Plasmodium spp. mixed infection leading to severe malaria: a systematic review and meta-analysis

Mixed Plasmodium malaria infections can lead to severe malaria. This systematic review and meta-analysis aimed to explore the prevalence of severe mixed Plasmodium malaria infection and to compare it with the prevalence of severe P. falciparum malaria mono-infection across the included studies. Original English-language research articles from PubMed, Scopus, and ISI Web of Science were identified and screened. Articles reporting the number of mixed infections and the number of severe mixed infections were used to determine the main outcome of this study, while the number of P. falciparum infections and the number of severe P. falciparum infections were used to determine the secondary outcome of this study. For the main outcome, the pooled prevalence and 95% confidence interval (CI) of severe mixed infections was analysed using STATA software version 15.0 (Stata Corp, College Station, TX, USA). For the secondary outcome, the rate of severe mixed infections compared to severe P. falciparum infections was analysed using the meta-analysis approach, and summary odds ratios (ORs) and 95% CIs were calculated. Random-effects models were used to produce the summary ORs. The Mantel–Haenszel method and calculated I2 were also reported to test whether there was heterogeneity among the included studies. Publication bias was also assessed using funnel plots. The meta-analysis of secondary outcomes was conducted using Review Manager 5.3 software (Cochrane Community). A total of 894,561 malaria patients were reported in all 16 included studies. Overall, a pooled analysis showed that 9% (2,006/35,768, 95% CI 7.0–12.0%) of patients with mixed Plasmodium infection had severe mixed infection. A meta-analysis of 14 studies demonstrated that patients with mixed Plasmodium infection (1,999/35,755) and patients with P. falciparum malaria (9,249/294,397) had an equal risk of developing severe malaria (OR 0.93, 95% CI 0.59–1.44). Both mixed infection and P. falciparum mono-infection showed a similar trend of complications in which severe anaemia, pulmonary failure, and renal impairment were the three most common complications found. However, patients with mixed infection had a higher proportion of severe anaemia and pulmonary complications than those with P. falciparum infection. Moreover, patients with mixed infection had a higher proportion of multiple organ failure than those with P. falciparum mono-infection. Mixed Plasmodium spp. infections were common but often unrecognized or underestimated, leading to severe complications among these malaria patients. Therefore, in routine clinical laboratories, using an accurate combination of diagnostic procedures to identify suspected patients with mixed infections is crucial for therapeutic decisions, prompt treatment, and effective patient management.

Definition of severe malaria. The major complications of severe mixed malaria were considered to be the same as those defined for P. falciparum by the World Health Organization (WHO) and included respiratory distress or acidosis (a base deficit of > 8 meq/L, a plasma bicarbonate of < 15 mM or venous plasma lactate > 5 mM), pulmonary oedema (radiologically confirmed, or oxygen saturation < 92% on room air with a respiratory rate > 30/min), impaired consciousness (a Glasgow Coma Score < 11 in adults or a Blantyre coma score < 3 in children), convulsions (more than two episodes within 24 h), prostration (generalized weakness so that the person is unable to sit, stand or walk without assistance), hypotension/shock (systolic blood pressure < 70 mmHg in children or < 80 mmHg in adults), jaundice [plasma bilirubin > 50 µM/L (3 mg/dL)], severe anaemia (A haemoglobin concentration < 5 g/dL), bleeding/Disseminated Intravascular Coagulation (DIC) (recurrent or prolonged bleeding from the nose, gums, or venepuncture sites; haematemesis or melaena), hyperparasitemia (P. falciparum parasitaemia > 10%), and hypoglycaemia [blood or plasma glucose < 2.2 mM (< 40 mg/dL)] 12 . Cerebral malaria, one criterion of severe P. falciparum malaria in the former version of the WHO definition, was assigned to the group "impaired consciousness" and described as "impaired consciousness/cerebral malaria" for further analysis and demonstration in the results section.
inclusion and exclusion criteria. Original research articles published in the English language were included in the current analysis if they met the following criteria: (1) malaria positivity confirmed by any combination of rapid diagnostic tests (RDTs), microscopy, or polymerase chain reaction (PCR); (2) enrolled both uncomplicated and complicated malaria; (3) the numbers of mixed infections and severe mixed infections were reported, and (4) all complications in the patients with severe mixed infections were reported. Case reports, animal studies, experimental studies, clinical trials, book or book chapters, letters to the editor, editorials, reviews or systematic reviews, conference papers, short surveys, and studies of co-infection of Plasmodium with other agents were excluded from the present study.
Data extraction. For all articles included in the analysis, the following information was extracted: name of the authors, year of publication, country of the participants, duration of the study, the total number of malaria patients, number of severe mixed infections, number of mixed infections, number of severe P. falciparum infections, number of P. falciparum infections, complications of severe mixed infections, and complications of P. falciparum infections. The number of mixed infections and the number of severe mixed infections was used to determine the main outcome of this study, while the number of P. falciparum infections and the number of severe P. falciparum infections were used to determine the secondary outcome of this study. For  www.nature.com/scientificreports/ lysed using the meta-analysis approach and summary odds ratios (ORs) and 95% CI were calculated. Randomeffects models were used to produce summary ORs as described previously 13 . The Mantel-Haenszel method and the calculated I 2 were also reported to determine whether there was heterogeneity among the included studies. Publication bias was also assessed using funnel plots and Egger's test as described elsewhere 14 . The meta-analysis of the secondary outcomes was conducted using Review Manager 5.3 software (Cochrane Community). www.nature.com/scientificreports/ 9/16) were descriptive studies or cross-sectional observational designs [15][16][17]20,23,24,26,27,30 . Six studies (37.5%, 6/16) were prospective studies or prospective cohort studies 18,19,21,22,28,29 . One study was a retrospective observational study 25 15,16,19 . Three studies demonstrated that patients with a mixed infection had a significantly higher risk of developing severe malaria than patients with a P. falciparum mono-infection 21,25,28 .

Results
In a subgroup analysis comparing the results from India and non-India areas in 13 studies, the pooled analysis showed that patients with mixed Plasmodium spp. infection and patients with P. falciparum mono-infection had an equal risk of developing severe malaria (OR 0.91, 95% CI 0.58-1.42) (Fig. 4). There was a subgroup difference (P value = 0.02, I 2 = 80.4%) in this subgroup analysis, indicating that the study area (India and non-India) was one source of heterogeneity in the present study. Further stratification by diagnostic technique (microscopy alone and microscopy with other techniques) also showed that patients with mixed infection had an equal risk of developing severe malaria compared to those with P. falciparum mono-infection (OR 0.72, 95% CI 0.45-1.15) (Fig. 5). Once again, there was a subgroup difference (P value = 0.02, I 2 = 82%) in this subgroup analysis, indicating that diagnostic technique (microscopy alone and microscopy with other techniques) was also a source of heterogeneity in the present study.  Fig. 6. Both mixed infection and P. falciparum mono-infection showed similar trends of severe complications by severe anaemia, pulmonary failure, and renal impairment, which were the three most common complications found in this study. Patients with mixed infection had a higher proportion of severe anaemia (65.8% vs 57.6%) and pulmonary complications (20.9% vs 14.6%) than those with P. falciparum mono-infection. Patients with mixed infection (13.1%) had a higher proportion of multiple organ failure than those with P. falciparum mono-infection (3.95%). The publication bias among studies was assessed by funnel plots (Fig. 7) and Egger's test for small-study effects. The result of Egger's test indicated that no publication bias was found in the present study (P value = 0.857, t = 0.18, 95% CI = − 2.57-3.04).

Discussion
The present study showed a high prevalence of severe mixed Plasmodium infection across the included studies (9%), demonstrating for the first time, to our knowledge, that mixed infection can cause a high rate of severe malaria. Although the mixed malaria prevalence was predominantly due to P. falciparum/P. vivax infection, the prevalence of non-P. falciparum/P. vivax mixed infections, such as P. falciparum/P. malariae mixed infections was also reported in our study, but they were not shown in their literature 24 . This high prevalence of severe mixed malaria partly explains why malaria remains one of the leading causes of morbidity and mortality worldwide despite available interventions, public health control, and management employed. These findings suggested that there is a need for continued detection and monitoring of mixed infection using species-specific RDTs in combination with routine microscopy, or even using PCR as soon as possible, to move towards malaria elimination and to protect against severe malaria resulting in death. A previous study indicated that the severity of mixed P. falciparum/P. vivax infection occurred when P. vivax superinfection occurred over an existing P. falciparum infection. However, P. falciparum superinfection over an existing P. vivax infection results in a lower risk of severe malaria 28  www.nature.com/scientificreports/ a significantly lower risk of developing severe malaria than patients with P. falciparum mono-infection 15,16,19 . These results were consistent with results from a previous study conducted in Thailand, which observed that severe malaria was less common among patients with mixed infections compared to those with P. falciparum mono-infection 31 . However, three other included studies demonstrated that patients with mixed infections had a significantly higher risk of developing severe malaria than patients with P. falciparum mono-infections 21,25,28 . This could be because mixed infections are often unrecognized or underestimated by microscopists 32,33 , leading to treatment failure, anti-malarial drug resistance, and the development of severe P. falciparum malaria 7 . Previous studies also demonstrated that age was associated with mixed infection 34,35 . They found that children under two years of age had a lower frequency of mixed Plasmodium malaria compared to those at an older age. They suggested that maternal antibodies could be the source of mixed infection malaria protection 34 . The major complications of severe malaria defined by the World Health Organization (WHO) included respiratory distress, acidosis, pulmonary oedema, death, impaired consciousness, convulsions, prostration, hypotension/shock, jaundice, severe anaemia, bleeding/DIC, hyperparasitemia, and hypoglycaemia 12 . The present study demonstrated that 9% of severe malaria was caused by mixed infection, whereas a previous study showed that severe malaria accounts for approximately 5% of total malaria-infected patients 36  www.nature.com/scientificreports/ www.nature.com/scientificreports/ severe mixed malaria in the present study was 0.9%, which was consistent with the case fatality rate in previously reported P. falciparum malaria mono-infection (0.6-3.8%) 4 . The present study also indicated that both mixed infection and P. falciparum mono-infection showed similar trends of complications in which severe anaemia, pulmonary failure, and renal impairment were the three most common complications. However, patients with mixed infection had a higher proportion of severe anaemia and pulmonary complications than those with P. falciparum mono-infection. Moreover, patients with mixed infection had a higher proportion of multiple organ failure than those with P. falciparum mono-infection. A study in Thailand indicated that mixed P. falciparum/P. vivax infection could reduce the risk of severe anaemia among patients with falciparum malaria by cross-species immunity 37 . In Southeast Asia, other possible reasons behind the reduction of the risk for severe anaemia among patients with malaria infections were haemoglobinopathies and enzymatic deficiencies 38 . Haemoglobinopathies related to the reduced risk of malaria infections or reducing the risk of severe malaria included sickle cell traits 39 , haemoglobin C 40 , haemoglobin E 41 , and thalassemia 40 . Enzymatic deficiencies related to the reduced risk of malaria infections include glucose-6-phosphate dehydrogenase (G6PD) deficiency 42 and pyruvate kinase deficiency 43 . In addition, individuals with blood type O were less susceptible to severe malaria than individuals who were not blood type O 44 . The expression of the host RBC surface protein called Duffy antigen receptor for   www.nature.com/scientificreports/ chemokines (DARC) has been shown to protect against malaria infections 38 . Moreover, altered RBC morphologies such as Southeast Asian ovalocytosis (SAO) could reduce the risk of malaria infection or severe malaria 45,46 .
The included studies conducted in Papua New Guinea (1997Guinea ( -2004 demonstrated that mixed infection caused more severe anaemia than did the Plasmodium mono-infection alone 19 . The results of our study were also consistent with the results of studies in India 9 and Indonesia 47 that reported a high prevalence of severe anaemia among patients with mixed infections. The higher proportion of severe mixed infection than that of P. falciparum and P. vivax mono-infection was due to mixed infection having higher parasite densities 19 . The present study had limitations. First, there was a high level of heterogeneity across the included studies. Second, except for the area of the study (India and non-India) and diagnostic method, the source(s) of heterogeneity could not be explored due to the incomplete data among the included studies. Third, a limited number of studies met the criteria for inclusion because many publications included patients with severe complications and infections with etiologic agents other than malaria. Fourth, most of the included studies used microscopy for malaria detection, which might have led to missed detection of Plasmodium mixed infections. The analysis of mixed-species infections compared with P. falciparum mono-infections needs to be carefully interpreted as it is highly likely to be confounded by a proportion of undiagnosed mixed infections in the P. falciparum monoinfection groups. Fifth, a large number of additional factors related to transmission intensity, host immunity, and vectors that likely influenced the large variance seen in the mixed-Plasmodium species infections could not be taken into account because of the inherent data limitations from each study. Lastly, the present Review submits analysis of data which is relevant for the asexual blood stages of Plasmodium spp. infections resulting to severe manifestation and does not take into account hypnozoites and/or submicroscopic co-infections.
conclusion Mixed Plasmodium spp. infections are common but often unrecognized or underestimated, leading to severe complications among malaria patients. Therefore, in routine clinical laboratories, using an accurate combination of diagnostic procedures or repeat blood film examinations by microscopists to identify mixed infection in suspected patients is crucial for therapeutic decisions, prompt treatment, and effective management among those patients.

Data availability
The datasets used during the current study are available from the corresponding author based on reasonable request.