A case of an asymptomatic Plasmodium falciparum infection followed by a symptomatic Plasmodium ovale infection in a soldier deployed to South Sudan

Abstract

Background

Asymptomatic malaria poses a significant challenge to malaria eradication efforts and delays global elimination strategies. Mixed infections are also a major concern, as they frequently relapse, increase the risk of severe malaria, require more accurate diagnosis for appropriate treatment, and contribute to the development of drug resistance.

Case presentation

A 25-year-old soldier was diagnosed with malaria following deployment in South Sudan. A comprehensive survey identified an asymptomatic Plasmodium falciparum infection, confirmed by peripheral blood smear and polymerase chain reaction (PCR). Despite being discharged after treatment, the patient developed fever and other symptoms one month later. Subsequent laboratory tests confirmed Plasmodium ovale infection based on peripheral blood smears and PCR.

Conclusion

This case underscores the importance of molecular detection for surveillance and vigilant follow-up in malaria management, particularly among patients with a history of deployment in endemic regions. The detection of P. ovale after treatment for P. falciparum highlights the need for increased awareness and testing for mixed infections to ensure effective malaria control strategies.

Background

Asymptomatic malaria infection poses risks to human health and malaria elimination efforts [1]. In the Central African Republic, 51.2% of children and 12.2% of adults are asymptomatic [2]. Moreover, patients with mixed infections are at a higher risk of relapse and progression to more severe disease [3, 4]. Furthermore, accurate diagnosis is crucial for providing appropriate and effective therapies [4, 5]. The global prevalence of severe mixed infections is approximately 9% [6]. Traditional methods such as microscopy are less sensitive than molecular methods for diagnosing mixed infections [5, 7].

South Sudan is one of the 22 countries with the highest malaria burden in the world. Between 2010 and 2020, Plasmodium falciparum was the most prevalent species, followed by Plasmodium vivax, and other Plasmodium species [8]. During this period, no mixed infections were reported [8]. Survey data from 1978, before the separation of South Sudan from Sudan, showed that Plasmodium ovale was very rarely found in South Sudan [9]. Plasmodium ovale is an understudied malaria species prevalent throughout sub-Saharan Africa. In a study of 18,149 adults tested, the researchers detected 143 cases of prevalent P. ovale infections, yielding an estimated prevalence of 0.8% [10].

Here, a case of malaria was identified in a soldier following deployment in South Sudan. A comprehensive survey was conducted among soldiers using peripheral blood (PB) smears and PCR. During the survey, three patients with asymptomatic P. falciparum infection were identified. Notably, one of these patients, who had previously received treatment for an asymptomatic P. falciparum infection was referred to the Infectious Disease Department. One month later, the patient presented with fever, and a symptomatic P. ovale infection was confirmed.

Case presentation

After confirmed malaria cases were reported among soldiers following their deployment to South Sudan, a comprehensive survey was conducted at Chosun University Hospital on March 23, 2023, to assess the malaria infection status among the deployed troops.

During the survey, a 25-year-old soldier was  diagnosed with asymptomatic malaria based on a positive PB smear. An initial examination revealed an asymptomatic P. falciparum infection, with the PB smear showing a few infected red blood cells (RBCs: 1/100 high-power field [HPF]) (Fig. 1A). Laboratory findings included white blood cells (WBC): 6.66 × 10³/µL, haemoglobin (Hgb): 13.1 g/dL, platelets (PLT): 313 × 10³/µL, C-reactive protein (CRP): 0.30 mg/dL, aspartate aminotransferase (AST): 24 U/L, alanine aminotransferase (ALT): 26 U/L, and glucose: 57 mg/dL.

Fig. 1

A. Peripheral blood smear of a 25-year-old soldier on March 23, 2023, during the first hospitalization. The smear shows low parasitemia with one infected red blood cell per 100 high-power fields, along with a single “banana-shaped” gametocyte. B. Peripheral blood smear from the same soldier on May 8, 2023, during the second hospitalization. The smear reveals numerous non-P. falciparum gametocytes, indicating a symptomatic P. ovale infection. C. Phylogenetic tree based on the partial 18S rRNA sequence (875 bp) of Plasmodium species. The tree was constructed using the CLUSTAL X software (http://www.clustal.org/clustal2/) with the neighbor-joining method and 1000 bootstrap replicates.

The patient had a history of military service in malaria-endemic regions (Incheon, Gyeonggi-do, and northern Gangwon, South Korea) from June to August 2021, and was deployed to South Sudan from April 7 to December 9, 2022. He stated that he had taken mefloquine (Lariam®) 250 mg (228 mg base), one tablet orally once a week, from March 2022 until January 2023. He began taking it one week before deployment, continued throughout the entire deployment period, and took it for four weeks after returning home.

Upon confirmation of the initial findings, the patient was hospitalized from April 3 to April 6, 2023, for further evaluation. Follow-up PB smears showed no abnormalities. Rapid diagnostic test (RDT) using immunochromatography was performed on blood samples using the Standard Q Malaria Pf/Pan Ag RDT (SD Biosensor, Inc., Suwon, South Korea). This kit was designed to detect histidine-rich protein 2 (HRP-2) specific to P. falciparum, and Plasmodium lactate dehydrogenase (pLDH), specific to P. falciparum, P. vivax, P. ovale, Plasmodium malariae and Plasmodium knowlesi. Laboratory tests confirmed a positive rapid antigen test for P. falciparum and  a confirmatory malaria polymerase chain reaction (PCR) test (Table 1). Blood tests during this admission revealed WBC: 7.54 × 10³/µL, RBC: 4.57 × 10⁶/µL, Hgb: 13.8 g/dL, PLT: 262 × 10³/µL, AST: 25 U/L, ALT: 22 U/L, creatinine kinase (CK): 743 U/L, CRP: 0.33 mg/dL, and glucose: 42 mg/dL.

Table 1 Laboratory test results of a 25-year-old soldier diagnosed with malaria

The patient exhibited no significant clinical symptoms and was treated with artesunate (180 mg) every 12 h (at 0, 12, and 24) for two days, from April 4 to April 5, 2023. After the treatment, the patient remained asymptomatic and was discharged on April 6, 2023. One week after discharge, on April 12, 2023, a PB smear showed no abnormalities; CK levels decreased from 743 to 238 U/L, and CRP levels were 0.08 mg/dL. Follow-up assessments over the next three weeks in the outpatient clinic revealed no significant issues.

However, on May 8, 2023, the patient developed a fever of up to 39 °C, accompanied by chills, dizziness, myalgia, and diarrhea. A PB smear revealed numerous gametocytes (infected RBCs: 1/1 HPF) (Fig. 1B), and qPCR confirmed positive P. ovale infection (Ct: 21.89). The patient was hospitalized for evaluation on May 10, 2023. Upon admission, a PB smear revealed numerous gametocytes (infected RBCs: 3/1 HPF). Blood tests indicated WBC: 7.64 × 10³/µL, Hgb: 14.0 g/dL, PLT: 48 × 10³/µL, AST: 31 U/L, ALT: 19 U/L, total bilirubin: 2.93 mg/dL, LDH: 586 U/L, CK: 34 U/L, and CRP: 16.1 mg/dL. The patient was initially treated with atovaquone and proguanil for two days but continued to experience high fever, chills, myalgia, and nausea/vomiting. Consequently, treatment with primaquine and artesunate was initiated on May 12, 2023, after which the patient’s fever subsided. Primaquine was administered orally at a dose of 15 mg once daily for 14 consecutive days, from May 11 to May 23, 2023.

On May 12, 2023, qPCR confirmed the presence of only P. ovale. By May 14, the patient was free of fever and chills, with CRP levels at 3.39 mg/dL and PLT recovering to 11 × 10³/µL. However, on May 15, the patient experienced a recurrence of fever (38.7 °C), with the PB smear showing a few gametocytes (infected RBCs: 1/10 HPF). By May 16, the PB smear indicated infected RBCs at 1/40 HPF, with the PLT recovering to 258 × 10³/µL, and the patient’s condition improved, leading to discharge on May 17, 2023.

Follow-up PB smear tests conducted one month later (June 7, 2023) and two months (July 3, 2023) later showed no significant abnormalities.

The PCR was performed using pathogen-specific primers. Real-time PCR using a LightCycler^® TaqMan^® Master (Roche, Switzerland) targeted the msp1 genes of P. falciparum and P. ovale. Meanwhile, conventional PCR (C-PCR) was designed to amplify all Plasmodium species by targeting the 18S rRNA gene. C-PCR was performed using AccuPower^® PCR PreMix (Bioneer, South Korea), and PCR was performed at 94 °C for 10 min, [94 °C for 30 s, 55 °C for 30 s, 72 °C for 45 s] for 35 cycles, and 72 °C for 7 min. The PCR products were electrophoresed on a 1.5% agarose gel (Seakem LE agarose). For C-PCR positivity, the PCR products were purified using the QIAquick PCR Purification Kit, followed by direct sequencing to analyze the nucleotide sequences. Subsequently, NCBI BLAST searches were performed to identify the pathogens. A phylogenetic tree was constructed from the sequences of the positive samples using CLUSTAL X and DNASTAR software). According to NCBI BLASTN results, the sample from April 3 matched P. falciparum 3D7 (XR_002273101) with 99.78% identity (891/893), whereas the sample from May 8 matched P. ovale (KF219559, KF219560, KF696363, and KF696359) with 99.77% identity (866/868) (Fig. 1C). In the 18S rRNA phylogenetic tree, each sequence clustered with P. falciparum and P. ovale.

Discussion

Globally, the high mortality and morbidity rates associated with malaria infections are primarily caused by P. falciparum [11]. This is due to its virulence factors, which enable cyto-adhesion, allowing infected erythrocytes to adhere to endothelial cells [12]. However, P. ovale generally causes milder disease with lower mortality but can lead to relapse several months or even years after the initial infection due to its ability to form dormant liver stages known as hypnozoites [13,14,15]. Systemic parasitic and bacterial infections-but not viral infections- can activate P. vivax hypnozoites [16]. A study in Papua New Guinea demonstrated that relapses contribute significantly to P. vivax and P. ovale infections, with approximately 80% of P. vivax and 60% of P. ovale infections attributed to relapses [17].

Of the 5,261 identified studies conducted between 1996 and 2016, data on triple mixed infections were available for 35 studies involving 601 patients from 22 countries. The overall pooled prevalence of triple-mixed infections was 4% (95% CI: 3–5%; I² = 92.5%). The pooled proportion of triple-mixed infections compared to double-mixed infections was 12% (95% CI: 9–18; I² = 91%). Most of the included studies (29 of 35, or 82.9%) reported a lower proportion of triple-mixed infections than double-mixed infections. Overall, 12,023 PCR-confirmed malaria cases and 3,059 double mixed infections were reported, including 188 cases (1.56%) of P. falciparum and P. ovale across 21 studies [6].

Based on data from the Korea National Infectious Disease Surveillance System, 601 imported malaria cases were recorded in the Republic of Korea between 2009 and 2018. Overall, 76.5% of these cases involved Korean citizens returning from endemic regions, particularly from countries in Asia and Africa, where P. falciparum (55.7%) and P. vivax (30.3%) were the predominant species [18]. Several case reports on imported malaria have been published, including a case of P. ovale imported from Ghana, West Africa, in 2004 and three cases of mixed infections, including P. falciparum/P. ovale from Cameroon and the Democratic Republic of the Congo in 2016, P. falciparum/P. vivax from Angola in 2012, and P. falciparum/P. vivax from Bangladesh in 2001 [19,20,21,22].

In this patient, the P. falciparum infection may have reactivated the P. ovale infection, highlighting the need for further research on this possibility.

According to Data from India and Ethiopia, traditional methods such as microscopy have lower sensitivity than PCR in diagnosing mixed infections [5]. This suggests that cryptic mixed infections are more commonly detected through PCR testing. In this patient, only P. falciparum PCR was positive during the asymptomatic phase, whereas P. ovale was confirmed only after symptoms developed, suggesting that hyponozoites of P. ovale may be difficult to detect by PCR. Nevertheless, PCR was positive for P. falciparum during the asymptomatic phase, and the patient received treatment for P. falciparum. It is crucial to consider the possibility of a mixed infection and to use primaquine for radical cure and preventing relapse from hypnozoites in the liver [23].

Long-term mefloquine chemoprophylaxis showed a high level of compliance and was well tolerated, as evidenced by questionnaire results and medical history [24]. Additionally, this was supported by blood concentration measurements, which indicated sufficient levels for prophylactic therapy with minimal side effects. However, mefloquine is not entirely effective, as cases of malaria infection caused by mefloquine-resistant Plasmodium species have been reported. The compliance rate for mefloquine was significantly higher than that for doxycycline [24]. This is also reflected in the current case, in which the patient adhered to mefloquine prophylaxis without experiencing any side effects. However, drug concentration was not measured, and resistance testing was not conducted. Therefore, infection in this case may have been due to mefloquine resistance, necessitating further research to explore this possibility. A case of P. ovale infection was also reported in a young soldier from Indonesia who received 250 mg/week mefloquine prophylaxis for one year [25]. The patient developed fever and chills two weeks after returning from a mission in the Congo, Central West Africa. It is suspected that the dormant stage of P. ovale, quinoline drug resistance, and the efficacy of mefloquine contributed to this phenomenon [25].

In conclusion, this is a case of mixed malaria infection with P. falciparum and P. ovale originating in South Sudan. This case highlights the challenges in diagnosing and managing asymptomatic malarial infections, particularly mixed infections. It is crucial to conduct surveillance for malaria infections and monitor resistance to prophylactic drugs in high-risk groups, such as individuals returning from malaria-endemic areas. Furthermore, the detection of P. ovale after treatment for P. falciparum underscores the importance of vigilant follow-up in malaria management.