Development and Clinical Evaluation of Rapid Diagnostic Reagents for Measles Kei Numazaki International University of Health and Welfare Takehiro Hasegawa, Takeshi Imoarai, and Yukio Hamaguchi Sysmex Corporation Overview Measles is a viral infectious disease caused by the measles virus, an enveloped negative-strand RNA virus of the Morbillivirus genus in the Paramyxoviridae. There is no specific treatment for measles and most people recover within 2–3 weeks. However, particularly in malnourished children and people with lowered immunity, measles can cause serious complications, including blindness, encephalitis, severe diarrhea, ear infection and pneumonia. Number of Reported Measles Cases with onset date from Dec 2010 to Jun 2011 (WHO measles Surveillance Data) Despite the great effort, an increased number of outbreaks of measles in the African and Western pacific regions were reported, and measles remains an important causes of death. Summary □There is no clinically-useful rapid diagnosis for measles over the world. □Since atypical cases of measles are not uncommon, rapid diagnosis based on laboratory testing is required. □The purpose of this study is to investigate the possibility of diagnosing measles infection with a lateral flow-based rapid diagnostic kit we developed. The target antigen for the lateral flow-based rapid diagnostic kit In order to select the target protein for the lateral flow-based rapid diagnostic kit, 25 reagents were prepared with previously-developed antibodies for measles H, F, M and N proteins, and the detection limits of them were examined using cultured virus of Edmonston strain. A. B. Rapid diagnostic kit for measles Anti-measles monoclonal antibody Absorption pad H Membrane Latex Membrane Measles virus antigen Evaluation of reactivity of kit with H,F,M or N proteins B12 B5 B7 B12 B5 B7 C130 B12 B5 B7 C130 B12 B5 B7 C130 B12 B5 B7 C130 X1 + + + + + + + + + + + + + + + + X10 - + + + + + + + - + + + + + + + X100 ND - - - - - - - ND - + - - + - - X250 ND ND ND ND ND ND ND ND ND ND - ND ND - ND ND X500 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND X1,000 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND X2,000 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND F Anti-measles monoclonal antibody conjugated color latex Membrane Sample pad Latex Latex pad C130 M B48 C527 N A23 A51 B4 B48 C527 B48 C527 A23 A51 A23 A51 B4 X1 + + + + + + + - + X10 + + + + + + - - + X100 - - + - - - - ND + X250 ND ND - ND ND ND ND ND + X500 ND ND ND ND ND ND ND ND + X1,000 ND ND ND ND ND ND ND ND + X2,000 ND ND ND ND ND ND ND ND - Sample dilution rate is indicated at left. +, positive reaction; -, negative reaction: ND, not done. B4 antibody to N protein exhibited 10 times higher sensitivity than the other antibodies. Development of antibodies against recombinant N proteins Method of antibody production Immunization of BALB/c mice with measles antigen (antigen: measles virus-infected cell extract or recombinant N protein) Fusion of a myeloma cell and the splenocyte from immunized mice Cloning of the hybridoma producing antibodies with high reactivity Incubation/antibody production in the abdominal cavity of mice Purification Reactivity of antibodies against recombinant N proteins Latex MV1-225 MV3-763 MV3-320 MV1-614 B4 MV-225 B4 MV2-3241 MV3-3892 MV1-1259 MV2-2214 MV3-1459 MV3-3892 MV2-3707 MV2-3241 MV3-3892 MV3-3892 MV2-1780 MV2-1780 MV2-1780 MV2-1780 MV2-1780 MV2-1780 MV2-2649 MV2-2649 MV2-2649 MV2-2649 H1 - - - - + - ++ ++ + + - - D3 ++ ++ ++ + ++ + ++ ++ ++ ++ ++ - D5 + + + + + + + + + + + - D9 + + - - - + + + + + + - A + + + + - - ++ + + + + - Membrane Genotype MV1-1259 The reactivity of the lateral flow-based kit was examined with 1.6 ng/test recombinant N protein of H1, D3, D5, D9 and A genotypes. Density of signal is indicated by ++, +: -, negative reaction. Clone MV3-320, MV2-3707, MV2-2649 and MV2-3241 were selected for the antibodies of the lateral flow-based kit. The recognition site of antibodies against measles N protein The recognition site was examined using N protein deletion mutant. A. B. Homology research for N protein of measles virus antigen C. Immunocytochemistry for evaluation of reactivity of antibodies The scheme of deletion mutant of N protein recognition site of antibody The result of immunocytochemistry showed that the antibodies reacted to the highly preserved region of the N protein. The antibodies were thought to react measles N proteins of various genotypes. The detection limits of the kits The detection limits with two kits were evaluated with the Edmonston Schwarz FF-8 strain virus. A. Antibodies kit-B kit-A Membrane MV2-3707 MV2-3241 MV2-2649 MV2-2649 Latex MV3-320 B. C. The detection limits for Edmonston Schwarz FF-8 strain kit-A kit-B Schema of the lateral flow system The detection limits were 5.1×103 copies/mL (kit-A) and 1.0×104 copies/mL (kit-B). We chose kit-A as a rapid diagnostic kit for further evaluation. Reactivity and cross-reactivity of the kit Reactivity to wild type and recombinant antigens, and cross-reactivity to other pathogen of both reagents were evaluated. A. C. Reactivity to the wild type antigens B. Reactivity to the recombinant antigens Detection limit (Log PFU/test) Kit Detection limit (ng/test) Kit Ys4(D5) Ys1(H1) IC-B(D3) SA203(D5) 2.4 2.8 2.7 2.5 H1 D3 D5 D9 0.75 1.6 1.6 1.6 Cross-reactivity to other pathogens Virus titer Rubella Mumps RS Adeno Influenza A Influenza B 1.5x104 1.5x103 1.2x103 5.7x104 9.6x105 1.4x106 TCID50/test TCID50/test PFU/test TCID50/test FFU/test FFU/test Kit － － － － － － The kit reacted with wild type and recombinant antigens. Cross-reactivity with other pathogens was not found. Evaluation of the kit using clinical samples ① 《Protocol》 □Summary ■ Duration: March 2008 - July 2010 ■ the number of facilities: 14 ■ the number of patients: 45 （positive 11, negative 34） ■ Age: 0 - 38 years （Average 8.1 years) ■ Sex: 24 males, 20 females, 1 unknown ■ Reference assay: PCR analysis □Method ■ In the collaboration facilities, throat samples of patients with suspected measles infection were collected and diagnosed with the kit. ■ The residual samples for the kit were analyzed with PCR in the laboratory Sampling and measurement of the kit Collect throat sample Extract viral antigen Drop the sample Insert the assay strip Detect (10 min) Evaluation of the kit using clinical samples ② We examined the correlation between the kit and PCR, and the symptoms in non-measles patients. A. Correlation with PCR PCR + Kit + - - 5 6 Total 0 5 35 41 Total 11 35 46 Positive predictive value ：45.5% Negative predictive value ：100% B. Clinical symptoms in non-measles patients Clinical Symptoms Fever Rash Cough Number of cases 35 25 6 Kit Koplik Conjunctival Nasal spot hyperemia discharge 21 12 12 Positive Negative 0 35 We confirmed that the kit could detect measles in clinical samples. Although non-measles patients showed measles-like symptoms, the kit did not exhibit false-positive. Evaluation of the kit using clinical samples ③ A. Clinical symptoms in measles patients Clinical sy mptoms 1 2 3 4 5 6 7 8 9 10 11 B. Age Sex TEMP 17.1 7.6 15.8 16 17.3 15.11 0.9 20 0.9 11.9 9.4 F F M F M F F M F F M ND 38.6 ND ND ND ND ND 39.7 ND 39.1 39.4 Koplik Fev er Rash spot + + + + + + + + + + + + + +/+ + + + + + + + + +/+ + + + + Laboratory result Nasal Conjunctiv al Ey e Cough Vaccination discharge hy peremia discharge + + + + + + + + + + + + + + + + + + + + + + 1 ND 1 0 1 0 0 0 1 1 0 Day s af ter onset of f ev er gene ty pe Log copies/ test 0.5 0.75 2 3 3 4 4 4 4 6 7 D5 D5 D5 D5 D5 D5 D5 ND ND D5 D5 1.9 2.6 5.0 4.9 5.2 5.1 5.3 -0.4 3.5 3.8 2.6 kit-A 10min 20min + + + + + - + + + + + - IgM IgG ND 5.7 3.1 5.7 9.6 23.1 1.6 9.7 24.5 25.0 24.8 ND <2 96.6 2.1 3.1 <2 14.8 <2 5.1 12.3 The period of disease 6.0 Log copies/test 5.0 4.0 Result of the kit ●Positive ●Negative 3.0 2.0 1.0 The kit detected 71% (5/7) of measles cases at 2-4 day after onset of fever. 0.0 -1.0 0 2 catarrhal period 4 6 measles period 8 Result and Conclusion Result ■ We developed the rapid diagnostic kit for measles from throat swabs in 10 min. ■ Negative predictive value of the kit was 100％ （35/35）, and positive predictive value was 45.5％ （5/11）. ■ The kit detected 71% (5/7) of measles cases at 2-4 day after onset of fever. ■ Non-measles patients showed measles-like symptoms. Conclusion ■ It is difficult to diagnose measles only based on clinical symptoms. ■ The kit showed good specificity ■ The kit needs to be improved in sensitivity.
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