B-257 Rapid Implementation of Monkeypox Detection in a Brazilian Diagnostic Laboratory
Authors: Andrade M S , Silva L , Hurtado F A , Belmok A , Ferreira A , Sousa C , Vieira A F , Correa R , Gil G , Sena T , Soares M , Santos M , Jácomo R , Velasco L , Nery L , Andrade R 
Published: September 2023

Abstract

Background
Monkeypox is a double-stranded DNA virus (approximately 197 kbp) belonging to the genus Orthopoxivirus. It is responsible for sporadic outbreaks in countries in Africa. Recently, in May 2022 a patient with a recent travel history to Nigeria tested positive for monkeypox in the UK. After that, people with symptoms (usually epithelial pustular lesions) were diagnosed with the virus in several countries, such as Portugal and the United States. In Brazil, the first case was confirmed in June 2022, the patient had traveled to Spain and Portugal. It was in this context that the Technical Operational Nucleus of our laboratory, Sabin Diagnosis and Health, located in Brasilia, capital of Brazil, validated and implemented a test to detect the genetic material of Monkeypox. Thus, in this work we present the implementation of this test.

Method
For the detection of Monkeypox viral DNA we chose to implement a real-time PCR (qPCR) methodology. The validated samples were swabs and wound scabs. The nucleic acid extraction chosen was using the Virus Pathogen kit from (QIAGEN), ‘midi’ protocol in the qiasymphony equipment (QIAGEN). Later we validated the extraction with the Maxwell RSC Viral Total Nucleic Acid Purification Kit extraction kit (Promega). For qPCR, we selected monkeypox-specific primers and probes, which were not able to recognize other Orthopoxiviruses (such as human pox) to ensure specificity. We used RNASEP-specific primers as an endogenous extraction control. As a positive control (PC) we used a synthetic double-stranded DNA fragment with the target sequence flanked by 10 bp. The limit of detection of the test was performed using serial dilutions of the positive control. Furthermore, given the initial absence of a positive sample for validation, we designed four pairs of primers that amplified four different regions of the viral genome for sequencing by the Sanger technique. The first positive samples were confirmed by sequencing these four regions.

Results
The protocol was designed in the first week after the confirmation of the first case in Brazil. The first tests with the PC showed the effectiveness of the primers. After adjusting the reaction conditions to assess the specificity of the test, we tested the protocol with samples known to be positive for other viral pathogens and obtained negative results for all of them, confirming the high specificity. After that we experimentally determined the limit of detection (25 copies per reaction). On July 8, 2022, one month after the first confirmed case in Brazil in the state of São Paulo, we released the Monkeypox detection test with the methodology described above. Since then, we have performed 193 tests, of which 59 (31%) were positive, most of them (56–95%) in men. The first 10 positive results were confirmed by genetic sequencing (Sanger).

Discussion
The implementation of the Monkeypox detection test by our laboratory allowed a quick response for Brazilian patients who needed the test after the virus entered the country.