nghiệmchẩnđoán nhanh kháng nguyên và kháng...

Hiểu và ứng dụng đúng kết quả xétnghiệm chẩn đoán nhanh kháng nguyên

và kháng thể đối với sốt xuất huyếtDengue

TS BS Nguyễn Minh TuấnBệnh viện Nhi Đồng 1

Khoa Y – Đại học Quốc gia TPHCMĐơn vị Nghiên cứu Lâm sàng Đại học Oxford

Hội nghị Khoa học Nhi khoa Bệnh viện Nhi Đồng 1TPHCM, 20-21/9/2019

Contents

• Virus Dengue

• Immune response to Dengue infection

• Performance of rapid Dengue diagnostictests

• Interpretation of results

• Take-home messages

Virus Dengue

Virus Dengue

• RNA Virus

• 10 genes (3 structural

and 7 non-structural )

Kuhn, R.J. et al. , 2002

Contents

• Virus Dengue





Sensitivity of dengue tests when dengue occurs for the first time

Sensitivity of dengue tests when dengue occurs for the second time

Courtersy of Dr. S Guanasena

Dengue serology

• IgM detection (qualitative)

In a suspected case of dengue, presence of dengue IgMindicates recent infection IgM capture ELISA (blood collected after 5th day)

50% + in 3-5 day, 70% on 7th day, 100% day 10-

14

• IgG detection (quantitative)

Diagnostic sero-conversion is defined as a four fold rise (or fall) in antibodies in paired sera (collected in the first 7 days & 10 – 14 days later)

HI assay / ELISA / Neutralization assay

• Primary infection: Characterized by detectable levels of IgM antibodies by days five or six after onset of fever; levels peak at about 14 to 15 days and can remain high up to 30 to 60 days later, and then decline gradually over time. IgG antibodies against dengue are elevated after the eighth or ninth day of the onset of fever and are detectable for life.• Secondary infection:Very high levels of IgG antibodies against dengue are observed from the second or third day of fever. IgM antibody levels can be less elevated in secondary infections and are sometimes not detectable.

Dengue serology

Contents

• Virus Dengue





Sensitivity by day of illness of two NS1 diagnostic tests, A) an ELISA-based assay and B) a lateral flow rapid test (BioRad)

in Vietnamese children

Hang VT et al., 2009

Ngày sốt DENV

RTPCR

dương

PlateliaTM

DENV NS1 Ag

SD DENV

NS1 Ag ELISA

SD BiolineTM

Dengue 2010

(NS1)

SD BiolineTM

Dengue Duo-

2010 (NS1/IgM)

< 5 ngày n=107 91,6% 90,7% 86,9% 87,9%

≥ 5 ngày n=69 66,7% 62,3% 56,5% 68,1%

Tổng cộng n=176 81,8%

(74,5%-89,1%)

79,5%

(71,9%-87,1%)

75,0%

(66,8%-83,2%)

80,1%

(72,6%-87,6%)

Loại mẫu Số XN PlateliaTM

DENV NS1 Ag

SD DENV

NS1 Ag ELISA

SD BiolineTM Dengue

Duo-2010 (NS1/IgM)

Không nhiễm

DENV

98 95,9% 95,9% 93,9%

JEV 20 100% 100% 100%

Tổng cộng 118 96,6%

(92,4%-100%)

96,6%

(92,4%-100%)

94,9%

(89,9%-100%)

Độ nhạy toàn bộ - Test nhanh

Độ đặc hiệu toàn bộ

Nguồn: PTN Arbovirus - PIHCMC

Diagnostic performance of NS1 rapid test in enrolment plasma samples within 72hrs of illness onset and odds of NS1

detection in relation to plasma viremia

Tuan NM et al., 2015

Duong V, et al. PLoS Negl Trop Dis 2011; 5(7): e1244

Sensitivity of NS1 assay for each DENV serotype

Tinh hinh SXHD khu vực phía Nam

0

2

4

6

8

10

12

14

16

18

20

0

50

100

150

200

250

300

350

400

450

500

19

96

19

97

19

98

19

99

20

00

20

01

20

02

20

03

20

04

20

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Tỉ lệ

vi r

út

lưu

hàn

h (

%)

Mắc

/ 1

00

.00

0 d

ân

Tương quan giữa mắc/100.000 dân với típ vi rút

Măc / 100.000 dân D1 D2 D3 D4

D1

D2

D4

D3

Nguồn: Viện Pasteur TPHCM - Tình hình & giải pháp phòng chống SXHD KVPN 2019

Contents

• Virus Dengue





CROSS REACTIVITYA POSITIVE RESULT ?

• NS1 glycoprotein: produced by all flavivirusesand is secreted from mammalian cells.

• IgM Dengue: Cross-reactivity with other circulating flaviviruses, such as Japanese encephalitis, St Louis encephalitis and yellow fever, does not seem to be a problem

Some false positives: malaria, leptospirosis and past dengue infection.

• IgG Dengue: lacks specificity within the flavivirusserocomplex groups.

INTERPRETATION OF DENGUE DIAGNOSTIC TESTS

Highly suggestive Confirmed

One of the following:

• IgM + in a single serum sample

• IgG + in a single serum sample with a HI titre of 1280 or greater

• NS1 +

One of the following:

• PCR +

• Virus culture +

• IgM seroconversion in paired sera

• IgG seroconversion in paired sera or fourfold IgGtiter increase in paired sera

WHO, 2019BYT, 2012

COMPARISON OF DIAGNOSTIC TESTS ACCORDING TO THEIR ACCESSIBILITY AND

CONFIDENCE

DIRECT METHODS INDIRECT METHODS

ACCESSIBILITY

CONFIDENCE

VIRUS ISOLATION

GENOMEDETECTION

NS1 ANTIGEN

SEROLOGYIgM

SEROLOGYIgG

WHO, 2019

“ROUGH” GUIDE FOR INTERPRETATION OF DENGUE SEROLOGY REPORTS

IgM IgG INTERPRETATION

Negative Negative Early sample/not dengue

Negative Positive (low titre) Past dengue infection

Negative Positive (high titre) Secondary dengue infection

Positive Negative Primary dengue infection

Positive Positive (low titre) Recent primary dengue infection

Positive Positive (high titre) Secondary dengue infection

IgG/IgM NS1 and IgG/IgM


NS1


Negative NS1 +IgM +IgG +

NS1 +IgM -IgG -

NS1 -IgM +IgG +

NS1 -IgM -IgG +

NS1 -IgM +IgG -

SCENARIO 1

Day of illness = 4

Ig M - Positive

Ig G - Negative

NS1 - Antigen Positive

Diagnosis – ?

Primary infection

Day of illness = 4

Ig M Negative

Ig G Positive

NS1 Negative

Diagnosis - ?

SCENARIO 2

Secondary infection OR Past infection

Day of illness = 3

Ig M Negative

Ig G Negative

NS1 Positive

Diagnosis - ?

SCENARIO 3

Primary infection

LIMITATIONS

A NEGATIVE RESULT ?

➢NS1 can be false negative:

• Secondary infection

• Late collection of blood sample

• DEN2

➢IgM Dengue can be false negative:

• Sometimes in secondary infection

TAKE-HOME MESSAGES

➢ Primary vs. Secondary infection.

➢Cross-reactivity.

➢A “rough” interpretation:

• NS1+: Early Dengue infection

• IgM+: Recent Dengue infection

• IgG+: Primary Dengue or past infection

➢A definitive diagnosis must be based on the test result + clinical evaluation.

➢ Evaluation of diagnostic accuracy and cost-effectivenessaccording to reference statndard tests.

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