Validation of a complete technological solution (DNA extraction + qpcr) to aid in the diagnosis of plasmodium falciparum or plasmodium vivax in environments with little infrastructure

The Porto Velho Region is located in the Amazon region, with an average temperature of around 30 oC throughout the year, frequently reaching 40 oC, and an average humidity of 50% in the dry months, June-October, and 90% in the months of rain between November and May. Recent data show that some 77 thousand people live in 82 settlements near Porto Velho, both in Rondônia and in the south of Amazonas. Between the states of Rondônia and Amazonas, a situation known as border malaria is established, with social disorganization and an increased risk for adults and children living close to the forests. In these regions, access to basic health services is greatly affected, requiring travel of several hours to obtain this public service. This difficulty in access results in delayed diagnosis and treatment of cases, predisposing the region to outbreaks and maintenance of the infection locally. For these populations, access to reliable diagnostic methods is of fundamental importance. Optical microscopy (OM) has always been the method of choice for use in areas with difficult access or with low infrastructure. MO is useful for using only a simple microscope, but it has the disadvantage of needing a well-trained technician who will be able to detect only concentrations of up to 100 parasites / µL of blood. However, OM is usually not useful in the diagnosis of asymptomatic patients, or those with low parasitemia, which can function as reservoirs of parasites, and this identification is crucial for achieving the objectives proposed by WHO for the elimination of malaria. Lateral flow chromatography tests (or rapid tests) are serological tests capable of detecting specific antigens of each parasite in a low sample volume, in just 15 minutes and without the use of equipment or electricity. However, the use of these tests has decreased due to the generation of false positive results, technical problems resulting from environmental conditions such as high humidity and / or temperature, in addition to low sensitivity (70-75% in the field) despite higher values reported for tests in the laboratory. Tests based on the detection of nucleic acids (NAT) are more sensitive and specific, being able to detect the levels of infection found in asymptomatic patients. Among the tests available, Real-Time PCR (qPCR) is the most used in reference laboratories and commercial tests, although other methods have been developed and are also available for diagnosing malaria. In recent developments, lateral flow techniques have been combined with nucleic acid amplification to detect infectious diseases in environments with little infrastructure. However, NAT tests require laborious sample preparation and sensitive equipment, which prevents them from being used in field situations. To mitigate the situation, several protocols for the storage and preparation of samples using simplified procedures have been proposed, usually coupled with portable equipment to perform the NAT test. In recent years, our group has worked to develop and validate a NAT test based on qPCR to aid in the diagnosis of malaria, composed of reagents produced in Brazil, both for use in laboratories and for use in a portable device, the Q3-Plus. The Q3-Plus is a lightweight and portable device (approx. 300 grams) that performs qPCR reactions on a silicon chip and transmits the results to software that automatically records and analyzes the data. However, as the qPCR reagents are thermolabile, gelation technology is used to store the reagents already at the reaction site (plate or chip). Gelation is a technique that mixes stabilizing and thermo-protective agents to the qPCR solution which, when subjected to vacuum, forms a gel structure that allows the reagents to be stored in a refrigerator or at room temperature (20-25 oC). The technique has already been used to gel qPCR reagents for detection of Campilobacter, T. cruzi, and also malaria. Recently, our group optimized and validated a qPCR for the detection of P. falciparum or P. vivax DNA in blood samples, developed with national reagents, which had been gelled on the equipment plate and stored at room temperature for up to 2 months. In parallel, a protocol was optimized to extract DNA from parasites from blood samples stored on FTA Micro Elute filter paper. FTA Micro Elute papers have chaotropic and solubilizing agents embedded in their fibers, which mix with the solution that is aliquoted, resulting in cell lysis and release of intracellular content. In these conditions, proteins will bind strongly to the paper fibers, while the DNA can be easily eluted into solution. This protocol was validated with> 100 samples and proved to be as efficient as a commercial DNA extraction kit. It is intended to combine the rapid DNA extraction protocol from filter paper with the portability of the Q3-Plus equipment and the practicality of the ‘ready-to-use’ qPCR reactions to compose a complete technological solution, capable of detecting the DNA of the causative parasite malaria in remote areas without infrastructure, such as settlements and mining in the Amazon region. Configured as a kit, the proposed technological solution encompasses all the necessary steps to perform a molecular-based test in the field, allowing health agents to initiate eventual treatments, even in persistently asymptomatic patients, without the need for the population to go to an outpatient clinic. diagnosis of malaria in the nearest urban center, usually a few hours away.

Diagnostics

Jan 2020 — Jan 2023

Brazil