Are respiratory complications of Plasmodium vivax malaria an underestimated problem?

Respiratory complications are uncommon, but often life-threatening features of Plasmodium vivax malaria. This study aimed to estimate the prevalence and lethality associated with such complications among P. vivax malaria patients in a tertiary hospital in the Western Brazilian Amazon, and to identify variables associated with severe respiratory complications, intensive care need and death. Medical records from 2009 to 2016 were reviewed aiming to identify all patients diagnosed with P. vivax malaria and respiratory complications. Prevalence, lethality and risk factors associated with WHO defined respiratory complications, intensive care need and death were assessed. A total of 587 vivax malaria patients were hospitalized during the study period. Thirty (5.1%) developed respiratory complications. Thirteen (43.3%) developed severe respiratory complications, intensive care was required for 12 (40%) patients and 5 (16.6%) died. On admission, anaemia and thrombocytopaenia were common findings, whereas fever was unusual. Patients presented different classes of parasitaemia and six were aparasitaemic on admission. Time to respiratory complications occurred after anti-malarials administration in 18 (60%) patients and progressed very rapidly. Seventeen patients (56.7%) had comorbidities and/or concomitant conditions, which were significantly associated to higher odds of developing severe respiratory complications, need for intensive care and death (p < 0.05). Respiratory complications were shown to be associated with significant mortality in this population. Patients with comorbidities and/or concomitant conditions require special attention to avoid this potential life-threatening complication.

especially regarding the prevalence of severe syndromes and fatal disease [9], with those in lower and unstable transmission settings, as in the Americas, presenting a wider variety of organ dysfunctions [10]. Underlying conditions and comorbidities have long been perceived as having a negative impact on vivax malaria outcomes. An autopsy series conducted among confirmed vivax infected individuals revealed that 76% of them presented some type of underlying condition [6]. In that study, 41% had developed lung complications [6]. However, no clear information is available regarding the general epidemiology, risk factors and prevalence of respiratory complications in vivax malaria, especially in the Brazilian Amazon.
A recent meta-analysis showed that the prevalence of acute respiratory distress syndrome (ARDS) among vivax malaria cases ranged between 2.2% (adults) and 2.8% (children), with nearly 50% mortality, confirming the significance and associated poor prognosis of this type of complication among P. vivax malaria patients [11]. Moreover, female sex, having any comorbidity, lower body temperature, lower haemoglobin, and low oxygen saturation values (hypoxaemia) were significantly associated with mortality. However, as only studies containing respiratory complications were included, the prevalence of respiratory complications may have been overestimated. Similarly, population-based studies are rarely reported in the literature, leading to a possible publication bias. The aims of this study were to estimate the prevalence and lethality of such complications among P. vivax malaria patients in the Western Brazilian Amazon and to identify variables associated with the development of severe respiratory complications according to WHO definitions, need for intensive care (ICU) and death.

Study site and case management
The Fundação de Medicina Tropical Doutor Heitor Vieira Dourado (FMT-HVD) is a tertiary care centre in Manaus (Western Brazilian Amazon) and a reference institution for infectious diseases in the north region of the country. Patients may either directly seek care at the hospital's outpatient clinic or be referred from other local hospitals and surrounding municipalities. At hospital presentation, patients with fever, or history of fever in the preceding days, are tested for malaria by thick blood smear. Admission/arrival to the hospital may happen after microscopic diagnosis and treatment initiation at primary level of health-care. Patient data are registered in the hospital's electronical medical charts (iDoctor ® ). Once at the hospital, all patients undergo medical consultation and a 7-day post treatment initiation follow-up visit if diagnosed with malaria. Patients presenting with any severe clinical sign according to WHO severe malaria definitions (impaired consciousness, jaundice, significant bleeding, shock, respiratory distress and others) [12] are immediately referred to the emergency department and managed accordingly. Additional investigation for concomitant infections is triggered by the presence of specific manifestations and/or at medical's discretion through clinical investigation and routine blood and urine testing. Comorbidities are sought based on patient's provided information and medical chart assessment. G6PD deficiency is not tested systematically.

Malaria diagnosis and treatment
In Brazil, thick blood smears are routinely performed for malaria diagnosis and occur at different health posts in the primary level of health care. All malaria tests (positive and negative) are recorded in the national epidemiological surveillance system for malaria (SIVEP), implemented by the Brazilian National Ministry of Health in 2002. Slides are read by local microscopists and results are given following a semi-quantitative system: 1/2 + (200-300 parasites/mm 3 ); 1 + (301-500 parasites/ mm 3 ); 2 + (501-10,000 parasites/mm 3 ); 3 + (10,001-100,000 parasites/mm 3 ); and 4 + (> 100,001 parasites/ mm 3 ). All positive slides and 10% of negative slides are routinely reviewed in a reference unit by senior microscopists. In case of divergence, the reviewed reading is updated in the SIVEP system. PCR diagnosis is not routinely performed for malaria diagnosis. Treatment is only provided after a positive smear for malaria. Anti-malarial treatment for P. vivax malaria occurs following WHO recommendations and Brazilian guidelines with chloroquine (CQ, 25 mg/kg divided in 3 days) for uncomplicated cases, or parenteral artemether or artesunate in patients suspected of severe malaria or severe vomiting, followed by primaquine (PQ, 3.5 mg/kg in 7 or 14 days) [13,14].

Study design, population and data collection
A retrospective search of charts was performed using the International Classification of Diseases aiming to identify all patients diagnosed with P. vivax malaria who were hospitalized. After identification, all were completely screened for respiratory complications prior to hospital presentation, upon admission or during hospitalization. These included respiratory complaints reported by the patient in the previous days to hospital presentation or at hospital presentation or by medical examination at admission or during the hospitalization period. All clinical and laboratory information was then retrieved from the patient's medical chart using a structured questionnaire completed by two independent investigators of the study. Data regarding parasitaemia, time of diagnosis, time of treatment start and anti-malarials used were also collected if available. Fever data (axillary temperature > 37.5 °C) was also collected if available. Patients anonymity was preserved through the analysis. No restriction of age was applied.

Respiratory complications and case definition
Respiratory distress (RD) was defined as the following: respiratory rate > 30/min and oxygen saturation < 92% on room air in adults [12] or respiratory rate > 40/min (children 12 months up to 5 years) or > 50/min (children aged from 2 to 12 months) and oxygen saturation < 90% [15]. Blood gas analysis was also used to further assist diagnosis and management when available. Both vivax and falciparum malaria share the same criteria for severe disease with exclusion of parasitaemia thresholds [12]. Severe WHO definitions for severe respiratory impairment in malaria, including acidotic breathing and pulmonary oedema (PE) were used [12]. Acute respiratory distress syndrome (ARDS) following Berlin definition was also used [16]. Definitions are presented in Table 1.

Statistical analysis
Statistical analyses were performed using independent t tests, Wilcoxon Mann-Whitney or χ 2 tests to compare variables across groups stratified by three independent outcomes (development of severe respiratory impairment composed by patients presenting PE and/or ARDS; patients in need for intensive care support and those who died). Difference in proportions were tested by Chi squared test (corrected by Fisher's test if necessary). Odds ratio (OR) with its 95% confidence interval (CI) was calculated for association between variables and outcomes using logistic regression. The diagnostic performance of laboratorial parameters for the three outcomes was assessed by means of receiver operating characteristic (ROC) curves. Significance was set as p < 0.05. Analyses were performed using STATA version 12.1 (Stata Corp LP, Texas USA). The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement was used as guidelines for reporting this study (Additional file 1) [17].

Ethical considerations
This study was approved by the Ethics Review Board (ERB) of FMT-HVD, Manaus, Brazil (1943/2008) and the National Brazilian Committee of Ethics (CONEP) (343/2009). All data analysed were anonymous. Since data was obtained exclusively from the medical charts, the ERB gave a waiver of informed consent.

Severe respiratory complications
Of the 30 patients with a respiratory complication, 13 (43.3%) patients developed severe respiratory complications (PE and/or ARDS). The proportion of patients presenting comorbidities and/or concomitant conditions was significantly increased in this group (p = 0.001). Four patients died. Lung deterioration occurred after anti-malarials in nine patients (69.2%) of this group and progressed rapidly in some cases. Further results are presented in Table 3. Radiological findings (including alveolar infiltrates and diffuse patchy bilateral opacities) of three cases developing ARDS are presented Fig. 2.   Table  S4).

Intensive care and death
Twelve (40%) patients needed intensive care support. Analysis revealed that these patients presented comorbidities and/or concomitant conditions (33.3% vs 91.6%, p = 0.002) more frequently and had lower haemoglobin values at admission (10.2 ± 0.4 g/dL vs 8.5 ± 0.6 g/dL, p = 0.003). Also, creatinine and urea at admission were found to be significantly elevated in this group, with four (33.3%, p = 0.046) patients requiring further haemodialysis ( Table 4).

Table 3 Demographics and baseline characteristics between patients with severe and non-severe respiratory complications
Completeness of data: a 80%; b 96.6%; c 83%; d 73%; e 63.3%. Values expressed in mean (± standard error) unless stated otherwise. T-test or Wilkoxon-Mann-Whitney according to normality were used. Pearson Chi squared test was used to compare proportions. Significant if p < 0.05

Discussion
Data from medical charts of 30 cases of hospitalized vivax malaria patients that developed respiratory complications in a tropical disease hospital of the western Brazilian amazon, between 2009 and 2016, were collected retrospectively. Anaemia and thrombocytopaenia at admission were common findings among these patients. Comorbidities and/or concomitant conditions were present in more than half of the studied population. Severe WHO defined respiratory complications and multi-organ failure developed in several cases with intensive care being necessary. Reported anti-malarial regimen consisted mainly of CQ plus PQ with a more than half of patients developing respiratory complications after treatment start.
A pooled analysis of clinical studies since the year 1900 describing complications in vivax patients showed a 0.27% prevalence of respiratory dysfunction in reports with and without severity signs [10]. Geographical stratification has shown that prevalence estimates of respiratory complications vary according to endemic regions [11], mostly due to a complex interplay of host and vector characteristics [9]. Nonetheless, part of the variation is because of definitions used to describe these respiratory complications. In the present study, prevalence of ARDS and other severe respiratory complications were found to be equivalent to studies of similar design originating from India [18][19][20][21] and other countries from South-East Asia, the Eastern Mediterranean and the Western-Pacific regions [22][23][24][25][26].
The presence of comorbidities and concomitant conditions and the extent they contribute to complicated disease and death in malaria has lingered for centuries and still remains a matter of debate. A recent study from India with 511 P. vivax patients showed no significant difference in the presence of non-infectious comorbidities between severe and non-severe patients [27]. On the other hand, a study conducted in India and Brazil with 778 patients showed that patients with comorbidities may present higher risk of complications and death [8]. Also, four out of seven patients with respiratory complications presented comorbidities in the Brazilian autopsy series [6]. Despite evidence of association between comorbidities and severe disease, this still needs elucidation in vivax malaria. It is hypothesized that comorbidities, co-infections and other concomitant conditions may become unstable due to anaemia and fever in acute vivax infections. Thus, a concurrent acute or chronic relatively controlled condition can lead to a severe and potentially life-threatening one through hypoxia exacerbation and haemodynamic decompensation [28]. G6PDd screening is not routinely performed in the Americas for vivax malaria placing this population at a high risk for haemolysis [29]. In the present study, five patients were screened for G6PD activity according to medical discretion. Due to this, G6PD status is unknown in the other patients. The precise pathogenic mechanisms involving G6PD deficiency, primaquine-induced haemolysis and the development of respiratory complications in vivax malaria deserves more research and elucidation.
Several studies have reported cases of respiratory complications in vivax malaria after treatment [11], with a tenfold increase in the risk for developing respiratory distress [8]. It is suggested that a post-treatment inflammatory phenomenon occurs within lung microvasculature in these cases leading to respiratory impairment [30,31]. This study has shown a possible association between treatment initiation and respiratory impairment. Importantly, chloroquine is known to have immune-modulatory proprieties [32][33][34], which could further prevent worsening of post-treatment inflammation and associated symptoms. Nonetheless, after taking into account increasing chloroquine-resistance, a change to artemisinin combination therapies for Plasmodium blood stage killing, which does not have an anti-inflammatory profile, could further increase the number of post-treatment inflammatory complications in vivax patients, such as respiratory distress [35]. Studies addressing this specific aspect of anti-malarial treatment switch are urgently needed.
The few published histopathological analyses in vivax malaria have revealed infiltrates of monocytes, lymphocytes, and neutrophils in pulmonary microvasculature along with phagocytosed pigment and diffuse alveolar damage [36,37] and hyaline membrane formation [38]. Severe alveolar oedema and congestion with infiltrates containing mononuclear cells and suggestive images of adhesion of Plasmodium-infected red cells to lung microvasculature have also been reported [39]. In P. vivax malaria, cytoadhesion of parasitized red blood cells also seems to occur, but to a much lesser extent and magnitude to what can be seen in P. falciparum [40,41]. Additionally, peripheral parasitaemia is generally much lower in this species, due to the preferential invasion of reticulocytes [38]. Instead the relatively much greater inflammatory and endothelial activation, as revealed by elevated cytokines and other inflammatory-inducing molecules [42][43][44], may be responsible for alveolar-capillary barrier loss and increased permeability [38], and is probably the basis for the particular respiratory complications in this species, as seen here.
This study has limitations that need to be addressed. The authors decided to focus on respiratory complications of P. vivax malaria and provide a more comprehensive description of its associated clinical manifestations in detriment of comparing epidemiological, clinical and laboratory aspects between this type of complication and other WHO-defined severe malaria syndromes. Therefore, no risk factor for developing respiratory complications in comparison to other malaria related complications were assessed. The retrospective nature of this study accounts for incomplete data collection due to a lack of prospective and detailed targeted investigation, including proper registration of respiratory rates, which may lead to underestimation of the prevalence of this complication, accurate registration of oxygenation rates and mechanical ventilation parameters, which may lead to lack of proper classification of ARDS according to the Berlin definition and also proper malaria diagnosis through molecular methods and systematic co-infection screening and comorbidity investigation. The decision to admit patients was made at the attending physician's discretion and may have resulted in selection bias, which was minimized through adoption of WHO severe malaria criteria. A great number of patients in this study had received anti-malarial treatment prior to hospital presentation with some of them presenting aparasitaemic, or probably with declining parasitaemia, at the time of admission. This could implicate in clinical improvement at this time and may have underestimated the rate of respiratory complication. In addition, the relatively low prevalence of respiratory complications, and therefore, the small sample size in this study does not support more robust predictive analyses, which may further hamper translational implications. Furthermore, pathogenic mechanisms of respiratory deterioration development could not be explored with such a study design.

Conclusions
Respiratory complications were found to have low prevalence among all vivax episodes in this study. Despite that, this complication was found to be a frequent clinical exacerbation among P. vivax malaria cases in need for hospital care. Comorbidities and/or concomitant conditions occurred in older patients, were associated with worsening of cases and remained associated to elevated odds for severe respiratory complication development, intensive care need and death. In this case, age may have influenced the presence of comorbidities, and therefore, be associated to poor outcome. Nonetheless, the presence of comorbidities, but not age, directly influenced the poor outcome in this study. The results from this study points to the importance of properly recognizing the existence of complications in vivax malaria infection, especially in patients with associated comorbidities and/or concomitant conditions, and further supports