Unveiling the overlooked burden of malaria misdiagnosis using lamp-based re-evaluation of routine malaria diagnosis in health centers: implications for public health and clinical practice in Northwest Ethiopia

Abstract

Background

Accurate malaria diagnosis is critical not only for prescribing correct treatment but also for ensuring effective case management, preventing drug resistance, and interrupting transmission. Despite this importance, laboratory diagnostic error driven by multifactorial causes yielding both false-positive and false-negative results remains a persistent yet underrecognized challenge, particularly in endemic regions. This study aimed to compare routine malaria microscopy with Loop-Mediated Isothermal Amplification (LAMP) to determine the prevalence of malaria misdiagnosis by routine microscopy in Northwest Ethiopia.

Methods

A health facility–based cross-sectional study was conducted at four selected health centers from July 1 to August 30, 2024. A convenience sampling technique was applied to recruit study participants. A total of 384 malaria-suspected individuals whose routine microscopy results recorded at health centers were re-evaluated using a LAMP molecular assay results performed on venous blood samples. The data were entered into Microsoft Office Excel and exported to SPSS version 27 for analysis. Descriptive statistics were used to estimate malaria misdiagnosis by routine microscopy, and the McNemar’s test was applied to assess statistical significance, with a p-value < 0.05 considered statistically significant.

Results

Among the 384 study participants (116 microscopy-positive and 268 microscopy-negative), 30.0% of routine malaria microscopy results were discordant when compared with LAMP findings. The discordant results comprised false-positive results (7.6%), false-negative results (18.8%), and species misidentification (3.6%). McNemar’s test demonstrated a statistically significant difference between microscopy and LAMP results (p < 0.001).

Conclusions and recommendations

The current study showed a high prevalence of malaria misdiagnosis; false negatives, false positives, and species misidentification by routine microscopy. Although this undermines malaria control efforts, microscopy remains the most accessible and WHO-endorsed diagnostic method in resource-limited settings. Strengthening its accuracy through continuous training, quality assurance, and supervision is therefore essential. While molecular methods such as LAMP offer superior sensitivity and specificity, microscopy continues to serve as the operational gold standard due to its practicality and affordability.

Clinical trial number

Not applicable.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12879-026-13231-8.

Introduction

Malaria is fatal vector-borne disease caused by protozoan parasites of the genus Plasmodium, transmitted through infected female Anopheles mosquitoes [1]. It predominantly affects individuals residing in tropical and subtropical regions, particularly in sub-Saharan Africa and Southeast Asia [2]. According to the World Health Organization (WHO) 2024 malaria report, nearly half of the world’s population was at risk of malaria in 2023, with 263 million cases and 597,000 deaths in 83 malaria-endemic countries—an increase of 11 million cases compared with 2022 [3]. Malaria remains a critical public health challenge in Ethiopia, with 75% of the country being malaria-endemic and 69% of the population at risk [4]. According to the WHO report, from January 1 to October 20, 2024, Ethiopia reported more than 7.3 million cases of malaria and 1,157 deaths, leading to a case fatality rate of 0.02% [5].

Despite the scale-up of interventions and control measures, which include prompt diagnosis and treatment of cases with artemisinin-based combination therapy (ACT), the use of long-lasting insecticidal nets (LLINs), and indoor residual spraying (IRS) [6], malaria remains a significant public health problem in Ethiopia [7, 8]. Apart from morbidity and mortality, it halted long-lasting socio-economic impacts primarily to more than 80% of the country’s rural community [9]. Accordingly, malaria stands to be one of the top priority programs in the national health and overall socioeconomic development agenda, and hence, the country launched a malaria elimination program, aiming to achieve nationwide malaria elimination by 2030 [10].

The World Health Organization (WHO) recommends prompt and effective diagnosis and treatment as one of the key interventions to control malaria [11]. Different methods can be used to diagnose malaria, including microscopy, rapid diagnostic tests (RDTs), and several nucleic acid amplification tests (NAATs) such as loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) [12, 13]. In routine diagnosis, microscopic examination of blood films and rapid diagnostic tests(RDT) are performed [13]. Accurate diagnosis of Plasmodium infections is crucial to reduce morbidity and mortality in tropical areas [14]. Diagnosis remains a considerable challenge in malaria control and elimination, and the performance of tools differs in various epidemiological settings [15]. Microscopic diagnosis of Plasmodium species in blood smears is the globally accepted “gold standard method” [16, 17]. It is regarded as the most suitable diagnostic instrument for malaria control because it is inexpensive to perform and allows for the determination of parasite densities, distinction between parasite stages, differentiation between Plasmodium species, and monitoring of parasite clearance [18]. However, microscopy has some drawbacks including slide reading is labor-intensive and prone to operator fatigue, leading to variability in reliability; risk of misdiagnosis in detecting mixed infections and the misidentification of P. ovale from P. vivax, and it depends on the level of parasitemia, which is lower detection limit of 4–20 parasites/µl, even for expert microscopists [15, 19, 20]. In field conditions, a threshold of about 50–100 parasites/µL of blood is more realistic but can be higher in remote settings with less skilled microscopists and poor equipment [21].

In high transmission areas, microscopy may only detect about 75% of malaria infections, and in low transmission areas, it may miss up to 88% of infections [22]. The accuracy of malaria diagnosis with microscopy can also be affected by potential factors such as technician expertise, equipment quality, workload, lack of training, and quality control programs [22]. Moreover, the reliability of microscopy diagnosis has been questioned due to false negative results at low parasitemia levels and errors in species identification in mixed infections [14, 23]. In an earlier study, only 45% of microscopists in hospitals and health centers accurately identified mixed infections of P. falciparum and P. vivax in Ethiopia, which needs attention to improve the malaria surveillance system for effective control and elimination [24, 25]. These challenges affect malaria control and elimination, leading to unnecessary drug use, resistance development, treatment delays, and potential fatalities [26]. Addressing misdiagnosed cases is crucial for successful case management and transmission interruption [27].

Several nucleic acid amplification tests (NAATs) have been developed for the detection of malaria infections in humans that can overcome microscopy limitations [28]. The Polymerase Chain Reaction (PCR) method is a better alternative for malaria diagnosis and epidemiological studies in endemic areas due to its high sensitivity and specificity [29]. However, despite being highly sensitive, PCR is expensive and cannot be utilized in routine practice in resource-limited settings [30]. Loop-mediated isothermal Amplification(LAMP) is a simpler molecular procedure and a better alternative to PCR, offering several operational advantages like acceptability of crude sample preparation, minimal capital equipment requirements, rapid result turnaround, lower cost, and less technical complexity [31]. The LAMP assay can detect Plasmodium species with a sensitivity of ten times greater than that of blood smear microscopy carried out by experienced microscopists [32]. A study in Ghana demonstrated that LAMP assays achieved 95.6% sensitivity and 100% specificity for detecting P. falciparum, significantly surpassing microscopy [33].

Despite the scale-up of malaria control and intervention practices, malaria continues to increase in Ethiopia. This may be related to the prevalence of routine blood film microscopy misdiagnosed malaria cases. However, data are scarce on the prevalence of blood film microscopy misdiagnosed malaria cases in the country in general and in the study area in particular. Therefore, this study aimed to assess the prevalence of microscopically misdiagnosed malaria cases using the LAMP assay at selected health centers in Northwest Ethiopia.

Methods

Study design, period and area

Multicenter cross-sectional study design was conducted from July 1 to August 30, 2024 at purposively selected malarious area (Teda, Addis Zemen, Yifag, and Woreta health centers), Northwest Ethiopia. All four study sites are situated in northwest Ethiopia’s Amhara Region, located between 625 km (Woreta) and 698 km (Teda) from the national capital, Addis Ababa. Demographically, Teda Health Center serves a catchment exceeding 20,000 people from its sub-city and neighboring Kebeles, while Woreta functions as the capital of Fogera District, which hosts a substantial population of 233,529 (26,812 urban). Addis Zemen and Yifag both fall within the Libokemkem District (South Gondar Zone), with Addis Zemen serving as the district’s administrative hub. Yifag is supported by six health posts and one health center. The sites occupy moderate highland elevations conducive to malaria transmission, ranging from 1,750 m (Fogera District’s lowlands) to 2,200 m (Teda’s highest point). Woreta town sits at 1,828 m, Addis Zemen at approximately 610 m (2,000 ft), and Yifag between 1,829 and 1,868 m. The areas share high annual rainfall (900–1,400 mm), peaking between June and October, and warm temperatures averaging 12.6–30 °C. This combination of ample rainfall, moderate elevations, and sustained warmth creates an environment highly favorable for malaria vectors. Malaria transmission across all sites exhibits distinct seasonality driven by rainfall patterns. A major transmission peak occurs from September to December, following the heaviest rains. Minor peaks in April–June are documented in Teda and Addis Zemen. Plasmodium falciparum and P. vivax are explicitly confirmed as the circulating parasites species [34–37].

Population

Study population

All malaria-suspected individuals who tested positive or negative with routine microscopy technique at selected health centers and voluntarily participated in the study, during the study period were study populations.

Inclusion and exclusion criteria

Inclusion criteria

All malaria-suspected individuals referred to the laboratory by clinicians and undergone microscopy examination for malaria and voluntary to participate were included in the study.

Exclusion criteria

Patients who had been treated for malaria three weeks before the study to minimize detection of residual parasite DNA after recent antimalarial treatment, as parasite DNA can persist for up to 2–3 weeks post-treatment [38], who were unable to respond due to severe illnesses and could not provide a blood sample were excluded.

Operational Definitions

Laboratory misdiagnosis

The combined occurrence of false-positive results, false-negative results, and species misidentification during microscopy laboratory malaria diagnostics.

False-positive result

A microscopy result positive for malaria when the comparative LAMP result is negative.

False-negative result

A microscopy result negative for malaria when the comparative LAMP result is positive.

Species misidentification

Incorrect classification of Plasmodium species by microscopy when malaria parasites are detected.

Sample size determination and sampling technique

Sample size determination

The total sample size was determined by single population proportion formula using 50% prevalence (to obtain maximum sample size), 95% confidence level, and 5% margin of error. Therefore, the final sample size was 384.

n = the initial sample size.

Z α/2 = Standardized normal distribution value for the 95% CI, = 1.96.

P= proportion of microscopically misdiagnosis malaria cases (50%).

d= margin of error 5%.

Sampling technique

The total sample size was proportionally allocated to the four purposively selected health centers based on the number of suspected patients served at the laboratory for malaria diagnosis during the ten days before the start of data collection. Therefore, 74 data from Woreta, 119 from Yifag, 86 from Addis Zemen, and 105 data from Teda health centers were collected, respectively. Finally, proportionally allocated malaria-suspected patients who fulfilled the inclusion criteria were conveniently recruited in the study until the allocated sample size was obtained in each of the four health centers.

Data collection tools and laboratory method

Data collection tools

Routine malaria microscopy, including blood film preparation and examination, was performed by health center laboratory professionals as part of their routine diagnostic services for febrile patients referred by clinicians. The study team did not perform smear preparation or microscopy reading. Simultaneously, 1 mL of venous blood was collected from the same participants into EDTA tubes by trained data collectors using standard aseptic procedures. Blood samples were maintained under appropriate cold-chain conditions and transported to the Amhara Public Health Institute (APHI) for storage and subsequent Loop-Mediated Isothermal Amplification (LAMP) molecular analysis. Prior to data collection, a two-day training was provided by the principal investigator to the data collectors, focusing on study procedures, accurate recording of routine microscopy results, venous blood sample collection, labeling, cold-chain handling, and sample transportation to APHI.

Laboratory method

Blood sample collection

A trained laboratory technologist collected 1 ml of venous blood in heparinized tubes using standard sample collection protocols from each patient for molecular analysis.

DNA extraction

Genomic DNA was extracted from whole blood using the Loopamp™ PURE DNA Extraction Kit (Eiken Chemical) following the manufacturer’s instructions [39, 40]. Briefly, 30 µL of whole blood mixed with sodium chloride solution was added to the extraction tube containing sodium hydroxide solution and incubated at 75 °C for 5 min to lyse cells and release DNA. The lysate was then purified using the kit’s adsorbent system, which removes proteins and other contaminants that could inhibit nucleic acid amplification. Finally, the purified DNA was eluted and used as the template for the LAMP assay.

LAMP amplification

The LAMP assay was conducted strictly according to the Eiken Loopamp™ MALARIA Pan and Pf/PV Detection Kit procedures (Eiken Chemical Company, Japan) [39, 40]. After mixing well, 30 µL of the PURE-extracted DNA solution was added to the malaria Pan or Pf/Pv reaction tube with one negative and one positive control included in each 16 reactions, mixed well by inversion to reconstitute primers in the lid of the reaction tubes and incubated at 65 °C for 40 min. The DNA was amplified by the strand displacement DNA polymerase in accordance with the LAMP reaction. LAMP-amplified DNA was assessed by naked eye by observing the turbid solution (qualitative). Turbidity caused by magnesium pyrophosphate, a by-product of the amplification reaction, is produced in proportion to the amount of amplified products. The presence of turbidity indicates the presence of amplicon. Alternatively, visual detection under UV light was being used. All reactions were also visualized the turbidity or the Fluorescent (Fig. 1), with a subsequent reading by a separate person blinded to previous LAMP readings.

Fig. 1.

Representative LAMP amplification results visualized under UV light. Positive reactions (two left tubes) exhibit bright green fluorescence due to amplification of Plasmodium DNA. The negative control shows no fluorescence, confirming absence of non-specific amplification or contamination. The positive control displays clear fluorescence, validating the performance of the assay. The visible fluorescence corresponds to accumulation of amplification by-products generated during the isothermal reaction

Data quality control and management

Training was given for data collectors by principal investigators two days before the beginning of data collection regarding the objective of the study, data collection approach, contents and relevance of the study, the confidentiality of the information as well as rights of participants. Data was collected by these trained personnel under supervision of the principal investigator. A pretest was done on 5% [19] of the sample size in Addis Zemen Primary Hospital which was not included in the study area to check for its clarity, simplicity, understandability, completeness, consistency, and coherency. Appropriate correction was taken on time for completeness and accuracy of the questionnaires. Every day after data collection, the questionnaires were reviewed and checked for completeness by the principal investigator. LAMP analysis was performed by PI at APHI by following a standard protocol. To ensure the quality of the LAMP, positive and negative controls were included in each set of LAMP runs according to manufacturers’ instructions [39, 40]. All LAMP analyses were interpreted blinded to the microscopy results. All LAMP reactions were also visualized with a subsequent reading by a separate person blinded to previous LAMP readings.

Data analysis and interpretation

All data were entered into Microsoft Office Excel and statistical analyses were performed with Statistical Package for Social Sciences (SPSS) version 27. Then, the study findings were explained in words, tables, and figures. Descriptive statistics (frequency and cross tabulations) were used. Diagnostic disagreement between microscopy and LAMP was assessed using McNemar’s test for paired binary outcomes. A p-value < 0.05 was considered statistically significant.

Results

Baseline characteristics and enrollment of study participants

A total of 384 individuals who had clinical sign and symptom and supposed to give blood sample for smear preparation and underwent malaria diagnosis via microscopy at selected health centers were enrolled in this study. Out of these, 179 (46.6%) were males and 205 (53.4%) were females, with a mean age of 28.95 (± 17.798 SD) years and an age range of 4–85 years. The ≥ 30 year’s group was the most represented, accounting for 42.9% (165/384) of the participants.

Sociodemographic characteristics of Laboratory professionals

A total of 18 laboratory professionals, 5 from Teda health center, 6 from Adiss Zemen health centers, 2 from Yifag health center, 5 from Woreta health center were included in this study. Of the total study participants, 39% were between 20 and 30 years. Compared to 50% of 31–40 and 11% above 40 years. For about 83% of laboratory professionals, their educational status was a diploma, followed by a degree. Of the laboratory professionals, 83% had 2–5 of work experience in malaria microscopy diagnosis. Sixteen (89%) took in-service training on malaria microscopy diagnosis at least once (Table 1).

Table 1.

Demography of laboratory professionals, North West Ethiopia, 2024

Characteristics	Category	Number of laboratory professional (%)
Age in years	20–30	7(39%)
	31–40	9 (50%)
	> 40	2 (11%)
Gender	Male	8 (44%)
	Female	10 (56%)
Educational Status	Bachelor degree	3 (16%)
	Diploma	15 (83%)
In-service training on malaria microscopy	Yes	2 (11%)
	No	16(89%)
Frequency of training	Once	4 (17%)
	Two times	5 (22%)
	Three times	9 (50%)
Experience in routine malaria diagnosis	< 2Yrs	1 (6%)
	2-5Yrs	15 (83%)
	> 5Yrs	2 (11%)

General laboratory setup

All four health centers maintained standardized registration books with accurate records and used request forms. Only thick blood films stained with freshly prepared Giemsa stain was consistently performed, and slides dried using heat rather than air dried. Each facility reported both the Plasmodium species and parasite developmental stage for positive samples and participated in an external quality assessment (EQA) program. However, none of the health centers reported parasite density, performed regular quality control (QC), or adhered to recommended slide reading times. Furthermore, slide examination exceeded 20 slides per day at each center, result validation was not conducted. Buffered distilled water was used to dilute Giemsa stain in only 25% of health centers, whereas 75% were using tap water (Table 2).

Table 2.

General laboratory setup, North West Ethiopia, 2024

Malaria microscopy laboratory format
Variables	Response category	Percent of study participants
Presence of a standard laboratory request paper	Yes	4(100%)
	No	0
Availability of SOP	Yes	1 (25%)
	No	3 (75%)
Presence of guideline for reagent preparation	Yes	2 (50%)
	No	2 (50%)
Availability of Internal quality control (IQC)log sheet	Yes	2 (50%)
	No	2 (50%)
Maintenance logbook for microscope	Yes	0
	No	4 (100%)
Malaria microscopy skill assessment
type of blood film they use	Thin	0
	Thick	4 (100%)
	Both	0
Blood smear drying system	Heat	4 (100%)
	Air	0
Number of fields of examined to report a negative result	≥ 100	1 (25%)
	< 100	3 (75%)
Perform a parasite count	Yes	0
	No	4 (100%)
Reporting species & stages	Yes	4 (100%)
	No	0
number of malaria slides examined daily	< 5	0
	5–10	0
	> 10	4 (100%)
Does the work load allow the recommended reading time	Yes	0
	No	4 (100%)
Information related to staining reagent & equipment
Method of diagnosis for malaria	Microscope	3 (75%)
	Microscope &RDT	1 (25%)
Labeling of stock solution	Yes	3 (75%)
	No	1 (25%)
Recommended Storage of staining solutions	Yes	4 (100%)
	No	0
How prepare/dilute working solution	Tape water	3 (75%)
	Distilled water	1 (25%)
Cleaning of Lense of microscope with recommended solution	Yes	2 (50%)
	No	2 (50%)
General Information on quality Assurance
Internal quality control programs conducted regularly	Yes	0
	No	4 (100%)
Slide rechecked when result released	Yes	0
	No	4 (100%)
Supervision from regional or national laboratories within a year	Yes	3 (75%)
	No	1 (25%)
Regular training program for microscopists available	Yes	1 (25%)
	No	3 (75%)
Participating in EQA program	Yes	4 (100%)
	No	0

Prevalence of microscopy compared to LAMP assay

Among all study participants, overall confirmed malaria cases were 30.2% (116/384) diagnosed through blood smear microscopy conducted by laboratory professionals at the health centers. Regarding species-level identification, out of 116 cases, 46 were reported as P. falciparum, 67 as P. vivax, and the remaining 3 were diagnosed as mixed P. falciparum and P. vivax (Fig. 2).

Fig. 2.

Flow diagram comparing routine malaria microscopy and LAMP assay results among suspected cases (N = 384). The figure illustrates the distribution of microscopy-positive and microscopy-negative cases and their corresponding pan-LAMP and species-specific LAMP outcomes, including false-negative, false-positive, and species misidentification results. Abbreviations: TN=true negative FN=false negative PF=Plasmodium falciparum PV=Plasmodium vivax PO=Plasmodium ovale PM=Plasmodium malaria LAMP=Loop mediated isothermal amplification

The study demonstrated a 30% (115/384) malaria misdiagnosis rate in routine microscopy using LAMP, consisting of 22.4% parasite detection errors (false negatives and positives) and 7.6% Plasmodium species misidentification. Specifically, 18.8% (72/384) were false negatives, where microscopy missed infections that were confirmed positive by LAMP. These included 37 cases of P. falciparum, 23 cases of P. vivax, 8 cases of mixed infections of P. falciparum and P. vivax, and 4 cases of other species (Po/Pm). Additionally, 3.6% (14/384) of cases were false positives, where microscopy incorrectly identified infections as positive, but LAMP results were negative. These false positives included 9 cases misidentified as P. vivax and 5 cases misidentified as P. falciparum by microscopy. Microscopy also demonstrated errors in species identification (7.6%), as highlighted by discrepancies with LAMP results. Among 46 microscopy-diagnosed P. falciparum cases, 5 were identified as P. vivax by LAMP, and 5 were identified as mixed infections (P. falciparum and P. vivax) by LAMP. Similarly, among 67 microscopy-diagnosed P. vivax cases, 11 were identified as P. falciparum by LAMP, 4 as mixed infections (P. falciparum and P. vivax), and 3 as other species (Pan LAMP positive). Furthermore, among 3 microscopy-diagnosed mixed infections (P. falciparum and P. vivax), 1 was identified as a single P. vivax infection by LAMP (Table 3 and Fig. 2).

Table 3.

Comparison of Microscopy and LAMP assay using McNemar Test

		PAN LAMP		Total		df	P-value
		Negative	Positive
Microscopy	Negative	196	72	268	McNemar Test	1	< 0.001
	Positive	14	102	116
Total		210	174	384

Comparison of Microscopy and LAMP assay

Of the 384 paired samples analyzed, routine microscopy and LAMP results showed substantial discordance. McNemar’s test demonstrated a statistically significant difference between microscopy and LAMP results (p < 0.001), indicating a significant level of routine microscopy misdiagnosis (Table 3).

Molecular prevalence of microscopically misdiagnosis malaria cases in each health center were in Teda (38%), Adiss Zemen (18.6%), Yifag (34.5%), Woreta (24.3%) (Table 4)

Table 4.

Prevalence of microscopically malaria misdiagnosis in each health center, northwest Ethiopia, 2024

Health centers	Result	Frequency (%)
Teda	Concordance	65 (61.9)
	Misdiagnosis	40 (38.1)
	Total	105 (100.0)
Woreta	Concordance	56 (75.7)
	Misdiagnosis	18 (24.3)
	Total	74 (100.0)
Yifag	Concordance	78 (65.5)
	Misdiagnosis	41 (34.5)
	Total	119 (100.0)
A/zemen	Concordance	70 (81.4)
	Misdiagnosis	16 (18.6)
	Total	86 (100.0)

NB: % =Percent

Discussion

Accurate malaria diagnosis in healthcare settings is critical for effective disease management and elimination efforts. This study identified a high level of diagnostic discrepancy between microscopy and LAMP assays, revealing key gaps in field performance. The statistically significant McNemar’s test result (p < 0.001) confirms that the diagnostic discrepancies observed between routine microscopy and LAMP were not due to random variation (Table 3). This finding underscores the limitations of routine microscopy under real-world conditions and highlights the need for strengthened quality assurance systems and complementary diagnostic approaches in malaria-endemic settings.

A significant proportion of misdiagnosis was observed, with 30% of cases being discordant between microscopy and LAMP. This misdiagnosis rate 30% (95% CI: 25.4–34.8%), including false negatives (18.8%), false positives (3.6%), and species misidentification (7.6%) (Table 5).

Table 5.

Frequencies of overall misdiagnosed malaria suspected cases by microscopy using LAMP test as the test reference, Northwest, Ethiopia, 2024

	Concordance(in both parasite detection & species identification)		Microscopy misdiagnosis: 30% (115/384) (95% CI: 25.4–34.8%)
						Misidentification of species 7.6%(29/384)
	✓ Negative = 196 ✓ Positive = 73 (PV = 40, PF = 31, Mixed = 2)	False negative (FN)		False positive (FP)
		✓ PV reported as negative(n = 23) ✓ PF as negative(n = 37) ✓ Po/Pm as negative(n = 4) ✓ Mixed reported as negative (n = 8)		✓ Negative as PV (n = 9) ✓ Negative as PF (n = 5)	✓ mixed reported as PF(n = 5) ✓ mixed infection as PV (n = 4) ✓ PV as PF (n = 5) ✓ PF as PV (n = 11) ✓ Po/Pm as PV (n = 3) ✓ PV as mixed (n = 1)
Total	70%(269/384)	18.8%(72/384) (95% CI: 15–23%)		3.6%(14/384), (95% CI: 2–6%)	7.6%(29/384) (95% CI: 5.1–11%)

n = number of blood samples examined, PF= Plasmodium falciparum, PV= Plasmodium vivax, Mixed=infected with both PF and PV

-Misdetection indicates the failure to detect the presence or absence of parasites (i.e., false negatives or false positives)

-Whereas misidentification refers to incorrect classification of the Plasmodium species after a positive result

The overall misdiagnosis rate of 30% in the study settings suggests that nearly one-third of the disease cases were inaccurately diagnosed, which has profound implications for individual patient care and public health efforts. This high level of misdiagnosis indicates a compromised diagnostic quality in real health settings, highlighting a substantial, yet often unnoticed, barrier to the success of ongoing disease elimination campaigns. Misdiagnosis contributes to the continual transmission of the disease, which undermines efforts to control and eventually eliminate the disease in the study area. The overall prevalence of misdiagnosed cases of this study align to studies conducted in Oromia Region (26%) [41] and Kenya (28%) [42]. However, it is higher than studies conducted in West Amhara region, Northwest Ethiopia, Nigeria and South Africa reported misdiagnosis of 8.12%, 17.2%, 5.5% and 13.8% respectively [17, 43–45].

In the current study, a substantial majority represented the false negative rate (18.8%), indicating malaria cases missed by microscopy but detected by LAMP. This indicates a high likelihood of misdiagnosing true infections or underreporting of actual cases. The national malaria guideline recommends a minimum of 100 fields should be observed in the thick smear before reporting negative results. However, in the study area all laboratory professionals responded that they frequently observe less than 100 fields before reporting negative slides due to high workload this may lead to false negative result [45, 46]. Other factors contributing for false negative results were lack of training, failure to follow standard operating procedures (SOP) [45].In this study 88.9% of health professionals did not take in service training on malaria microscopy and 75% of health centers did not follow SOP therefore, this all may be leads to false negative result.

In this study, the false negativity rate 18.8% (95% CI: 15–23%) (Table 5) matched results from Ethiopia, North-West Ethiopia, Equatorial Guinea, Thailand, Tanzania, and Egypt, with false negative rates of 16.8%, 16.3%, 19.4%, 16%, 18%, and 20%, respectively [47–52]. However, our findings were higher than those from North Gondar (13.1%) [53], the West Amhara region (1.3%) [45], Ethiopia (11.5%) [54], Oromia Región (7.17%) [41], Aribaminch (2.8%) [55], and Nigeria (7.5%) [56]. It was lower than a study in Ethiopia with a rate of 42.7% [47]. False-negative results can have an impact on malaria control and elimination because they prolong parasite transmission when untreated. Additionally, it can lead to delayed treatment, exposure to unnecessary treatment with other drugs, and increased morbidity and mortality. Moreover, false negatives can significantly undermine both clinical confidence in laboratory results and the credibility of the health services within the community [57].

From the total of reported slides, our findings show that the false positivity rate (microscopy identified malaria where LAMP did not) was 3.6% (95% CI: 2–6%) (Table 2) which was similar to the study conducted in Bahir Dar city administration (2.7%) [58], in Addis Ababa (5.08%) [59], Oromia region (4.05) [41], in Southern Ethiopia (2%) [46], in Ethiopia (2.9%) [47], and in Canada (2.0%) [60]. This finding was higher than the finding of a study conducted in Ethiopia where the false positive rate was 0.8% [54], and 0.7% in Northwest Ethiopia [49]. However, it is lower than 9.5% reported in Oromia Region [41], 24.4% reported in Western Oromia [61], 6.9% reported in Hawassa town [24], 12% reported in Hawassa, Ethiopia [62], 9.9% reported in Ethiopia [47],10% reported in Tigray [63],19% reported in Democratic Republic of Congo [64], and 38.5% in Nigeria [56].These false positive results might be due to misidentifying artifacts as parasites in smears because of poor reagent quality, incorrect staining procedures, failure to scan the recommended field for low parasite concentrations, or lack of training [41]. False Positive results often, lead to other diseases to be overlooked, or not treated timely manner, contributing to significant morbidity and mortality due to non-malaria illness. False positive results also lead to misuse of antimalarial drugs, exposure of parasites to sub-therapeutic blood levels of the drugs and development of resistance, increased cost to health services, and patient dissatisfaction [57]. Therefore, improved training and higher-quality of smear preparation and staining are required to reduce false positive readings [21].In the current study, there were significant challenges in species identification with the overall species misidentification was 7.6% (95% CI: 5.1–11%). Specifically, P. vivax was misidentified as P. falciparum in 1.3% (5/384) of cases, while P. falciparum was misidentified as P. vivax in 2.9% (11/384) of cases microscopically. Many of these errors were may be due to less-experienced field readers, specific diagnosis often based on only the thick smear, and variability in smear preparation and stain quality [65]. The gold standard for parasite detection is thick and thin blood smears, stained with Giemsa [66]. However, all of the technician in the study area used thick smear only this may lead to incorrect reporting of P. falciparum, P. vivax and mixed infection [67]. Additionally, 75% of health centers in the study area did not use buffer for preparing the Giemsa staining solution; instead, they used tap water. This may contribute to the high incidence of species mismatch and the low sensitivity of microscopy [46]. In this study, Giemsa microscopy identified only 0.5% (2/384) mixed infections of P.falciparum and P.vivax correctly. However, an additional 4.4% (17/384) cases were identified as mixed infections by LAMP but reported as either negative or mono infection by microscopy. Of the 17(4.4%) participants who had coinfection infections with P.vivax and P.falciparum with LAMP, eight mixed infections were reported as negative, 4 cases were reported as P. vivax mono-infection and 5 cases as P. falciparum mono-infection by microscopy. The reason why microscopy failed to detect mixed infections may be due to the presence of higher numbers of parasites of one species relative to the other [68]. Moreover, in the study area as we observed health center laboratories had high patient flows, the workload was not proportional to the staff available, and this lead to insufficient time for examining blood films. This might be one of the reasons for the low sensitivity of microscopy in the detection of mixed infections [46]. An accurate estimate of malaria parasite coinfection prevalence is essential for the successful implementation of malaria control and elimination programs [69]. If P. falciparum coinfection suppresses P. vivax parasitemia, effective management of P. falciparum infection in an area may trigger P. vivax transmission, which is more challenging to control [70]. If mixed infection is misdiagnosed as P. vivax single infection, treatment of P.vivax increases P.falciparum parasitaemia. Mixed infections aggravate the emergence of anti-malarial drug resistance [71].Therefore, accurate diagnosis of malaria parasites in mixed infections, followed by prompt treatment, is crucial to preventing relapses or recrudescence and associated severe complications. Our findings showed that microscopy did not identify any cases of other less common Plasmodium species infections (Po/Pm). Out of 7 (1.8%) Pan positive LAMP results, three cases were reported as negative and four cases were reported as P. vivax by microscopy. These cases, which were positive for Pan LAMP (but negative for Pf/Pv LAMP), might be other Plasmodium species, particularly P. ovale infection, because it was reported before in North Gondar, Ethiopia [53]. Accurate species identification, especially between P.vivax and P.ovale, has long been a bottleneck in microscopy [72]. Rely on microscopy for identification of parasite species, it may be argued that confusion between and P.vivax may lead to a degree of misdiagnosis [73]. Therefore, Pan LAMP positive samples need further analysis with specific molecular techniques or sequencing to classify them under specific species to know the epidemiology in the study area.

Limitation of study

This study provides valuable contributions by evaluating the quality of malaria diagnosis in real-world healthcare settings, offering critical insights into the current state of diagnostic services and their implications for malaria control efforts.

However, several limitations must be acknowledged when interpreting the findings. For instance, the study was unable to identify specific factors contributing to routine microscopy misdiagnosis due to the inclusion of a limited number of health centers and a restricted number of professionals, primarily because of resource constraints. Additionally, the lack of primers for all Plasmodium species meant that pan-LAMP positive results, which were negative for species-specific LAMP (Pf/Pv LAMP), could not be fully characterized.

Furthermore, the absence of parasitic density assessment hindered the interpretation of submicroscopic results, and the study did not identify which stages of Plasmodium species were overlooked by routine blood film microscopy. Despite these limitations, the findings underscore the need for further research to address these gaps and enhance the accuracy and reliability of malaria diagnostics. The study’s strengths lie in its real-world applicability and its contribution to understanding the challenges in malaria diagnosis, which are crucial for improving diagnostic services and malaria control strategies.

Conclusions and recommendations

This study revealed a significant prevalence of malaria misdiagnosis in routine health center settings, characterized by high rates of false-negative results, false positives, and species misidentification. These findings highlight the urgent need for improved real-world health center diagnostic practices in malaria-endemic regions. While molecular techniques such as the LAMP assay demonstrate superior sensitivity and specificity, their implementation in resource-limited settings remains constrained by cost and technical requirements.

Therefore, immediate efforts should focus on strengthening routine microscopy through enhanced training, regular quality control, and strict adherence to standardized operating procedures (SOPs) for smear preparation, slide examination, result recording, and storage. Additionally, governments, national and regional laboratories, and health bureaus must collaborate to establish robust laboratory quality assurance programs, secure funding for rigorous quality control, and create regional networks of laboratory supervisors to ensure consistent diagnostic accuracy.

Future research should prioritize multicenter studies with larger sample sizes to identify specific factors contributing to routine microscopy misdiagnosis, thereby informing targeted interventions to improve malaria diagnosis and reduce diagnostic errors.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations and Acronyms

ACT

Artemisinin-based combination therapy

APHI

Amhara Public Health Institute

CI

Confidence Interval

DNA

Deoxyribose Nucleic Acid

LAMP

Loop Mediated Isothermal Amplification

NPV

Negative Predictive Values

PCR

Polymerase Chain Reaction

PPV

Positive Predictive Values

WHO

World Health Organization

SPSS

Statistical Package for Social Sciences

Author contributions

M.T.A.: made substantial contribution to conceptualization, writing the original document, formal analysis, design, data curation, collection, and Interpretation. M.T.A., T.E., W.L., and A.S.: served in formal analysis, data curation, and review. M.T.A., B.T., A.A., D.R. P., and M.Y.; Participated in Molecular investigation and analysis. M.T.A., M.W., S.G., A.A., A.A., M.T., B.M., and A.D.: participated in the data collection. Finally all the authors were read and approved the final manuscript.

Funding

This study was fully granted by University of Gondar.

This fund was provided only for covering and supports us in the expense of the thesis work. The funding body has no any other role regarding to the study design, date collection, analysis, interpretation, and manuscript write up.

Data availability

The data supporting the findings of this study are not deposited into the publicly available repository. The data used in this study are available from the corresponding author and will be accessible upon reasonable request.

Declarations

Ethics approval and consent to participate

Ethical approval for this study was obtained from the Institutional Review Board (IRB) of the University of Gondar, College of Medicine and Health Sciences. Permission letters were also obtained from the Amhara Public Health Institute and the respective health centers. Written informed consent was obtained from all study participants or their legal guardians before sample collection. The study was conducted in accordance with the Declaration of Helsinki. This study was conducted after Ethical Approval was obtained from research and Ethics committee of School of Biomedical and Laboratory Sciences, College of Medicine and Health Sciences, University of Gondar. Permission letters were also obtained from the Amhara Public Health Institute and the respective health centers. Moreover, letter of support was submitted to the Addis Zemen health center, Teda health center, Yifag health center and, Woreta health center. Additionally, after explaining the purpose, benefits, and the possible risks of the study, written informed consent from the age of 16 and above and/or assent from those less than 16 years old study participants along with written informed consent from their respective parents/ caregiver/guardians was obtained. And also, written informed consent from illiterate study participants was obtained from their respective parents/guardians. The data was kept in codes instead of personal identifiers, and the confidentiality of all participants was maintained throughout the study. All microscopy-confirmed malaria-positive patients were treated in accordance with the national malaria treatment guidelines in the health centers. Cases of microscopy misdiagnosis were traced back to the respective health centers based on the patients’ residential addresses. The study was carried out according to the declaration of Helsinki.

Consent for publication

Not applicable.

Patients or the public involvement

Patients or the public were not involved in the design, or conduct, or reporting, or dissemination plans of our research.

Competing interests

The authors declare no competing interests.