PHWR Public Health Weekly Report

Malaria is a parasitic infection transmitted by female mosquitoes of the genus Anopheles and considered endemic in tropical and subtropical regions. In the Republic of Korea (ROK), malaria is prevalent near the demilitarized zone, the border area with North Korea [1].

The severity of malaria varies depending on the Plasmodium species. Plasmodium vivax is known to be the only endemic species causing malaria in the ROK [1]. P. vivax is transmitted from infected mosquitoes to humans. It undergoes an incubation period during which it divides and multiplies in hepatocytes. The initial symptoms of malaria is similar to common cold, making it difficult to immediately suspect a P. vivax infection [2]. This leads to a diagnostic delay (DD), where the time from “symptom onset to medical institution visit and ultimately to diagnosis” is delayed. During this period, the number of malaria gametocytes in the body rises, which in turn leads to an increased probability of occurrence of infectious mosquitoes and, consequently, an elevated risk of malaria transmission to the community [3].

The Korea Disease Control and Prevention Agency (KDCA) released “The Second Malaria Re-elimination Action Plan (2024–2028)” in April 2024, with the goal of eliminating malaria by 2030. A total of 44 countries have been certified of malaria elimination to date (as of December, 2024), and it is known that the eradication certification process requires thousands to hundreds of thousands of tests to find a positive case [4]. In the ROK, passive surveillance of malaria detection is the primary method, relying mainly on patient reporting and symptom-based approaches. Achieving malaria elimination necessitates the implementation of active surveillance strategies, such as the establishment of large-scale screening systems.

A recent study has demonstrated that the detection of infected red blood cells (iRBCs) using hematology analyzers can serve as an adjunct to malaria diagnosis. Huh et al. [5] reported an iRBC sensitivity of 83.6% and specificity of 100.0% in 221 febrile patients (including 67 with P. vivax infection) in Ilsan, Gyeonggi-do, ROK. Khodaiji et al. [6] found an iRBC sensitivity of 88.7% and specificity of 100.0% in 575 patients (including 187 with P. vivax) in a multicenter collaborative study in India. They further confirmed that the iRBC detection was not affected by other conditions (anemia, reticulocytosis, thrombocytopenia, eosinophilia, or dengue fever). In addition, a recent meta-analysis of 15 studies evaluating the accuracy of diagnosing malaria using a hematology analyzer showed very high diagnostic accuracy, with an overall sensitivity of 95.0% and specificity of 99.0% [7].

These findings indicate the feasibility of expediting malaria diagnosis by diminishing the medical diagnostic delay (MDD) period between patient visits and malaria diagnosis. Complete blood count testing is commonly performed in medical institutions as a routine diagnostic procedure and enables large-scale hematological test. Confirmatory diagnosis of iRBCs can be used to promptly identify patients with malaria.

Therefore, the Division of Vectors and Parasitic Diseases at the KDCA conducted a pilot program for early malaria diagnosis using hematology analyzers in cooperation with medical institutions. This article aims to summarize the key outcomes and limitations of the pilot program, which can be used for future early diagnosis projects to eliminate malaria.

1. Recruitment of Participating Organizations

In February 2024, a project plan was shared with 130 medical institutions in malaria-risk areas, requesting their participation in the pilot project. This was carried out with the support of four cities and provinces (Gangwon, Gyeonggi, Seoul and Incheon), and the Armed Forces Medical Command.

2. Operation System of the Program

The program commenced with business briefings and public health center training in March. If iRBC were detected in blood samples from visitors (including those undergoing medical examinations), a microscopic examination was conducted at medical institutions to confirm malaria. If a definitive diagnosis could not be made, a genetic test was referred to the KDCA or Institute of Health and Environment. The test request and result notification were made through the integrated disease control information system (eid.kdca.go.kr). The KDCA analyzed the results by aggregating the number of blood cell analyses, abnormal blood cell detections, confirmed malaria diagnoses, and positive malaria cases for each month from April to October 2024 (Figure 1).

Figure 1. Malaria early diagnosis pilot project system diagram
iRBC=infected red blood cells.

3. Detection of Infected Red Blood Cells

The detection of iRBCs was performed through the implementation of flow cytometry, utilizing sysmex hematology analyzers (XN-550, 1000, 1500, 2000, 9000, and 9100). The hematology analyzer measurement modalities include side fluorescence light, forward scattered light, and side scattered light. Notably, the iRBCs are measured and classified through the white cell nucleated channel (WNR) and white cell differential channel (WDF). When specific abnormalities were identified in the WNR and WDF scatterplot, iRBC flags were automatically generated. It was determined as an iRBC if the number of flags equaled or exceeded 100 [4,5].

4. Diagnosis of Malaria

Microscopic and genetic tests are used to confirm malaria. Microscopic tests were performed by preparing tongue-shaped thin smear slide, staining with Giemsa or a commercially available Hemacolor^Ⓡ Rapid Staining Kit (Merck Millipore), and performing species identification at high magnification (×100). Genetic tests were performed by extracting DNA from blood, followed by nested polymerase chain reaction to determine positivity and negativity via an automated electrophoresis system (QIAxcel; QIAGEN).

The pilot program involved 47 medical institutions (6 health centers, 22 hospitals, 12 military hospitals, and 7 commercial reference laboratories) out of 130 that have hematology analyzers capable of detecting iRBCs in malaria-endemic areas. The participating health centers and hospitals were distributed as follows: 46.4% (13) in Gyeonggi-do, 28.6% (8) in Incheon, 17.9% (5) in Seoul, and 7.1% (2) in Gangwon-do, while all 12 military hospitals participated nationwide. The commercial referral laboratories were mostly located in the Seoul metropolitan area (Figure 2).

Figure 2. Participating institutions in the malaria early diagnosis program in the Republic of Korea

A total of 7.27 million hematology analyses were performed during the pilot project, detecting 1,359 iRBCs. The number of referrals for confirmatory diagnostic testing was 499, of which 239 were actually confirmed positive for malaria. In the public health centers, all cases with iRBCs referred for malaria confirmatory diagnostic testing. However, the hospitals and commercial reference laboratories had low referral rates of 65.9% (195/296 cases) and 24.6% (247/1,006 cases), respectively (Table 1). A monthly hematology analysis revealed an increase in the detection of iRBCs from June to September, which corresponded to the peak malaria season. This finding was similar to the 2024 malaria outbreak pattern in ROK (Figure 3).

Figure 3. Monthly blood cell analysis status (2024)
iRBC=infected red blood cells.

Table 1 Malaria test referral rate by institution

Institution	No. of blood cell analyses	No. of iRBC detections	No. of confirmatory tests for malaria (%)	No. of malaria positive cases
Public health centers	13,281	14	14 (100.0)	2
Hospitals	2,045,012	296	195 (65.9)	93
Commercial reference laboratories	5,126,021	1,006	247 (24.6)	101
Military hospitals	87,306	43	43 (100.0)	43
Total	7,271,620	1,359	499 (37.0)	239

iRBC=infected red blood cells..

In addition, as a result of investigating the reason for the visit with the cooperation of six public health centers for some cases where malaria infection was confirmed through iRBCs, 11 out of 19 cases were asymptomatic or unsuspected cases of malaria. Two cases were diagnosed in asymptomatic individuals, one with a leg fracture and another with shock. The remaining nine cases exhibited fever as the primary symptom; however, malaria was not suspected at the time of presentation. These cases were confirmed to be malaria-positive through confirmatory diagnosis following the detection of infected iRBC in hematology analysis (Table 2). Those who tested positive for malaria were treated at the medical institution according to the malaria control guideline [8].

Table 2 Malaria early diagnosis cases

Public health center	Hospital	Malaria positive cases	Suspected malaria symptoms (%)	Non-suspected or asymptomatic (%)	Reason for visit
Gangseo-gu, Seoul	Ewha Womans University Medical Center	9	4 (44.4)	5 (55.6)	Fever (ED visit)
Jungnang-gu, Seoul	Seoul Medical Center	2	1 (50.0)	1 (50.0)	Shock (ED visit)
Seo-gu, Incheon	Geomdan Top Hospital	1	1 (100.0)	0 (0.0)	-
Seo-gu, Incheon	Onnuri Hospital	2	1 (50.0)	1 (50.0)	Leg fracture (ED visit)
Paju, Gyeonggi-do	Paju Hospital	2	0 (0.0)	2 (100.0)	Fever (ED visit)
Bucheon, Gyeonggi-do	Bucheon Sejong Hospital	2	1 (50.0)	1 (50.0)	Fever 40℃ (ED visit)
Uijeongbu, Gyeonggi-do	Gyeonggi Provincial Medical Center Uijeongbu Hospital	1	0 (0.0)	1 (100.0)	Fever (ED visit). Discharged with cold medication; iRBC later dectected via hematology..
Total		19	8 (42.1)	11 (57.9)

ED=emergency department; iRBC=infected red blood cells; -=not available..

The symptoms associated with P. vivax infection are often mild and can be confused with common cold. Consequently, individuals infected with P. vivax frequently experience a delay between the onset of symptoms and their voluntary visit to a medical institution, referred to as patient delay (PD). DDs cause delays treatment for malaria-infected individuals, increasing the risk of spreading infection in the community [3,9]. Early detection of infected individuals and the provision of appropriate treatment are critical to the elimination of malaria. Therefore, minimizing PD and MDD is considered a key strategy for the effective management of malaria. Detection of iRBCs using hematology analyzers effectively reduced the turnaround time for MDD and contributed to identifying malaria-infected individuals among patients visiting medical institutions.

The pilot program detected 239 malaria-positive patients, representing 36.3% of the 659 malaria cases reported nationwide in 2024 [8]. Although it is difficult to assume that all of these cases were entirely unsuspected of malaria, the results of the in-depth epidemiologic investigation of the 19 malaria-positive cases suggest that the pilot program contributed to reducing the time to diagnosis in a significant number of cases. This pilot project demonstrated a new approach for the early detection of malaria using hematology analyzers and confirmed its feasibility and field applicability by utilizing existing medical systems without requiring additional budget and human resources.

However, there were some limitations, as it was conducted only with the cooperation of medical institutions. First, the referral rate for confirmed malaria diagnoses was 37.0% (499/1,359 cases), which was insufficient to ensure the effectiveness of the early diagnosis system. The low referral rates may be due to the cost burden of testing and a lack of awareness among healthcare providers. In addition, it may have been difficult to implement confirmatory diagnostic tests in commercial reference laboratories as the detection of iRBC required patient consent through the referring hospital. Second, the number of cases analyzed for reasons for visiting medical institutions to identify associations with early diagnosis among asymptomatic or unsuspected malaria patients was limited to 19, which was insufficient to represent all malaria-positive cases. To address this, asymptomatic or unsuspected cases should be distinguished from suspected malaria cases by accurately recording “iRBC detection” in epidemiological reports.

In the future, the limitations of these pilot projects will be improved, and confirmatory diagnosis of malaria will be conducted for all cases of iRBC detection through budget support such as malaria testing costs. The effect of early diagnosis will be analyzed by ensuring accurate documentation of the reason for the visit through the education of epidemiological investigators. This pilot project may contribute to achieving the national goal of malaria eradication certification by conducting early diagnosis and treatment of malaria using hematology analyzers.

Ethics Statement: Not applicable.

Funding Source: None.

Acknowledgments: We are grateful to the medical institutions, including hospitals, public health centers, military hospitals, commercial reference laboratories, and the Public Health and Environment Research Institute, for their participation in this project.

Conflict of Interest: The authors have no conflicts of interest to declare.

Author Contributions: Conceptualization: HIL. Funding acquisition: HIL. Investigation: HIS. Data analysis: HES, MRL, HIS. Visualization: HES, HIS. Writing – original draft: HES. Writing – review & editing: MRL, JWJ, HIL.