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登革熱及其他病媒性傳染病

快速診斷試劑之開發與應用快速診斷試劑之開發與應用

Jyh-Hsiung Huang

Research and Diagnostic CenterResearch and Diagnostic Center

Taiwan CDC

2008.7.19

IntroductionDengue Diagnosis

IgM/IgG ELISA/ rapid testIgM/IgG ELISA/ rapid testNS1 Ag ELISA/ rapid test

Fever screening at airports (NS1 Ag rapid test)Chik ng n a JapaneseChikungunya, Japanese encephalitis, Scrub typhus, and Hantavirus infection.

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Major ArbovirusesFlavivirus/Flaviviridae

Alphavirus/Togaviridae

Phlebovirus/Bunyaviridae

Reovirus/Reoviridae

Y ll f Chik C lif i C l d ti kYellow fever Chikungunya California encephalitis

Colorado tick fever

Dengue Ross river Rift Valley fever

Japanese encephalitis

Eastern equine encephalitis

Sandfly fever

West Nile Western equine encephalitis

Crimean-Congo haemorrhagic fever

St. Louis encephalitis

Venezuelan equine encephalitis

Tick-Borne encephalitis

Classification of Rickettsiae

Orientia

DiseaseSpeciesGenus

Scrub typhus

FamilyOrderClass

Alpha-

RickettsiaceaeRickettsiales

Rhizobiales rickettsii

Murine typhus (TG)typhi

Epidemic typhus (TG)prowazekii

Scrub typhus (STG) tsutsugamushi

Rocky mountain

Rickettsia

pproteobacteria

henselaeBartonella

Bartonellaceae

Q feverburnetiiCoxiella

Legionellales

Cat scratch disease

Coxiellaceae

mountain spotted fever

Gamma-proteobacteria

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Ideal Diagnostic TestEarly diagnosis (acute phase)Rapid test (Point-Of-Care)p ( )Simple and Easy to use (no equipment) Highly sensitive (antigen, mAbs or primers)Highly specific (accurate)Various antibody isotypesCost effective (inexpensive)

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Cost effective (inexpensive)Automatic to handle large samplesScreening vs. ConfirmationQualitative vs. Quantitative

Temperature, Virus Positivity and Temperature, Virus Positivity and AntiAnti--Dengue IgM , by Fever DayDengue IgM , by Fever Day

100100

ve39 5elsi

us)

its)300300

20

40

60

80

20

40

60

80

rcen

t Vir

us P

ositi

v39.5

39.0

38.5

38.0

37.5ratu

re (d

egre

es C

e

ngue

IgM

(EIA

uni

150

75

225

150

75

225病毒血症期(Viremia)

偵測病毒抗原或核酸 偵測 IgM 及 IgG抗體

66Anti-Dengue IgMMean Max. Temperature Virus

Vaugh et al., J Infect Dis. 1997. 176:322

Day of defervescence

00Per

-4 -3 -2 -1 0 1 2 3 4 5 637.0

Tem

pe Den

00

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Dengue Dengue Onset Virus Real-time RT-PCR E/M-specific

Representative results of routine diagnosis of serum samples from reported dengue cases.

infection serotype Days isolation Ct value Capture IgM and IgG ELISA

OD 405nm

Flavi-

specific

Dengue-

specific

Serotype-

specific

Dengue-

IgM

JE-

IgM

Dengue-

IgG

JE-

IgG

Primary DEN-1 3 + 28 25 25 0.526 0.237 0.200 0.330

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16 2.791 0.951 1.687 1.453

Secondary DEN-1 1 + 27 28 24 0.166 0.151 0.226 0.176

16 1.949 0.469 3.606 1.699

Early and Rapid detection and iff i i f i f i i

Ideal Dengue Diagnostic Test

differentiation of DENV infection in the acute-phase serum samples of patients can help to reduce the introduction of DENVs and their l l t i i id ti llocal transmission, provide timely clinical treatment and initiate emergency control measures.

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Flavivirus Genome (~11Kb)

黃病毒傳染病黃病毒傳染病分子診斷系統分子診斷系統

Flavivirus Genome ( 11Kb)

CAP Structural Nonstructural

5‘NCR 3‘NCR

Cotranslational processing

C prM E NS1 2A 2B NS3 NS4A 4B NS5

Pr M

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ImmunoChromatography Test

ImmunoChromatography Test (ICT) (or calledImmunoChromatography Test (ICT) (or called “Rapid Test”) is technology platform that has enabled the development of affordable, easy-to-use, dipstick-format diagnostic tools.The products are Pregnancy, Drug abuse, and Infectious disease test.Infectious disease test. It’s a low-cost, accurate, fast and simple testing.

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Whole blood, serum

or plasma

>90% sensitivity

and specificityand specificity

Results in 15

minutes

Differentiation

between primary and

secondary dengue cansecondary dengue can

help predict disease

severity

25 tests

(54) (70)

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Dengue Duo IgM and IgG Rapid Strip Test(Sensitivity: Analysis of paired sera)

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登革熱快速檢驗試紙的使用上限制

只能做為定性偵測

不可用於區辨初次感染與二次感染

只適用於出現臨床症狀的登革熱疑似患者

陽性結果仍需進一步用病毒分離, 抗原偵測或PCR進行再確認工作 (screening test)

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The kit, called Platelia Dengue Antigen Assay, ELISA testsfor the NS1 dengue virus antigen in a patient and renders

Cheaper, faster dengue test kits on the way

for the NS1 dengue virus antigen in a patient and renders the results in one day.It is expected that the kit will not only help doctors diagnose the dengue fever during the first 5 days of the disease, but also facilitate the identification of possible dengue breeding sites and the prevention of the spread of the disease.The test kit is manufactured by Bio-Rad Laboratories and costs about 19~25 U.S. dollars. (There is another test on the market which is good, but costs $175 and is not used as often.)

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Platelia Dengue NS1 Ag test sensitivity according to dengue virus serotype detected by RT-PCR and/or virus isolation (n = 239)

DEN serotypeNo. of sera

testedNo. of positive NS1 Ag tests Sensitivity (%) 95% CI

DEN-1 42 38 90.5 77.4-97.3

DEN-2 42 37 88.1 74.4-96.0

DEN-3 107 94 87.9 80.1-93.4

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DEN-4 48 43 89.6 77.3-96.5

All serotypes 239 212 88.7 84.0-92.4

Days after onset of fevera No. of sera tested

Platelia Dengue NS1 Ag test MAC-ELISA

No. of positive No. of positive

Sensitivities of the Platelia Dengue NS1 Ag test and of the MAC-ELISA related to number of days after onset of fever (n = 266)

tests Sensitivity (%) 95% CI ELISA Sensitivity (%) 95% CI0 15 15 100.0 78.2-100.0b 0 0.0 0.0-23.2b

1 70 61 87.1 77.0-93.9 1 1.4 0.0-7.6

2 59 54 91.5 81.3-97.2 3 5.1 1.1-14.2

3 40 34 85.0 70.2-94.3 6 15.0 5.7-29.8

4 25 19 76.0 54.9-90.6 8 32.0 15.0-53.5

5 19 8 42 1 20 3 66 5 16 84 2 60 4 96 65 19 8 42.1 20.3-66.5 16 84.2 60.4-96.6

6 18 17 94.4 75.7-99.8 18 100.0 81.0-100.0b

≥7 20 9 45.0 23.1-68.5 20 100.0 83.1-100.0b

Total 266c 217 81.6 76.4-86.1 72 26.7 21.8-32.8

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2222

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There is Need for Antigen-Based Rapid Diagnostic Tests to Identify Common Acute Tropical Illnesses

Enteric fever typhus leptospirosis dengue melioidosisEnteric fever, typhus, leptospirosis, dengue, melioidosis, and tuberculous meningitis present urgent diagnostic problems that require experience and clinical judgment to make early evidence-based management decisions.

Established international firms see little profit in such diagnostic kits since they would be used in poor countries with little prospects for return of investment capital. p p p

We believe that rapid diagnostic technology could prevent significant delay in starting appropriate therapy, reduce hospital expenses, and even save lives.

Dengue NS1 Ag Assay for Early Diagnosis of the Dengue Virus Infection

Dengue NS1 Rapid Test Kitg pBio-Rad Dengue NS1 kit Standard Diagnostics

Dengue NS1 kit

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Relative sensitivity of Dengue NS1 Ag STRIPaccording to DENV serotype (Bio-Rad)

Type Number of Sensitivity of NS1 STRIP

Sensitivity of I Msera NS1 STRIP IgM

1 14 92.9% 21.4%

2 7 100% 14.3%

3 5 100% 0%

4 27 92 6% 37%

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4 27 92.6% 37%

Not Typed 8 87.5% 100%

TOTAL 61 93.4% 36.1%

Fever Screening at Airports

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快速血清試劑 5分鐘驗出60％登革熱

疾管局表示，目前在機場設有發燒篩檢站，民眾入境時經過紅外線監測儀 體溫若超過攝氏37 5℃ 就會被攔下經過紅外線監測儀，體溫若超過攝氏37.5℃，就會被攔下

來，再進行耳溫槍確認（超過38.0℃ ），一旦確認發燒，就會進行抽血，並將血清檢體送往實驗室，估計去年約有五千多人次。

去年(2007) 台灣有179例登革熱境外移入案例，其中42％在機場入境時因發燒被發現，但必須抽血後送檢1~2天才能確認，衛生署疾病管制局為加速篩檢，6月起在桃園國際機場採用快速血清試劑，只要5~30分鐘就可以當場確認約60％~70%的患者。

Evaluation of Dengue NS1 Rapid Test Kiton Acute-phase Serum Samples of Confirmed

Dengue cases from Airport Screening (Bio-Rad)

Dengue infection ELISA (%) Rapid test (%)

Primary 94 72

Secondary 66 48

Total fever patient

85 65

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評估在機場使用登革熱快速檢驗試劑(Dengue NS1 Antigen rapid test)

進行疑似登革熱發燒病患之即時篩檢進行疑似登革熱發燒病患之即時篩檢5-30 minutes test

CDC登革熱快速檢驗機場驗出首例個案

衛生署疾病管制局桃園國際機場檢疫站，於6月27日下午利用登革熱快速檢驗試劑篩檢出一名陽性病例。午利用登革熱快速檢驗試劑篩檢出一名陽性病例疾管局專案進口的登革熱快速檢驗試劑，在桃園機場試用10天後，篩到1名自越南經商返國的本國男子呈陽性反應，且檢驗時間只花了17分鐘。個案才剛離開機場，就接到篩檢站電話通知應儘快前往醫療院所就醫，並通報轄區衛生局啟動個案追蹤管理措施。快速檢驗試劑較傳統實驗室檢驗(PCR and ELISA)需1-快速檢驗試劑較傳統實驗室檢驗(PCR and ELISA)需12天快速許多，可大幅減少患者潛伏期四處「趴趴走」傳染他人的機率。6月28日經昆陽實驗室確認為感染第一型登革病毒，由於即時通報個案返家放置行李後立即前往醫院就醫，並由醫院採取隔離與防蚊措施，無須執行緊急噴藥消毒。

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Dengue NS1 Ag Rapid Test Kit (Bio-Rad)

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OnSite Rapid Test for thedetection of Chikungunya Fever

OnSite Chikungunya Ab Test The first lateral flow rapid test on the market for Chikungunya Detects antibodies for Chikungunya IgMA hi hi h iti it d ifi itAchieves high sensitivity and specificity with the recombinant antigens used

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Future Development of Rapid Diagnosis of Virus Infection

Multiplex real-time RT-PCRRapid micro-electro-mechanical-system- or MEMS-based miniaturized devices and systems Rapid, sensitive and cheap NS1 Ag screening test (ELISA, ICT or Biosensor)Recombinant protein (such as VLP) based ELISAELISADengue virus serotyping Microarrays for fever Rapid test reader