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Diagnosis of dengue
Diagnosis of dengue
Diagnosis of dengue
Diagnosis of dengue
Diagnosis of dengue
Diagnosis of dengue
Diagnosis of dengue
Diagnosis of dengue
Diagnosis of dengue
Diagnosis of dengue
Diagnosis of dengue
Diagnosis of dengue
Diagnosis of dengue
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Diagnosis of dengue

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  • 1. 10.1586/ERI.12.76 895ISSN 1478-7210www.expert-reviews.comReview© 2012 Kin Fai TangDengue is endemic throughout the tropicalworld. The WHO has estimated that approxi-mately 3 billion people live at risk of infectioneach year. Infection produces a spectrum ofclinical manifestation, from mild influenza-likeillness to dengue fever (DF) or severe dengueillness. The latter comprise of either plasma leak-age, which leads to hypovolemic shock or dengueshock syndrome and internal hemorrhage, orother organ failure, including encephalo­pathy [1].The disease is caused by dengue virus (DENV),which belongs to the genus Flavivirus of theFlaviviridae family [2]. DENV is a positive-sense,single-stranded RNA virus (approximately 11 kbin length). The genome is transcribed as a singleopen reading frame encoding three structural(C, prM and E) and seven nonstructural (NS1,NS2A, NS2B, NS3, NS4A, NS4B and NS5)proteins [3] that are subsequently cleaved intoindividual components by proteolytic cleavage[4]. The untranslated terminal regions at bothends of the viral genome (3′ and 5′ untranslatedterminal regions) are important in the regula-tion of translation and replication of the viralgenome [5].DENV is composed of four antigenicallydistinct serotypes. Infection by a specific sero-type confers lifelong immunity against thespecific serotype but not to the remainingthree, although transient crossprotection hasbeen observed within 2–3 months followingacute dengue infection [6]. Secondary infectioncarries a higher risk of plasma leakage that, ifnot appropriately supported clinically with fluidmanagement, can lead to shock [7]. This asso-ciation between secondary infection and severedengue is thought to be mediated by the bind-ing of crossreactive, neutralizing antibodies atsubneutralizing concentration that enhances theinfection of monocytes and dendritic cells (DCs)via the Fc receptors, a process termed antibody-dependent enhancement (ADE) [8–10]. BesidesADE, other factors that could influence severeclinical outcome in a dengue infection includeboth human host [11–14] and viral factors [15–18].DENV is transmitted to humans primarilyby infected Aedes aegypti – the predominant epi-demic vector – while Aedes albopictus and Aedespolynesiensis have also caused dengue outbreaks[19–21]. Current methods of disease preventionrely on reducing the vector population density.However, given the experience of countriessuch as Singapore, where periodic epidemics ofdengue continue despite concerted public healthefforts to control the vector population [22], acost-effective vaccine remains the most viableoption for dengue prevention.The development of a dengue vaccine has beencomplicated by the concern that subneutralizinglevels of antibodies may paradoxically increasethe risk of severe dengue in the form of denguehemorrhagic fever (DHF) and dengue shocksyndrome through ADE [8–10]. Hence, while adengue vaccine was initially advocated in theKin Fai Tang*1andEng Eong Ooi1,21Program in Emerging InfectiousDisease, Duke-NUS Graduate MedicalSchool Singapore, 8 College Road,169857 Singapore2DSO National Laboratories, Singapore*Author for correspondence:Tel.: +65 651 67406Fax: +65 622 12529kinfai.tang@duke-nus.edu.sgEarly diagnosis of dengue, the most common mosquito-borne disease globally, remainschallenging. Dengue presents initially as undifferentiated fever, with symptoms becomingmore pathognomonic in the later stages of illness. This limits the timeliness in the delivery ofappropriate supportive interventions. Laboratory tests are useful for diagnosis although theshort-lived viremia and the presence of secondary infection with one of the four heterologousviral serotypes collectively complicate the choice and interpretation of laboratory tests. In thisarticle, the authors review the various approaches for diagnosis of dengue and discuss theappropriate tests to use, including when a dengue vaccine, which is in the late stages ofdevelopment, is licensed for use. The ensuing reduced dengue prevalence could make diagnosisfor vaccine efficacy and escape-mutant monitoring even more challenging.Diagnosis of dengue:an updateExpert Rev. Anti Infect. Ther. 10(8), 895–907 (2012)Keywords: dengue • diagnostics • early diagnosis • surveillance • vaccineFor reprint orders, please contact reprints@expert-reviews.com
  • 2. Expert Rev. Anti Infect. Ther. 10(8), (2012)896Review1940s [23], it was not until 1971 that the feasibility of a denguevaccine in preventing DHF was studied [10,24]; and based on initialdata [25–29], a vaccine search was initiated, endorsed and discussedby the SEARO/WHO Research Study Group and experts in thefield [24]. Despite initial optimism [30,31], more than three decadeshave passed without a licensed dengue vaccine in the market.However, current developments are promising and six tetravalentcandidate vaccines are in Phase I–III trials. Optimistically, alicensed vaccine can be anticipated in the next 5–7 years [32–34].Meanwhile, apart from vector control, the burden of dengue onsociety can also be reduced through appropriate and timely clini-cal interventions to prevent severe morbidity and mortality. Thisrelies on early and accurate diagnosis of dengue. Even when a vac-cine or an antiviral drug becomes available, the need for accuratediagnosis would not be diminished. Instead, the requirement foraccurate diagnosis could become more demanding, as surveillancefor dengue in vaccinated individuals would be needed to deter-mine vaccine efficacy and for the early detection of vaccine-escapemutants. The goal of this review is thus to determine the state ofthe art in diagnosis of dengue and identify areas where improve-ments through research are needed to prepare for the quality ofdiagnostics needed in a postvaccination world.Current status in diagnosis of dengueClinical diagnosisDiagnosis of dengue starts with a clinical suspicion, prompted bythe recognition of a collection of presenting symptoms and signs. Inthe early acute febrile phase of illness, dengue patients often presentwith a history of sudden onset fever, which is often accompaniedby nausea, aches and pains. Unfortunately, these symptoms are notunique to dengue and are reported with other febrile illnesses (OFI).The onset of a maculopapular rash, retro-orbital pain, petechiae orbleeding nose or gums are more pathognomonic of dengue andwould more probably trigger a differential diagnosis of dengue,although these symptoms usually appear in the later stages of illness,nearer the phase of fever defervescence, when plasma leakage occurs[1].Their usefulness for early diagnosis would thus be more limited.A list of the commonly reported symptoms is shown in Table 1.As each of the individual symptoms cannot accurately differenti-ate dengue from OFIs, an alternative approach to clinical diagnosisis to use a permutation of a list of symptoms or signs. The WHOguidelines for dengue are such examples [1,35]. Indeed, when appliedto prospectively recruited patients who presented with acute febrileillness less than 72 h from fever onset, both the 1997 and 2009WHOguidelines showed a similar sensitivity of over 95% in young adults,albeit with poor specificities of less than 40% [12]. Consequently, theWHO classification schemes can be useful to trigger a suspicion ofdengue. During epidemics, when the prevalence of dengue is high,cases that fit these definitions could be treated as presumptive denguewhile awaiting other test results. However, they cannot be used fora confirmatory diagnosis of dengue. Furthermore, caution must beexercised in places where dengue infection occurs in older adults.The same study showed that adults who are 56 years of age and olderhad greatly reduced sensitivities, from over 95 to 73.7% and 81.6%for the 1997 and 2009 WHO classification schemes, respectively.In such cases, the study suggested that leukopenia in patients withfebrile illness should trigger a suspicion of dengue [12].Besides the WHO classification schemes, others have attemptedto develop diagnostic algorithms for dengue. Tanner et al. useda data mining approach to identify a group of symptoms andhematologic measurements to differentiate dengue from OFIs [36].The resultant algorithm had a sensitivity and specificity of 71.2and 90.1%, respectively. Another comprehensive multivariablelogistic regression model was also developed and validated fordistinguishing DHF from DF, DHF from DF or OFIs, denguefrom OFIs and severe dengue from nonsevere dengue. The modelwas found to have a sensitivity that ranged from 89.2 (denguefrom OFIs) to 79.6% (DHF from DF) [37]. This model alsoprovides a tool for probability calculation and classification ofpatients based on readily available clinical and laboratory data.However, the usefulness of such algorithms remains to be testedin different populations with different circulating DENV strains.An important limitation in the development of useful clinicalapproaches to diagnosis of dengue is the lack of standardizationwith regard to study design, diagnostic criteria and data collec-tion [38]. While this is not surprising as these studies were per-formed by various laboratories in different countries, it limits thedevelopment of diagnostic classification or algorithms that canbe applied internationally. Indeed, the need for more prospectivestudies to construct a valid and generalizable algorithm to guidethe differential diagnosis of dengue in endemic countries remainsurgent [38].Laboratory diagnosisAs clinical diagnosis lacks specificity, a definitive diagnosis ofdengue infection requires laboratory confirmation. A number ofdifferent laboratory tools are available for diagnostic use. A sum-mary of laboratory diagnostic methods used in dengue infectionis shown in Table 2 and the approximate time from illness onset atwhich these diagnostic tests should be used is shown in Figure 1.Virus isolationDengue viremia can be detected from 2 to 3 days prior to theonset of fever to up to 5.1 and 4.4 days after the onset of thedisease for primary and secondary infection, respectively [39].During this viremic period, blood, serum or plasma samples canbe used for virus isolation.Mosquito inoculation remains the most sensitive method forvirus isolation. The isolation rate of the four serotypes of DENVis in the range of 71.5–84.2% [40]. Various mosquito specieshave been found to be useful and sensitive in dengue isolation,including A. aegypti, A. albopictus and Toxorhynchites splendens,where both male and female mosquitoes are susceptible [6,41–44].These mosquitoes are inoculated intrathoracically with serum orplasma specimens [40–44]. Specimens collected early in the courseof illness have a greater isolation rate (85.3% before day 4 of ill-ness) than those collected later (65.4% after day 4 of illness) [40].Furthermore, the isolation rate in patients with primary dengue(91.0%) was higher than those with secondary dengue (77.6%).This observation could be due to the interference of crossreactiveTang & Ooi
  • 3. 897www.expert-reviews.comReviewantibodies with virus isolation or a faster rate of viral clearance inpatients with secondary DENV infection [39]. Either explanation,however, suggests that the prevalence of primary or secondaryDENV infection may influence the overall virus isolation [40,45].Mouse brain inoculation has also been used to isolate andamplify DENV. Generally, unweaned mice (2–4 days of age)are inoculated intracerebrally with serum or plasma specimensand observed daily. Moribund mice are then sacrificed to harvestthe isolate [46,47].Both mosquito and mouse brain inoculation techniques arenot routinely used in day-to-day diagnosis owing to their highlyspecialized technical, safety and facility requirements as well ashigh maintenance costs. Instead, virus isolation using cell linesis more widely used. The most commonly used cell line forDENV isolation is C6/36, which was derived from A. albopictus.Alternatively, mammalian cell lines such as Vero, LLC-MK2 orBHK-21 could also be used, although these offer lower sensitiv-ity than C6/36 [41,42,48–53]. Besides diagnosis, virus isolationoffers the advantage of providing a virus isolate that may becharacterized during subsequent in vitro studies, such as genome­sequencing, virus neutralization and infection studies.A successful isolation of DENV on mosquito or cell culturecan be confirmed and serotyped by an immunofluorescenceassay using DENV- and serotype-specific monoclonal anti­bodies,respectively [41,53]. Virus isolation is highly specific and has atheoretical detection limit of a single viable virus, although inpractice, the sensitivity is only approximately 40.5% in cell line-based virus isolation [54]. It also requires highly trained operators,a dependence on sample integrity and a short viremia period, thusproviding a narrow window of opportunity from illness onset [54].Therefore, despite its advantages, this approach is not widely usedin routine diagnostic laboratories.Viral RNA detectionReverse transcriptase PCR (RT-PCR) detection of dengue viralRNA extracted from blood, serum or plasma provides a rapid,sensitive and specific method for dengue infection confirmation.Various primers and protocols have been developed, validated andused in conventional RT-PCR [1,54–61] and real-time RT-PCR,either using SYBR®Green as a fluorescent detection marker orlabeled oligonucleotide probes [58,59,62–79]. A technique using asingle reaction mixture at a constant temperature (nucleic acidTable 1. Symptoms differentiating dengue infection from other febrile illnesses.Symptoms Den|OFIs p-value Children (<15 years)|adults(>15 years)p-value Ref.Nausea 50.0|28.9%68.0|49.0%†51.3|30.5%<0.00001<0.05<0.00150.2|76.4% <0.001 [12,103,145,146]Vomitting 16.4|8.4%16.2|8.5%57.0–64.0|31.0–46.0%†70.0|52.0%†<0.000010.03<0.01<0.0550.2|76.4% <0.001 [12,103,145–148]Retro-orbital pain 26.0|15.9%26.6|13.5%10.01§<0.000010.0030.0018.7|29.1% <0.001 [12,103,146,148]Aches/pains 1.4§<0.0001 20.3|36.4% 0.012 [12,146]Rash 11.2–41.2|3.0–6.4% <0.003/0.007 NA NA [12,103,134,149]Tourniquet test positive 34.0|19.0%42.0|5.0%†43.0–65.0|21.0–39.0%1.86§0.02<0.01<0.1<0.001NA NA [12,103,134,149]Leukopenia 3.8 × 103|7.3 × 103/µl4.5 × 103|8.1 × 103/μl<4.5 × 103/μl: 72.1|11.5%<0.0001<0.1<0.001NA NA [12,37,103,145]Thrombocytopenia(platelet/mm3)16|4% (≤100,000)†16|82%‡(≤100,000)66|95%‡(≤100,000)14.9|1.5% (≤100,000)32,000|96,500163,500|239,00070,000|104,000¶NANA<0.01<0.001<0.001<0.0001NANA NA [12,103,145,147,150]†Studies perfomed in children younger than 15 years.‡Dengue and severe dengue comparison, performed in children younger than 15 years.§Risk ratio.¶Average.Den: Confirmed dengue cases; NA: Not applicable; OFI: Other febrile illness.Diagnosis of dengue: an update
  • 4. Expert Rev. Anti Infect. Ther. 10(8), (2012)898Reviewsequence-based amplification [NASBA]) [80] was found to behighly sensitive (98.5%) and specific (100%) [81,82]. NASBA maybe highly useful and applicable during outbreak field diagnosiswhere thermocyclers are not readily available.Sensitivity of conventional RT-PCR ranges from 48.4 to98.2% and has a detection limit of 1–50 plaque forming units(PFUs) [54–56,59]. These assays employ primers that bind toknown conserved regions of the DENV genome to avoid falsenegative findings due to spontaneous mutations expected inthe replication of the RNA viral genome. The use of in silicomethods to develop a cocktail of primers that bind to almostall DENV with known sequences has also been explored [58],although validation in a clinical setting remains to be carriedout. The sensitivity of RT-PCR is also highly dependent on theshort window of opportunity that coincides with the viremicperiod, which can last up to 8 days from illness onset (Figure 1).However, RT-PCR is rarely positive in a case of dengue after6 days from illness onset [1].Fluorescence-based real-time RT-PCR has a better reported sen-sitivity (58.9–100%) and detection limit (0.1–3.0 PFUs) owing tothe sensitivity of the fluorescence detector within the thermocycler[59,62–67,71,74,75,79]. Multiplex RT-PCRs that differentiate DENVserotypes in a single assay have also been developed [56,59,65,69,76].The experience with NASBA is more limited compared withRT-PCR, although a study has shown thatit can be as sensitive as RT-PCR, with adetection limit of <25 PFUs/ml [81]. RNAextraction from whole blood may be moresensitive (90.0%) than serum or plasma(62.0%) in the same pool of samples [78].Besides blood samples, RT-PCR can alsobe used to detect DENV RNA in tissues,including formalin-fixed specimens [82].Although RT-PCR usually requires exper-tise in molecular techniques and expensiveequipment [83], modified protocols usingfast-ramping thermocyclers can be usedin conjunction with newly trained opera-tors during emergency settings, such as indifferentiating dengue from SARS duringan outbreak [62], provided a strict standardoperating procedure is followed.Dengue viral RNA can also be detectedin urine [68,72] and saliva [72] samples usingreal-time RT-PCR. In urine, samples–2 –1 0Diseaseonset3Time (Days)1 2 4 5 6 7 8 9 10 11 20 30 40 60 90lgMlgMlgGlgG2* dengue1* dengue>90ViremiaNS1Virus isolationViral RNA detectionFigure 1. Approximate window of detection for dengue diagnostics.NS1: Non-structural protein 1.Data taken from [1].Table 2. Laboratory diagnostics for dengue: sensitivity and specificity.Category Technique Parameters Ref.Sensitivity (%) Specificity (%) Detection limitViral detection Virus isolation (mosquitoes) 71.5–84.2 100 NA [6,40–42,44]Virus isolation (mouse intrecerebral inoculation) NA NA NA [46,47]Virus isolation (cell culture) 40.5 100 ≥1 viable virus [54]Viral RNA RT-PCR (conventional) 48.4–100 100 1–50 PFUs [54–57]Viral RNA RT-PCR (real-time detection) 58.9–100 100 0.1–3.0 PFUs [54,57,63–67,79]Viral RNA RT-PCR (NASBA) 98.5 100 <25 PFUs/ml [81]Viral antigen detection (NS1 detection) 54.2–93.4 92.5–100 0.2 ng/ml†[79,91–103,110]Antibody detection IgM detection 61.5–100 52.0–100 NA [54,102,115,116]IgG detection 46.3–99.0 80.0–100 NARapid IgM detection (strips) 20.5–97.7 76.6–90.6 NA [115]Antigen/antibodycombined detectionNS1 and IgM 89.9–92.9 75.0–100 NA [91,100–102]NS1 and IgM/IgG 93.0 100 NA [101,151]NA: Not applicable; NASBA: Nucleic acid sequence-based amplification; PFU: Plaque forming unit; RT-PCR: Reverse transcriptase PCR.†Data taken from [110].Tang & Ooi
  • 5. 899www.expert-reviews.comReviewcollected between day 6 and day 16 after illness onset werefound to have higher rates of detection (50–80%) comparedwith day 1 to 3 samples (25–50%) [68]. RT-PCR for DENVin urine may thus extend the window of opportunity for viralRNA detection compared with blood specimens (up to day 8).However, the level of viral RNA in urine and saliva samples is low(1 × 101–5 × 101PFUs/ml) compared with the corresponding serumsamples (7.9 × 102–1.9 × 105PFUs/ml) [72].Antigen detectionDengue NS1 is a highly conserved glycoprotein essential forDENV viability and is secreted from infected cells as a solublehexamer [84,85]. Serum or plasma DENV NS1 level has been foundto correlate with viremia titer and disease severity [86–88]. It canbe found in the peripheral blood circulation for up to 9 daysfrom illness onset [89–91], but can persist for up to 18 days fromillness onset in some cases [92]. NS1 detection thus offers a largerwindow of opportunity for diagnosis of dengue compared withvirus isolation, RT-PCR or NASBA [68,72]. Commercially availableNS1 capture-based detection kits with sensitivities that rangedfrom 54.2 to 93.4% have been comprehensively evaluated (Table 2)[66,79,91,93–103] and found to be able to confirm dengue infectionin serum specimens that were RT-PCR negative and secondarydengue infection [97]. However, NS1 detection is less sensitive insecondary dengue infection (67.1–77.3%) compared with primarydengue cases (94.7–98.3%) [93,94,96,103], probably owing to thepresence of crossreactive anti-NS1 antibodies that impedes thedetection of free NS1 proteins in the serum or plasma [86,89].Anti-NS1 antibodies can also be used to detect infection inother sample sources, such as tissues, including liver, lung andkidney [104], through immunohistochemistry. This could be use-ful in postmortem studies. Although highly conserved, serotype-specific NS1 monoclonal antibodies have been raised and appliedfor NS1-based dengue serotype identification assays [105–109]. Astudy by Puttikhunt et al. has shown an overall sensitivity of 76.5%and specificity of 100% for diagnosis of dengue while having sero­typing sensitivities of 100% for DENV 1, 3 and 4 and 82.4% forDENV2 [109]. However, the sensitivity of these tests may differwith different strains of DENV as the magnitude of NS1 secretionappears to be strain dependent [110].Antibody detection (IgM & IgG)Detection of antidengue antibodies (IgM and IgG) is the mostwidely used test in diagnosis of dengue [111].These kits are either inthe form of Ig capture or direct Ig detection and are configured todetect IgM, IgG or both simultaneously [102,112–115].There are twoversions of these tests: ELISA or strip format (rapid test). WhileELISA provides greater sensitivity, the strip format is amenablefor bedside use [116].Antibody response in the form of antidengue IgM can bedetected as early as 3–5 days after illness onset. Levels of IgMcontinue to increase for approximately 2 weeks thereafter and maypersist for approximately 179 and 139 days following primary andsecondary infection, respectively [117]. Thus, while a single IgMraises the likelihood that a febrile patient has dengue, a definitivediagnosis may require the use of paired sera to demonstrate risingIgM titers.A multinational and multicenter study of ten IgM kits hasconcluded that ELISA-based detection kits have higher sensi-tivities (61.5–99.0%) compared with the rapid test formats(20.5–97.7%). The specificities are in the range of 79.9–97.8%and 76.6–90.6% for ELISA and rapid tests, respectively [115].Other evaluation studies have also reported similar sensitivitiesand specificities [62,102,116]. The wide ranges of these values areprobably due to the timing of sample collection [118].Early antidengue IgM response (<2 months) has been found tobe crossreactive to all four DENV serotypes [119] and other flavi­viruses [115]. Hence, epidemiological information on the preva-lence of other flaviviruses would be useful to guide the interpre-tation of a positive IgM finding. False positives have also beenobserved in patients with previous dengue or malaria infection[116]. However, more efficient algorithms can be developed tomitigate this ­problem, as shown by Prince et al. [120].During primary infection, IgG can only be detected after10 days from illness onset, making it less useful for early diagno-sis. However, the rapid increase of IgG levels during secondaryinfection (as early as day 4 from illness onset) [1] can be suggestiveof dengue when the ratio of IgM and IgG is used [62,102,115–117].Dengue neutralizing antibody detectionNeutralizing antibodies inhibit DENV infection and can thusprovide greater specificity in distinguishing antibodies to DENVfrom other crossreactive flavivirus antibodies [121,122]. These anti-bodies can be measured by using plaque reduction neutralizingtests (PRNTs), first developed by Russell et al. [122] based on theprotocol from Dulbecco et al. [123]. To date, PRNT remains themost widely used assay for immunity studies [124,125]. However, itis labor intensive, time consuming and has low throughput [124],and is therefore not routinely used in dengue diagnostics.New tests such as the ELISA-based microneutralization test(ELISA-MN) [126], the fluorescent antibody cell sorter-basedDendritic Cell-Specific Intercellular adhesion molecule-3-Grab-bing Non-integrin expressor DC assay [127] and the enzyme-linkedimmuno­sorbent spot microneutralization assay [128] have beendeveloped to overcome the limitations associated with PRNT.These new tests have been separately validated, compared and eval-uated [124,126–130] against PRNT and found to have good agreement(false-positive rate <10%) in cases with primary DENV infection[124,130]. However, Putnak et al. reported poor agreement amongthe tests in association with vaccination or secondary infection[124]. This result could have been influenced by the use of differentcell lines or different strains of DENV [129]. Others have suggestedthe use of Fcγ receptor (FcγR)-positive cells for these assays sinceDENV infects monocytes and DCs that express such receptors[131,132]. The use of such cells may also provide information onwhether the antibodies were able to neutralize DENV intra­cellularly or whether neutralization was only mediated throughthe coligation of FcγRIIB, which inhibits FcγR-mediated phago-cytosis and hence DENV entry into monocytes [133]. A limitedobservation suggests that this could provide greater specificity onDiagnosis of dengue: an update
  • 6. Expert Rev. Anti Infect. Ther. 10(8), (2012)900Reviewthe DENV serotype responsible for the infection [133]. However,detailed validation is required for all of these assays before theycan be used clinically.Combined antigen/antibody detectionGiven the dynamic nature of the NS1 antigen, antidengue IgMand IgG antibody levels during the course of acute illness, effortshave been made to combine all three tests into a single reactionfor ease of use. Some of these have been evaluated and shown tohave promising diagnostic sensitivity (89–93%) and specificity(75.0–100%) [91,100–102,134].Advances in rapid diagnostic toolsWhile rapid bedside diagnosis formats are available for antigenor antibody detection or both simultaneously, the sensitivitiesand specificities of the available tests have been uniformly lowerthan the equivalent laboratory-based assays. A complete review ofthese assays is provided elsewhere [102,118]. These limitations maybe due to the use of the lateral flow dipstick approach. The newlab-on-a-chip platform could offer a way to improve the perfor-mance of these bedside diagnostic tools [135–137]. This platformmakes use of a number of new technologies; some in combina-tion, such as microfluidics [135–137] and grating couplers [137] toimprove multiplexing, accommodate better mixing of reagentswith test samples as well as achieving greater sensitivity for detect-ing positive signals [138]. This platform could feature prominentlyin dengue diagnostics in the coming years.Disease prognosticationProgression of mild dengue infection to severe dengue(DHF/dengue shock syndrome) is difficult to predict owing to anincomplete understanding of disease pathogenesis. Symptoms andsigns of severe dengue have a sudden onset at the time of deferves-cence [1,19]. Careful monitoring of hematocrit as well as signs ofcirculatory failure or internal hemorrhage needs to be carried outfor at least 2 days after fever defervescence. Specifically, patientsshould be observed for signs such as severe abdominal pain, passageof black stools, bleeding into the skin, nose or gums, sweating orcold skin, which could indicate the devel-opment of DHF [7]. Depending on diseaseprogression, should DHF, occurs, oral rehy-dration therapy is sufficient for milder DHFwhile intravenous fluid therapy is suggestedfor more severe manifestations, and blood­transfusion is suggested for ­critical cases [7].However, hospitalization for close mon-itoring of all patients in dengue-endemiccountries is often not feasible, particularlyduring outbreaks, as it stresses the lim-ited medical healthcare resources. Undersuch circumstances, an ability to predictthe development of severe dengue at theearly stages of illness could thus be use-ful for triaging patients. Various clinicalmarkers have been proposed as warningsigns of severe dengue progression, asshown in Table 3. How well these clinicalsymptoms/signs perform in predicting theonset of severe dengue remains to be fullydetermined.Besides monitoring individual symptomsor signs, several groups have also evaluatedthe usefulness of combining these into analgorithm for predicting severe dengue. Leeet al. explored the use of a probability equa-tion that combines four simple clinical lab-oratory observations, including bleeding,lymphocyte proportion, increased serumurea and low total serum protein [139]. Theyreported a sensitivity of 100% and speci-ficity of 46%. They estimated that 43.9%of the mild dengue cases could have beenprevented from hospitalization in 2004.The authors followed up this study with aTable 3. Warning signs and symptoms leading to potential severedengue (dengue hemorrhagic fever/dengue shock syndrome).Warning signs for severe disease progressionSigns Den|OFIs p-value Ref.Abdominal pain & tenderness 63.3|22.6% <0.05 [150,152]78.0|22.0%†0.03Persistent vomiting NA NA [153]Clinical fluid accumulation 46.4|7.1% 0.002 [154]Mucosal bleed/ spontaneous bleeding 84.1|65.9% 0.008 [150,153]26.6|10.4% 0.05Lethargy, restlessness 12.7|2.2% 0.05 [150,152]54.0|20.0%†0.002Liver enlargement >2 cm 91.6|72.4%‡0.01 [146]Severe dengue (DHF/DSS)Symptoms Mild|severe p-value Ref.Severe plasma leakage leading toshock and fluid accumulation withrespiratory distress92.9–100% in severe den NA [154]Severe bleeding (evaluated by clinician) 10.0–35.7% in severe den <0.01 [139,154]Gums: 5.7–6.0|65.0–67.8%Nose: 0.9|12.0–16.9%Severe organ involvement AST: 1293|196 IU/l 0.015 [37,145,153,155]  Liver: increased AST/ALT ALT: 309|132 IU/l 0.075  CNS: impaired consciousness AST: 76|12 U/l <0.01  Heart and other organs ALT: 30|20 U/l <0.01Thrombocytopenia ≤100,000/mm316|82% NA [145]†Dengue and severe dengue comparison, performed in children younger than 15 years of age.‡Children (younger than 15 years of age) versus adults (older than 15 years of age).ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; Den: Confirmed dengue cases;DHF: Dengue hemorrhagic fever; DSS: Dengue shock syndrome; IU/l: International units per liter;NA: Not applicable; OFI: Other febrile illness.Tang & Ooi
  • 7. 901www.expert-reviews.comReviewprospective validation of their equation in the same hospital andfound similar levels of accuracy [140].Another algorithm using platelet count, crossover thresholdof PCR-positive results (viremia in blood) and pre-existing anti­dengue IgG (secondary infection) measured during the first 72 hof illness was shown to predict hospitalization with a sensitivityand specificity of 78.2 and 80.2%, respectively [36]. Likewise,as discussed earlier, the algorithm that distinguishes DHF fromdengue infection is able to achieve a sensitivity of 79.6% [37]. Theability to calculate the probability of development of severe den-gue based on routinely performed clinical tests could also be use-ful to guide prognostication [37]. However, further prospectiveclinical studies are needed to validate their usefulness.Quality assuranceWhile the authors have reviewed the sensitivities and specificitiesof the various tools for diagnosis of dengue, how these tests actu-ally perform can be affected by a number of variables that differfrom laboratory to laboratory, or from region to region. Thus,quality assurance programs should be instituted in all diagnos-tic and reference laboratories that offer services in diagnosis ofdengue. This ensures that the tests perform at the expected levelsin different laboratories and in different hands. Details on suchquality assurance programs have been reviewed elsewhere [141].Diagnosis of dengue in a vaccinated populationWhile an effective and safe vaccine against dengue is anticipated,its introduction could also provide fresh challenges for diagnosisof dengue. Even though the goal of vacci-nation is to eliminate dengue cases entirely,there are currently no data that indicatethat a complete elimination of DENV isfeasible with vaccination programs. On thecontrary, there remains a concern that theantibodies generated by vaccination mayenhance dengue, particularly when anti-body levels wane in the years following vac-cination. Furthermore, vaccination may notprevent infection against all strains [142] ormay drive the emergence of vaccine-escapemutants [143], as encountered with otherinfections, such as hepatitis B [144]. A com-prehensive surveillance of dengue amongcases of febrile illness would thus be neededto determine the true efficacy of ­vaccinationand to monitor for ­vaccine failure.Epidemiologically, vaccination wouldreduce DENV transmission and hence theprevalence of dengue. Under such circum-stances, diagnostic approaches or tests withhigh sensitivity but poor specificity wouldresult in a high false-positive rate. However,a low sensitivity could lead to false-negativefindings, which could result in an inabilityto detect the emergence of vaccine failureor escape mutants early enough to trigger the necessary publichealth responses.Given these requirements, clinical diagnosis using symptoms,signs and standard routine hematological or biochemical tests isunlikely to provide sufficient specificity (Figure 1). Furthermore,vaccinated individuals may also present with a milder illnessthan classical dengue infection, making approaches such as theuse of the WHO dengue classification schemes less sensitive.Diagnosis of acute DENV infection must thus rely even moreon the laboratory.Serologically, DENV infection in vaccinated individuals wouldalso resemble that of a secondary infection, where a rise in IgMtiters is not a consistent feature but a rapid rise in IgG titers orthe ratio of IgM and IgG could be suggestive of acute DENVinfection [117]. In this respect, collection of a convalescent serumsample to demonstrate rising antibody titers would be very use-ful in interpreting these serological tests. Caution will need to beexercised in places where another flavivirus, such as West Nileor Japanese encephalitis virus, circulates. Overall, however, sero-logical approaches will probably lack the specificity required fora definitive diagnosis of dengue in the low prevalence settingexpected in vaccinated populations (Figure 2).Detection of DENV or components of DENV are likely toprovide the necessary sensitivity and specificity needed (Figure 2).While NS1 antigen detection is easy to use and is suited to point-of-care diagnosis, the presence of vaccine-induced antidengue IgGantibodies, as with secondary DENV infection, could lower theoverall sensitivity of this test. Likewise, while virus isolation may be0.81.000.950.900.850.60.4PPVNPV0.20.00 10 20 30 0 10 20WHO (<56 yrs)WHO (≥56 yrs)PCRNS1lgM (ELISA)lgM (Rapid)30Prevalence PrevalenceFigure 2. Positive- and negative- predictive values of the various diagnosticapproaches for dengue at different rates of prevalence. Results were generatedfrom median values of sensitivity and specificity presented in Table 2 for PCR(conventional and real time), IgM (ELISA), IgM (Rapid) and NS1 detection, respectively.A specificity of 96% was assumed for conventional PCR and real-time PCR as moststudies have limited population sizes. Diagnosis using the WHO 2009 classification wasused to represent clinical diagnosis. Data for WHO criteria were obtained from Low et al.[12] and separated into two age groups (<56 and ≥56 years).NPV: Negative-predictive value; NS1: Non-structural protein 1; PPV: Positive-predictive value.Diagnosis of dengue: an update
  • 8. Expert Rev. Anti Infect. Ther. 10(8), (2012)902Reviewhighly specific, it lacks sufficient sensitivity, especially since in mostplaces, a suitable insectary for mosquito inoculation is not likely tobe available and laboratories will have to rely on cell cultures.However, virus isolation will not be redundant and would needto be done in all RT-PCR-positive specimens, as isolation ofvaccine-escape mutants would be needed to characterize theseviruses. Such information could be useful in updating vaccinecomposition through the development or selection of appropri-ate vaccine strains or even updating the primers and probes usedin ­molecular diagnostic assays [143].Nucleic acid detection offers the highest sensitivity and speci-ficity, and would thus be the most appropriate approach for acutediagnosis of dengue in vaccinated populations with low diseaseprevalence (Figure 2). Emphasis should be on those assays thathave been carefully validated in different laboratories servingdifferent populations. The availability of panels of standard-ized positive and negative controls, along with an internation-ally coordinated quality assurance program, would be neededto ensure consistency in the performance of the diagnosticassays. Presently, such a molecular diagnostic assay is lacking.RT-PCR method used in different laboratories differ in termsof primers/probes, enzymes and buffers as well as cycling con-ditions. The method of detection of the RT-PCR amplicon,whether as an end point or real-time assay, is also likely to bedifferent, as with the method of viral RNA extraction from clini-cal specimens. These limitations need to be addressed urgentlyif we are to be prepared for diagnosis of dengue and surveillancein a postvaccination world.Expert commentaryDENV and its mosquito vectors have expanded geographicallythroughout the tropical world and are now encroaching intosubtropical regions. These trends make dengue a global healthconcern. In the absence of either a licensed vaccine or antiviraldrug, reduction of the disease burden relies on early clinicalrecognition of dengue and the timely initiation of supportivetherapy. As differentiation between dengue and other causes offebrile illnesses is difficult based on presenting symptoms andsigns, laboratory tests are needed for a confirmatory diagnosis.This review summarizes the current knowledge on clinical aswell as laboratory diagnosis of dengue. It reveals that clinicalapproaches generally have high sensitivities but poor specificitiesand discusses the various decision algorithms that have beendesigned to improve the specificity of clinical diagnosis. Forconfirmatory diagnosis, a range of laboratory tools are avail-able and the main consideration on which tool to use is thetime from illness onset. A central theme of this review is theneed for a systematic validation of the performance of both thedecision algorithms and laboratory assays in different popula-tions and diagnostic laboratory settings, respectively. This needfor quality-assured standardized performance could, paradoxi-cally, become more acute when a dengue vaccine or antiviraldrug becomes available. The consequent reduction in dengueprevalence necessitates the use of the most sensitive and specificmethod to derive useful positive and negative predictive val-ues to support clinical decisions in treatment and public healthresponses.Five-year viewWe speculate that a dengue vaccine will be near licensing in5 years and that potential antiviral drugs against dengue willalso enter late stages of clinical trials. The implementation ofeither countermeasure against dengue would shift the emphasisof diagnosis of dengue from serological to virological. Tools thatdetect either the viral genome or antigen, particularly at the bed-side, would gain favor. These tools are better able to distinguishdengue from other flaviviral infections and are also useful inthe early phases of illness, when initiation of antiviral therapywould probably exert its maximal effect. Furthermore, definitivediagnosis of dengue in vaccinated populations would becomeeven more important as it could herald waning immunity oremergence of vaccine-escape mutants; either scenario wouldtrigger a public health emergency. Hence, the need for improve-ments to existing approaches for the diagnosis of dengue wouldnot be diminished with the advent of either vaccination or anti-viral drug therapy, but rather the demand for tests that achievenear-perfect sensitivity and specificity will increase in the next5 years.‍Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with anyorganization or entity with a financial interest in or financial conflict withthe subject matter or materials discussed in the manuscript. This includesemployment, consultancies, honoraria, stock ownership or options, experttestimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.Key issues• Clinical diagnosis using the 1997–2009 WHO dengue classification schemes has high sensitivity but lacks specificity.• Decision algorithms for diagnosis have been proposed but lack prospective validation.• Choice of diagnostic assays should be guided by the time from illness onset.• The presence of pre-existing antibodies from a previous heterologous dengue virus infection or a previous flavivirus infection can affect the sensitivity or specificity of many diagnostic assays.• Postvaccination surveillance would face the same challenges for diagnostics as currently encountered with secondary dengue infection.• Despite the above, diagnosis of dengue in vaccinated individuals is critical for the surveillance of vaccine failure and escape mutants.• Diagnostic assays with high sensitivity and specificity will be in particular demand in the low dengue prevalence setting followingvaccination.Tang & Ooi
  • 9. 903www.expert-reviews.comReviewReferencesPapers of special note have been highlighted as:• of interest•• of considerable interest1 WHO. DENGUE: Guidelines for Diagnosis,Treatment, Prevention and Control – NewEdition. WHO, Geneva,Switzerland (2009).•• This publication consolidates exceptionalefforts put in by the experts in the field.It contains extensive information fordiagnosis, treatment, prevention andcontrol of dengue.2 Westaway EG, Blok J. Taxonomy andevolutionary relationships of flavivirus.In: Dengue and Dengue HemorrhagicFever. Gubler DJ, Kuno G (Eds). CABInternational, London, UK (1997).3 Chambers TJ, Weir RC, Grakoui A et al.Evidence that the N-terminal domain ofnonstructural protein NS3 from yellowfever virus is a serine protease responsiblefor site-specific cleavages in the viralpolyprotein. Proc. Natl Acad. Sci. USA87(22), 8898–8902 (1990).4 Rice CM, Lenches EM, Eddy SR, Shin SJ,Sheets RL, Strauss JH. Nucleotide sequenceof yellow fever virus: implications forflavivirus gene expression and evolution.Science 229(4715), 726–733 (1985).5 Harris E, Holden KL, Edgil D, Polacek C,Clyde K. Molecular biology of flaviviruses.Novartis Found. Symp. 277, 23–39; discussion40, 71–73, 251–253 (2006).6 Sabin AB. Research on dengue duringWorld War II. Am. J. Trop. Med. Hyg. 1(1),30–50 (1952).7 WHO-SEARO. Guidelines for Treatment ofDengue Fever/Dengue Haemorrhagic Feverin Small Hospitals. WHO-SEARO,New Delhi, India (1999).8 Halstead SB, O’Rourke EJ. Antibody-enhanced dengue virus infection in primateleukocytes. Nature 265(5596),739–741 (1977).9 Halstead SB, Venkateshan CN, GentryMK, Larsen LK. Heterogeneity of infectionenhancement of dengue 2 strains bymonoclonal antibodies. J. Immunol.132(3), 1529–1532 (1984).10 Halstead SB. Neutralization and antibody-dependent enhancement of dengue viruses.Adv. Virus Res. 60, 421–467 (2003).11 Sessions OM, Barrows NJ, Souza-Neto JAet al. Discovery of insect and human denguevirus host factors. Nature 458(7241),1047–1050 (2009).12 Low JG, Ong A, Tan LK et al. The earlyclinical features of dengue in adults:challenges for early clinical diagnosis.PLoS Negl. Trop. Dis. 5(5), e1191 (2011).13 Khor CC, Chau TN, Pang J et al.Genome-wide association study identifiessusceptibility loci for dengue shocksyndrome at MICB and PLCE1. Nat.Genet. 43(11), 1139–1141 (2011).14 Pastorino B, Nougairède A, Wurtz N,Gould E, de Lamballerie X. Role of hostcell factors in flavivirus infection:implications for pathogenesis anddevelopment of antiviral drugs. AntiviralRes. 87(3), 281–294 (2010).15 Martina BE, Koraka P, Osterhaus AD.Dengue virus pathogenesis: an integratedview. Clin. Microbiol. Rev. 22(4),564–581 (2009).16 Rico-Hasse R. Dengue virus virulenceand transmission determinants. Curr.Trop. Microbiol. Immunol. 338,45–55 (2010).17 Tuiskunen A, Wahlström M, Bergström J,Buchy P, Leparc-Goffart I, Lundkvist A.Phenotypic characterization of patientdengue virus isolates in BALB/c micedifferentiates dengue fever and denguehemorrhagic fever from dengue shocksyndrome. Virol. J. 8, 398 (2011).18 OhAinle M, Balmaseda A, Macalalad ARet al. Dynamics of dengue disease severitydetermined by the interplay between viralgenetics and serotype-specific immunity. Sci.Transl. Med. 3(114), 114ra128 (2011).19 Gubler DJ. Dengue and denguehemorrhagic fever. Clin. Microbiol. Rev.11(3), 480–496 (1998).20 Xu G, Dong H, Shi N et al. An outbreak ofdengue virus serotype 1 infection in Cixi,Ningbo, People’s Republic of China, 2004,associated with a traveler from Thailandand high density of Aedes albopictus. Am. J.Trop. Med. Hyg. 76(6), 1182–1188 (2007).21 Lambrechts L, Scott TW, Gubler DJ.Consequences of the expanding globaldistribution of Aedes albopictus for denguevirus transmission. PLoS Negl. Trop. Dis.4(5), e646 (2010).22 Ooi EE, Goh KT, Gubler DJ. Dengueprevention and 35 years of vector control inSingapore. Emerging Infect. Dis. 12(6),887–893 (2006).23 Sabin AB, Schlesinger RW. Production ofimmunity to dengue with virus modifiedby propagation in mice. Science 101(2634),640–642 (1945).24 WHO. Dengue Vaccine Development: theRole of the WHO South-East Asia RegionalOffice. WHO, Geneva, Switzerland (2010).25 Halstead SB, Diwan AR, Marchette NJ,Palumbo NE, Srisukonth L. Selection ofattenuated dengue 4 viruses by serialpassage in primary kidney cells. I.Attributes of uncloned virus at differentpassage levels. Am. J. Trop. Med. Hyg.33(4), 654–665 (1984).26 Halstead SB, Marchette NJ, Diwan AR,Palumbo NE, Putvatana R. Selection ofattenuated dengue 4 viruses by serialpassage in primary kidney cells. II.Attributes of virus cloned at different dogkidney passage levels. Am. J. Trop. Med.Hyg. 33(4), 666–671 (1984).27 Halstead SB, Marchette NJ, Diwan AR,Palumbo NE, Putvatana R, Larsen LK.Selection of attenuated dengue 4 viruses byserial passage in primary kidney cells. III.Reversion to virulence by passage of clonedvirus in fetal rhesus lung cells. Am. J. Trop.Med. Hyg. 33(4), 672–678 (1984).28 Halstead SB, Eckels KH, Putvatana R,Larsen LK, Marchette NJ. Selection ofattenuated dengue 4 viruses by serialpassage in primary kidney cells. IV.Characterization of a vaccine candidate infetal rhesus lung cells. Am. J. Trop. Med.Hyg. 33(4), 679–683 (1984).29 Halstead SB, Marchette NJ. Biologicproperties of dengue viruses followingserial passage in primary dog kidney cells:studies at the University of Hawaii. Am. J.Trop. Med. Hyg. 69(6 Suppl.), 5–11 (2003).30 Halstead SB. Studies on the attenuation ofdengue 4. Asian J. Infect. Dis. 2,112–117 (1978).31 Russell PK. Progress toward denguevaccines. Asian J. Infect. Dis. 2,118–120 (1978).32 Swaminathan S, Batra G, Khanna N.Dengue vaccines: state-of-the-art. ExpertOpin. Ther. Pat. 20(6), 819–835 (2010).33 Gubler DJ. Emerging vector-borne flavivirusdiseases: are vaccines the solution? ExpertRev. Vaccines 10(5), 563–565 (2011).34 Coller BA, Clements DE. Dengue vaccines:progress and challenges. Curr. Opin.Immunol. 23(3), 391–398 (2011).35 WHO. Dengue Haemorrhagic Fever:Diagnosis, Treatment, Prevention andControl (2nd Edition). WHO, Geneva,Switzerland (1997).36 Tanner L, Schreiber M, Low JG et al.Decision tree algorithms predict thediagnosis and outcome of dengue fever inDiagnosis of dengue: an update
  • 10. Expert Rev. Anti Infect. Ther. 10(8), (2012)904Reviewthe early phase of illness. PLoS Negl. Trop.Dis. 2(3), e196 (2008).37 Potts JA, Thomas SJ, Srikiatkhachorn Aet al. Classification of dengue illness basedon readily available laboratory data. Am. J.Trop. Med. Hyg. 83(4), 781–788 (2010).•• Points out the fundamental shortcomingsand limitations of clinical studies indengue and the need for a consolidatedclinical and laboratory data collection forbetter disease management.38 Potts JA, Rothman AL. Clinical andlaboratory features that distinguish denguefrom other febrile illnesses in endemicpopulations. Trop. Med. Int. Health 13(11),1328–1340 (2008).39 Vaughn DW, Green S, Kalayanarooj Set al. Dengue viremia titer, antibodyresponse pattern, and virus serotypecorrelate with disease severity. J. Infect. Dis.181(1), 2–9 (2000).40 Jarman RG, Nisalak A, Anderson KB et al.Factors influencing dengue virus isolationby C6/36 cell culture and mosquitoinoculation of nested PCR-positive clinicalsamples. Am. J. Trop. Med. Hyg. 84(2),218–223 (2011).41 Kuberski TT, Rosen L. A simple techniquefor the detection of dengue antigen inmosquitoes by immunofluorescence. Am. J.Trop. Med. Hyg. 26(3), 533–537 (1977).42 Kuberski TT, Rosen L. Identification ofdengue viruses using complement fixingantigen produced in mosquitoes. Am. J.Trop. Med. Hyg. 26(3), 538–543 (1977).43 Gubler DJ, Nalim S, Tan R, Saipan H,Sulianti Saroso J. Variation in susceptibilityto oral infection with dengue virusesamong geographic strains of Aedes aegypti.Am. J. Trop. Med. Hyg. 28(6),1045–1052 (1979).44 Thet W. Detection of dengue virus byimmunofluorescence after intracerebralinoculation of mosquitoes. Lancet 1(8262),53–54 (1982).45 Yeh WT, Chen RF, Wang L, Liu JW, ShaioMF, Yang KD. Implications of previoussubclinical dengue infection but not virusload in dengue hemorrhagic fever. FEMSImmunol. Med. Microbiol. 48(1),84–90 (2006).46 Meiklejohn G, England B, Lennette EH.Propagation of dengue virus strains inunweaned mice. Am. J. Trop. Med. Hyg.1(1), 51–58 (1952).47 Sabin AB. The dengue group of viruses andits family relationships. Bacteriol. Rev.14(3), 225–232 (1950).48 Yuill TM, Sukhavachana P, Nisalak A,Russell PK. Dengue-virus recovery bydirect and delayed plaques in LLC-MK2cells. Am. J. Trop. Med. Hyg. 17(3),441–448 (1968).49 Matsumura T, Stollar V, Schlesinger RW.Studies on the nature of dengue viruses. V.Structure and development of dengue virusin Vero cells. Virology 46(2),344–355 (1971).50 Fujita N, Tamura M, Hotta S. Denguevirus plaque formation on microplatecultures and its application to virusneutralization (38564). Proc. Soc. Exp. Biol.Med. 148(2), 472–475 (1975).51 Igarashi A. Isolation of a Singh’s Aedesalbopictus cell clone sensitive to dengue andchikungunya viruses. J. Gen. Virol. 40(3),531–544 (1978).52 Tesh RB. A method for the isolation andidentification of dengue viruses, usingmosquito cell cultures. Am. J. Trop. Med.Hyg. 28(6), 1053–1059 (1979).53 Gubler DJ, Kuno G, Sather GE, Velez M,Oliver A. Mosquito cell cultures andspecific monoclonal antibodies insurveillance for dengue viruses. Am. J.Trop. Med. Hyg. 33(1), 158–165 (1984).54 Chua KB, Mustafa B, Abdul Wahab AHet al. A comparative evaluation of denguediagnostic tests based on single-acuteserum samples for laboratory confirmationof acute dengue. Malays. J. Pathol. 33(1),13–20 (2011).55 Lanciotti RS, Calisher CH, Gubler DJ,Chang GJ, Vorndam AV. Rapid detectionand typing of dengue viruses from clinicalsamples by using reverse transcriptase-polymerase chain reaction. J. Clin.Microbiol. 30(3), 545–551 (1992).56 Harris E, Roberts TG, Smith L et al. Typingof dengue viruses in clinical specimens andmosquitoes by single-tube multiplex reversetranscriptase PCR. J. Clin. Microbiol. 36(9),2634–2639 (1998).57 Raengsakulrach B, Nisalak A, Maneekarn Net al. Comparison of four reversetranscription-polymerase chain reactionprocedures for the detection of dengue virusin clinical specimens. J. Virol. Methods105(2), 219–232 (2002).58 Gijavanekar C, Añez-Lingerfelt M, Feng Cet al. PCR detection of nearly any denguevirus strain using a highly sensitive primer‘cocktail’. FEBS J. 278(10), 1676–1687(2011).59 Yong YK, Thayan R, Chong HT, Tan CT,Sekaran SD. Rapid detection andserotyping of dengue virus by multiplexRT-PCR and real-time SYBR greenRT-PCR. Singapore Med. J. 48(7),662–668 (2007).60 Upanan S, Cabrera-Hernandez A,Ekkapongpisit M, Smith DR. A simplifiedPCR methodology for semiquantitativelyanalyzing dengue viruses. Jpn. J. Infect. Dis.59(6), 383–387 (2006).61 Dash PK, Parida M, Santhosh SR et al.Development and evaluation of a 1-stepduplex reverse transcription polymerasechain reaction for differential diagnosis ofchikungunya and dengue infection. Diagn.Microbiol. Infect. Dis. 62(1), 52–57 (2008).62 Barkham TM, Chung YK, Tang KF,Ooi EE. The performance of RT-PCRcompared with a rapid serological assay foracute dengue fever in a diagnosticlaboratory. Trans. R. Soc. Trop. Med. Hyg.100(2), 142–148 (2006).63 Chutinimitkul S, Payungporn S,Theamboonlers A, Poovorawan Y. Denguetyping assay based on real-time PCR usingSYBR Green I. J. Virol. Methods 129(1),8–15 (2005).64 Chien LJ, Liao TL, Shu PY, Huang JH,Gubler DJ, Chang GJ. Development ofreal-time reverse transcriptase PCR assays todetect and serotype dengue viruses. J. Clin.Microbiol. 44(4), 1295–1304 (2006).65 Lai YL, Chung YK, Tan HC et al.Cost-effective real-time reversetranscriptase PCR (RT-PCR) to screen forDengue virus followed by rapid single-tubemultiplex RT-PCR for serotyping of thevirus. J. Clin. Microbiol. 45(3),935–941 (2007).66 Pok KY, Lai YL, Sng J, Ng LC. Evaluationof nonstructural 1 antigen assays for thediagnosis and surveillance of dengue inSingapore. Vector Borne Zoonotic Dis.10(10), 1009–1016 (2010).67 Hue KD, Tuan TV, Thi HT et al.Validation of an internally controlledone-step real-time multiplex RT-PCR assayfor the detection and quantitation ofdengue virus RNA in plasma. J. Virol.Methods 177(2), 168–173 (2011).68 Hirayama T, Mizuno Y, Takeshita N et al.Detection of dengue virus genome in urineby real-time reverse transcriptase PCR: alaboratory diagnostic method useful afterdisappearance of the genome in serum.J. Clin. Microbiol. 50(6),2047–2052 (2012).69 Tripathi NK, Shrivastava A, Dash PK,Jana AM. Detection of dengue virus.Methods Mol. Biol. 665, 51–64 (2011).Tang & Ooi
  • 11. 905www.expert-reviews.comReview70 Leparc-Goffart I, Baragatti M, Temmam Set al. Development and validation ofreal-time one-step reverse transcription-PCRfor the detection and typing of dengueviruses. J. Clin. Virol. 45(1), 61–66 (2009).71 Gurukumar KR, Priyadarshini D, Patil JAet al. Development of real-time PCR fordetection and quantitation of dengueviruses. Virol. J. 6, 10 (2009).72 Poloni TR, Oliveira AS, Alfonso HL et al.Detection of dengue virus in saliva andurine by real-time RT-PCR. Virol. J. 7,22 (2010).73 Sadon N, Delers A, Jarman RG et al. A newquantitative RT-PCR method for sensitivedetection of dengue virus in serum samples.J. Virol. Methods 153(1), 1–6 (2008).74 Singh K, Lale A, Eong Ooi E et al. Aprospective clinical study on the use ofreverse transcription-polymerase chainreaction for the early diagnosis of denguefever. J. Mol. Diagn. 8(5), 613–616; quiz617 (2006).75 Kong YY, Thay CH, Tin TC, Devi S. Rapiddetection, serotyping and quantitation ofdengue viruses by TaqMan real-timeone-step RT-PCR. J. Virol. Methods138(1–2), 123–130 (2006).76 Saxena P, Dash PK, Santhosh SR,Shrivastava A, Parida M, Rao PL.Development and evaluation of one stepsingle tube multiplex RT-PCR for rapiddetection and typing of dengue viruses.Virol. J. 5, 20 (2008).77 Dos Santos HW, Poloni TR, Souza KPet al. A simple one-step real-time RT-PCRfor diagnosis of dengue virus infection.J. Med. Virol. 80(8), 1426–1433 (2008).78 Klungthong C, Gibbons RV,Thaisomboonsuk B et al. Dengue virusdetection using whole blood for reversetranscriptase PCR and virus isolation.J. Clin. Microbiol. 45(8), 2480–2485(2007).79 Watthanaworawit W, Turner P, Turner CLet al. A prospective evaluation of diagnosticmethodologies for the acute diagnosis ofdengue virus infection on the Thailand–Myanmar border. Trans. R. Soc. Trop. Med.Hyg. 105(1), 32–37 (2011).80 Compton J. Nucleic acid sequence-basedamplification. Nature 350(6313), 91–92(1991).81 Wu SJ, Lee EM, Putvatana R et al. Detectionof dengue viral RNA using a nucleic acidsequence-based amplification assay. J. Clin.Microbiol. 39(8), 2794–2798 (2001).82 Bhatnagar J, Blau DM, Shieh WJ et al.Molecular detection and typing of dengueviruses from archived tissues of fatal casesby rt-PCR and sequencing: diagnostic andepidemiologic implications. Am. J. Trop.Med. Hyg. 86(2), 335–340 (2012).83 Peeling RW, Artsob H, Pelegrino JL et al.Evaluation of diagnostic tests: dengue.Nat. Rev. Microbiol. 8(12 Suppl.),S30–S38 (2010).84 Winkler G, Maxwell SE, Ruemmler C,Stollar V. Newly synthesized dengue-2 virusnonstructural protein NS1 is a solubleprotein but becomes partially hydrophobicand membrane-associated after dimerization.Virology 171(1), 302–305 (1989).85 Flamand M, Megret F, Mathieu M,Lepault J, Rey FA, Deubel V. Dengue virustype 1 nonstructural glycoprotein NS1 issecreted from mammalian cells as a solublehexamer in a glycosylation-dependentfashion. J. Virol. 73(7), 6104–6110 (1999).86 Libraty DH, Young PR, Pickering D et al.High circulating levels of the dengue virusnonstructural protein NS1 early in dengueillness correlate with the development ofdengue hemorrhagic fever. J. Infect. Dis.186(8), 1165–1168 (2002).87 Avirutnan P, Punyadee N, Noisakran Set al. Vascular leakage in severe denguevirus infections: a potential role for thenonstructural viral protein NS1 andcomplement. J. Infect. Dis. 193(8),1078–1088 (2006).88 Hang VT, Nguyet NM, Trung DT et al.Diagnostic accuracy of NS1 ELISA andlateral flow rapid tests for denguesensitivity, specificity and relationship toviraemia and antibody responses. PLoSNegl. Trop. Dis. 3(1), e360 (2009).89 Young PR, Hilditch PA, Bletchly C,Halloran W. An antigen capture enzyme-linked immunosorbent assay reveals highlevels of the dengue virus protein NS1 inthe sera of infected patients. J. Clin.Microbiol. 38(3), 1053–1057 (2000).90 Alcon S, Talarmin A, Debruyne M,Falconar A, Deubel V, Flamand M.Enzyme-linked immunosorbent assayspecific to dengue virus type 1nonstructural protein NS1 revealscirculation of the antigen in the bloodduring the acute phase of disease inpatients experiencing primary or secondaryinfections. J. Clin. Microbiol. 40(2),376–381 (2002).91 Dussart P, Labeau B, Lagathu G et al.Evaluation of an enzyme immunoassay fordetection of dengue virus NS1 antigen inhuman serum. Clin. Vaccine Immunol.13(11), 1185–1189 (2006).92 Xu H, Di B, Pan YX et al. Serotype1-specific monoclonal antibody-basedantigen capture immunoassay for detectionof circulating nonstructural protein NS1:implications for early diagnosis andserotyping of dengue virus infections.J. Clin. Microbiol. 44(8),2872–2878 (2006).93 Kumarasamy V, Wahab AH, Chua SKet al. Evaluation of a commercial dengueNS1 antigen-capture ELISA for laboratorydiagnosis of acute dengue virus infection.J. Virol. Methods 140(1–2), 75–79 (2007).94 Kumarasamy V, Chua SK, Hassan Z et al.Evaluating the sensitivity of a commercialdengue NS1 antigen-capture ELISA forearly diagnosis of acute dengue virusinfection. Singapore Med. J. 48(7),669–673 (2007).95 McBride WJ. Evaluation of dengue NS1test kits for the diagnosis of dengue fever.Diagn. Microbiol. Infect. Dis. 64(1),31–36 (2009).96 Bessoff K, Delorey M, Sun W, HunspergerE. Comparison of two commerciallyavailable dengue virus (DENV) NS1capture enzyme-linked immunosorbentassays using a single clinical sample fordiagnosis of acute DENV infection. Clin.Vaccine Immunol. 15(10),1513–1518 (2008).97 Blacksell SD, Mammen MP Jr,Thongpaseuth S et al. Evaluation of thePanbio dengue virus nonstructural1 antigen detection and immunoglobulinM antibody enzyme-linked immunosorbentassays for the diagnosis of acute dengueinfections in Laos. Diagn. Microbiol. Infect.Dis. 60(1), 43–49 (2008).98 Bessoff K, Phoutrides E, Delorey M,Acosta LN, Hunsperger E. Utility of acommercial nonstructural protein1 antigen capture kit as a dengue virusdiagnostic tool. Clin. Vaccine Immunol.17(6), 949–953 (2010).99 Lima Mda R, Nogueira RM, SchatzmayrHG, dos Santos FB. Comparison of threecommercially available dengue NS1 antigencapture assays for acute diagnosis of denguein Brazil. PLoS Negl. Trop. Dis. 4(7),e738 (2010).100 Wang SM, Sekaran SD. Evaluation of acommercial SD dengue virus NS1 antigencapture enzyme-linked immunosorbentassay kit for early diagnosis of dengue virusinfection. J. Clin. Microbiol. 48(8),2793–2797 (2010).101 Tricou V, Vu HT, Quynh NV et al.Comparison of two dengue NS1 rapid testsfor sensitivity, specificity and relationshipDiagnosis of dengue: an update
  • 12. Expert Rev. Anti Infect. Ther. 10(8), (2012)906Reviewto viraemia and antibody responses. BMCInfect. Dis. 10, 142 (2010).102 Blacksell SD, Jarman RG, Bailey MS et al.Evaluation of six commercial point-of-caretests for diagnosis of acute dengueinfections: the need for combining NS1antigen and IgM/IgG antibody detectionto achieve acceptable levels of accuracy.Clin. Vaccine Immunol. 18(12), 2095–2101(2011).• Comprehensive evaluation of diagnostickits for dengue point-of-care tests.103 Chaterji S, Allen JC Jr, Chow A, Leo YS,Ooi EE. Evaluation of the NS1 rapid testand the WHO dengue classificationschemes for use as bedside diagnosis ofacute dengue fever in adults. Am. J. Trop.Med. Hyg. 84(2), 224–228 (2011).104 Lima MDA R, Noguieira RM, SchatzmayrHG, de Pilippis AM. A new approach todengue fatal cases diagnosis: NS1 antigencapture in tissues. PLoS Negl. Trop. Dis.5(5), e1147 (2011).105 Ding X, Hu D, Chen Y et al. Fullserotype- and group-specific NS1 captureenzyme-linked immunosorbent assay forrapid differential diagnosis of dengue virusinfection. Clin. Vaccine Immunol. 18(3),430–434 (2011).106 Shu PY, Chen LK, Chang SF et al. DengueNS1-specific antibody responses: isotypedistribution and serotyping in patients withdengue fever and dengue hemorrhagicfever. J. Med. Virol. 62(2),224–232 (2000).107 Shu PY, Chen LK, Chang SF et al. Potentialapplication of nonstructural protein NS1serotype-specific immunoglobulin Genzyme-linked immunosorbent assay in theseroepidemiologic study of dengue virusinfection: correlation of results with thoseof the plaque reduction neutralization test.J. Clin. Microbiol. 40(5),1840–1844 (2002).108 Shu PY, Chen LK, Chang SF et al. Denguevirus serotyping based on envelope andmembrane and nonstructural protein NS1serotype-specific capture immunoglobulinM enzyme-linked immunosorbent assays.J. Clin. Microbiol. 42(6),2489–2494 (2004).109 Puttikhunt C, Prommool T, U-thainual Net al. The development of a novelserotyping-NS1-ELISA to identifyserotypes of dengue virus. J. Clin. Virol.50(4), 314–319 (2011).110 Watanabe S, Tan KH, Rathore AP et al.The magnitude of dengue virus NS1protein secretion is strain dependent anddoes not correlate with severe pathologiesin the mouse infection model. J. Virol.86(10), 5508–5514 (2012).111 De Paula SO, Fonseca BA. Dengue: areview of the laboratory tests a clinicianmust know to achieve a correct diagnosis.Braz. J. Infect. Dis. 8(6), 390–398 (2004).112 Innis BL, Nisalak A, Nimmannitya S et al.An enzyme-linked immunosorbent assay tocharacterize dengue infections wheredengue and Japanese encephalitisco-circulate. Am. J. Trop. Med. Hyg. 40(4),418–427 (1989).113 Kuno G, Gómez I, Gubler DJ. An ELISAprocedure for the diagnosis of dengueinfections. J. Virol. Methods 33(1–2),101–113 (1991).114 Lam SK, Fong MY, Chungue E et al.Multicentre evaluation of dengue IgM dotenzyme immunoassay. Clin. Diagn. Virol.7(2), 93–98 (1996).115 Hunsperger EA, Yoksan S, Buchy P et al.Evaluation of commercially availableanti-dengue virus immunoglobulin Mtests. Emerging Infect. Dis. 15(3),436–440 (2009).116 Groen J, Koraka P, Velzing J, Copra C,Osterhaus AD. Evaluation of siximmunoassays for detection of denguevirus-specific immunoglobulin M and Gantibodies. Clin. Diagn. Lab. Immunol.7(6), 867–871 (2000).117 Prince HE, Matud JL. Estimation ofdengue virus IgM persistence usingregression analysis. Clin. Vaccine Immunol.18(12), 2183–2185 (2011).118 Blacksell SD, Doust JA, Newton PN,Peacock SJ, Day NP, Dondorp AM. Asystematic review and meta-analysis of thediagnostic accuracy of rapidimmunochromatographic assays for thedetection of dengue virus IgM antibodiesduring acute infection. Trans. R. Soc. Trop.Med. Hyg. 100(8), 775–784 (2006).• Comprehensive review on denguediagnostics.119 Tomashek KM. Dengue fever & denguehemorrhagic fever. In: CDC HealthInformation for International Travel 2010,The Yellow Book, Centers for Disease Controland Prevention. Gw B (Ed.). OxfordUniversity Press, NC, USA (2010).120 Prince HE, Yeh C, Lapé-Nixon M.Development of a more efficient algorithmfor identifying false-positive reactivityresults in a dengue virus immunoglobulinM screening assay. Clin. Vaccine Immunol.15(8), 1304–1306 (2008).121 Russell PK, Nisalak A. Dengue virusidentification by the plaque reductionneutralization test. J. Immunol. 99(2),291–296 (1967).122 Russell PK, Nisalak A, Sukhavachana P,Vivona S. A plaque reduction test fordengue virus neutralizing antibodies.J. Immunol. 99(2), 285–290 (1967).123 Dulbecco R, Vogt M, Strickland AG. Astudy of the basic aspects of neutralizationof two animal viruses, western equineencephalitis virus and poliomyelitis virus.Virology 2(2), 162–205 (1956).124 Putnak JR, de la Barrera R, Burgess T et al.Comparative evaluation of three assays formeasurement of dengue virus neutralizingantibodies. Am. J. Trop. Med. Hyg. 79(1),115–122 (2008).125 Roehrig JT, Hombach J, Barrett AD.Guidelines for plaque-reductionneutralization testing of human antibodiesto dengue viruses. Viral Immunol. 21(2),123–132 (2008).126 Vorndam V, Beltran M. Enzyme-linkedimmunosorbent assay-formatmicroneutralization test for dengue viruses.Am. J. Trop. Med. Hyg. 66(2), 208–212(2002).127 Martin NC, Pardo J, Simmons M et al. Animmunocytometric assay based on dengueinfection via DC-SIGN permits rapidmeasurement of anti-dengue neutralizingantibodies. J. Virol. Methods 134(1–2),74–85 (2006).128 Rodrigo WW, Alcena DC, Rose RC, Jin X,Schlesinger JJ. An automated dengue virusmicroneutralization plaque assay performedin human Fc{γ} receptor-expressing CV-1cells. Am. J. Trop. Med. Hyg. 80(1),61–65 (2009).129 Thomas SJ, Nisalak A, Anderson KB et al.Dengue plaque reduction neutralizationtest (PRNT) in primary and secondarydengue virus infections: how alterations inassay conditions impact performance. Am.J. Trop. Med. Hyg. 81(5), 825–833 (2009).130 Liu L, Wen K, Li J et al. Comparison ofplaque- and enzyme-linked immunospot-based assays to measure the neutralizingactivities of monoclonal antibodies specificto domain III of dengue virus envelopeprotein. Clin. Vaccine Immunol. 19(1),73–78 (2012).131 Moi ML, Lim CK, Kotaki A, Takasaki T,Kurane I. Discrepancy in dengue virusneutralizing antibody titers between plaquereduction neutralizing tests with Fcγreceptor (FcγR)-negative and FcγR-expressing BHK-21 cells. Clin. VaccineImmunol. 17(3), 402–407 (2010).Tang & Ooi
  • 13. 907www.expert-reviews.comReview132 Moi ML, Lim CK, Chua KB, Takasaki T,Kurane I. Dengue virus infection-enhancing activity in serum samples withneutralizing activity as determined by usingFc?R-expressing cells. PLoS Negl. Trop. Dis.6(2), e1536 (2012).133 Chan KR, Zhang SL, Tan HC et al.Ligation of Fc γ receptor IIB inhibitsantibody-dependent enhancement ofdengue virus infection. Proc. Natl Acad.Sci. USA 108(30), 12479–12484 (2011).134 Ramos MM, Tomashek KM, Arguello DFet al. Early clinical features of dengueinfection in Puerto Rico. Trans. R. Soc.Trop. Med. Hyg. 103(9), 878–884 (2009).135 Aytur T, Foley J, Anwar M, Boser B, HarrisE, Beatty PR. A novel magnetic beadbioassay platform using a microchip-basedsensor for infectious disease diagnosis.J. Immunol. Methods 314(1–2),21–29 (2006).136 Lee YF, Lien KY, Lei HY, Lee GB. Anintegrated microfluidic system for rapiddiagnosis of dengue virus infection. Biosens.Bioelectron. 25(4), 745–752 (2009).137 Duval D, González-Guerrero AB, Dante Set al. Nanophotonic lab-on-a-chip platformsincluding novel bimodal interferometers,microfluidics and grating couplers. Lab Chip12(11), 1987–1994 (2012).138 Fang X, Tan OK, Tse MS, Ooi EE. Alabel-free immunosensor for diagnosis ofdengue infection with simple electricalmeasurements. Biosens. Bioelectron. 25(5),1137–1142 (2010).139 Lee VJ, Lye DC, Sun Y, Leo YS. Decisiontree algorithm in deciding hospitalizationfor adult patients with denguehaemorrhagic fever in Singapore. Trop.Med. Int. Health 14(9), 1154–1159 (2009).140 Thein TL, Leo YS, Lee VJ, Sun Y, Lye DC.Validation of probability equation anddecision tree in predicting subsequentdengue hemorrhagic fever in adult dengueinpatients in Singapore. Am. J. Trop. Med.Hyg. 85(5), 942–945 (2011).141 Peeling RW, Smith PG, Bossuyt PM. Aguide for diagnostic evaluations. Nat. Rev.Microbiol. 8(12 Suppl.), S2–S6 (2010).142 Shrestha B, Brien JD, Sukupolvi-Petty Set al. The development of therapeuticantibodies that neutralize homologous andheterologous genotypes of dengue virustype 1. PLoS Pathog. 6(4),e1000823 (2010).143 Gromowski GD, Roehrig JT, DiamondMS, Lee JC, Pitcher TJ, Barrett AD.Mutations of an antibody binding energyhot spot on domain III of the dengue 2envelope glycoprotein exploited forneutralization escape. Virology 407(2),237–246 (2010).144 Ma Q, Wang Y. Comprehensive analysis ofthe prevalence of hepatitis B virus escapemutations in the major hydrophilic regionof surface antigen. J. Med. Virol. 84(2),198–206 (2012).145 Kalayanarooj S, Vaughn DW,Nimmannitya S et al. Early clinical andlaboratory indicators of acute dengueillness. J. Infect. Dis. 176(2),313–321 (1997).146 Kittigul L, Pitakarnjanakul P, Sujirarat D,Siripanichgon K. The differences of clinicalmanifestations and laboratory findings inchildren and adults with dengue virusinfection. J. Clin. Virol. 39(2),76–81 (2007).147 Phuong CX, Nhan NT, Kneen R et al.;Dong Nai Study Group. Clinical diagnosisand assessment of severity of confirmeddengue infections in Vietnamese children:is the World Health Organizationclassification system helpful? Am. J. Trop.Med. Hyg. 70(2), 172–179 (2004).148 Chau TN, Anders KL, Lien le B et al.Clinical and virological features of denguein Vietnamese infants. PLoS Negl. Trop.Dis. 4(4), e657 (2010).149 Nunes-Araújo FR, Ferreira MS, NishiokaSD. Dengue fever in Brazilian adults andchildren: assessment of clinical findingsand their validity for diagnosis. Ann.Trop. Med. Parasitol. 97(4),415–419 (2003).150 Alexander N, Balmaseda A, Coelho IC et al.;on behalf of the European Union, WorldHealth Organization (WHO-TDR) supportedDENCO Study Group. Multicentreprospective study on dengue classification infour South-east Asian and three LatinAmerican countries. Trop. Med. Int. Health16(8), 936–948 (2011).151 Fry SR, Meyer M, Semple MG et al. Thediagnostic sensitivity of dengue rapid testassays is significantly enhanced by using acombined antigen and antibody testingapproach. PLoS Negl. Trop. Dis. 5(6),e1199 (2011).152 Giraldo D, Sant’Anna C, Périssé AR et al.Characteristics of children hospitalizedwith dengue fever in an outbreak in Rio deJaneiro, Brazil. Trans. R. Soc. Trop. Med.Hyg. 105(10), 601–603 (2011).153 Binh PT, Matheus S, Huong VT, DeparisX, Marechal V. Early clinical and biologicalfeatures of severe clinical manifestations ofdengue in Vietnamese adults. J. Clin. Virol.45(4), 276–280 (2009).154 Leo YS, Thein TL, Fisher DA et al.Confirmed adult dengue deaths inSingapore: 5-year multi-centerretrospective study. BMC Infect. Dis. 11,123 (2011).155 Ong A, Sandar M, Chen MI, Sin LY. Fataldengue hemorrhagic fever in adults duringa dengue epidemic in Singapore. Int. J.Infect. Dis. 11(3), 263–267 (2007).Diagnosis of dengue: an update

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