RAPID DIAGNOSTIC TESTS - A BOON TO THE CLINICIAN
Dr. Saranya Nandakumar, Consultant Microbiologist*
Director, Suryodaya Centre for Health Information Pvt. Ltd, Chennai.*
Although diseases of "affluence" such as diabetes and cardiovascular disease have been increasing in developing countries, infectious diseases still continue to impose the greatest health burden. The last decade has seen tremendous strides in the field of diagnostics. With newer infections emerging and older almost forgotten ones re-emerging with greater force, the need for simple, rapid, equally if not more effective diagnostic tools is often felt strongly.

These rapid tests are specially useful in certain situations where good laboratory facilities are non-existent, where the clinical picture is confusing and or critical, when an outbreak is suspected or in field studies.

Rapid diagnostic tests are available either as single or multiple step formats and can be used to detect antigen or antibody. They can be classified as:
  1. Tests used to detect either antigen or antibody - ELISA, Agglutination, Western Blot, Chromatography
  2. Rapid culture techniques - TB
  3. Molecular methods - PCR, bDNA, etc. and most recently
  4. Micro-array technology is just finding clinical application

When dealing with an infectious disease in pediatric practice, a very sick child, anxious parents together with a rapidly deteriorating situation necessitates that the clinical findings need to be substantiated so as to be able to start treatment as early as possible. Some of the situations where rapid diagnostic tests (RDT) have been of particular relevance are dealt with below.
Dengue
A major problem in the pediatric age group needs a quick diagnosis and treatment to avoid the possibility of DHF or shock from setting in. While culture will offer a conclusive diagnosis, it is time-consuming, labour-intensive, not sensitive and available only in specialized laboratories. Rapid tests with improving accuracy are available as follows and are increasingly being used. They are:

  1. Immuno-chromatography with the result available in 5-7 mins and
  2. Capture ELISA (IgG and IgM) - 2 hrs
  3. IgM and IgG ELISA
  4. *Hemagglutination inhibition
  5. PCR for dengue

(a-d) identify antibody presence and (*d) is not used routinely in diagnostic labs.
Advantages of Serological assays to identify antibody
  1. These assays are able to differentiate correctly between primary and secondary dengue
  2. Sensitivity around 100%
  3. Specificity around 89%
  4. Relatively inexpensive
  5. Can be done in any laboratory and
  6. No specialist training is required

Evaluation of the RDTs available have yielded conflicting results by various workers. One study that evaluated 8 different RDTs found that sensitivity ranged from 6-65% and specificity from 69-100%. In another study, the Immunochromatographic card test and capture IgM and IgG ELISA yielded very promising results with sensitivities of 100% and specificities of around 89%.

With the recent re-emergence of Chikungunya that has assumed epidemic proportions and mimics dengue, it is critical to identify dengue as soon as possible and these tests together with hematological parameters such as platelet counts should be useful in arriving at a definitive diagnosis. However, if the initial result is inconclusive, a convalescent sample is a must.
Leptospirosis
This spirochetal infection has the dubious distinction of being unrecognized usually or under-reported. In the small percentage of people in whom this is manifested, most often spontaneous remission takes place; while just around 5-10% have serious complications. In these situations, the sequelae range from multi-organ failure and death to morbidity as a result of neurological, cardiac, or ophthalmologic complications. While culture is the gold standard, the numerous disadvantages of culture tip the balance in favor of the rapid diagnostic tests. The Microscopic Agglutination Titre test (MAT) that was once considered the cornerstone of lab diagnosis of Leptospirosis no longer occupies its pride of position. The rapid diagnostic tests that have gained acceptance are those that detect antibody and are the following:
  1. Micro-titre IgM ELISA from various manufacturers
  2. IgM Dot ELISA
  3. Dipstick - Detects IgM presence
  4. Indirect Hemagglutination Test (IHA)
  5. Latex Agglutination
  6. Indirect Fluorescent Antibody test (IFA)
  7. Lateral flow


Test name

Sensitivity

Specificity

IgM ELISA
Dipstick
Latex Agglutination
Lateral Flow

26-51%
26-51%
85-90%
53-86%

85-100%
85-100%
10%
89-94%



Sensitivities of all assays improved after the second week of illness, but this has proved to be the bugbear of as detection in the first week is critical.

Molecular assays that identify antigen presence like PCR, RAPD and Hybridization techniques, while being extremely sensitive and specific are available only in a few specialized and mainly research labs and need trained manpower and expensive equipment.

Dark Field Microscopy (DFM) a simple procedure, that is rapid that can be used in field conditions by an adequately trained microscopist has unfortunately won favor, but still remains a very reliable tool in good hands.
Malaria
In remote areas where laboratory support is lacking, symptomatic treatment for malaria is the norm. While microscopy is available in better-equipped laboratories, the evolution of rapid diagnostic tests for malaria has helped to make accurate tests available for a wider population of people.

RDT for malaria are mainly in two formats:
  1. Immunochromatography and
  2. Lateral Flow

Immunochromatography targets different antigens of the malaria parasite.
  1. Histidine rich protein 2 of P. Falciparum (pfHRP-2)
  2. Plasmodium aldolase - all malaria parasites and
  3. Parasite lactate dehydrogenase (pLDH) - all malaria parasites

The pfHRP-2 test can detect asexual parasitemia of > 40 parasites/ul, while the pLDH test detects parasitemia of >100-200 parasites/ul.

The falciparum specific tests are less expensive and more accurate and with the growing resistance of P. Falciparum to anti malarial drugs in Asia, Africa and S. America, the need for accurate and rapid diagnosis is felt.

Other assays available are that are more expensive are:
  1. Quantitative Buffy Coat (QBC)
  2. ELISA
  3. Multiplex PCR
  4. Qualitative NASBA - detects 50 parasites/ml of blood
Typhoid
Typhoid fever remains a major public health risk with around 13 million cases occurring annually. Blood culture while remaining the gold standard has several disadvantages starting with proper sampling. The Widal test is the one commonly used in spite of the problems with interpretation and standardization. With the advent of rapid tests, diagnosis has become considerably simple. Most are for antibody detection and are as follows:
  1. Dot ELISA - Typhidot - Both IgM and IgG
    Dip-S-ticks - IgG detection
    TUBEX - utilizes somatic 09 antibody - IgM
  2. ELISA - monoclonal antibodies to capture somatic antigen 9 (09), flagellar antigen d (Hd), and the Vi capsular polysaccharide antigen (Vi) of S. typhi in urine.

Antigen detection rapid tests are:
(i) PCR - one pair of primers using the flagellin gene of S.Typhi and (ii) Typhigen using recombinant DNA technology - S. Typhi antigen detected within 1-3 minutes.

Sensitivity and specificity of these tests vary depending on the population it is used in and the timing of sampling as is indicated by studies in different parts of the world.

A rough guide however would be as follows:

Sensitivity

Specificity

Typhidot
Dip-S-Ticks
TUBEX

79% (96 compared to BC)
89% (86 compared to BC)
89% 53%
78% 89%



A similar study from CMC Vellore showed that the Typhidot had a sensitivity of 100% with a specificity of 80%. Studies from Pakistan and Malaysia showed almost identical observations.

In another study from Indonesia, the PCR results (63% positive cases) were compared with those of blood culture (13.7% positive cases) and the Widal test (35.6% positive cases), using the same samples from each of the 73 patients admitted to the General Hospital in Makassar.

The diagnostic value of the Widal test especially in single samples remains an issue of contention. The sensitivity of the Typhidot assay even in early infections together with its high NPV even in high incidence populations makes it an ideal test in low incidence areas.
Tuberculosis
AFB smear and culture have been the mainstay of TB diagnosis since time immemorial. An unprocessed sample of sputum that contained > 100,000 AFB/ml was usually positive on staining and the sensitivity of the technique could be improved to detect AFB in concentrations as low as 1000 bacilli/ml.

Culture continues to remain the only definitive diagnostic tool for TB and the newer broth and subsequent automated rapid culture methods have brought down the period required for isolation of AFB. From smear positive samples, it takes around 10 days and from smear negative samples it takes three weeks. With these culture techniques it is possible to differentiate between atypical and typical mycobacteria and to have a sensitivity report in a week's time.

Newer more rapid tests available are:
  1. Accuprobe test (Gen Probe, SanDiego, CA) - HPLC
  2. The Gen-Probe AMPLIFIEDTM Mycobacterium tuberculosis Direct (MTD) Test - Direct Amplification Technique (DAT)
  3. Roche AMPLICOR Mycobacterium tuberculosis (MTB) Test (DAT)
  4. Stand Displacement Amplification test (BD)

There is again a degree of conflict regarding the usefulness of these tests. Some studies quote a 96% sensitivity and 100% specificity for tests (2) and (3) for respiratory and non-respiratory samples, including CSF. This should be relevant particularly in cases of meningitis.

Other studies conducted by the Mycobacteriology laboratory, Jewish Medical and Research Centre, Denver; show that the direct amplification techniques such as test numbers 2 and 3 are in no way superior to a proper smear examination.

Advantages of DAT: (a) Able to differentiate between atypical and typical mycobacteria and (b) More rapid than culture techniques.

Disadvantages: (a) False negatives (positive in only 50% of smear negatives that were subsequently confirmed positive) and (b) False positives (dead as well as live bacilli).

Some groups feel that concordance between smear and DATs gives a conclusive report, while when the results are not concordant, the overall clinical picture together with results of repeat testing must be considered.

Still others feel that rapid DATs can never replace standard lab protocols of diagnosis and can only be used in addition to them at this point in time. They also feel that local epidemiological data and the cost factor need to be considered.

Currently another team from UK are looking for "biomarkers" or molecular signatures of the TB bacilli, which they hope will provide the much-needed accurate, inexpensive and rapid diagnostic tool for TB.
Hepatitis
The maximum number of Rapid Diagnostic Tests in the market today are for the detection of Hepatitis B. However in the pediatric age group, a rapid diagnosis in a child with hepatitis is required in very specific situations such as a) A PUO with Hepatitis, b) in a child who has received several units of blood or blood products, c) prior to an emergency blood transfusion, d) to identify the agent responsible in congenital hepatitis and e) or in a particularly severe Hepatitis.

For most other forms of Hepatitis, the micro-titre ELISA is the usual method used for diagnosis. For Hepatitis B however, RDTs belong to the following formats:
  1. Agglutination
  2. Lateral flow
  3. Flow-through
  4. Immunoblots
  5. Immunochromatography.

Three tests passive hemagglutination (PHA) and Immunochromatography (IC) when compared to classic EIAs displayed comparable sensitivity and specificity and fulfilled WHO criteria for use in clinical situations.

Sensitivity and specificity of the IC tests was 100% while sensitivity of the PHA test was 97.5% with a specificity of 100%.

Another rapid IC test that has been evaluated is for simultaneous detection of Hep B surface and envelope antigen.

The sensitivity and specificity for the detection of HBsAg were 95 and 100%, and the corresponding positive and negative predictive values were 100 and 99.7%, respectively. The sensitivity and specificity for the detection of HBeAg were slightly less and were 80 and 98%, with positive and negative predictive values of 91 and 94%, respectively.

The usefulness of this test is because it helps to identify those patients who would benefit from treatment from those who do not and also those who require immunization.
HIV
The lag time between clinical examination to laboratory confirmation of a HIV positive status of an individual together with the simultaneous and effective anti-retroviral therapy, heightened the need for tests that gave conclusive results far sooner.

Situations when initial rapid testing is most required in pediatric practice are as follows:
  • To check the maternal HIV status in the immediate post partum period to rule out a congenital HIV infection
  • When there is a clinical suspicion of HIV in a child under 18 months
  • H/O sexual abuse/exposure in a child or adolescent
  • When the child is unlikely to return for follow up and a HIV result is required

The Rapid tests for HIV, detect the presence of antibody and hence are not useful to detect a HIV infection soon after exposure. All rapid tests detect HIV-1, while a few also identify HIV-2 infections. Those that have got US FDA approval have sensitivities ranging from 95.3% - 100% and specificities ranging from 96.7% to 100%. Most rapid tests have similar accuracy to classical EIAs and hence do not need confirmation with an EIA. However as the positive predictive value varies depending on the population studied, supplemental confirmation by WB or Immunofluorescence is required.

Rapid testing is NOT indicated in a child under one year of age because:
  • There is transfer of maternal IgG across the placenta
  • There is admixture of small quantities of maternal and fetal blood
  • Tiny placental leaks

These will result in a false antibody positive result in the child. If a conclusive diagnosis is required for example prior to adoption, a HIV DNA PCR should be performed. If two such assays are negative prior to 18 months of age, it is safe to assume that the child is not infected with HIV. In infants older than 4 weeks of age, HIV DNA PCR show a consistent sensitivity between 91-99%.
Rapid culture methods
Rapid Culture Methods are now being increasingly used in reference labs to identify viral infections. The Shell Vial Culture technique is now available for Respiratory Viruses, HSV, CMV and VZV. While they do give quick results, are economical and easy to use and can be interpreted without any specialist training, blind passages cannot be done and they remain beyond the scope of routing diagnostic laboratories.

Rapid testing devices are a great innovation, do save considerable time and can be used in certain places where sophisticated equipment or trained personnel are not available. While rapid diagnostic tests, whether serological, molecular based or culture techniques evolve with increasing sensitivity and specificity, the clinician needs to be certain of what exactly needs to be identified. They need to be interpreted after taking into account the prevalence of that particular infection/disease in the population under study. RDTs can confirm the clinical diagnosis and in very rare situations alone, make the diagnosis. The skill of the clinician together with clinical correlation of every test result is critical at every juncture.
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