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GeNeViSTA
Types of specimen collected for microbiological
studies |
1. Respiratory tract infections: nasal and bronchial washings, throat and nasal swabs, sputum |
2. Eye infections: throat and conjunctival swab/scraping |
3. Gastrointestinal tract infections: stool and rectal swabs |
4. Vesicular rash: vesicle fluid, skin scrapings |
5. Maculopapular rash: throat, stool, and rectal swabs |
6. CNS infection: stool, tissue, saliva, brain biopsy, cerebrospinal fluid |
7. Genital infections: vesicle fluid or swab |
8. Urinary tract infections: urine |
9. Blood-borne infections: blood |
The gold standard in bacteriology largely remains culture, primarily due to cost factors and the complex nature of infections but there could be shortcomings like:
Similarly there could be limitations in cases of serological tests like:
There are a large number of molecular techniques which can be used for detection and quantitation of pathogens.2 Some
of these techniques include:
⋅ Nucleic acid amplification: Nucleic acid amplification includes not only polymerase chain reaction (PCR) and
its variants, but also alternate technologies, such as strand displacement amplification and transcription-mediated
amplification. The technique involves selecting a specific genomic target in the pathogen of interest and designing specific
primers for amplification of this target using Taq polymerase. Presence of a band of amplification of a specific size would
indicate presence of the organism (Figure 2).
The applications of molecular techniques in clinical microbiology depend on the organism being studied. The various
applications are detailed below:
⋅ For bacterial diseases: In case of fastidious bacteria like Mycobacterium tuberculosis, Chlamydia
trachomatis, Neisseria gonorrheae and Bordetella pertussis, molecular testing has reduced the time taken by
conventional culture to allow early detection and treatment. Although molecular methods have helped
mycobacteriology, it is important to note that conventional culture still remains more sensitive. Despite this
limitation, molecular detection of M. tuberculosis allows confirmation of acid-fast bacilli (AFB) seen on
microscopy with up to 98% sensitivity within a day compared to approximately four weeks using phenotypic
methods.
In the management of sexually transmitted diseases (STDs), traditional screening methods require invasive methods
which are less acceptable as they cause embarrassment and discomfort, thus reducing compliance. Molecular methods offer
more convenience and acceptance, enhancing the compliance. This has indirectly led to increase in laboratory confirmed
cases of STDs. Molecular methods have the advantage of being performed on dry swabs with little degradation of the
DNA (DNA is stable) during transit compared to the difficulties in maintaining viability during transport, which make
them very useful for samples collected from remote and rural areas. In addition, molecular methods can detect multiple
pathogens such as C. trachomatis, N. gonorrhoeae, Haemophilus ducreyi and the genital mycoplasma from the same
swab.
Some bacteria can only be detected by molecular means as culture is either difficult or represents a significant
occupational risk to the laboratory personnel. Examples include Whipple’s disease due to Tropheryma whipplei, cat
scratch disease due to Bartonella henselae, Q fever due to Coxiella burnetii, and male urethritis due to Mycoplasma
genitalium. Molecular methods have the advantage here. In case of meningococcal disease, detection can be done on the
same day from specimens arising from sterile sites. Similarly, multiplex PCR methods have been developed for detection
of other common bacterial causes of meningitis like Streptococcus pneumoniae and Haemophilus influenza type
B.4
⋅ For mycology and parasitology: Molecular testing can be helpful in certain circumstances. The diagnosis of
Pneumocystis jiroveci pneumonia in immunosuppressed patients is limited to microscopy of respiratory tract specimens.
Immunofluorescence is more sensitive than microscopy but is more expensive and needs specialized facilities. PCR can be
useful but the specificity of PCR is limited because this organism is a ubiquitous commensal and can be detected in the
absence of pneumonia. Another example is the use of 18S rRNA gene PCR to detect Aspergillus species infection in
neutropenic patients.
In parasitological diagnosis, Toxoplasma gondii can be detected by PCR from amniocentesis fluid to confirm fetal
infection and from cerebrospinal fluid (CSF) to diagnose toxoplasma encephalitis. Microscopy remains the mainstay of
malaria diagnosis but Plasmodium species PCR, because of its superior sensitivity compared to microscopy, can diagnose
malaria in patients whose thick and thin blood films test negative due to administration of chemoprophylaxis or
partial immunity. Plasmodium species PCR can also detect mixed infections that can be difficult to detect
microscopically.
⋅ For viral diseases: a. Meningitis and encephalitis – The diagnosis of HSV encephalitis previously required brain
biopsy in certain cases due to the low sensitivity of cerebrospinal fluid (CSF) culture and serology. PCR now allows the
detection of HSV DNA from CSF with 95% sensitivity.5 HSV PCR can be multiplexed with other pathogens responsible
for meningitis.
b. Blood borne virus infection – Active hepatitis C viral (HCV) infections are diagnosed by the presence of
HCV RNA, since the detection of antibody to HCV cannot distinguish between past and present infection.
Early HIV infection and vertical transmission of HIV infection can be detected by the presence of HIV
pro-viral DNA. Some blood banking services screen pooled samples from all donations for HIV and HCV using
transcription mediated amplification assays, reducing the window period from 22 and 66 days to 9 and 7 days
respectively.6
c. Genital /intrauterine infections - Cytomegalovirus (CMV), rubella, varicella zoster virus, and genital ulceration due
to HSV type 2 infection are now routinely being detected by PCR.
d. Respiratory viral pathogens - Molecular detection of respiratory viral pathogens is cost-effective due to
avoidance of unnecessary testing and hospitalization. It also helps reduce unnecessary antibiotic use. New assays
of multiplex PCR are now available for testing all the common respiratory viruses along with fastidious
bacterial causes of pneumonia. Uncommon viruses such as the severe acute respiratory syndrome- associated
coronavirus (SARS-CoV) and influenza A/H5N1 (avian influenza) virus can also be incorporated into these
tests.
e. Viral diarrhea - Viruses cause more infectious diarrhoea worldwide than bacteria and other pathogens. The method
of choice for microbiological diagnosis of rotavirus from stool samples is PCR. Norovirus, responsible for large outbreaks
both in the community and health care facilities is amenable for diagnosis by PCR, which is the most sensitive
and rapid method. PCR is also the most sensitive method for the diagnosis of astroviruses and enteric
adenoviruses.
⋅ For treatment monitoring: Monitoring viral DNA or RNA load has become the standard of care for several
chronic viral infections and is an integral component of the management of HIV, HCV and hepatitis B virus (HBV)
infections. Measurement of the viral load is performed by real-time PCR. Patients who remain negative for HCV
RNA 6 months after completing combination therapy for HCV infection almost always remain free of the
virus.
⋅ For sensitivity testing:
Bacterial pathogens: Detection of antibiotic resistance by molecular methods has become routine in many
laboratories. This is done by detection of mutations in the organisms which confer resistance. Examples of genes studied
include:
Summary of uses for molecular methods
|
1. Identification - nearly all possible pathogens |
2. Viral load monitoring - Cytomegalovirus, Epstein-Barr virus, Hepatitis B, Hepatitis C, HIV |
3. Viral genotyping - HIV, Hepatitis B, Hepatitis C, Human papillomavirus |
4. Bacterial resistance detection - MRSA, VRE, ESBL producing E. coli, K. pneumonia, M. tuberculosis |
5. Bacterial genotyping - M. tuberculosis, N. meningitides |
6. Broad-range PCR - Infective endocarditis, bacterial meningitis |
MRSA: Methicillin resistant staphylococcus aureus
VRE: Vancomycin resistant enterococcus
ESBL: Extended spectrum beta lactamase
Limitations of Molecular Methods |
1. Unlike bacterial culture, which can detect a large number of cultivable bacteria without initially knowing the specific organism, PCR can only detect the organism whose DNA is complementary to the primers used. |
2. Differentiation between infection and disease, since the presence of nucleic acid does not necessarily mean the presence of viable microorganisms |
3. Molecular tests are often subject to false positive results due to their high sensitivity |
4. High level of staff training and skill is required for performing and interpreting these tests |
Molecular technology has gone beyond the era of research and has now become an integral part of any microbiological laboratory. The introduction of molecular methods in clinical microbiology laboratories not only depends on the performance of the test for each individual microorganism, but also on the clinical relevance of the diagnosis, the disease load and whether the new methods are supplementary to the procedures in use or their replacements. Therefore, strategies have to be devised for each infectious agent or clinical syndrome based on the available phenotypic information.
Dr. Ashish Bahal is a registered PhD student of Manipal University, Karnataka, India.
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