methods have been commonly employed, PCR-RE (restriction
enzymes ) and PCR-single-strand-conformation polymorphism
(PCR-SSCP) analysis.
The former is also termed PCR-restriction fragment length
polymorphism (PCR-RFLP) analysis and is restriction enzyme
digestion of PCR amplicons.PCR-SSCP employs a nondenaturing
polyacrylamide gel to compare melted single strands of amplicons.
In theory a strand with a single base mutation will migrate
differently from the non-mutant type. In practice this degree of resolution
requires meticulous technique.
4.4 Quantitative viral estimation
Quantification of viral load is becoming increasingly important as a
means of monitoring antiviral therapy of viruses such as HIV and hepatitis C. It is also used, in HIV infection, as a means for deciding
when to start therapy. PCR, nucleic acid sequence-based amplification
(NASBA) and branched chain DNA (bDNA) amplification methods
have all been applied to quantification of viral load in the clinical setting. Quantitative competitive PCR includes a target sequence mimic
which contains a template (control sequence) which is amplified as
efficiently as the actual target. Thus a comparison of, for example, HIV-1 target amplification with that of the control allows for
a value to be extrapolated. There are a number of different methods
that allow differentiation between the two sets of amplicons. The insertion of a restriction site, the inclusion of internal deletions or insertions, or replacement of a portion of the sought sequence by a novel
sequence in the control are all used. The last is used in the commercially available system from Roche.
This can be enhanced by the use of ‘real-time’ PCR to 50 copies ml-1.
NASBA is an isothermal RNA amplification system which has a
similar lower limit of detection to PCR. It is available commercially
with a linear dynamic range of 102 -107 copies ml-1 when applied to
HIV-1 quantification. This is a transcription-based amplification
system (TAS), which utilizes 3 enzyme activities:
RT, RNAse H and T7 RNA polymerase.
An oligonucleotide probe primer is bound to target RNA and the RT
makes a DNA copy. RNAse H removes the RNA portion of
the RNA-DNA hybrid and allows a second probe primer to anneal
downstream. RT then acts as a DNA-dependent DNA polymerase to
extend from one probe binding site to the other. One probe primer has
a T7 promoter site incorporated so that this enzyme can then produce
a further RNA copy to allow the process to start again. Typically a
108 - 109 amplification can be achieved.
bDNA amplification does not require an internal control template to be
quantifiable. It is a signal amplification method that uses branched
chain DNA probes that can then act as substrates for further
hybridization reactions if the template is present initially. The
technology is licensed by Chiron and has a linear range for HIV-1 of
104 –106 copies ml-1.
4.5 Measurement of antiviral resistance
Genotypic determination of viral resistance relies on identification of
mutations that confer this state. Thus, it is known that mutations in
the UL97 phosphotransferase gene and the UL54 DNA polymerase
genes of cytomegalovirus confer resistance to the antiviral drugs,
ganciclovir and/or foscarnet. For some viruses, such as HIV which
mutates frequently like most other RNA viruses, a battery of probes
could be used to look for the common mutations that confer
resistance in the RT and protease genes.
4.6 Detection of novel agents of disease
The identification of a virus, or any other micro-organism, from patients
with a disease is, of course, not sufficient grounds to establish cause and
effect. Although not conclusive, the finding of nucleic acid within a diseased cell, but not a normall cell, provides convincing evidence of aetiology. In-situ hybridization enable this.
As with other probe methods, in-situ PCR is becoming the advancement
of choice. In-situ PCR can be direct or indirect.
Indirect methods involve adding standard PCR reagents to the fixed
cells and then post-amplification of the products are detected by
standard in-situ hybridization.
Direct in-situ PCR involves the use of a labeled dNTP in the PCR
reaction so that it can detected: a biotin-or digoxigenin dUTP detected
by labeled avidin or antidigoxigenin are common examples.
VIROLOGICAL METHODS
1. Virus Isolation
Viruses are obligate intracellular parasites that require living cells in
order to replicate. Cultured cells, eggs and laboratory animals may be
used for virus isolation. Although embryonated eggs and laboratory
animals are very useful for the isolation of certain viruses, cell
cultures are the sole system for virus isolation in most laboratories.
The development of methods for cultivating animal cells has been
essential to the progress of animal virology. To prepare cell cultures,
tissue fragments are first dissociated, usually with the aid of trypsin
or collagenase. The cell suspension is then placed in a flat-bottomed
glass or plastic container (petri dish, a flask, a bottle, test tube)
together with a suitable liquid medium. e.g. Eagle's, and an animal
serum. After a variable lag, the cells will attach and spread on the
bottom of the container and then start dividing, giving rise to a
primary culture. Attachment to a solid support is essential for the
growth of normal cells.
Primary and Secondary Cultures
Primary cultures are maintained by changing the fluid 2 or 3 times a
week. When the cultures become too crowded, the cells are detached
from the vessel wall by either trypsin or EDTA, and portions are used to
initiate secondary cultures. In both primary and secondary cultures, the
cells retain some of the characteristics of the tissue from which they are
derived.
Cell Strains and Cell Lines
Cells from primary cultures can often be transferred serially a number of
times. The cells may then continue to multiply at a constant rate over
many successive transfers. Eventually, after a number of transfers, the
cells undergo culture senescence and cannot be transferred any longer.
For human diploid cell cultures, the growth rate declines after about 50
duplications. During the multiplication of the cell strain, some cells
become altered in that they acquire a different morphology, grow faster,
and become able to start a cell culture from a smaller number of cells.
These cells are immortalized and have an unlimited life-span.
Cell Cultures
Cell cultures are separated into 3 types:1. Primary cells - prepared directly from animal or human tissues
and can be subcultured only once or twice e.g. primary monkey
kidney
2. Semi-continuous diploid cells - which are derived from e.g.
human fetal tissue and can be subcultured 20 to 50 times e.g.
human diploid fibroblasts such as MRC-5
3. Continuous cells - derived from tumours of human or animal
tissue e.g. Vero, Hep2
• Cell cultures vary greatly in their susceptibility to different viruses. It is of
utmost importance that the most sensitive cell cultures are used for a particular
suspected virus. Specimens for cell culture should be transported to the
laboratory as soon as possible upon being taken. Swabs should be put in a vial
containing virus transport medium. Bodily fluids and tissues should be placed
in a sterile container.
• Upon receipt, the specimen is inoculated into several different types of cell
culture depending on the nature of the specimen and the clinical presentation.
The maintenance media should be changed after 1 hour or if that is not
practicable, the next morning. The inoculated tubes should be incubated at 3537oC in a rotating drum. Rotation is optimal for the isolation of viruses and
result in an earlier appearance of the CPE for many viruses. If stationary tubes
are used, it is critical that the culture tubes be positioned so that the cell
monolayer is bathed in nutrient medium.
• The inoculated tubes should be read at least every other day for the presence of
cytopathic effect. Certain specimens, such as urine and faeces, may be toxic to
cell cultures that may produce a CPE-like effect. If toxic effects are extensive, it
may be necessary to passage the inoculated cells. Cell cultures that are
contaminated by bacteria should either be put up again or passed through a
bacterial filter. Cell cultures should be kept for at least one to two weeks (longer
in the case of CMV).
•Cell cultures should be refed with fresh maintenance medium at regular intervals or if
required should the culture medium become too acidic or alkaline. When CPE is seen, it
may be advisable to passage infected culture fluid into a fresh culture of the same cell
type. For cell-associated viruses such as CMV and VZV, it is necessary to trypsinize
and passage intact infected cells. Other viruses such as adenovirus can be subcultured
after freezing and thawing infected cells.
Cytopathic effects of enterovirus 71, HSV, and CMV in cell culture: note
the ballooning of cells.
Cytopathic effects of mumps and measles viruses in cell culture: note the
formation of syncytia.
Influenza and parainfluenza viruses do not ordinarily induce CPE, however they
possess haemagglutinins and thus the ability to absorb guinea pig RBCs as they
bud from the cell. This phenomenon is known as haemadsorption.
Commonly employed cell cultures include primary monkey kidney, LLC-MK2
and MDCK cells. The cell cultures are incubated with a suspension of guinea pig
RBCs at 4oC or RT for 30 minutes. The unabsorbed RBCs are then removed and
the cell sheet observed microscopically for the presence of haemadsorption.
Presumptive identification of virus isolates can usually be made on the basis of the
type of CPE, haemadsorption, and selective cell culture susceptibility. For final
identification, immunofluorescence, neutralization, haemadsorption inhibition,
electron microscopy, or molecular tests are normally carried out.
Rapid Culture Techniques e.g. DEAFF test
.One of the most significant contributions to rapid diagnosis has been the
application of centrifugation cultures to viral diagnosis. The cell culture is stained
by monoclonal antibodies for the presence of specific viral antigens 24-48 hours
later. The best known example of this technique is the DEAFF test used for the
early diagnosis of CMV infection. In the DEAFF test, the specimen is inoculated
onto human embroyonic fibroblasts and then spun at a low speed. After a period
of 24-48 hours, the cells are then stained by monoclonal antibodies against CMV
early antigen. Therefore a rapid diagnosis of CMV infection can be made without
having to wait 1-3 weeks for the CPE to appear
Haemadsorption of red blood cells onto the
surface of a cell sheet infected by mumps virus.
Also note the presence of syncytia
Susceptible Cell Lines
1.Herpes Simplex
HEL),human amnion
2.VZV
3.CMV
4.Adenovirus
5.Poliovirus
6.Coxsackie B
7.Echo
8.Influenza A
9.Influenza B
10.Parainfluenza
11.Mumps
12.RSV
13.Rhinovirus
14.Measles
15.Rubella
Vero Hep-2, human diploid (HEK and
human diploid (HEL, HEK)
human diploid fibroblasts
Hep2, HeLa,
MK, BGM, LLC-MK2, human diploid,Vero,
Hep-2, Rhadomyosarcoma
MK, BGM, LLC-MK2, vero, hep-2
MK, BGM, LLC-MK2, human diploid, Rd
MK, LLC-MK2, MDCK
MK, LLC-MK2, MDCK
MK, LLC-MK2
MK, LLC-MK2, HEK, Vero
Hep-2, Vero
human diploid
MK, Vero
Vero, RK13
2. Electron Microscopy
Virus diagnosis by electron microscopy relies on the detection and identification of
viruses on the basis of their characteristic morphology. A major advantage of virus
diagnosis by EM is the ability to visualize the virus.
Speed is another advantage of EM as the specimen can be processed within
minutes of receipt and thus EM can be used as a rapid diagnostic method.
On the other hand, the main disadvantage of EM is its inability to examine
multiple specimens coincidentally.
Secondly, there must be a minimum number of virus particles present (around 106
virus particles per ml for detection) Some viruses may give a non-distinct
morphological appearance which may make detection very difficult.
Finally, EM is a very expensive service to provide and requires highly skilled
personnel.
EM has found a particular niche in the detection of fastidious gastroenteritis
viruses such as rota, adeno, astro, Norwalk, and Caliciviruses.
In addition EM may be used to confirm the results of virus isolation by cell
culture such as for parainfluenza viruses.
There are two types of EM methods;- direct or immunoelectron microscopy
(IEM). With direct methods, negative staining is normally used which requires
little special equipment, in contrast to thin sectioning techniques. The specimens
may be used directly or the virus particles may be concentrated before negative
staining. Immunoelectron microscopy is a means of increasing the sensitivity and
specificity of EM and is particularly useful in the following situations;1.The number of virus particles present is small.
2.Many different viruses have different morphology e.g. herpesviruses and
picornaviruses. IEM may identify the virus
There are 2 types of IEM, simple IEM, where the specimen is incubated with
specific antibody before staining in the hope that the antibody will agglutinate the
specimen, and solid phase IEM (SPIEM), where the copy grid is coated with
specific antibody which is used to capture virus particles from the specimen.
Electronmicrographs of viruses commonly found in stool specimens from patients
suffering from gastroenteritis. From left to right: rotavirus, adenovirus,
astroviruses, Norwalk-like viruses.
ELISA
ELISA was developed in 1970 and became rapidly accepted. A wide variety of
assay principles can be used in ELISA techniques. Currently the most important
ones are;
1.Competitive methods
2.Sandwich methods
3.Antibody capture methods
Competitive methods
One component of the immune reaction is insolubilized and the other one labeled
with an enzyme. The analyte can then be quantified by its ability to prevent the
formation of the complex between the insolublized and the labelled reagent.
Advantages of this approach are that only one incubation step is necessary and
that the "prozone effect" at high analyte concentrations cannot occur.
Disadvantages are that the concentration range in which the analyte can be
quantified without sample dilution is rather narrow and that the antigen or
antibody therefore cannot be distinguished in a one step assay
Sandwich method
The method in which the same component of the immune reaction (e.g. the
antibody) is used in the insolubilized and the enzyme in the labelled form.
Indirect Method
1.The method in which one component (usually the antigen) is used in an
insolubilized form to bind the analyte from the sample (the antibody),which is
subsequently determined by addition of labelled second antibody against the
same class of antibody as the analyte antibody .
Modification of the test in so that antibodies of a specific class such as IgM,
can be detected in the sample.
Also RF ( rheumatoid factor ) is known to be a potentially interfering factor.
Antibody capture methods
These methods used to detect antibodies of specific immunoglobulin subclasses, by
first reacting the sample with e.g. insolubilized anti-IgM,and subsequently with
either enzyme labelled antigen or with antigen followed by enzyme linked
antibody. Neither antibodies from other immunoglobulin subclasses nor
rheumatoid factor interfere significantly in such assays. They are widely used for
the diagnosis of acute infections by IgM detection. These assays may be used for
detecting IgG and IgA.
Direct Competitive
ELISA:
operate on the basis
of competition
between the
horseradish
peroxidase (HRP)
enzyme conjugate
and the analyte in
the sample for a
limited number of
specific binding
sites on the
precoated
microplate
Competitive or Blocking ELISA
In a competitive ELISA, a patient
serum and an Ag-specific conjugate
(pink) are co-incubated with a
captured Ag.
The amount of colour developed is
inversely proportional to the
amount of Ag-specific patient Ig
present. Careful standardisation is
required to interpret the results
In a blocking ELISA, the patient
serum is added first, incubated and
the excess washed off.
Next an Ag-specific conjugate is
added and the results interpreted
as above. Titres here may be lower
if the conjugate is of a high enough
titer to displace patient Ab.
In a variation of this format, a conjugated Ag is used the
competitor.
Types of
ELISA
Direct
ELISA
Direct ELISA is the most basic of ELISA
configurations.
It is used to detect an Ag (red triangle;
virus/bacteria/fungus, recombinant
peptide/protein, or another Ab) after it has been
attached to the solid phase (eg. a membrane or
Types of ELISA
polystyrene microwell or dipstick).
Direct ELISA
An Ab (green), conjugated with a label (yellow
star; eg. HRPO, AP, FITC) is then incubated with
the captured antigen. After washing off excess
conjugate and incubating with a substrate and
chromogen, the presence of an expected colour
indicates a specific Ab-Ag interaction.
The conjugate could be a commercial prepartion
specific for the Ag of interest, or an in-house
conjugated monoclonal or polyclonal Ab, or even
patient serum.
Indirect
Once again an Ag is adsorped onto a solid
phase. The first, or primary Ab (green) is
incubated with the Ag, then the excess is
washed off. A second or secondary Ab
(blue), the conjugate, is then incubated with
the samples.
The excess is again removed by washing.
For colour to develop, a primary Ab that is
specific for the Ag must have been present in
the sample (eg. human serum, CSF or saliva
or the supernatant from a hybridoma
culture).
This indiactes a positive reaction.
It is important, during assay optimisation, to
ensure that the secondary Ab does not bind
non-specifically to the Ag preparation or
impurities within it, nor to the solid phase.
Capture ELISAs
Antigen Capture
In this, more specific approach, a capturing Ab
(orange) is adsorped onto the solid phase. The
capture antibody may be the reagent to be
tested (eg. the titre of a patients immune
response to a known Ag). However, the Ab
may be a standard reagent and the antigen
the unknown(as when a patients serum is
being investigated for the presence of a
microbial infection).
The same stringent optimisation is required as
for Indirect ELISA. This will ensure that the Ab
do not cross-react in the absence of Ag, or
non-specifically bind to the solid phase.
It is also important, when detecting the Ag, to
use Ab from different animal species to
prevent same-species Ab binding.
Antibody Capture
In this approach, a capturing Ab (orange) is
adsorped onto the solid phase. The Ab is
designed to capture a class of human Ab
(green; eg. IgG, IgA or IgM). Next the sample
is applied, containing the Ab under
investigation. After washing, an Ag (red)
specific for the Ab is added and finally an antiAg conjugate (blue) provides the signal.
Assay Characteristics
The use of monoclonal antibodies has lead to many improvements in ELISA
systems.
1.Higher sensitivity ;- either by selection of antibodies with a extremely high
affinity, which makes very low concentrations of analyte more readily
detectable.
2.Higher specificity ;- by avoiding the presence of any antibody in the assay
system with specific reactivity against non-analyte epitopes, and by selecting
combinations of monoclonal antibodies which may further increase specificity.
The enzyme label ;
-Most of the assays employ horse-radish peroxidase, alkaline phosphatase, or B-Dgalactosidase. Methods are available to detect horse radish peroxidase by means
of chemilumininescence.
- TMB is gradually replacing mutagenic substrates such as OPD, leading to
increased sensitivity and safety.
Microplate ELISA: coloured wells indicate reactivity. The darker the colour, the
higher the reactivity
Immunofluorescense
Immunofluorescence (IF) is widely used for the rapid diagnosis of virus infections
by the detection of virus antigen in clinical specimens, as well as the detection of
virus-specific IgG or IgA or IgM antibody. The technique makes use of a
fluorescein- labelled antibody to stain specimens containing specific virus antigens,
so that the stained cells fluoresces .
In the case of direct IF, the specimen is probed directly with a specific labelled
antibody against a particular virus antigen.
In the case of indirect IF, the specimen is first probed with a non-labelled specific
antibody, followed by a labelled antibody against the first antibody. Direct or
indirect IF can be used for the detection of virus antigen, whereas indirect IF is
virtually always used for the detection of antibody. Indirect IF possess the
advantage of an extra amplification step for the signal, however, it requires an
extra step in comparison to direct IF.
Detection of viral antigens
Nasopharyngeal aspirates are the best specimens to use and is usually collected
from babies less than 12 months old. A number of respiratory viruses can be
detected by direct or indirect IF, including RSV, influenza A and B, adenoviruses
and parainfluenza viruses. However, the sensitivities vary greatly between
different viruses. The method is most useful in the case of RSV ,is also widely used
for the detection of HSV infections, from vesicle lesions and for VZV.
A typical indirect IF procedure for the detection of viral antigens is as follows;
-cells from the clinical specimen are immobilized onto individual wells on a slide.
Specific polyclonal or monoclonal sera is then added to each well and the slide is
incubated at 37oC for 30 to 60 minutes. The slide is then washed 3 times for 5
minutes each with PBS and fluorescein labelled antibody against the first antibody
is added.
-The slide is further incubated at 37oC for 30 to 60 minutes and washed again. The
slide is then prepared for microscopy. Specific monoclonal or polyclonal sera
raised against the viral antigen can be used.
Monoclonal sera offer the advantage of increased sensitivity and specificity.
However, one must be certain that it can detect all the different strains of the virus.
Positive immunofluorescense tests of HSV antigen from epithelial cell and CMV
pp65 antigen from peripheral blood neutrophils. Right: Positive
immunofluorescense test of rabies virus antigen (CDC)
Detection of viral antibodies
IF is probably the simplest serological assay to set up. It simply requires virally
infected cells that express viral antigens and a fluorescein-labelled antiserum
against human immunoglobulin. IF can be used to detect IgG, IgM and IgA. IF is
extensively used for the diagnosis of EBV infections .
8. Neutralization
Neutralization of a virus is defined as the loss of infectivity through reaction of the
virus with specific antibody. Virus and serum are mixed under appropriate
condition and then inoculated into cell culture. The presence of unneutralized
virus may be detected by reactions such as CPE. The loss of infectivity is bought
about by interference by the bound Ab with any one of the steps leading to the
release of the viral genome into the host cells.
Stable neutralization - with time, Ag-Ab complexes usually become more
stable (several hours) . Such neutralization is generally produced by Ab molecules
that establish contact with 2 antigenic sites on different monomers of a virion,
greatly increasing the stability of the complexes. An example of stable
neutralization is the neutralization of polioviruses, whereby, the attachment of the
antibody to the viral capsid stabilizes the capsid and inhibits the uncoating and
release of viral nucleic acid.
Viral evolution must tend to select for mutations that change the antigenic
determinants involved in neutralization. In contrast, other antigenic sites would
tend to remain unchanged because mutations affecting them would not be selected
for and could even be detrimental. Because of its high immunological specificity,
the neutralization test is often the standard against which the specificity of the
other serological techniques is evaluated.
Before the neutralization test is carried out, the components that are to be used
must be standardized.
To identify a virus isolate, a known pretitred antiserum is used. Conversely, to
measure the antibody response of an individual to a virus, a known pretitred virus
is used.
To titrate a known virus, serial tenfold dilutions of the isolate is prepared and
inoculated into a susceptible host system such as cell culture . The virus endpoint
titre is the reciprocal of the highest dilution of virus that infects 50% of the host
system eg. 50% of cell cultures develop CPE.
This endpoint dilution contains one 50% tissue culture infecting dose (TCID50) or
one 50% lethal dose (LD50) of virus per unit volume. The concentration of virus
generally used in the neutralization test is 100 TCID50 or 100 LD50 per unit
volume.
The antiserum is titrated in the neutralization test against its homologus virus.
Serial twofold dilutions of serum is prepared and mixed with an equal volume
containing 100TCID50 of virus. The virus and serum mixtures are incubated for 1
hour at 37oC. The time and temperature for incubation varies with different
viruses. The mixtures are then inoculated into a susceptible host system.
The endpoint titration contains one antibody unit and is the reciprocal of the
highest dilution of the antiserum protecting against the virus.
Generally 20-100 antibody units of antiserum is used in the neutralization tests
Serology
Following exposure, the first antibody to appear is IgM, which is followed by a
much higher titre of IgG. In cases of reinfection, the level of specific IgM either
remain the same or rises slightly. But IgG shoots up rapidly and far more earlier
than in a primary infection. Many different types of serological tests are available.
With some assays such as EIA , one can look specifically for IgM or IgG, whereas
with other assays such as CFT and HAI, one can only detect total antibody, which
comprises mainly IgG.
Newer techniques such as EIAs offer better sensitivity, specificity and
reproducibility than classical techniques such as CFT and HAI. The sensitivity and
specificity of the assays depend greatly on the antigen used.
Assays that use recombinant protein or synthetic peptide antigens tend to be more
specific than those using whole or disrupted virus particles.
Criteria for diagnosing Primary Infection
1.A significant rise in titre of IgG/total antibody between acute
and convalescent sera
- however, a significant rise is very difficult to define and depends greatly on
the assay used. In the case of CFT and HAI, it is normally taken as a four-fold
or greater increase in titre.
The main problem is that diagnosis is usually retrospective because by the
time the convalescent serum is taken, the patient had probably recovered.
1.
Presence of IgM - EIA and IF are used for the detection of IgM. This
offers a rapid means of diagnosis.
2.
However, there are many problems with IgM assays, such as interference
by rheumatoid factor, re-infection by the virus, and unexplained
persistence of IgM years after the primary infection.
3.
Seroconversion - this is defined as changing from a previously antibody
negative state to a positive state e.g. seroconversion against HIV following
a needle-stick injury, or against rubella following contact with a known
case.
4.
A single high titre of IgG (or total antibody) - this is a very unreliable
means of serological diagnosis since the cut-off is very difficult to define.
Serological events following
primary infection and reinfection.
Note that in reinfection, IgM may
be absent or only present
transiently at a low level.
Criteria for diagnosing re-infection/reactivation
It is often very difficult to differentiate
re-infection/re-activation from a
primary infection. Under most
circumstances, it is not important to
differentiate between a primary
infection and re-infection. However, it
is very important under certain
situations, such as rubella infection in
the first trimester of pregnancy:
primary infection is associated with a
high risk of fetal damage whereas reinfection is not. In general, a sharp
large rise in antibody titres is found in
re-infection whereas IgM is usually
low or absent in cases of reinfection/re-activation.
Limitations of serological diagnosis
How useful a serological result is depends on the individual virus.
1.For viruses such as rubella and hepatitis A, the onset of clinical symptoms
coincide with the development of antibodies. The detection of IgM or rising
titres of IgG in the serum of the patient would indicate active disease.
2.However, many viruses often produce clinical disease before the appearance
of antibodies such as diarrhoeal viruses. So in this case, any serological
diagnosis would be retrospective and therefore will not be so useful.
3.There are also viruses which produce clinical disease months or years after
seroconversion e.g. HIV and rabies. In the case of these viruses, the mere
presence of antibody is sufficient to make a definitive diagnosis.
There are a number of problems associated with serology:
1.long length of time required for diagnosis for paired acute and convalescent
sera
2.mild local infections such as HSV genitalis may not produce a detectable
humoral immune response
3.Extensive antigenic cross-reactivity between related viruses e.g. HSV and
VZV, may lead to false positive results
4.immunocompromised patients often give a reduced or absent humoral
immune response.
Antibody in the CSF
In a healthy person, there should be little or no antibodies in the CSF.
Where there is a viral meningitis or encephalitis, antibodies may be produced
against the virus by lymphocytes in the CSF.
The finding of antibodies in the CSF is said to be significant when ratio between
the titre of antibody in the serum and that in the CSF is less than 50-80.
But this does depend on an intact blood-brain barrier. The problem is that in
many cases of meningitis and encephalitis, the blood-brain barrier is damaged, so
that antibodies in the serum can actually leak across into the CSF.
This also happens where the lumbar puncture was traumatic in which case the
spinal fluid would be bloodstained.
So really, one should really check the integrity of the blood-brain barrier before
making a definite diagnosis.
One way to check the integrity of the blood brain barrier is to use a surrogate
antibody that most individuals would have, such as measles virus, since most
people would have been vaccinated.
If the blood-brain barrier is intact, there should be little or no measles antibodies
in the CSF.
Human infectious diseases II
bacteria, fungi, and protozoa
5.1 Introduction
It is estimated that up to 95% of bacteria present in a sample are noncultivable. Cheaper cultural methods will, no doubt, be used as a standard
but there are four areas where nucleic acid based diagnosis will be the
method of choice:
• The identification and characterization of fastidious organisms;
•Rapid typing of isolates for epidemiological and other purposes;
•Rapid determination of antimicrobial resistance;
• Identification of organisms in a noncultivable state
Bacteria are responsible for the majority of currently treatable infectious
diseases and because of this an accurate rapid diagnosis and determination of susceptibility to antibiotics is important. In addition, typing is important for epidemiology and, ultimately, control of these organisms.
5.2 Specimen collection and preparation
Material collected may range from the sterile, relatively acellular, environment of cerebrospinal fluid to the highly complex milieu of food.
The more complex the environment, the more likely that there will be
substances that interfere with hybridization or inhibit amplification.
Thus, specimens need to be collected in a manner likely to stabilize nucleic acids for transport to the laboratory . Apart from the special circumstance when differentiation between viable and nonviable bacteria
is required, DNA is the target and RNAase inhibition is not usually required. Obviously procedures should be as sterile as possible to prevent
the addition of exogenous micro-organisms. Further purification before
probing or PCR may not be required when bacterial load is likely to be
high such as with acute meningitis. If significant nonbacterial contamination is likely, then additional steps to ‘purify’ the bacterial DNA may
be required.
Proteinase K digestion, alkaline lysis are commnonly used.
5.3 Identification
The bacterial genome consists of a circular chromosome. In addition
there may be extra-chromosomal elements on plasmids. Methods have
been developed which facilitate the detection of chromosomal, plasmid
or total DNA in a wide range of bacteria. In addition the advent of more
advanced genotypic methods, such as PCR, has enabled the detection of
previously undiscovered pathogens. Indeed molecular methods have
been important in the understanding of pathogenesis. The use of probes
to 16S ribosomal RNA sequences is peculiar to bacterial diagnostics
enabling both detection and phylogenetic analysis.
CULTURE AND IDENTIFICATION OF INFECTIOUS AGENTS
Bacterial identification in the diagnostic laboratory versus taxonomy
Isolation and identification of bacteria from patients aids treatment
since infectious diseases caused by different bacteria have a variety
of clinical courses and consequences. Susceptibility testing of isolates
(i.e. establishing the minimal inhibitory concentration or MIC) can
help in selection of antibiotics for therapy. When patients are
suspected of having a bacterial infection, it is usual to isolate visible
colonies of the organism in pure culture (on agar plates), and then
speciate the organism. The identification is based on taxonomic
principles applied to the clinical microbiological situation. In the
diagnostic laboratory, many samples must be characterized each day
and results obtained as quickly as possible.
Classical methods for speciation of bacteria are based on
morphological and metabolic characteristics. There are numerous
different tests for each of the many target pathogens.
Additionally, molecular biology techniques (for characterization of specific
genes or gene segments) are now commonplace in the clinical laboratory.
Taxonomic terms (classification)
Family: a group of related genera.
Genus: a group of related species.
Species: a group of related strains.
Type: sets of strain within a species (e.g. biotypes, serotypes).
Strain: a single isolate of a particular species.
The most commonly used term is the species name (e.g.
Streptococcus pyogenes - abbreviation S.pyogenes). There are always
two parts to the species name, one defining the genus in this case
"Streptococcus" and the other the species (in this case "pyogenes").
The genus name is always capitalized but the species name is not.
Both species and genus are underlined or in italics.
Steps in diagnostic isolation and identification of bacteria
Step 1. Samples of body fluids (e.g. blood, urine, cerebrospinal fluid)
are streaked on culture plates and isolated colonies of bacteria
appear after incubation for one to several days . Each colony consists
of millions of bacterial cells.
Observation of these colonies for size, texture, color, and (if grown
on blood agar) hemolysis reactions, is highly important as a first step
in bacterial identification.
Whether the organism requires oxygen for growth is another
important differentiating characteristic.
Step 2. Colonies are Gram stained and individual bacterial cells
observed under the microscope.
Step 3. The bacteria are speciated using these isolated colonies. This
often requires an additional 24 hours of growth.
THE GRAM STAIN
A colony is dried on a slide and treated as follows :
Step 1. Staining with crystal violet.
Step 2. Fixation with iodine stabilizes crystal violet staining. All
bacteria remain purple or blue.
Step 3. Extraction with alcohol or other solvent. Decolorizes some
bacteria (Gram negative) and not others (Gram positive).
Step 4. Counterstaining with safranin.
Gram positive bacteria are already stained with crystal violet and
remain purple. Gram negative bacteria are stained pink.
Taxonomic characterization of bacteria
There is considerable diversity even within a species.
Comparisons are primarily based on chemical or molecular analysis.
Chemical and Molecular Approaches:
•Cell wall composition.
•Membrane lipid signatures.
•Electrophoretic comparison of proteins.
•Nucleic acid hybridisation.
•Gene sequence comparisons.
Molecular analysis
It would be ideal to compare sequences of entire bacterial
chromosomal DNA, but this is currently not feasible. Millions of
nucleotides have to be sequenced for each strain.
In the past several years, sequencing of the entire genomes of one
representative (i.e. a strain) of a few bacterial species has been
achieved. In each case, this has involved massive amounts of work by
large research groups dedicated to the task of sequencing.
Alternatively, genomic similarity has been historically assessed by
the content of guanine (G) plus cytosine (C), usually expressed as a
percentage (% GC). This has been replaced by two alternatives hybridization and sequencing (most commonly of the gene coding for
16S rRNA).
DNA-DNA homology is employed to compare the genetic relatedness
of bacterial strains/species. If the DNA from two bacterial strains
display a high degree of homology , the strains are considered to be
members of the same species. DNA from different bacterial species
(unless closely related) display no homology.
In the last few years, sequencing of 16S ribosomal RNA molecules
(16S rRNA) has become the "gold standard" in bacterial taxonomy.
The molecule is approximately sixteen hundred nucleotides in
length. The sequence of 16S rRNA provides a measure of genomic
similarity above the level of the species allowing comparisons of
relatedness across the entire bacterial kingdom.
Closely related bacterial species often have identical rRNA
sequences.
Bacterial DNA sequences can be amplified directly from human
body fluids (the polymerase chain reaction, PCR). For example,
great success has been achieved in rapid diagnosis of tuberculosis.
Serologic identification of an antibody response (in patient's serum)
to the infecting agent can only be successful several weeks after an
infection has occurred.
Genotypic Identification
Differences in the DNA base sequences between different organisms
can be determined quantitatively, such that a phylogenetic tree can
be constructed to illustrate probable evolutionary relatedness
between the organisms.
The nucleotide base sequence of the gene which codes for 16S
ribosomal RNA is becoming an important standard for the definition
of bacterial species. Comparisons of the sequence between different
species suggest the degree to which they are related to each other; a
relatively greater or lesser difference between two species suggests a
relatively earlier or later time in which they shared a common
ancestor.
A comparison between species of gram-negative bacteria is
illustrated.When the sequences are aligned such that similarities and
differences can be readily seen . Gaps and insertions of nucleic acid
bases (the result of "frame-shift" mutations occuring over eons of
time as the organisms diverge from common ancestors) which affect
long stretches of DNA have to be taken into account for a proper
alignment.
The organisms are as follows:
•Neisseria sicca .
•Pseudomonas fluorescens.
•Three enteric-like organisms: Vibrio cholerae, Photobacterium
phosphoreum and Plesiomonas shigelloides.
•Three relatively obscure enterics: our new organism (shown by
initials "AH" ), Budvicia aquatica and Edwardsiella tarda.
•Escherichia coli – the archetypal enteric – with two closelyrelated species, Shigella dysenteriae and Escherichia hermannii.
"AH" (our new organism)
gataactactggaaacggtagctaataccgcataacgtcgcaagaccaaa
gcgggggaccttagggcctcgcgccatcagatgaacccagatgggattag
ctagtaggtggggtaatggctcacctaggcgacgatccctaactggtctg
agaggatgaccagtcacactgggactgagacacggcccagactcctacgg
gaggcagcagtggggaatattgcacaatgggcgcaagcctgatgcagcca
tgccgcgtgtgtgaagaaggccttcgggttgtaaagcactttcagtgagg
aggaaggcgttgcagttaatagctgtagcgattgacgttactcacagaag
aagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaa
gcgttaatcggaattactgggcgtaaagcgcacgcaggcggtttgttaag
tcagatgtgaaatccccgggctcaacctgggaactgcatttgaaactggc
aggctagagtctcgtagaggggggtagaattccaggtgtagcggtgaaat
gcgtagagatctggaggaataccggtggcgaaggcggccccctggacgaa
gactgacgctcaggtgcga.agcgtggggagcaaacaggattagataccc
tggtagtccacgctgtaaacgatgtcgatttggaggttgtgcccttgagg
cgtggcttccggagctaacgcgttaaatcgaccgcctggggagtacggcc
gcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggag
catgtggtttaattcgatgcaacgcgaagaaccttacctactcttgacat
ccagagaaggttccagagatgggactgtgccttcgggagctctgagacag
gtgctgcatggctgtcgtcagctcgtgttgtgaaatgttgggttaagtcc
cgcaacgagcgcaacccttatcctttgttgccagc.acttcgggtgggaa
ctcaagggagactgccggtgataaaccggaggaaggtggggatgacgtca
agtcatcatggcccttacgagtagggctacacacgtgctacaatggcgca
tacaaagagaagcgaacttgcgagagtaagcggacctcataaagtgcgtc
gtagtccggattggagtctgcaactcgactccatgaagtcggaatcgcta
gtaatcgtagatcagaatgctacggtgaatacgtccccgggccttgtaca
caccgccc
When a 1308-base stretch of that part of the chromosome which
codes for 16S ribosomal RNA was lined up and analyzed to find the
extent to which four of the above organisms differed from each
other, the percent difference between any two organisms was
determined, and the results are summarized as follows:
PF
PF
AH
14.8*
AH
BA
14.5
3.2
BA
ET
14.9
4.3
5.0
ET
* An example: The same bases appear in the same sequence, position by
position, for each of the two organisms except for 14.8% of the time.
With the percent differences used to denote probable evolutionary
distances between the organisms, a phylogenetic tree was roughed out
to illustrate the relationships. The distances between any two organisms,
when read along the horizontal lines, corresponds closely to the percent
differences. (The bar at the bottom signifies approximately 1% base
difference.)
Databases of various gene sequences are found on the web.
Genbank's database was used as the source of the above sequences.
And rather than having to line up the sequences and determine the
differences manually, a set of programs to analyze sequence data and
plot trees are available. Numerical taxonomy, the use of computers.
The similarity matrix and conversion to dendrogram
•
•Sequence determinations and construction of gene probes.
•Phylogenetic trees from complete sequences.
•Prokaryote phylogeny and comparison with conventional
classification:
Phylogeny
rRNA genes are found in all bacteria and mutate at a slow rate. The
use of rRNA probes for DNA fingerprinting has been termed ribotyping: labeled probes are generally derived from E. coli 23S, 16S, and
5S rRNA sequences. Current developments use gene amplification.
Primers to conserved regions are used to PCR amplify regions that are
then identified using genus-specific and species-specific probes. With
the proper design of primers it is possible to amplify all genes of a range of bacteria, so-called universal primers. Such primers have been designed to detect a broad range of bacteria, with subsequnt genus and
species identification using probes to intervening variable sequences
(Figure 5.1). The use of 16S rRNA gene probes has enabled the identification of noncultivable bacteria and, indeed, suggests that only a fraction of the total bacterial species have been identified by cultural methods.
Ribotyping
It uses so-called universal probes targeted at specific conserved
domains of ribosomal RNA coding sequences, allowing
characterization with only limited sequence information.
Depending on the protocols used, the resultant band patterns can
be compared with known species and strains of organisms to
determine genetic and evolutionary relationships.
Basis of Methodology
Strategies can vary widely depending on the sequences selected,
the level of information required, and the target organism.
Here are some of the key advantages that differentiate this
method of DNA typing:
1. The genes for rRNA appear in several different copies at
different loci within the genome having different flanking
restriction site locations.
2.There is variability among the rRNA genes (16S and 23S).
3.There is variability in the spacer region between 16S and 23S
rRNA genes.
Related Strategies
The amplified fragments are subsequently cut with one or
more restriction enzymes and separated by native
polyacrylamide gel electrophoresis. The band patterns are
compared .
This method is also called amplified ribosomal DNA
restriction analysis (ARDRA).
Direct sequencing of 16S RNA is also commonly employed for
similar species and subspecies identification purposes,
because it is fairly direct and uncomplicated. Direct
sequencing of 16S ribosomal RNA, however, has the
disadvantage of the highly conserved nature of the 16S
sequence.
Thus useful polymorphism in the 16S region is
frequently absent at the subspecies level.
RAPD
Random amplification of polymorphic DNA (RAPD) is a very
general method for obtaining a molecular fingerprint of a strain or
species. It is a convenient and sensitive method that is finding
increasing application in such fields as epidemiology, molecular
genetics, microbial ecology, molecular evolution, and taxonomy.
Low-stringency PCR amplification of genomic DNA using a single
short primer (10–15 bases) of arbitrary sequence is used to generate
a set of fragments that is characteristic of the species or strain from
which the DNA was prepared. Each fragment in the set results from
the fortuitous hybridization of a pair of primers on opposite DNA
strands in the appropriate orientation, and separated by such a
distance allowing efficient PCR (<1,500 bp).
Following electrophoresis and staining, the number of bands
in common between a known and an unknown DNA sample
can be used for visual estimation of the molecular
relatedness of two individuals. By performing similar
experiments with different primers and many strains,
quantitative data can be derived which can then be used to
prepare dendrograms for taxonomic studies. A particular band
can also be considered to be a Mendelian "trait" of an
organism and can be used as a molecular marker to study the
segregation of other traits of economic or clinical significance.
In food production and clinical settings, RAPD has been found
to be a particularly quick and sensitive alternative to standard
bacteriological methods for strain identification, which may
take days to perform.
The principal advantage of RAPD is that, unlike most other
methods of strain identification, it requires no prior
knowledge of the organism under investigation. A primer of
arbitrary sequence and low-stringency PCR conditions is all
that is required to generate information. Of course, some
effort is necessary to optimize the amount of information
obtained in an experiment, since a particular primer and set
of PCR conditions may result in too few or too many bands to
be particularly useful. The method is sufficiently simple and
rapid, however, that such optimization can be performed in a
short time.
Because RAPD depends on the identification of common bands
between two samples based on their size, the spatial resolution of
the electrophoresis method employed contributes significantly to the
method's accuracy. The thin polyacrylamide gels however, offer
significantly improved resolution over agarose gels for the size
range of fragments generated in RAPD.
Silver staining is generally recognized to offer greater sensitivity
than ethidium bromide.
A more serious problem, related to the sensitivity of the method, is
reproducibility. Amplification under low-stringency conditions is
very sensitive to such factors as quality and quantity of the DNA,
primer and magnesium concentration, quality of the polymerase,
non accurate quantification of small amounts of DNA, each of
which can lead to loss of reproducibility.
Digitized RAPD patterns and dendrogram of 18 Acinetobacter genomic species (1 to 18) and 13 clinical Acinetobacter
isolates (19* to 31*) obtained after separate PCRs with six different primers. For each strain, all six RAPD patterns
have been combined into one single lane (1 to 6). The dendrogram was constructed with Gelcompar cluster analysis by
UPGMA. Percentages of similarity and molecular weights are shown above the dendrogram.
VNTR/ STR
Tandemly repeated sequences are powerful tools for
genotyping and linkage analysis. The sequences that are the
core of the repeated units represent a relatively wide
diversity of size (2–80 bp), sequence, and genome
distribution. They are extremely valuable as genetic markers
because of their highly polymorphic and abundant presence
in the genomes of higher organisms.
Tandemly repeated sequences can be broadly categorized
into two groups:
— VNTRs
and STRs
based primarily on the size range of the core sequence that is
repeated.
VNTRs represent repeated sequences that can typically range
in length from 10 to 80 bps and are highly polymorphic as to
the number of repeats at a given locus. They occur fairly
frequently in the genome, but there are relatively few
different types.
Occurring every few kilobases on average, they are not evenly
dispersed throughout the genome, tending to cluster toward
the telomeric ends of chromosomes
A working VNTR fragment size range is 50 to 1,500 base
pairs. These sequences are typically identified using a
strategy similar to that of RFLP analysis, where genomic DNA
is digested with an endonuclease and separated by
electrophoresis. The gels are then analyzed by southern
blotting, using sequence-specific probes to the repeated core
sequence.
Short tandem repeat (STR) sequences, or microsatellites,
consist of much shorter (2–10 bp) core sequences whose
allelic variants are tandemly repeated as many as hundreds
of times at different genetic loci. They are typically more
evenly dispersed throughout the genome as compared with
the larger VNTRs, and they represent ideal tools as genetic
markers because of their rich diversity, wide distribution, and
polymorphism.
Common repeats used for typing and linkage analysis are
"CA" or "ACTT" sequences.
Specifically designed as amplification-based detection
methods, STR and microsatellite-based DNA typing offer some
practical advantages over typing methods based on larger
repeat sequences. For example, PCR amplification using
primers targeted to a specific STR sequence typically
generates 50-to-500-bp-sized fragments without
compromising allelic diversity.
This allows for easier sizing of a wider range of alleles on a
single electrophoretic separation, as compared with larger
tandem repeat sequences that typically produce an order-ofmagnitude greater range in fragment size diversity.
In addition, the smaller average size of STR and microsatellite
alleles allows the technique to exhibit a greater tolerance for
crude or partially degraded genomic DNA samples, because
the need for longer intact sequence domains is reduced.
Variable Number of Tandem Repeat (VNTR) loci are chromosomal
regions in which a short DNA sequence motif (such as GC or AGCT)
is repeated a variable number of times at a single location (tandem
repeat). In this example, Locus A is a tandem repeat of the motif GC:
there are four alleles, with two, three, four, or five repeats (A2, A3,
A4, and A5, respectively). Locus B is a tandem repeat of the motif
AGCT: there are only two alleles, with two or three repeats (B2 and
B3, respectively).
The example shows a DNA fingerprint that examines both loci
simultaneously. Individual #1 is heterozygous at Locus A (A2 / A5)
and homozygous at Locus 2 (B2 / B2: note that this genotype gives a
single band phenotype in the fingerprint). Individual #2 is
heterozygous at both loci: (A4 / A3 and B3 / B2) respectively). The
two individuals are distinguishable at either locus.`
Locus QUB-11a was amplified by PCR and the products were resolved by agarose gel
electrophoresis. Length polymorphisms correspond to multiples of a 69 bp tandem repeat
unit. Lanes: 1, PCR negative control; Ma, 100 bp DNA ladder (Promega); 2, M. tuberculosis
H37Rv (allele 2); 3, M. bovis AF2122/97 (allele 9); 4, M. bovis 028 (allele 10); 5, M. bovis 029
(allele 10); 6, M. bovis 030 (allele 10); Mb, 100 bp DNA ladder; 7, M. bovis 031 (allele 10); 8,
M. bovis 032 (allele 10); 9, M. bovis 033 (allele 9); 10, M. bovis 034 (allele 10); 11, M. bovis
035 (allele 9); Ma, 100 bp DNA ladder; 12, M. bovis 036 (allele 5).
5.3.1 Mycobacteria
Slow-growing or fastidious bacteria are particularly appropriate targets
for molecular diagnostics . The prime example of the former
is Mycobacterium tuberculosis. Traditional methods of diagnosis, as
with other bacterial pathogens, have relied on microscopy and culture.
Culture on specialized media, such as Lowenstein-Jensen, takes several
weeks.
Neither method is, however, as fast as PCR-based methods.
In the main, PCR-based methods for M. tuberculosis utilize probes
to either rRNA or to a repetitive DNA sequence, IS6110.
Utilization of the IS6110 target has an inherent advantage, in that,
although both M. tuberculosis and the related M. bovis BacilleCalmette-Guerin strain are detected, the differences in copy number
of the target sequences within the two organisms results in differing
patterns of amplification products.
5.3.2 Other bacteria
Many bacteria can exist in both a pathogenic and nonpathogenic state.
In this scenario, genotypic methods can be used to detect virulence determinants. An example of this would be the use of toxin gene-specific
probes to detect toxigenic strains of Clostridium difficile, a cause of a
particular type of diarrhea associated with the use of antibiotics, termed
antibiotic-associated colitis . Not all virulence determinants will be
chromosomally mediated but probes are equally suited to detect toxin
genes on plasmids.
Escherichia coli is a Gram-negative bactetium which constitutes
much of the bacterial flora of the normal human gastrointestinal
tract. Certain strains however carry plasmids that code for toxins
that cause diarrhea.
5.3.3 Genotypic vs phenotypic
It is now recognised that specific virulence genes are only expressed
in the host and not under laboratory conditions.The main
drawback of most probe methods that are described in the
literature is that they do not differentiate between ‘live’ and
‘dead’ bacteria.
The most common approach to resolve this is to detect mRNA by
RT-PCR.
False positives due to contamination with minute quantities of DNA can be minimized by DNAase digestion of template.
5.4 Commercial systems
Most methods are based on PCR for which the world-wide patents are
held by Hofmann La Roche, and accrue a royalty payment. These systems (Amplicor®) are semi-automated, based on a PCR-ELISA methodology.
Such systems do not require precultivation of organisms. A
commercial typing system, Enviro-Amp® for Legionella species in the
environment is also available. This kit uses a set of biotinylated primers to conserved regions of 5S rDNA sequences, then species-specific primers which anneal to the Mip gene of Legionella pneumophilia.
Identification of amplicons is by hybridization to membrane bound
capture probes and subsequent detection by incubation with streptavidin/horse radish peroxidase conjugate in the presence of the substrate,
33΄,55΄ tetramethylbenzidine (TMB). A colorless to blue color change
indicates a positive reaction.
LCR methods have been developed by Abbott laboratories and have
become a commonly used methodology in, for example, the detection of
Chlamydia trachomatis in urine.
Transcription-mediated amplification (TMA) systems are also available. They can use both RNA and ssDNA targets.
5.5 Typing of isolates
5.5.1 Epidemiology
Epidemiological typing of isolates is useful for tracking an outbreak of
infection prospectively and for finding a source retrospectively as the
aim is to abort the spread of infectious disease.
In identification, there are a number of targets for probes:
• Chromosomal DNA
• Plasmid DNA
•
Repetitive DNA
5.5.2 Chromosomal DNA
Chromosomal DNA is extracted from, ideally, a pure bacterial culture
and digested with frequent-cutting restriction enzymes. Subsequent electrophoresis of the fragments results in an RFLP pattern. This technique is not as easily applied to bacteria as it is for some viruses because the much larger genome produces a large number of restriction
fragments, some of which may be difficult to resolve on a gel. Instead
of using standard gel elecrtophoresis, PFGE has also been used as it
results in smaller number of bands and an easier to interpret fingerprint. Gene amplification techniques are now more commonly employed for the typing of microbial isolates. The first described was arbitrarily-primed PCR (AP-PCR) in which an arbitrary primer is used
to detect amplification length polymorphisms . This has now been
adapted as the RAPD (Random Amplified Polymorphic DNA) method.
A short single primer oligonucleotide, is used so that binding is of
low specificity to a large number of loci in the target DNA.
These primers are typically of random sequence and greater
than 60% G:C base ratio. After PCR amplification, complex banding
patterns can be visualized in polyacrylamide gels. In theory
it is possible to obtain a specific fingerprint for an organism but there are some practical difficulties.
The use of short primers necessitates low annealing temperatures.
Ramp rates and primer concentrations are crucial to reproducibility. The
target DNA may also be polymorphic, and a single point mutation can
lead to a different banding pattern. For large genomes, the banding pattern may be also very complex and be difficult to interpret.
5.5.3 Plasmid DNA
The plasmids usually characterized are those that confer
antimicrobial resistance, termed R (for resistance) factors.
These have been shown to be useful epidemiological markers of the
spread of nosocomial infection within hospital settings.
5.5.4 Repetitive DNA
Repetitive DNA is common in bacterial genomes. The identification of
the differing frequency of repetitive sequences at differing positions
within the chromosomal DNA allow a ‘fingerprint’ to be established.
Rep-PCR involves the PCR amplification of regions of the genome between repetitive elements. Differing sizes of product occur as a result of
distance variations between the repeat sequences.
The repetitive sequences that code for tRNA are conserved and may
have variable intervening genome lengths.
With judicious use of primer probes to tRNA gene it is then possible to
produce DNA fingerprints with multiple bands or single bands of
differing size which can be separated by migration rate on agarose gel
electrophoresis.
It has been possible to type streptococcal and staphylococcal species by
this method.
5.6 Antimicrobial resistance
Conventional determination of antimicrobial resistance consists of culture and identification of the organism and determination of growth in the
presence of antibiotic as separate procedures. Gene probes can be used
to determine the presence of genetic sequences coding for resistance
which is plasmid or chromosomally-mediated; if the latter it may be possible to combine detection of the organism and determination of antibiotic resistance simultaneously.
A number of genes responsible for antibiotic resistance have now
been identified (Table 5.3). One of the best characterized is the mecA
gene of Staphylococcus aureus. This gene encodes a penicillinbinding protein and is present in the majority of methicillin-resistant
isolates of S. aureus ( MRSA ) but absent in those that are
susceptible.
In one study, in which the mecA gene was detected using PCR, 42 of
46 isolates of S. aureus and S. epidermidis that were mecA-positive
were also methicillin resistant .
Such detection enables appropriate infection control measures and antibiotic therapy, if required, to be instituted more rapidly than by conventional methods.
5.8 Fungi
The majority of the common fungal causes of human diseases can be rapidly diagnosed by Gram stain. This is cheap and sensitive and would
remain the method of choice for candidoses and dermatophyte infections.
Molecular probes will, however, have a limited role in diagnostic
mycology. Life-threatening infections due to fungi are becoming
more prevalent because of acquired immunosuppression due to
AIDS and transplantation procedures. Rapid diagnosis of
disseminated candidiasis and invasive aspergillosis could be
improved by molecular methods.
Currently diagnosis depends on culture and/or serology. Both of
these are insensitive, slow or both. The use of antigen detection
tests speeds up the process but has less than 100% sensitivity or
specificity, neither exceeding 70% in practice.
Any of the probe technologies that have been described for viruses and
bacteria could be applied to the detection of fungi.
There are some problems which have particular relevance to
the application of such methods to the mycological diagnosis.
• The first is that with the serious acute infections, blood is often the
most appropriate sample. Blood contains a number of inhibitors
of PCR, such as hemoglobin.
• Secondly, fungal cell walls are hardy structures and resist
release of nucleic acid targets.
• The third aspect to be considered in the interpretation of
molecular assays is that fungi are very common environmental
contaminants and indeed may be present in specimens without
probable clinical significance.
5.9 Protozoa
The diagnosis of protozoal infection is conventionally made by
microscopy or serology. When parasite load is high, microscopy is
an adequate methodology for the diagnosis of acute infection. The
most frequent protozoal infection world-wide is malaria which is
caused by one of four Plasmodium species.
Probes, when allied to amplification, have advantages over
conventional microscopy when low-level parasitemia occurs and
when it is useful to identify drug-resistant mutants. In the vast
majority of situations, microscopy will remain the method of
choice.
Probes to rRNA genes can be genus-or species-specific. They can also
be employed to detect mixed infections which are not infrequent.
Oligonucleotide rRNA probes are able to detect down to 10-50
parasites in blood spots.
PCR detection enables even greater sensitivity. Probes have also
been described which detect a 21 bp repetitive sequence,
and a number of parasite specific genes. PCR assays have been
developed that probe for specific mutations in the dihydrofolate
reductase gene that confer resistance to the antifolate drugs (such
as proguanil and pyrimethamine).
These assays seem to be clinically useful . Techniques such as
RAPD-PCR can also be applied to understand the epidemiology of these organisms. Protozoa have hypervariable minisatellite tandem repeat
sequences which can be exploited by mini-satellite variant repeat PCR
(MVR-PCR) for epidemiological purposes.
5.10 Future prospects
The next major advance will be the incorporation of DNA chip technology into routine diagnostics. Probes are bound to a solid phase for detection of unknown, ‘interrogated’, DNA. The use of a solid phase to
bind probes is not novel, being used in commercial PCR systems, but
DNA chips involve a very large array of probes of known sequence on
a solid surface, ‘the chip’.