A study involving 330 participants in Ethiopia examined the prevalence and diagnosis of malaria using microscopy, CareStart RDT, and 18S nested PCR. Among the participants, 63.94% were males, with 202 symptomatic and 128 asymptomatic individuals. The 18S nested PCR detected the highest prevalence of malaria (24.55%), followed by microscopy (10.91%) and RDT (9.4%). P. vivax was the most common parasite, accounting for 63% of infections, while P. falciparum and mixed infections were less frequent. PCR showed superior sensitivity for detecting asymptomatic cases, though it had lower specificity and predictive values compared to RDT and microscopy. The study highlights the effectiveness of 18S nested PCR in identifying asymptomatic malaria cases, particularly for P. vivax.
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Sociodemographic Characteristics of Study Participants
The study included a total of 330 participants, comprising 128 asymptomatic and 202 symptomatic individuals. The objective was to detect and identify malaria parasites using three diagnostic methods: microscopy, CareStart TMpf/pan RDT, and nested polymerase chain reaction (18S nested PCR). Of the participants, 211 (63.94%) were male, and 119 (36.06%) were female.
Among the 202 symptomatic participants, 55 tested positive for malaria parasites via 18S nested PCR, with 46 males and 9 females. Similarly, 26 of the 128 asymptomatic participants tested positive, including 12 males and 14 females. Detailed prevalence data are presented in Table 2.
Prevalence of Plasmodium Species Detected by RDTs, Microscopy, and PCR
From the total study population, 81 individuals (24.55%) tested positive via 18S nested PCR. Of these, 56 (69.14%) were infected with P. vivax, 19 (23.46%) with P. falciparum, and 6 (7.41%) with mixed infections. For the CareStart TM Pf/Pv RDT, 31 (9.4%) tested positive, with 24 (77.42%) infected with P. vivax, 5 (15.63%) with P. falciparum, and 2 (6.45%) with mixed infections. Microscopy identified 36 (10.91%) positive cases, with 26 (72.22%) P. vivax infections, 8 (22.2%) P. falciparum, and 2 (5.56%) mixed infections. Full distribution details are available in Table 3.
Representative Species-Specific PCR Amplification Results
The study included PCR amplification results for Plasmodium species. Lane designations for PCR results are described in Figures 2 and 3.
Diagnosis of Symptomatic and Asymptomatic Plasmodium Infections
Among the 202 symptomatic participants at Adama Malaria Control Center, 55 (27.23%) tested positive via 18S nested PCR. Additionally, 26 (12.87%) and 28 (13.86%) tested positive via CareStart TMpf/pan RDT and microscopy, respectively. When microscopy was used as the gold standard, the CareStart TMpf/pan RDT exhibited 100% sensitivity, specificity, and PPV. The 18S nested PCR had 100% sensitivity and NPV but lower specificity (84.5%) and PPV (50.9%). Results for comparative diagnostic performance are detailed in Table 4.
For asymptomatic participants, the detection rates were 20.31% (18S nested PCR), 6.25% (microscopy), and 4.1% (CareStart TMpf/pan RDT). Detailed diagnostic performance metrics are presented in Table 5.
Diagnosis of Total Malaria Cases and Species-Specific Analysis
Among the 330 participants, 81 (24.55%) tested positive via 18S nested PCR, compared to 36 (10.91%) via microscopy and 31 (9.4%) via CareStart TMpf/pan RDT. Notably, 51 PCR-positive cases were missed by RDT, and 3 microscopy-positive cases were not detected by PCR. Species-specific analysis revealed 55 P. vivax, 20 P. falciparum, and 6 mixed infections in PCR-positive cases. Table 5 provides comprehensive diagnostic performance metrics for each method.
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