10.1586/ERI.12.76 895ISSN 1478-7210www.expert-reviews.comReview© 2012 Kin Fai TangDengue is endemic throughout the tropica...
Expert Rev. Anti Infect. Ther. 10(8), (2012)896Review1940s [23], it was not until 1971 that the feasibility of a denguevac...
897www.expert-reviews.comReviewantibodies with virus isolation or a faster rate of viral clearance inpatients with seconda...
Expert Rev. Anti Infect. Ther. 10(8), (2012)898Reviewsequence-based amplification [NASBA]) [80] was found to behighly sens...
899www.expert-reviews.comReviewcollected between day 6 and day 16 after illness onset werefound to have higher rates of de...
Expert Rev. Anti Infect. Ther. 10(8), (2012)900Reviewthe DENV serotype responsible for the infection [133]. However,detail...
901www.expert-reviews.comReviewprospective validation of their equation in the same hospital andfound similar levels of ac...
Expert Rev. Anti Infect. Ther. 10(8), (2012)902Reviewhighly specific, it lacks sufficient sensitivity, especially since in...
903www.expert-reviews.comReviewReferencesPapers of special note have been highlighted as:• of interest•• of considerable i...
Expert Rev. Anti Infect. Ther. 10(8), (2012)904Reviewthe early phase of illness. PLoS Negl. Trop.Dis. 2(3), e196 (2008).37...
905www.expert-reviews.comReview70	 Leparc-Goffart I, Baragatti M, Temmam Set al. Development and validation ofreal-time on...
Expert Rev. Anti Infect. Ther. 10(8), (2012)906Reviewto viraemia and antibody responses. BMCInfect. Dis. 10, 142 (2010).10...
907www.expert-reviews.comReview132	 Moi ML, Lim CK, Chua KB, Takasaki T,Kurane I. Dengue virus infection-enhancing activit...
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Diagnosis of dengue

  1. 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. 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. 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. 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. 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. 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. 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. 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
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