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Dengue: Guidelines for Diagnosis, Treatment, Prevention and Control: New Edition. Geneva: World Health Organization; 2009.

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Dengue: Guidelines for Diagnosis, Treatment, Prevention and Control: New Edition.

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4LABORATORY DIAGNOSIS AND DIAGNOSTIC TESTS

4.1. OVERVIEW

Efficient and accurate diagnosis of dengue is of primary importance for clinical care (i.e. early detection of severe cases, case confirmation and differential diagnosis with other infectious diseases), surveillance activities, outbreak control, pathogenesis, academic research, vaccine development, and clinical trials.

Laboratory diagnosis methods for confirming dengue virus infection may involve detection of the virus, viral nucleic acid, antigens or antibodies, or a combination of these techniques. After the onset of illness, the virus can be detected in serum, plasma, circulating blood cells and other tissues for 4–5 days. During the early stages of the disease, virus isolation, nucleic acid or antigen detection can be used to diagnose the infection. At the end of the acute phase of infection, serology is the method of choice for diagnosis.

Antibody response to infection differs according to the immune status of the host (1). When dengue infection occurs in persons who have not previously been infected with a flavivirus or immunized with a flavivirus vaccine (e.g. for yellow fever, Japanese encephalitis, tick-borne encephalitis), the patients develop a primary antibody response characterized by a slow increase of specific antibodies. IgM antibodies are the first immunoglobulin isotype to appear. These antibodies are detectable in 50% of patients by days 3-5 after onset of illness, increasing to 80% by day 5 and 99% by day 10 (Figure 4.1). IgM levels peak about two weeks after the onset of symptoms and then decline generally to undetectable levels over 2–3 months. Anti-dengue serum IgG is generally detectable at low titres at the end of the first week of illness, increasing slowly thereafter, with serum IgG still detectable after several months, and probably even for life (24).

Figure 4.1. Approximate time-line of primary and secondary dengue virus infections and the diagnostic methods that can be used to detect infection.

Figure 4.1

Approximate time-line of primary and secondary dengue virus infections and the diagnostic methods that can be used to detect infection.

During a secondary dengue infection (a dengue infection in a host that has previously been infected by a dengue virus, or sometimes after non-dengue flavivirus vaccination or infection), antibody titres rise rapidly and react broadly against many flaviviruses. The dominant immunoglobulin isotype is IgG which is detectable at high levels, even in the acute phase, and persists for periods lasting from 10 months to life. Early convalescent stage IgM levels are significantly lower in secondary infections than in primary ones and may be undetectable in some cases, depending on the test used (5). To distinguish primary and secondary dengue infections, IgM/IgG antibody ratios are now more commonly used than the haemagglutination-inhibition test (HI) (68).

A range of laboratory diagnostic methods has been developed to support patient management and disease control. The choice of diagnostic method depends on the purpose for which the testing is done (e.g. clinical diagnosis, epidemiological survey, vaccine development), the type of laboratory facilities and technical expertise available, costs, and the time of sample collection.

In general, tests with high sensitivity and specificity require more complex technologies and technical expertise, while rapid tests may compromise sensitivity and specificity for the ease of performance and speed. Virus isolation and nucleic acid detection are more labour-intensive and costly but are also more specific than antibody detection using serologic methods. Figure 4.2 shows a general inverse relationship between the ease of use or accessibility of a diagnostic method and the confidence in the results of the test.

Figure 4.2. Comparison of diagnostic tests according to their accessibility and confidence.

Figure 4.2

Comparison of diagnostic tests according to their accessibility and confidence.

4.2. CONSIDERATIONS IN THE CHOICE OF DIAGNOSTIC METHODS

4.2.1. Clinical management

Dengue virus infection produces a broad spectrum of symptoms, many of which are non-specific. Thus, a diagnosis based only on clinical symptoms is unreliable. Early laboratory confirmation of clinical diagnosis may be valuable because some patients progress over a short period from mild to severe disease and sometimes to death. Early intervention may be life-saving.

Before day 5 of illness, during the febrile period, dengue infections may be diagnosed by virus isolation in cell culture, by detection of viral RNA by nucleic acid amplification tests (NAAT), or by detection of viral antigens by ELISA or rapid tests. Virus isolation in cell culture is usually performed only in laboratories with the necessary infrastructure and technical expertise. For virus culture, it is important to keep blood samples cooled or frozen to preserve the viability of the virus during transport from the patient to the laboratory. The isolation and identification of dengue viruses in cell cultures usually takes several days. Nucleic acid detection assays with excellent performance characteristics may identify dengue viral RNA within 24–48 hours. However, these tests require expensive equipment and reagents and, in order to avoid contamination, tests must observe quality control procedures and must be performed by experienced technicians. NS1 antigen detection kits now becoming commercially available can be used in laboratories with limited equipment and yield results within a few hours. Rapid dengue antigen detection tests can be used in field settings and provide results in less than an hour. Currently, these assays are not type-specific, are expensive and are under evaluation for diagnostic accuracy and cost-effectiveness in multiple settings. Table 4.1 summarizes various dengue diagnostic methods and their costs.

Table 4.1. Summary of operating characteristics and comparative costs of dengue diagnostic methods (9).

Table 4.1

Summary of operating characteristics and comparative costs of dengue diagnostic methods (9).

After day 5, dengue viruses and antigens disappear from the blood coincident with the appearance of specific antibodies. NS1 antigen may be detected in some patients for a few days after defervescence. Dengue serologic tests are more available in dengue-endemic countries than are virological tests. Specimen transport is not a problem as immunoglobulins are stable at tropical room temperatures.

For serology, the time of specimen collection is more flexible than that for virus isolation or RNA detection because an antibody response can be measured by comparing a sample collected during the acute stage of illness with samples collected weeks or months later. Low levels of a detectable dengue IgM response – or the absence of it – in some secondary infections reduces the diagnostic accuracy of IgM ELISA tests. Results of rapid tests may be available within less than one hour. Reliance on rapid tests to diagnose dengue infections should be approached with caution, however, since the performance of all commercial tests has not yet been evaluated by reference laboratories (10).

A four-fold or greater increase in antibody levels measured by IgG ELISA or by haemagglutination inhibition (HI) test in paired sera indicates an acute or recent flavivirus infection. However, waiting for the convalescent serum collected at the time of patient discharge is not very useful for diagnosis and clinical management and provides only a retrospective result.

4.2.1.1. Differential diagnosis

Dengue fever can easily be confused with non-dengue illnesses, particularly in non-epidemic situations. Depending on the geographical origin of the patient, other etiologies – including non-dengue flavivirus infections – should be ruled out. These include yellow fever, Japanese encephalitis, St Louis encephalitis, Zika, and West Nile, alphaviruses (such as Sinbis and chikungunya), and other causes of fever such as malaria, leptospirosis, typhoid, Rickettsial diseases (Rickettsia prowazeki, R. mooseri, R. conori, R. rickettsi, Orientia tsutsugamushi, Coxiella burneti, etc.), measles, enteroviruses, influenza and influenza-like illnesses, haemorrhagic fevers (Arenaviridae: Junin, etc.; Filoviridae: Marburg, Ebola; Bunyaviridae: hantaviruses, Crimean-Congo haemorrhagic fever, etc.).

Both the identification of virus/viral RNA/viral antigen and the detection of an antibody response are preferable for dengue diagnosis to either approach alone (see Table 4.2).

Table 4.2. Interpretation of dengue diagnostic tests [adapted from Dengue and Control (DENCO) study].

Table 4.2

Interpretation of dengue diagnostic tests [adapted from Dengue and Control (DENCO) study].

Unfortunately, an ideal diagnostic test that permits early and rapid diagnosis, is affordable for different health systems, is easy to perform, and has a robust performance, is not yet available.

4.2.2. Outbreak investigations

During outbreaks some patients may be seen presenting with fever with or without rash during the acute illness stage; some others may present with signs of plasma leakage or shock, and others with signs of haemorrhages, while still others may be observed during the convalescent phase.

One of the priorities in a suspected outbreak is to identify the causative agent so that appropriate public health measures can be taken and physicians can be encouraged to initiate appropriate acute illness management. In such cases, the rapidity and specificity of diagnostic tests is more important than test sensitivity. Samples collected from febrile patients could be tested by nucleic acid methods in a well-equipped laboratory or a broader spectrum of laboratories using an ELISA-based dengue antigen detection kit. If specimens are collected after day 5 of illness, commercial IgM ELISA or sensitive dengue IgM rapid tests may suggest a dengue outbreak, but results are preferably confirmed with reliable serological tests performed in a reference laboratory with broad arbovirus diagnostic capability. Serological assays may be used to determine the extent of outbreaks.

4.2.3. Surveillance

Dengue surveillance systems aim to detect the circulation of specific viruses in the human or mosquito populations. The diagnostic tools used should be sensitive, specific and affordable for the country. Laboratories responsible for surveillance are usually national and/or reference laboratories capable of performing diagnostic tests as described above for dengue and for a broad range of other etiologies.

4.2.4. Vaccine trials

Vaccine trials are performed in order to measure vaccine safety and efficacy in vaccinated persons. The plaque reduction and neutralization test (PRNT) and the microneutralization assays are commonly used to measure protection correlates.

Following primary infections in non-flavivirus immunes, neutralizing antibodies as measured by PRNT may be relatively or completely specific to the infecting virus type (11,12). This assay is the most reliable means of measuring the titre of neutralizing antibodies in the serum of an infected individual as a measure of the level of protection against an infecting virus. The assay is based on the principle that neutralizing antibodies inactivate the virus so that it is no longer able to infect and replicate in target cells.

After a second dengue virus infection, high-titre neutralizing antibodies are produced against at least two, and often all four, dengue viruses as well as against non-dengue flaviviruses. This cross reactivity results from memory B-cells which produce antibodies directed at virion epitopes shared by dengue viruses. During the early convalescent stage following sequential dengue infections, the highest neutralizing antibody titre is often directed against the first infecting virus and not the most recent one. This phenomenon is referred to as “original antigenic sin” (13).

The disadvantages of PRNT are that it is labour-intensive. A number of laboratories recently developed high through-put neutralization tests that can be used in large-scale surveillance studies and vaccine trials. Variable results have been observed in PRNTs performed in different laboratories. Variations can be minimized if tests are performed on standard cell lines using the same virus strains and the same temperature and time for incubation of virus with antibody. Input virus should be carefully calculated to avoid plaque overlap. Cell lines of mammalian origin, such as VERO cells, are recommended for the production of seed viruses for use in PRNT.

The microneutralization assay is based on the same principle as PRNT. Variable methods exist. In one, instead of counting the number of plaques per well, viral antigen is stained using a labelled antibody and the quantity of antigen measured colorimetrically. The test may measure nucleic acid using PCR. The microneutralization assay was designed to use smaller amounts of reagents and for testing larger numbers of samples. In viral antigen detection tests the spread of virus throughout the cells is not limited because, in PRNTs using semisolid overlays, the time after infection must be standardized to avoid measuring growth after many cycles of replication. Since not all viruses grow at the same rate, the incubation periods are virus-specific. As with standard PRNTs, antibodies measured by micromethods from individuals with secondary infections may react broadly with all four dengue viruses.

In drug trials, patients should have confirmed etiological diagnosis (see Table 4.2 for highly suggestive and confirmed diagnosis).

Table 4.3 summarizes the advantages and limitations of each of the diagnostic methods for each purpose.

Table 4.3. Advantages and limitations of dengue diagnostic methods (9).

Table 4.3

Advantages and limitations of dengue diagnostic methods (9).

4.3. CURRENT DENGUE DIAGNOSTIC METHODS

4.3.1. Virus isolation

Specimens for virus isolation should be collected early in the course of the infection, during the period of viraemia (usually before day 5). Virus may be recovered from serum, plasma and peripheral blood mononuclear cells and attempts may be made from tissues collected at autopsy (e.g. liver, lung, lymph nodes, thymus, bone marrow). Because dengue virus is heat-labile, specimens awaiting transport to the laboratory should be kept in a refrigerator or packed in wet ice. For storage up to 24 hours, specimens should be kept at between +4 °C and +8 °C. For longer storage, specimens should be frozen at -70 °C in a deep-freezer or stored in a liquid nitrogen container. Storage even for short periods at −20 °C is not recommended.

Cell culture is the most widely used method for dengue virus isolation. The mosquito cell line C6/36 (cloned from Ae. albopictus) or AP61 (cell line from Ae. pseudoscutellaris) are the host cells of choice for routine isolation of dengue virus. Since not all wild type dengue viruses induce a cytopathic effect in mosquito cell lines, cell cultures must be screened for specific evidence of infection by an antigen detection immunofluorescence assay using serotype-specific monoclonal antibodies and flavivirus group-reactive or dengue complex-reactive monoclonal antibodies. Several mammalian cell cultures, such as Vero, LLCMK2, and BHK21, may also be used but are less efficient. Virus isolation followed by an immunofluorescence assay for confirmation generally requires 1–2 weeks and is possible only if the specimen is properly transported and stored to preserve the viability of the virus in it.

When no other methods are available, clinical specimens may also be inoculated by intracranial route in suckling mice or intrathoracic inoculation of mosquitoes. Newborn animals can develop encephalitis symptoms but with some dengue strains mice may exhibit no signs of illness. Virus antigen is detected in mouse brain or mosquito head squashes by staining with anti-dengue antibodies.

4.3.2. Nucleic acid detection

RNA is heat-labile and therefore specimens for nucleic acid detection must be handled and stored according to the procedures described for virus isolation.

4.3.2.1. RT-PCR

Since the 1990s, several reverse transcriptase-polymerase chain reaction (RT-PCR) assays have been developed. They offer better sensitivity compared to virus isolation with a much more rapid turnaround time. In situ RT-PCR offers the ability to detect dengue RNA in paraffin-embedded tissues.

All nucleic acid detection assays involve three basic steps: nucleic acid extraction and purification, amplification of the nucleic acid, and detection and characterization of the amplified product. Extraction and purification of viral RNA from the specimen can be done by traditional liquid phase separation methods (e.g. phenol, chloroform) but has been gradually replaced by silica-based commercial kits (beads or columns) that are more reproducible and faster, especially since they can be automated using robotics systems. Many laboratories utilize a nested RT-PCR assay, using universal dengue primers targeting the C/prM region of the genome for an initial reverse transcription and amplification step, followed by a nested PCR amplification that is serotype-specific (14). A combination of the four serotype-specific oligonucleotide primers in a single reaction tube (one-step multiplex RT-PCR) is an interesting alternative to the nested RT-PCR (15). The products of these reactions are separated by electrophoresis on an agarose gel, and the amplification products are visualized as bands of different molecular weights in the agarose gel using ethidium bromide dye, and compared with standard molecular weight markers. In this assay design, dengue serotypes are identified by the size of their bands.

Compared to virus isolation, the sensitivity of the RT-PCR methods varies from 80% to 100% and depends on the region of the genome targeted by the primers, the approach used to amplify or detect the PCR products (e.g. one-step RT-PCR versus two-step RT-PCR), and the method employed for subtyping (e.g. nested PCR, blot hybridization with specific DNA probes, restriction site-specific PCR, sequence analysis, etc.). To avoid false positive results due to non-specific amplification, it is important to target regions of the genome that are specific to dengue and not conserved among flavi- or other related viruses. False-positive results may also occur as a result of contamination by amplicons from previous amplifications. This can be prevented by physical separation of different steps of the procedure and by adhering to stringent protocols for decontamination.

4.3.2.2. Real-time RT-PCR

The real-time RT-PCR assay is a one step assay system used to quantitate viral RNA and using primer pairs and probes that are specific to each dengue serotype. The use of a fluorescent probe enables the detection of the reaction products in real time, in a specialized PCR machine, without the need for electrophoresis. Many real-time RT-PCR assays have been developed employing TaqMan or SYBR Green technologies. The TaqMan real-time PCR is highly specific due to the sequence-specific hybridization of the probe. Nevertheless, primers and probes reported in publications may not be able to detect all dengue virus strains: the sensitivity of the primers and probes depends on their homology with the targeted gene sequence of the particular virus analyzed. The SYBR green real-time RT-PCR has the advantage of simplicity in primer design and uses universal RT-PCR protocols but is theoretically less specific.

Real-time RT-PCR assays are either “singleplex” (i.e. detecting only one serotype at a time) or “multiplex” (i.e. able to identify all four serotypes from a single sample). The multiplex assays have the advantage that a single reaction can determine all four serotypes without the potential for introduction of contamination during manipulation of the sample. However the multiplex real-time RT-PCR assays, although faster, are currently less sensitive than nested RT-PCR assays. An advantage of this method is the ability to determine viral titre in a clinical sample, which may be used to study the pathogenesis of dengue disease (16).

4.3.2.3. Isothermal amplification methods

The NASBA (nucleic acid sequence based amplification) assay is an isothermal RNA-specific amplification assay that does not require thermal cycling instrumentation. The initial stage is a reverse transcription in which the single-stranded RNA target is copied into a double-stranded DNA molecule that serves as a template for RNA transcription. Detection of the amplified RNA is accomplished either by electrochemiluminescence or in real-time with fluorescent-labelled molecular beacon probes. NASBA has been adapted to dengue virus detection with sensitivity near that of virus isolation in cell cultures and may be a useful method for studying dengue infections in field studies (17).

Loop mediated amplification methods have also been described but their performance compared to other nucleic acid amplification methods are not known (18).

4.3.3. Detection of antigens

Until recently, detection of dengue antigens in acute-phase serum was rare in patients with secondary infections because such patients had pre-existing virus-IgG antibody immunocomplexes. New developments in ELISA and dot blot assays directed to the envelop/membrane (E/M) antigen and the non-structural protein 1 (NS1) demonstrated that high concentrations of these antigens in the form of immune complexes could be detected in patients with both primary and secondary dengue infections up to nine days after the onset of illness.

The NS1 glycoprotein is produced by all flaviviruses and is secreted from mammalian cells. NS1 produces a very strong humoral response. Many studies have been directed at using the detection of NS1 to make an early diagnosis of dengue virus infection. Commercial kits for the detection of NS1 antigen are now available, though they do not differentiate between dengue serotypes. Their performance and utility are currently being evaluated by laboratories worldwide, including the WHO/TDR/PDVI laboratory network.

Fluorescent antibody, immunoperoxidase and avidin-biotin enzyme assays allow detection of dengue virus antigen in acetone-fixed leucocytes and in snap-frozen or formalin-fixed tissues collected at autopsy.

4.3.4. Serological tests

4.3.4.1. MAC-ELISA

For the IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) total IgM in patients' sera is captured by anti-μ chain specific antibodies (specific to human IgM) coated onto a microplate. Dengue-specific antigens, from one to four serotypes (DEN-1, -2, -3, and -4), are bound to the captured anti-dengue IgM antibodies and are detected by monoclonal or polyclonal dengue antibodies directly or indirectly conjugated with an enzyme that will transform a non-coloured substrate into coloured products. The optical density is measured by spectrophotometer.

Serum, blood on filter paper and saliva, but not urine, can be used for detection of IgM if samples are taken within the appropriate time frame (five days or more after the onset of fever). Serum specimens may be tested at a single dilution or at multiple dilutions. Most of the antigens used for this assay are derived from the dengue virus envelope protein (usually virus-infected cell culture supernatants or suckling mouse brain preparations). MAC-ELISA has good sensitivity and specificity but only when used five or more days after the onset of fever. Different commercial kits (ELISA or rapid tests) are available but have variable sensitivity and specificity. A WHO/TDR/PDVI laboratory network recently evaluated selected commercial ELISAs and first-generation rapid diagnostic tests, finding that ELISAs generally performed better than rapid tests.

Cross-reactivity with other circulating flaviviruses such as Japanese encephalitis, St Louis encephalitis and yellow fever, does not seem to be a problem but some false positives were obtained in sera from patients with malaria, leptospirosis and past dengue infection (10). These limitations have to be taken into account when using the tests in regions where these pathogens co-circulate. It is recommended that tests be evaluated against a panel of sera from relevant diseases in a particular region before being released to the market. It is not possible to use IgM assays to identify dengue serotypes as these antibodies are broadly cross-reactive even following primary infections. Recently, some authors have described MAC-ELISA (Figure 4.3) that could allow serotype determination but further evaluations are required (19).

Figure 4.3. Principle of a MAC-ELISA test.

Figure 4.3

Principle of a MAC-ELISA test.

4.3.4.2. IgG ELISA

The IgG ELISA is used for the detection of recent or past dengue infections (if paired sera are collected within the correct time frame). This assay uses the same antigens as the MAC-ELISA. The use of E/M-specific capture IgG ELISA (GAC) allows detection of IgG antibodies over a period of 10 months after the infection. IgG antibodies are lifelong as measured by E/M antigen-coated indirect IgG ELISA, but a fourfold or greater increase in IgG antibodies in acute and convalescent paired sera can be used to document recent infections. Test results correlate well with the haemagglutination-inhibition test. An ELISA inhibition method (EIM) to detect IgG dengue antibodies (20) is also used for the serological diagnosis and surveillance of dengue cases. This system is based in the competition for the antigen sites by IgG dengue antibodies in the sample and the conjugated human IgG anti-dengue.

This method can be used to detect IgG antibodies in serum or plasma and filter-paper stored blood samples and permits identification of a case as a primary or secondary dengue infection (20,21,22). In general, IgG ELISA lacks specificity within the flavivirus serocomplex groups. Following viral infections, newly produced antibodies are less avid than antibodies produced months or years after infection.

Antibody avidity is used in a few laboratories to discriminate primary and secondary dengue infections. Such tests are not in wide use and are not available commercially.

4.3.4.3. IgM/IgG ratio

A dengue virus E/M protein-specific IgM/IgG ratio can be used to distinguish primary from secondary dengue virus infections. IgM capture and IgG capture ELISAs are the most common assays for this purpose. In some laboratories, dengue infection is defined as primary if the IgM/IgG OD ratio is greater than 1.2 (using patient's sera at 1/100 dilution) or 1.4 (using patient's sera at 1/20 dilutions). The infection is secondary if the ratio is less than 1.2 or 1.4. This algorithm has also been adopted by some commercial vendors. However, ratios may vary between laboratories, thus indicating the need for better standardization of test performance (8).

4.3.4.4. IgA

Positive detection for serum anti-dengue IgA as measured by anti-dengue virus IgA capture ELISA (AAC-ELISA) often occurs one day after that for IgM. The IgA titre peaks around day 8 after onset of fever and decreases rapidly until it is undetectable by day 40. No differences in IgA titres were found by authors between patients with primary or secondary infections. Even though IgA values are generally lower than IgM, both in serum and saliva, the two methods could be performed together to help in interpreting dengue serology (22,23). This approach is not used very often and requires additional evaluation.

4.3.4.5. Haemagglutination-inhibition test

The haemagglutination-inhibition (HI) test (see Figure 4.4) is based on the ability of dengue antigens to agglutinate red blood cells (RBC) of ganders or trypsinized human O RBC. Anti-dengue antibodies in sera can inhibit this agglutination and the potency of this inhibition is measured in an HI test. Serum samples are treated with acetone or kaolin to remove non-specific inhibitors of haemagglutination, and then adsorbed with gander or trypsinized type O human RBC to remove non-specific agglutinins. Each batch of antigens and RBC is optimized. PH optima of each dengue haemagglutinin requires the use of multiple different pH buffers for each serotype. Optimally the HI test requires paired sera obtained upon hospital admission (acute) and discharge (convalescent) or paired sera with an interval of more than seven days. The assay does not discriminate between infections by closely related flaviviruses (e.g. between dengue virus and Japanese encephalitis virus or West Nile virus) nor between immunoglobulin isotypes. The response to a primary infection is characterized by the low level of antibodies in the acute-phase serum drawn before day 5 and a slow elevation of HI antibody titres thereafter. During secondary dengue infections HI antibody titres rise rapidly, usually exceeding 1:1280. Values below this are generally observed in convalescent sera from patients with primary responses.

Figure 4.4. Haemagglutination-inhibition assay.

Figure 4.4

Haemagglutination-inhibition assay.

4.3.5. Haematological tests

Platelets and haematocrit values are commonly measured during the acute stages of dengue infection. These should be performed carefully using standardized protocols, reagents and equipment.

A drop of the platelet count below 100 000 per μL may be observed in dengue fever but it is a constant feature of dengue haemorrhagic fever. Thrombocytopaenia is usually observed in the period between day 3 and day 8 following the onset of illness.

Haemoconcentration, as estimated by an increase in haematocrit of 20% or more compared with convalescent values, is suggestive of hypovolaemia due to vascular permeability and plasma leakage.

4.4. FUTURE TEST DEVELOPMENTS

Microsphere-based immunoassays (MIAs) are becoming increasingly popular as a serological option for the laboratory diagnosis of many diseases. This technology is based on the covalent bonding of antigen or antibody to microspheres or beads. Detection methods include lasers to elicit fluorescence of varying wavelengths. This technology is attractive as it is faster than the MAC-ELISA and has potential for multiplexing serological tests designed to identify antibody responses to several viruses. MIAs can also be used to detect viruses.

Rapid advances in biosensor technology using mass spectrometry have led to the development of powerful systems that can provide rapid discrimination of biological components in complex mixtures. The mass spectra that are produced can be considered a specific fingerprint or molecular profile of the bacteria or virus analysed. The software system built into the instrument identifies and quantifies the pathogen in a given sample by comparing the resulting mass spectra with those in a database of infectious agents, and thus allows the rapid identification of many thousands of types of bacteria and viruses. Additionally, these tools can recognize a previously unidentified organism in the sample and describe how it is related to those encountered previously. This could be useful in determining not only dengue serotypes but also dengue genotypes during an outbreak. Identification kits for infectious agents are available in 96-well format and can be designed to meet specific requirements. Samples are processed for DNA extraction, PCR amplification, mass spectrometry and computer analysis.

Microarray technology makes it possible to screen a sample for many different nucleic acid fragments corresponding to different viruses in parallel. The genetic material must be amplified before hybridization to the microarray, and amplification strategy can target conserved sequences as well as random-based ones. Short oligonucleotides attached on the microarray slide give a relatively exact sequence identification, while longer DNA fragments give a higher tolerance for mismatches and thus an improved ability to detect diverged strains. A laser-based scanner is commonly used as a reader to detect amplified fragments labelled with fluorescent dyes. Microarray could be a useful technology to test, at the same time, dengue virus and other arboviruses circulating in the region and all the pathogens responsible for dengue-like symptoms.

Other approaches have been tested but are still in the early stages of development and evaluation. For instance, the luminescence-based techniques are becoming increasingly popular owing to their high sensitivity, low background, wide dynamic range and relatively inexpensive instrumentation.

4.5. QUALITY ASSURANCE

Many laboratories use in-house assays. The main weakness of these assays is the lack of standardization of protocols, so results cannot be compared or analysed in aggregate. It is important for national or reference centres to organize quality assurance programmes to ensure the proficiency of laboratory staff in performing the assays and to produce reference materials for quality control of test kits and assays.

For nucleic acid amplification assays, precautions need to be established to prevent contamination of patient materials. Controls and proficiency-testing are necessary to ensure a high degree of confidence (24).

4.6. BIOSAFETY ISSUES

The collection and processing of blood and other specimens place health care workers at risk of exposure to potentially infectious material. To minimize the risk of infection, safe laboratory techniques (i.e. use of personal protective equipment, appropriate containers for collecting and transporting samples, etc.) must be practised as described in WHO's Laboratory biosafety manual (25).

4.7. ORGANIZATION OF LABORATORY SERVICES

In a disease-endemic country, it is important to organize laboratory services in the context of patients' needs and disease control strategies. Appropriate resources should be allocated and training provided. A model is proposed in Table 4.4. Examples of good and bad practice can be found in Table 4.5.

Table 4.4. Proposed model for organization of laboratory services.

Table 4.4

Proposed model for organization of laboratory services.

Table 4.5. Dengue laboratory diagnosis: examples of good and bad practice.

Table 4.5

Dengue laboratory diagnosis: examples of good and bad practice.

4.8. REFERENCES

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