Production and validation of durable, high quality standardized malaria microscopy slides for teaching, testing and quality assurance during an era of declining diagnostic proficiency

Background: Sets of Giemsa-stained, blood smear slides with systematically verified composite diagnoses would contribute substantially to development of externally validated quality assurance systems for the microscopic diagnosis of malaria. Methods: whole blood from Plasmodium-positive donors in Cambodia and Indonesia and individuals with no history of risk for malaria was collected. Using standard operating procedures, technicians prepared Giemsa-stained thick and thin smears from each donor. One slide from each of the first 35 donations was distributed to each of 28 individuals acknowledged by reputation as having expertise in the microscopic diagnosis of malaria. These reference readers recorded presence or absence of Plasmodium species and parasite density. A composite diagnosis for each donation was determined based on microscopic findings and species-specific small subunit ribosomal RNA (ssrRNA) DNA polymerase chain reaction (PCR) amplification. Results: More than 12, 000 slides were generated from 124 donations. Reference readers correctly identified presence of parasites on 85% of slides with densities < 100 parasites/mu l, which improved to 100% for densities > 350 parasites/mu l. Percentages of agreement with composite diagnoses were highest for Plasmodium falciparum (99%), followed by Plasmodium vivax (86%). Conclusion: Herein, a standardized method for producing large numbers of consistently high quality, durable Giemsa-stained blood smears and validating composite diagnoses for the purpose of creating a malaria slide repository in support of initiatives to improve training and competency assessment amidst a background of variability in diagnosis is described.