# Diagnostic challenges in malaria detection: A comparative diagnostic performance of HRP2-based rapid diagnostic tests, microscopy, and PCR at Bichena primary hospital, Northwest Ethiopia

## TL;DR

This study compares the effectiveness of different malaria diagnostic tools in Ethiopia, finding that PCR is more reliable than rapid tests and microscopy.

## Contribution

The study provides a comparative analysis of diagnostic tools in a highland region, highlighting the limitations of HRP2-based RDTs and microscopy.

## Key findings

- PCR detected 23.4% Plasmodium infections, higher than microscopy (20.1%) and RDTs (19%).
- HRP2-based RDTs had lower sensitivity and accuracy compared to PCR, especially for P. falciparum and mixed infections.
- Platelet count was significantly lower in PCR-confirmed cases, suggesting hematological parameters could support clinical diagnosis.

## Abstract

Accurate malaria diagnosis is crucial for effective case management, strong surveillance, and progress toward elimination. However, in highland regions, diagnostic tools are underutilized or yield suboptimal performance. While hematological alterations are frequently observed in malaria, their role remains largely supportive rather than diagnostic. This study aimed to evaluate diagnostic challenges by comparing the performance of HRP2-based rapid diagnostic tests, microscopy, and PCR at Bichena Primary Hospital, Northwest Ethiopia, with hematological profiles examined as supportive indicators to help contextualize diagnostic performance.

A facility-based cross-sectional study was conducted between 31/12/2024 to 28/02/2025, with 274 participants enrolled through consecutive sampling. Socio-demographic data were collected using semi-structured questionnaires. The diagnostic evaluation used nested polymerase chain reaction (PCR) (from dried blood spots), microscopy (capillary and venous blood), histidine rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs), and hematological profiling. Data analysis was carried out with Statistical Package for the Social Sciences (SPSS) version 25.0, assessing diagnostic accuracy through sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), while inter-test agreement was measured using Cohen's Kappa coefficient. Results were summarized in text, figures, and tables.

Higher prevalence of Plasmodium infections was detected in 23.4% of participants by PCR, 20.1% by microscopy and 19% by HRP2-antigen-based RDT. The HRP2-antigen based RDT showed lower sensitivity (79.1%), NPV (94.1%), and test accuracy (94.9%) compared to PCR. Similarly, microscopy exhibited high specificity and PPV (100%); however, the sensitivity was 85.9%, indicating that some true positives are missed compared to PCR. Moderate test agreement was observed between PCR and microscopy (κ = 0.904; P = 0.00) but weak agreement between PCR and RDTs (κ = 0.847). Hematological analysis revealed a significantly lower platelet count among PCR-confirmed malaria cases (P < 0.05), suggesting a supportive association rather than diagnostic utility.

Both HRP2-antigen based RDTs and microscopy demonstrated lower sensitivity compared to PCR. RDTs showed the lowest diagnostic potential for P. falciparum, mixed and even P. vivax infections, this may be due to low parasitemia and possible pfhrp2 deletions. Hematological parameters, particularly platelet count, may serve as complementary indicators to support clinical suspicion but should not replace parasitological or molecular diagnosis. Further investigation of pfhrp2/pfhrp3 deletions is critical to inform the selection of appropriate diagnostic tools in the area.