Formalin-fixed and paraffin-embedded tissues and control blocks were cut into 3-μm sections and mounted on slides. Sections were dried overnight at 37°C and then processed through deparaffinization in xylene and subsequently hydrated through a series of graded ethanol (100 to 50%), finishing in water. Antigen retrieval was performed by incubating slides at approximately 95°C in a steamer for 30 min using 1× Antigen Retrieval Reagent Universal (R&D Systems). Following antigen retrieval, slides were blocked using a universal blocking buffer (Thermo Fisher Scientific) for 20 min, followed by 10 min each of avidin- and biotin-blocking buffers (Invitrogen) to block endogenous biotin and avidin, respectively. Antibody incubation [mouse anti-KAHRP (Knob-associated histidine rich protein) combined with rabbit anti-CD31] was then performed overnight at 4°C diluted in blocking buffer (1:100, 1:100, and 1:20 dilutions, respectively, in universal blocking buffer). Wash steps in between each step thereafter were performed with tris-buffered saline + 5% Tween for 3 × 5 min. A secondary goat anti-rabbit horseradish peroxidase–conjugated and a biotin conjugate of the F(ab′)2 fragment of goat anti-mouse immunoglobulin G (IgG) (H + L) antibodies were used (Invitrogen) (diluted to 1:500 in universal blocking buffer), followed by streptavidin conjugated to alkaline phosphatase (Thermo Fisher Scientific) (diluted 1:3000 in universal blocking buffer). For the development of signal, a Fast Red 4 TR/Naphthol AS-MX substrate reagent (Sigma-Aldrich) was applied. Slides were subsequently rinsed in water and counterstained in Mayer’s hematoxylin and mounted in aqueous mounting medium. Slides were blinded to patient identification (ID), and parasites were counted in 100 consecutive high power fields, starting in the upper left corner of each section.

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