RATIONAL DIAGNOSTICS
Rapid Diagnostic Tests in Childhood Infections
RITABRATA KUNDU
From Department of Pediatrics, Institute of Child Health, Kolkata, India. rkundu22@[Link]
Presumptive treatment of infections often results in irrational antimicrobial use resulting in detrimental spread of drug resistance and
untoward side effects. A rapid diagnostic test (RDT) is a test that delivers a result earlier than conventional testing methods employed in
the past to identify the offending microorganism. RDTs help in early definitive therapy, reduction in hospital stay and cost, and in degree of
morbidity and mortality associated with the infection. To select a proper RDT, one should consider how specific and sensitive the test is.
Most RDTs gives a qualitative result not quantitative; hence disease severity, monitoring of the disease, prognostication and therapeutic
efficacy cannot be assessed. A RDT should be easy to perform, should not require sophisticated machines, and kits should be stable in
extremes of temperature. RDTs may be of immense help in remote places where conventional diagnostic facilities are unavailable or lack
quality. RDTs hold promise of reasonable diagnostic accuracy if done in a optimal clinical background. They should never be ordered as
a shotgun approach to exclude all possible infections but should be used judiciously with appropriate interpretation.
Keywords: Diagnosis, Dengue, Tuberculosis, Typhoid, Serology, Scrub typhus.
T
raditionally, patients with infectious diseases may be available at the point-of-care setting i.e., in the
are treated with antimicrobials empirically, and office or in the emergency room. This has immense value
definitive therapy is started only after the in deciding whether the patient will need inpatient care or
offending organism grows in cultures and may be treated as an outpatient [4]. The qualities of an
sensitivity test results are available, which takes ideal RDT are outlined in Box 1.
considerable time. The clinical distinction between viral
COMMON METHODS USED IN RDT
and bacterial infection may not be possible always, which
frequently results in overuse of antibiotics. Almost half of Pathogenic organisms in rapid tests are detected by
the patients in outpatient clinics with upper respiratory identifying the non-visual biological signal they generate.
infection receive antibiotics [1,2]. Overuse of antibiotics These signals include structural components of bacteria,
causes development of resistance, reducing the already viruses, protozoa and fungi, specific antigen and
dwindling number of effective antibiotics, drug toxicity antibodies, metabolic end products, DNA and RNA base
and increases the chances of resilient and opportunistic sequence, enzymes, toxins or surface polysaccharides [5].
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, BOX 1 QUALITIES OF AN IDEAL RAPID DIAGNOSTIC TEST
adequate antimicrobial stewardship and limit further • Rapid turnaround time from sample collection to
outbreak. result.
Conventional tests for diagnosis of infectious diseases • Can be performed in usual samples such as
blood, urine, stool and cerebrospinal fluid.
and for determining drug-susceptibility of
microorganisms were based upon microscopy and culture • High sensitivity, specificity and predictive values.
methods. These tests are more trustworthy but are time • Diagnosis should be pathogen-specific with
consuming, cumbersome and also require logistics and information about drug resistance.
skills, which may not be available in peripheral health • Should be available at different settings like
care setting. Average time required to identify the hospitals, outpatient clinics, and in remote places.
microorganism and its drug-susceptibility to antibiotics is • Results should be reproducible.
about 40 hours [3]. Rapid diagnostic tests (RDTs) are • Test should be cost-effective.
quick, easy to perform, and results are available earlier • It should utilize minimal logistics, and require
than conventional methods, with a usual turn-around time minimal skill to perform.
of few hours. For rapid immunochromatic card test it • It should withstand extremes of temperature.
takes 15 to 20 minutes whereas ELISA test requires 5 to 6
• Results are obtained in a single visit.
hours time. Due to the ease and feasibility of RDTs, they
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KUNDU R RAPID DIAGNOSTIC TESTS FOR INFECTIONS
Detectors are used to pick up the right signal from with Nucleic acid amplification test (NAAT), which uses
numerous background signals in the sample into Polymerase chain reaction (PCR). Quantitative PCR
meaningful information to clinch the diagnosis. (qPCR) is a significant advancement where one is able to
detect the number of targets in the original clinical
Antigen Detection Methods
specimen. This helps to establish the disease burden,
Agglutination test: Patient’s specimen containing the assess effectiveness of therapy, prognosticate and monitor
bacterial antigen with antibodies directed against it will disease progression. In multiplex PCR, more than one
result in visible precipitation. If quantity of antibody is primer or probe is used to detect different target in one
significantly higher or lower than antigen there will be no reaction; eg, multiplex PCR-containing probes for herpes
precipitation – known as prozone and postzone effect, simplex and enteroviruses to be used to detect the
respectively. As these tests have low sensitivity, they have responsible organism in viral meningitis.
been mostly abandoned.
Gas Liquid Chromatography
Latex agglutination assay: Latex coated antibodies are
Once the pathogen is grown, it can be identified quickly
used to detect the antigen.
by its metabolic end products, such as short-chain fatty
Enzyme immunoassay (EIA) or Enzyme linked acids, with use of gas liquid chromatography. It can also
immunoassay (ELISA): These are highly sensitive rapid be used to identify the long-chain fatty acids present in the
tests to detect viral antigens, toxins and organisms. cell wall and membranes of different organisms.
Antibodies are bonded to enzymes, and if antigen-
Matrix Assisted Laser Desorption Ionization Time of
antibody reaction occurs, the enzyme catalyzes the
Flight Mass Spectrometry (MALDI – TOFMS)
reaction to produce a visible colored end product.
In this technique, the organism grown is mixed with a
Detection of Antibodies by Serological Methods
chemical matrix, and laser is applied, which cause
EIA and other serological methods for detection of desorption of proteins and ionization. These ions are
antibodies can be used but have their own limitations. separated on the basis of time they travel in a charged
Presence of antibodies may indicate either a recent or past vacuum tube, known a flight tube, to a detector. The
infection. A 4-fold rise in antibody titers following patterns they produce are compared to known patterns of
infection or raised IgM antibodies can indicate early acute different organisms stored in the database.
phase response. However, this response may be inhibited
RAPID DIAGNOSTIC TESTS IN COMMON DISEASES
by elevated IgG, which competes with IgM for binding at
the same antigen site, thereby resulting in false negative Malaria
test.
Immunochromatographic tests are available to detect
Nucleic Acid Tests malaria antigen by monoclonal antibodies targeted to it.
They are simple to perform with results in few minutes,
These are used for the following purposes : (i) direct
without any sophisticated devices. The antigens targeted
detection and quantification of pathogens in patients’
in current test kits are:
specimen; (ii) identification of microorganism grown in
culture; (iii) characterization of microorganism beyond Histidine rich protein 2 (HRP2): This is specific to P.
basic identification eg, detecting genes encoding for falciparum produced by asexual stage and young
resistance; and (iv) identification of species of organisms gametocytes [7]. Antigen positivity may persist for weeks
and strain typing useful to locate the source of an outbreak even after successful treatment.
of infection.
Parasite lactate dehydrogenase (pLDH): It is present
Nucleic acid hybridization method: This method involves both in asexual and sexual (gametocyte) stage. Different
coupling of a probe that contains known nucleic acid isomers of this antigen are present. One is found in all the
sequences against target nucleic acid regions present in four species of malaria known as pan-specific. Other
patient’s specimen [6]. A negative hybridization test antigens are either P. falciparum or P. vivax specific.
indicates either the absence of a target organism or Unlike HRP2, these disappear following successful
presence below the limit of detection by hybridization. treatment but as gametocytes also produce pLDH, it may
remain positive after clearance of the asexual stage [8].
Nucleic acid amplification methods: At times, the target
in the specimen might not have sufficient nucleic acid to Plasmodium aldolase: This is an enzyme of the glycolytic
be detected by the above technique. Hence amplification pathway produced by all four species of malaria parasite;
of the target to greater number of copies can be performed the kits based on this test are hence panspecific [9].
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KUNDU R RAPID DIAGNOSTIC TESTS FOR INFECTIONS
P. falciparum and P. vivax malaria occur nearly in (TB) in children is multiple. Children have difficulty in
equal numbers as a single species infection in India. Their expectorating sputum, and they usually suffer from
treatment differs; hence, differentiation between these paucibacillary disease in childhood making the
two species with an appropriate RDT is essential. bacteriological diagnosis difficult. The expert MTB / RIF
Accuracy of detection should also be noted. World Health test is a major advance in the diagnosis of TB and
Organization (WHO) has suggested the detection score detection of resistance to rifampicin. It is a cartridge-
against P. falciparum and P. vivax to be at least 75% at based fully automated NAAT for TB case detection. It
parasite level of 200/µL. False positive rate should be less purifies, concentrates, amplifies and identifies the
than 10% and invalid result should be less than 5% [10]. targeted nucleic acid sequences in the organisms genome
These tests can even detect parasites sequestered in deep in a short time, providing results within 2 hours. It is a real
vascular compartment where microscopy would fail. As time PCR molecular testing as it fully integrates and
these antigens might persist even after successful automatizes three processes – sample preparation,
treatment, these are not useful to monitor treatment or to amplification and detection. WHO policy recommen-
quantify the parasite. Thus, they are not useful to dation states that it should be used as an initial diagnostic
prognosticate or judge therapeutic efficacy of antimalarial test in all children suspected of having TB,
drugs. They are useful where microscopic diagnosis is not acknowledging resource implication. It should be done in
available or is not of acceptable standard. RDT can also children suspected of having multidrug resistant TB
serve as screening diagnosis where microscopy can be (MDR) or HIV-associated TB [16]. The test has high
reserved for resolution of doubtful cases or confirmation sensitivity (88%) in detecting TB with negative predictive
of a negative RDT in spite of high clinical suspicion of value more than 98% in both high and low prevalence TB
malaria. Serological methods that detect antibodies settings. The false positive results are linked to detection
against malaria are also available but should never be of dead bacilli, which would not be picked up by culture
used for treatment decisions [11]. [17]. The test can be performed in respiratory specimens
Typhoid Fever like sputum, induced sputum and gastric aspirate. For
extra pulmonary TB, CSF, lymph nodes and other tissues
Widal test: This test, used extensively to diagnose may be used. Performance of this test is relatively poor in
typhoid, is an outdated particle agglutination test. It pleural fluid. This test is not suitable for monitoring TB
detects antibodies in the patient’s serum against the O an patients as they detect both live and dead bacilli. The test
H antigen of Salmonella typhi and H antigens of paratyphi is not recommended for stool, urine or blood samples.
A and B [12]. Conventionally, a positive Widal test
implies rising titer in paired blood sample 10 to 14 days Dengue
apart, which is too long period for diagnosis. This test has
Dengue produces symptoms and signs that resemble other
suboptimal sensitivity and specificity [13]. Poor
viral infections and are non-specific. Patients may
sensitivity is due to negative results early in infection,
progress to severe dengue very early and supportive
prior antibiotic therapy and poor immune response in
measures taken early can be life-saving; hence an early
certain individuals. Poor specificity is due to presence of
laboratory diagnosis is helpful. Virus isolation and
baseline antibodies in endemic areas, cross reactivity with
nucleic acid detection is time consuming and needs
other gram negative enterobactericae and non typhoidal
sophisticated instruments, which are mostly not available.
salmonella, and anamnestic reaction with unrelated
NS1 antigen is non structural protein that can be detected
infections like malaria.
as early as first day of onset of symptoms to about 7 days,
Typhidot test: It measures both IgM and IgG antibodies both in primary and secondary dengue. In secondary
against 50kDa outer membrane protein (OMP) in ELISA dengue, as patients have pre-existing IgG, they form
format. If IgM is positive, the test is considered positive immune complexes with the viral antigen. Two types of
whereas if IgM is negative and IgG is positive, it is rapid tests are available for diagnosis of dengue infections
considered indeterminate. In TyphidotM, only IgM [18-20]:
antibodies are measured against the above mentioned
antigen after completely removing the IgG molecules. Card test: This is a rapid agglutination test where results
Overall typhidot tests have a sensitivity and specificity are available in 20-30 minutes. It is a solid phase
close to 80%. However, these tests are not accurate enough immunochromatographic test for qualitative detection of
to replace the gold standard blood culture for typhoid [15]. NS1 antigen and IgM and IgG antibody against dengue.
In secondary dengue, as immune complex are formed
Tuberculosis with NS1 antigen, it may fail to detect the antigen. This
The problem of bacteriological diagnosis of tuberculosis test has poor performance as compared to ELISA test.
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KUNDU R RAPID DIAGNOSTIC TESTS FOR INFECTIONS
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.
ELISA test: It takes longer time, about 5 to 6 hours, to hepatitis E is not endemic, nucleic acid testing must be
detect NS1 antigen and IgG and IgM antibodies against done to confirm.
dengue. This test can detect antigens that have formed
Diarrhea
immune complex with IgG antibodies, and thus has much
higher sensitivity as compared to card test. There are high Immunochromatographic tests are used to detect group A
IgM and low IgG in primary dengue infection unlike in rotavirus antigens and adenovirus antigens from stools
secondary infection. A 4-fold rise in IgG between acute samples. False negative results are possible and test does
and convalescent sera is diagnostic but has the drawback not exclude coinfection with other pathogens. For
of need of two paired samples that results in delay in Clostridium difficile associated diarrhea, detection of
confirmation. Serum IgM may be undetectable in cytotoxin by ELISA test is done, but it does not establish
secondary dengue making the interpretation of the test the diagnosis unequivocally.
difficult. The best time to test for antibodies is five days or
Meningitis and Meningoencephalitis
more after the onset of symptoms.
Latex agglutination test in the CSF is used for rapid
TORCH Infections
detection of capsular polysaccharides of common
TORCH is an acronym which groups together congenital meningeal pathogens – pneumococcus, H. influenzae type
or perinatally transmitted non-bacterial infections in b and meningococcus. One of the advantages is that prior
neonates that share certain clinical and laboratory features. administration of antibiotics does not influence the test.
As detection of ‘other infections’ – which stand for the However, both false positive and negative results can
letter ‘O’ in TORCH – is increasing with time, doubts have occur [23]. These tests may be recommended for patient
been raised whether indiscriminate testing for a big group with negative gram stain and CSF culture [24]. For
needs to be done. Immunochromato-graphic tests are Japanese encephalitis, detection of IgM antibodies in the
available that detect IgG and IgM antibodies against the CSF by ELISA is diagnostic.
organisms [21]. However, these are not useful for CMV
Others
and HSV. If IgG is positive in the baby, it can be due to
infection or could be transplacental antibody from the Rapid test are available to detect antibodies to HIV 1/2
mother without true infection. On the other hand, if IgM is and ‘O’ subtypes and HIV1 p24 antigen in serum or
positive, it indicates recent infection. A negative test does plasma. However, they should never be used alone to
not rule out infection as IgM may not be positive for weeks diagnose or exclude HIV infection. Detection of scrub
in infections like Herpes. It is always prudent to look for typhus by IgM ELISA test is available but should be
specific signs of individual infection and direct the interpreted in the context of clinical presentation and
investigation accordingly. other laboratory findings. Rapid tests are also done to
detect antibodies to recombinant antigen RK39 for Kala
Viral Hepatitis
azar. Tests are also available to detect Mycoplasma IgM,
ELISA tests to detect IgG and IgM antibodies to hepatitis amebic liver abscess, Hydatid disease and Chikungunya.
A virus may be done of which IgM indicates current active Rapid tests can diagnose respiratory syncytial virus
disease [22]. For Hepatitis B detection, multiple antigen (RSV) from nasopharyngeal samples to prevent
and antibodies are required to establish the disease type. unnecessary antibiotics in infants with respiratory
ELISA testing to detect antibodies against Hepatitis C distress. Similarly Group A streptococcus (GAS) can be
denotes chronic disease but cannot diagnose acute diagnosed from pharyngeal specimens; though RDTs
disease. For Hepatitis E, both IgG and IgM antibodies can cannot distinguish between true infection and
be detected in acute disease by ELISA test. In areas where colonization.
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KUNDU R RAPID DIAGNOSTIC TESTS FOR INFECTIONS
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