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| **          | [Background:]{.underline} Serologic immunoassays for   |
| Abstract:** | detection of measles-specific IgM are currently most   |
|             | commonly used for laboratory confirmation of measles.  |
|             | In many settings, specimen processing and logistics    |
|             | are often an obstacle to achieving adequate measles    |
|             | surveillance. The 2003 measles outbreak in the         |
|             | Marshall Islands is an example of a setting in which   |
|             | the inability of the local lab to perform the          |
|             | necessary testing was further aggravated by            |
|             | difficulties in serum collection, transportation and   |
|             | storage thus causing significant delays in obtaining   |
|             | laboratory results.                                    |
|             |                                                        |
|             | [Study objective:]{.underline} To examine whether the  |
|             | filter paper blood-based assay for detection of        |
|             | measles-specific IgM antibodies is a feasible          |
|             | alternative to serum-based assays for routine measles  |
|             | surveillance.                                          |
|             |                                                        |
|             | [Methods:]{.underline} Patients with a                 |
|             | clinician-diagnosed case of acute measles infection    |
|             | who meet specified clinical criteria (rash and fever)  |
|             | will be asked to participate in the study.             |
|             | Anticipating the lowest concordance between the filter |
|             | paper-based and serum-based assay result (i.e. both    |
|             | results positive or both negative) of 80%, for a       |
|             | desired precision of 0.05, α=0.01, and a design effect |
|             | of 1.00, a total of 425 study participants need to be  |
|             | enrolled in this study. For each participant enrolled, |
|             | a serum sample and a filter paper blood spot will be   |
|             | obtained by finger stick on the day of presentation at |
|             | the clinic. Serum and filter paper blood spots will be |
|             | tested for measles using commercial measles-IgM kits   |
|             | (Dade Behring Enzygnost^R^).                           |
|             |                                                        |
|             | [Analysis]{.underline}: Results will be analyzed both  |
|             | to determine the correlation of raw optical density    |
|             | values as well as to determine the relative            |
|             | sensitivity and specificity of filter paper EIA        |
|             | results compared with serum results.                   |
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| **Specific  | 1\) To examine concordance of dried capillary          |
| aims:**     | blood-based vs. serum-based assays for detection of    |
|             | measles virus-specific IgM antibodies in field         |
|             | conditions;                                            |
|             |                                                        |
|             | 2\) To assess feasibility of using dried capillary     |
|             | blood specimens collected on filter paper for routine  |
|             | measles surveillance.                                  |
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| **Ba        | Serologic immunoassays for detection of                |
| ckground:** | measles-specific IgM are most commonly used for        |
|             | laboratory confirmation of measles. Serum-based tests  |
|             | usually require taking blood by phlebotomy, processing |
|             | the blood to separate serum, and appropriate logistics |
|             | to store and transport serum specimen to a lab where   |
|             | the testing can be performed.                          |
|             |                                                        |
|             | In many international settings, specimen processing    |
|             | and logistics are often an obstacle to achieving       |
|             | adequate measles surveillance. The 2003 measles        |
|             | outbreak in Marshall Islands is an example of a        |
|             | setting, in which the inability of the local lab to    |
|             | perform the necessary testing was further aggravated   |
|             | by complicated specimen logistics, causing significant |
|             | delays in obtaining laboratory results.                |
|             |                                                        |
|             | A number of studies have demonstrated that dried blood |
|             | specimens collected on filter paper, air-dried and     |
|             | transported without refrigeration or freezing can be   |
|             | used for detection of measles-specific IgM antibodies  |
|             | and characterization of measles virus genome. Most     |
|             | recently, CDC's Global Measles Branch and Measles      |
|             | Virus Section conducted a study in Uganda, a high      |
|             | measles prevalence setting, aiming to (1) adapt and    |
|             | fine-tune the commercially available IgM enzyme        |
|             | immunoassays (EIA) for detection of measles virus      |
|             | specific IgM and IgG antibodies in dried filter paper  |
|             | blood spots, (2) compare detection of measles-specific |
|             | IgM and IgG antibodies in filter paper blood specimens |
|             | to the same assay performed on serum specimens and (3) |
|             | identify measles virus by RT-PCR from filter paper     |
|             | blood specimens. For purpose of that study, filter     |
|             | paper blood specimens were being dried, individually   |
|             | wrapped, frozen at -20^o^C and transported to the CDC  |
|             | Measles Lab in Atlanta on dry ice. The laboratory      |
|             | phase of the Uganda study is still ongoing, but the    |
|             | preliminary results look promising.                    |
|             |                                                        |
|             | We therefore propose to conduct a follow-up study in   |
|             | field conditions to examine performance of the         |
|             | filter-paper based assay in laboratory diagnosis of    |
|             | rash and fever disease, and assess the feasibility of  |
|             | using the filter paper blood spot-based assay for      |
|             | routine measles surveillance.                          |
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| **Study     | The goal of the proposed study is to examine whether   |
| des         | the filter paper blood-based assay for detection of    |
| ign/Plan:** | measles-specific IgM antibodies is a feasible          |
|             | alternative to serum-based assays for routine measles  |
|             | surveillance.                                          |
|             |                                                        |
|             | The primary objective of the study is to compare       |
|             | detection of measles-specific IgM by commercially      |
|             | available EIA in filter paper blood spots, and         |
|             | determine the relative sensitivity and specificity of  |
|             | these compared to the same EIA performed in serum. The |
|             | secondary objective is to identify measles virus by    |
|             | RT-PCR from filter paper blood specimens.              |
|             |                                                        |
|             | The study will be implemented in a country with        |
|             | relatively low measles prevalence (e.g. during an      |
|             | inter-epidemic interval) and an adequate routine       |
|             | surveillance system, including a local laboratory      |
|             | experienced in using the commercial measles-IgM and    |
|             | rubella-IgM EIA tests for routine measles and rubella  |
|             | diagnostics. Patients with a clinician-diagnosed case  |
|             | of acute measles infection who meet specified clinical |
|             | criteria (rash and fever) will be asked to participate |
|             | in the study. Anticipating the lowest concordance      |
|             | between the filter paper-based and serum-based assay   |
|             | result (i.e. both results positive or both negative)   |
|             | of 80%, for a desired precision of 0.05, α=0.01, and a |
|             | design effect of 1.00, a total of 425 study            |
|             | participants need to be enrolled in this study. For    |
|             | each participant enrolled, a serum sample and a filter |
|             | paper blood spot will be obtained by finger stick on   |
|             | the day of presentation at the clinic. Filter paper    |
|             | blood specimens will be dried at the room temperature, |
|             | individually wrapped, and sent to the lab either by    |
|             | mail or by a courier service without refrigeration or  |
|             | freezing; serum specimens will be transported for      |
|             | testing according to the regular procedures for        |
|             | routine measles/rubella surveillance. A standardized   |
|             | questionnaire will be administered to the patient to   |
|             | collect basic demographic data and information         |
|             | regarding vaccination history, illness                 |
|             | characteristics, and exposures of interest. Serum and  |
|             | filter paper blood spots will be tested for measles    |
|             | using commercial measles-IgM kits (Dade Behring        |
|             | Enzygnost^R^). Results will be analyzed both to        |
|             | determine the correlation of raw optical density       |
|             | values as well as to determine relative sensitivity    |
|             | and specificity of filter paper EIA results compared   |
|             | with serum results. In addition, RT-PCR testing will   |
|             | be performed on the filter paper blood specimens to    |
|             | determine the rate of detection of measles virus       |
|             | genome in these.                                       |
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