Traditionally, patients with infectious
diseases are treated with antimicrobials empirically, and
definitive therapy is started only after the
offending organism grows in cultures and sensitivity test results are
available, which takes considerable time. The clinical distinction
between viral and bacterial infection may not be possible always, which
frequently results in overuse of antibiotics. Almost half of the
patients in outpatient clinics with upper respiratory infection receive
antibiotics [1,2]. Overuse of antibiotics causes development of
resistance, reducing the already dwindling number of effective
antibiotics, drug toxicity and increases the chances of resilient and
opportunistic infections like C difficile. Therefore, it is vital
to diagnose the causative organism and its sensitivity/resistance
pattern in order to ensure positive patient management, adequate
antimicrobial stewardship and limit further outbreak.
Conventional tests for diagnosis of infectious
diseases and for determining drug-susceptibility of microorganisms were
based upon microscopy and culture methods. These tests are more
trustworthy but are time consuming, cumbersome and also require
logistics and skills, which may not be available in peripheral health
care setting. Average time required to identify the microorganism and
its drug-susceptibility to antibiotics is about 40 hours [3]. Rapid
diagnostic tests (RDTs) are quick, easy to perform, and results are
available earlier than conventional methods, with a usual turn-around
time of few hours. For rapid immunochromatic card test it takes 15 to 20
minutes whereas ELISA test requires 5 to 6 hours time. Due to the ease
and feasibility of RDTs, they may be available at the point-of-care
setting i.e., in the office or in the emergency room. This has
immense value in deciding whether the patient will need inpatient care
or may be treated as an outpatient [4]. The qualities of an ideal RDT
are outlined in Box 1.
BOX 1 Qualities of an Ideal Rapid
Diagnostic Test
• Rapid turnaround time from sample
collection to result.
• Can be performed in usual samples such as
blood, urine, stool and cerebrospinal fluid.
• High sensitivity, specificity and
predictive values.
• Diagnosis should be pathogen-specific with
information about drug resistance.
• Should be available at different settings
like hospitals, outpatient clinics, and in remote places.
• Results should be reproducible.
• Test should be cost-effective.
• It should utilize minimal logistics, and
require minimal skill to perform.
• It should withstand extremes of
temperature.
• Results are obtained in a single visit.
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Common Methods Used in RDT
Pathogenic organisms in rapid tests are detected by
identifying the non-visual biological signal they generate. These
signals include structural components of bacteria, viruses, protozoa and
fungi, specific antigen and antibodies, metabolic end products, DNA and
RNA base sequence, enzymes, toxins or surface polysaccharides [5].
Detectors are used to pick up the right signal from numerous background
signals in the sample into meaningful information to clinch the
diagnosis.
Antigen Detection Methods
Agglutination test: Patient’s specimen containing
the bacterial antigen with antibodies directed against it will result in
visible precipitation. If quantity of antibody is significantly higher
or lower than antigen there will be no precipitation – known as prozone
and postzone effect, respectively. As these tests have low sensitivity,
they have been mostly abandoned.
Latex agglutination assay: Latex coated
antibodies are used to detect the antigen.
Enzyme immunoassay (EIA) or Enzyme linked immunoassay
(ELISA): These are highly sensitive rapid tests to detect viral
antigens, toxins and organisms. Antibodies are bonded to enzymes, and if
antigen- antibody reaction occurs, the enzyme catalyzes the reaction to
produce a visible colored end product.
Detection of Antibodies by Serological Methods
EIA and other serological methods for detection of
antibodies can be used but have their own limitations. Presence of
antibodies may indicate either a recent or past infection. A 4-fold rise
in antibody titers following infection or raised IgM antibodies can
indicate early acute phase response. However, this response may be
inhibited by elevated IgG, which competes with IgM for binding at the
same antigen site, thereby resulting in false negative test.
Nucleic Acid Tests
These are used for the following purposes : (i)
direct detection and quantification of pathogens in patients’ specimen;
(ii) identification of microorganism grown in culture; (iii)
characterization of microorganism beyond basic identification eg,
detecting genes encoding for resistance; and (iv) identification
of species of organisms and strain typing useful to locate the source of
an outbreak of infection.
Nucleic acid hybridization method: This
method involves coupling of a probe that contains known nucleic acid
sequences against target nucleic acid regions present in patient’s
specimen [6]. A negative hybridization test indicates either the absence
of a target organism or presence below the limit of detection by
hybridization.
Nucleic acid amplification methods: At times, the
target in the specimen might not have sufficient nucleic acid to be
detected by the above technique. Hence amplification of the target to
greater number of copies can be performed with Nucleic acid
amplification test (NAAT), which uses Polymerase chain reaction (PCR).
Quantitative PCR (qPCR) is a significant advancement where one is able
to detect the number of targets in the original clinical specimen. This
helps to establish the disease burden, assess effectiveness of therapy,
prognosticate and monitor disease progression. In multiplex PCR, more
than one primer or probe is used to detect different target in one
reaction; eg, multiplex PCR-containing probes for herpes simplex
and enteroviruses to be used to detect the responsible organism in viral
meningitis.
Gas Liquid Chromatography
Once the pathogen is grown, it can be identified
quickly by its metabolic end products, such as short-chain fatty acids,
with use of gas liquid chromatography. It can also be used to identify
the long-chain fatty acids present in the cell wall and membranes of
different organisms.
Matrix Assisted Laser Desorption Ionization Time of
Flight Mass Spectrometry (MALDI – TOFMS)
In this technique, the organism grown is mixed with a
chemical matrix, and laser is applied, which cause desorption of
proteins and ionization. These ions are separated on the basis of time
they travel in a charged vacuum tube, known a flight tube, to a
detector. The patterns they produce are compared to known patterns of
different organisms stored in the database.
Rapid Diagnostic Tests in Common Diseases
Malaria
Immunochromatographic tests are available to detect
malaria antigen by monoclonal antibodies targeted to it. They are simple
to perform with results in few minutes, without any sophisticated
devices. The antigens targeted in current test kits are:
Histidine rich protein 2 (HRP2): This is specific
to P. falciparum produced by asexual stage and young gametocytes
[7]. Antigen positivity may persist for weeks even after successful
treatment.
Parasite lactate dehydrogenase (pLDH): It is
present both in asexual and sexual (gametocyte) stage. Different isomers
of this antigen are present. One is found in all the four species of
malaria known as pan-specific. Other antigens are either P.
falciparum or P. vivax specific. Unlike HRP2, these disappear
following successful treatment but as gametocytes also produce pLDH, it
may remain positive after clearance of the asexual stage [8].
Plasmodium aldolase: This is an enzyme of
the glycolytic pathway produced by all four species of malaria parasite;
the kits based on this test are hence panspecific [9].
P. falciparum and P. vivax malaria occur
nearly in equal numbers as a single species infection in India. Their
treatment differs; hence, differentiation between these two species with
an appropriate RDT is essential. Accuracy of detection should also be
noted. World Health Organization (WHO) has suggested the detection score
against P. falciparum and P. vivax to be at least 75% at
parasite level of 200/µL. False positive rate should be less than 10%
and invalid result should be less than 5% [10]. These tests can even
detect parasites sequestered in deep vascular compartment where
microscopy would fail. As these antigens might persist even after
successful treatment, these are not useful to monitor treatment or to
quantify the parasite. Thus, they are not useful to prognosticate or
judge therapeutic efficacy of antimalarial drugs. They are useful where
microscopic diagnosis is not available or is not of acceptable standard.
RDT can also serve as screening diagnosis where microscopy can be
reserved for resolution of doubtful cases or confirmation of a negative
RDT in spite of high clinical suspicion of malaria. Serological methods
that detect antibodies against malaria are also available but should
never be used for treatment decisions [11].
Typhoid Fever
Widal test: This test, used extensively to
diagnose typhoid, is an outdated particle agglutination test. It detects
antibodies in the patient’s serum against the O an H antigen of
Salmonella typhi and H antigens of paratyphi A and B [12].
Conventionally, a positive Widal test implies rising titer in paired
blood sample 10 to 14 days apart, which is too long period for
diagnosis. This test has suboptimal sensitivity and specificity [13].
Poor sensitivity is due to negative results early in infection, prior
antibiotic therapy and poor immune response in certain individuals. Poor
specificity is due to presence of baseline antibodies in endemic areas,
cross reactivity with other gram negative enterobactericae and non
typhoidal salmonella, and anamnestic reaction with unrelated infections
like malaria.
Typhidot test: It measures
both IgM and IgG antibodies against 50kDa outer membrane protein (OMP)
in ELISA format. If IgM is positive, the test is considered positive
whereas if IgM is negative and IgG is positive, it is considered
indeterminate. In TyphidotM, only IgM antibodies are measured
against the above mentioned antigen after completely removing the IgG
molecules. Overall typhidot tests have a sensitivity and specificity
close to 80%. However, these tests are not accurate enough to replace
the gold standard blood culture for typhoid [15].
Tuberculosis
The problem of bacteriological diagnosis of
tuberculosis (TB) in children is multiple. Children have difficulty in
expectorating sputum, and they usually suffer from paucibacillary
disease in childhood making the bacteriological diagnosis difficult. The
expert MTB / RIF test is a major advance in the diagnosis of TB and
detection of resistance to rifampicin. It is a cartridge-based fully
automated NAAT for TB case detection. It purifies, concentrates,
amplifies and identifies the targeted nucleic acid sequences in the
organisms genome in a short time, providing results within 2 hours. It
is a real time PCR molecular testing as it fully integrates and
automatizes three processes – sample preparation, amplification and
detection. WHO policy recommen-dation states that it should be used as
an initial diagnostic test in all children suspected of having TB,
acknowledging resource implication. It should be done in children
suspected of having multidrug resistant TB (MDR) or HIV-associated TB
[16]. The test has high sensitivity (88%) in detecting TB with negative
predictive value more than 98% in both high and low prevalence TB
settings. The false positive results are linked to detection of dead
bacilli, which would not be picked up by culture [17]. The test can be
performed in respiratory specimens like sputum, induced sputum and
gastric aspirate. For extra pulmonary TB, CSF, lymph nodes and other
tissues may be used. Performance of this test is relatively poor in
pleural fluid. This test is not suitable for monitoring TB patients as
they detect both live and dead bacilli. The test is not recommended for
stool, urine or blood samples.
Dengue
Dengue produces symptoms and signs that resemble
other viral infections and are non-specific. Patients may progress to
severe dengue very early and supportive measures taken early can be
life-saving; hence an early laboratory diagnosis is helpful. Virus
isolation and nucleic acid detection is time consuming and needs
sophisticated instruments, which are mostly not available. NS1 antigen
is non structural protein that can be detected as early as first day of
onset of symptoms to about 7 days, both in primary and secondary dengue.
In secondary dengue, as patients have pre-existing IgG, they form immune
complexes with the viral antigen. Two types of rapid tests are available
for diagnosis of dengue infections [18-20]:
Card test: This is a rapid agglutination test
where results are available in 20-30 minutes. It is a solid phase
immunochromatographic test for qualitative detection of NS1 antigen and
IgM and IgG antibody against dengue. In secondary dengue, as immune
complex are formed with NS1 antigen, it may fail to detect the antigen.
This test has poor performance as compared to ELISA test.
ELISA test: It takes longer time, about 5 to 6
hours, to detect NS1 antigen and IgG and IgM antibodies against dengue.
This test can detect antigens that have formed immune complex with IgG
antibodies, and thus has much higher sensitivity as compared to card
test. There are high IgM and low IgG in primary dengue infection unlike
in secondary infection. A 4-fold rise in IgG between acute and
convalescent sera is diagnostic but has the drawback of need of two
paired samples that results in delay in confirmation. Serum IgM may be
undetectable in secondary dengue making the interpretation of the test
difficult. The best time to test for antibodies is five days or more
after the onset of symptoms.
TORCH Infections
TORCH is an acronym which groups together congenital
or perinatally transmitted non-bacterial infections in neonates that
share certain clinical and laboratory features. As detection of ‘other
infections’ – which stand for the letter ‘O’ in TORCH – is increasing
with time, doubts have been raised whether indiscriminate testing for a
big group needs to be done. Immunochromato-graphic tests are available
that detect IgG and IgM antibodies against the organisms [21]. However,
these are not useful for CMV and HSV. If IgG is positive in the baby, it
can be due to infection or could be transplacental antibody from the
mother without true infection. On the other hand, if IgM is positive, it
indicates recent infection. A negative test does not rule out infection
as IgM may not be positive for weeks in infections like Herpes. It is
always prudent to look for specific signs of individual infection and
direct the investigation accordingly.
Viral Hepatitis
ELISA tests to detect IgG and IgM antibodies to
hepatitis A virus may be done of which IgM indicates current active
disease [22]. For Hepatitis B detection, multiple antigen and antibodies
are required to establish the disease type. ELISA testing to detect
antibodies against Hepatitis C denotes chronic disease but cannot
diagnose acute disease. For Hepatitis E, both IgG and IgM antibodies can
be detected in acute disease by ELISA test. In areas where hepatitis E
is not endemic, nucleic acid testing must be done to confirm.
Diarrhea
Immunochromatographic tests are used to detect group
A rotavirus antigens and adenovirus antigens from stools samples. False
negative results are possible and test does not exclude coinfection with
other pathogens. For Clostridium difficile associated diarrhea,
detection of cytotoxin by ELISA test is done, but it does not establish
the diagnosis unequivocally.
Meningitis and Meningoencephalitis
Latex agglutination test in the CSF is used for rapid
detection of capsular polysaccharides of common meningeal pathogens –
pneumococcus, H. influenzae type b and meningococcus. One of the
advantages is that prior administration of antibiotics does not
influence the test. However, both false positive and negative results
can occur [23]. These tests may be recommended for patient with negative
gram stain and CSF culture [24]. For Japanese encephalitis, detection of
IgM antibodies in the CSF by ELISA is diagnostic.
Others
Rapid test are available to detect antibodies to HIV
1/2 and ‘O’ subtypes and HIV1 p24 antigen in serum or plasma. However,
they should never be used alone to diagnose or exclude HIV infection.
Detection of scrub typhus by IgM ELISA test is available but should be
interpreted in the context of clinical presentation and other laboratory
findings. Rapid tests are also done to detect antibodies to recombinant
antigen RK39 for Kala azar. Tests are also available to detect
Mycoplasma IgM, amebic liver abscess, Hydatid disease and Chikungunya.
Rapid tests can diagnose respiratory syncytial virus (RSV) from
nasopharyngeal samples to prevent unnecessary antibiotics in infants
with respiratory distress. Similarly Group A streptococcus (GAS) can be
diagnosed from pharyngeal specimens; though RDTs cannot distinguish
between true infection and colonization.
Multiplex PCR, which combines several probes, can
identify 25 or more common bacterial and fungal pathogens responsible
for blood stream infections. In addition, it can point out presence of
resistance genes like mec A, van A/B and carbapenem
resistance [4]. This not only reduces the use of multiple antibiotics
but also promotes targeted therapy.
Conclusion
Numerous rapid tests are now available, which can
rationalize antibiotic therapy and halt the relentless march of drug
resistance. With more and more RDTs being available, it is the duty of
treating physician to select the right RDT based on the clinical
presentation, prevalence of the organism in the community or hospital,
and the cost and time to result. Shot gun approach to do the entire
battery of tests at one go will only defeat the purpose of the tests.
Key Messages
• Rapid diagnostic tests ensure definitive
diagnosis of infections, thereby preventing unnecessary
presumptive treatment.
• These usually provide qualitative results,
and are not useful in monitoring treatment.
• These tests require minimum skill making
them useful in places without sophisticated laboratory
infrastructure.
• These tests must always be ordered on the basis of clinical
setting, and not as a battery.
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