Imported severe malaria and risk factors for intensive care: A single-centre retrospective analysis.

OBJECTIVES: This study aims to identify the risk factors for intensive care (IC) in severe malaria patients admitted to the "Lazzaro Spallanzani" National Institute for Infectious Diseases, Rome, Italy.
METHODS: All patients with confirmed severe malaria and hospitalized between 2007 and 2015 were included in the analysis and stratified into two groups: those requiring IC and those who did not. Five prognostic malaria scores were estimated; clinical severity at IC unit admission was assessed using the Sequential Organ Failure Assessment and the quick-Sequential Organ Failure Assessment scores. Univariate and multivariate analysis were performed to assess factors independently associated to IC.
RESULTS: A total of 98 severe malaria patients were included; 10 of them required IC. There were no deaths or sequelae. Patients requiring IC had higher severity scores. At the multivariate analysis, only the number of World Health Organization criteria and the aspartate aminotransferase value were independently associated with the need of IC.
CONCLUSIONS: An early and accurate assessment of the severity score is essential for the management of severe malaria patients.

Introduction

Imported malaria is a substantial clinical and epidemiological problem in many European countries currently considered as malaria-free. In Europe, approximately 6000 imported malaria cases are reported annually, with 10% of them progressing towards severe malaria [1]. The case-fatality rate of adequately treated severe malaria, due to Plasmodium falciparum is about 10–20% while it is almost 100% if left untreated [2]; among patients admitted to intensive care unit (ICU), such case-fatality rate ranges between 5 to 10% [3]. Individuals from non-endemic countries travelling to high malaria endemic countries are immunologically naïve as they have not been previously exposed to the infection. For this reason, when infected, they may evolve rapidly towards severe malaria with high parasite densities, multi-organ involvement, and eventually death [4-6]. Therefore, identifying among these patients those with a higher risk of adverse outcome may improve their management and thus reduce the risk of death. The aim of this study was to assess the risk factors associated with the need of IC. For this purpose, the clinical records of a cohort of severe malaria patients admitted over 8 years at the “Lazzaro Spallanzani” National Institute for Infectious Diseases (INMI), Rome, Italy were analysed.

Methods

In this single-centre cross sectional study, we retrospectively recruited a total number of 98 patients consecutively hospitalized with confirmed severe malaria at the INMI Spallanzani, Rome, Italy, from January 2007 to December 2015. The INMI Spallanzani is a 200-bed hospital dealing mainly with infectious and tropical diseases with a 10-bed ICU. This study was approved by the Ethic Committee of the INMI Spallanzani (ethics number 38/2016). Inclusion criteria: age >18 years old, written informed consent at hospital admission from the patient or next of kin if patient unable, severe confirmed malaria diagnosis. Severe malaria was diagnosed in presence of P. falciparum asexual parasitaemia and at least one clinical (impaired consciousness or unarousable coma; prostration; failure to feed; multiple convulsions–more than two episodes in 24 hours; respiratory distress; circulatory collapse or shock; clinical jaundice and evidence of other vital organ dysfunctions; haemoglobinuria; abnormal spontaneous bleeding; pulmonary oedema) or laboratory (hypoglycaemia; metabolic acidosis; severe anaemia; hyperparasitaemia; renal impairment hyperlactataemia) criterion in absence of any other obvious cause [7]. For Plasmodium vivax, all the criteria were applied with the only exception of hyperparasitaemia [7]. Demographic characteristics, medical and travel history, clinical presentation, anti-malarial and supportive treatment, parasitaemia before and during treatment, complications during treatment, adverse drug reactions, clinical outcome (survival, death or sequelae) at day 28 post-treatment were collected for all patients from the clinical record. In addition, the time to reduce parasite density below 1% and parasite clearance were also collected. Patients were divided into two groups, namely those requiring and those not requiring intensive care (ICU and non-ICU, respectively). The ICU criteria for admission were: Glasgow Coma Scale <11, presence of multiple convulsions, respiratory distress with a PaO2/FiO2 Ratio <100, conditions requiring mechanical or non-invasive ventilation, circulatory collapse or shock and/or mean artherial pressure <70 mmHg requiring vasopressor drugs, all those conditions requiring close monitoring and/or prolonged sedation. For each patient, five prognostic malaria scores were calculated: the Malaria Score for Adults (MSA) [8], the Coma Acidosis Malaria score (CAM) [9], the Respiratory rate-based CAM score (R-CAM) [9], the Malaria Severity Score (MSS) [10] and the GCRBS (Glasgow coma scale, Creatinine, Respiratory rate, Bilirubin, Systolic blood pressure) Score [11]. Clinical severity at ICU admission was also assessed using the Sequential Organ Failure Assessment (SOFA) score [12] and quick-SOFA (q-SOFA) score [13].

Statistical analysis

Comparison of continuous data between 2 groups were analysed with the Student's t test or Mann-Whitney test, as appropriate. Fisher exact test was used to evaluate age, gender and WHO criteria distribution. Pearson’s correlation and linear regression were used to evaluate the correlation between malarial and non-malarial scores. Data were expressed as median (interquartile range, IQR) or mean ± standard deviation (M±SD), as appropriate. Univariate and multivariate analyses were performed to analyse the factors independently associated to admission in ICU. The final multivariate model was selected with a forward stepwise variable selection procedure. In the multivariate analysis, a stepwise logistic regression model was fitted. A p value of <0.05 was considered statistically significant. Statistical analyses were performed using GraphPad PRISM and Stata Corp. 2013. Stata Statistical Software: Release 13. College Station, TX: StataCorp LP.

Results

Study population

From 2007 to 2015, a total of 98 patients with severe malaria were admitted; 10 of them requiring IC (Table 1).

Table 1

Severe malaria patients by Intensive Care need (%).

Variables	ICU	NON-ICU	p
	(n = 10)	(n = 88)
Male	8 (80)	71 (80.7)	0.421
Age (years) (M±SD)	45 ± 14	38 ± 13	0.142
Country of origin
- European	6 (60)	43 (49)
- Immigrant	4 (40)	41 (46.5)	0.223
- Visitors from endemic country	0	4 (4.5)
Comorbidities
- HIV	1 (10)	4 (4.5)
- Chronic Viral Hepatitis	1 (10)	1 (1.1)
- Arterial Hypertension	1 (10)	10 (11.4)
- COPD	0	4 (4.45)	0.443
- Splenectomy	1 (10)	0
- Cancer	0	1 (1.1)
- Hypothyroidism	0	2 (2.3)
Antimalarial chemoprophylaxis (n, %)	0	9 (10.2)	0.552
Area of infection (n, %):
West Africa	10 (100)	65 (73.9)	0.944
Purpose of travel (n, %)
- VRFs	2 (20)	30 (34.1)
- Tourism	3 (30)	25 (28.4)	0.876
- Business	4 (40)	13 (14.8)
- Humanitarian aid	1 (10)	10 (11.4)
- Other	0	10 (11.4)
Plasmodium (n, %)
- Falciparum	9 (90)	83 (94.3)
- Vivax	1 (10)	4 (4.5)	0.554
- Mixed Infection	0	1 (1.1)
Delay of diagnosis (days) (M±SD)	4.5 ± 0.8	4.2 ± 0.5	0.851
Delay of treatment (days) (M±SD)	4.9 ± 0.7	4.8 ± 0.5	0.951
Days of hospitalization (M±SD)	25.6 ± 7.4	7.1 ± 0.5	0.0001
Basal % parasitemia (Median, IQR)	7 (2–7)	5 (0.9–7)	0.811
Time to parasitaemia <1% (hours) (M±SD)	67.2 ± 11.7	51.5 ± 3.0	0.122
Time to parasite clearance (hours) (M±SD)	127.2 ± 19.6	84.7 ± 4.6	0.006
Antimalarial Treatment during Hospitalization
- Artesunate (iv)	6 (60)	17 (19.3)
- Quinine (iv)	4 (40)	17 (19.3)
- Arthemeter (im)	0	11 (12.5)	0.434
- Quinine (oral)	0	17 (19.3)
- Dihydroartemisinin/piperaquine	0	24 (27.4)
- Cloroquine	0	2 (2.2)

ICU: Intensive Care Unit; im: intramuscular; iv: intravenous; M: mean; SD: Standard deviation; IQR: interquartile range, COPD: chronic obstructive pulmonary disease, VRFs:Visiting Relatives and Friends.

Patients requiring IC showed longer length of hospitalization and parasite clearance time compared to the other group. Notably, there were 5 cases of P. vivax severe malaria, including one requiring IC. Overall, the reasons for IC were cerebral malaria (7 cases), shock (6 cases) and acute respiratory distress syndrome (4 cases). The presence of 2 concurrent criteria for IC was observed in seven patients. All patients who required IC were treated with either intravenous artesunate (2.4 mg/kg at 0, 12, 24 and 48 hours, in 6 cases) or quinine (20 mg/kg loading dose followed by 10 mg/kg for a total of 48 hours, in 4 cases). Moreover, all ICU patients received antibiotic treatment for bacterial infections (3 central venous catheter-associated blood stream infections due to multidrug resistant Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus, and Candida albicans, 6 ventilator associated pneumoniae, 1 catheter-associated urinary tract infection). In addition, eight patients needed mechanical ventilation for acute lung injury (6 cases) and acute respiratory distress syndrome (2 cases), 6 patients were treated with vasopressor drugs for shock and 2 patients with continuous veno-venous hemodiafiltration for acute renal failure. At 28-day follow-up, no deaths or documented sequelae were reported.

Malarial and non-malarial severity scores

The different malarial scores were estimated for all 98 severe malaria patients; those who required IC had higher scores than the others (Table 2).

Table 2

Malarial score: ICU vs non-ICU.

Malarial Scores (mean)	ICU	NON-ICU	p
MSA	5.4	0.9	<0.001
CAM	1.6	0.4	0.008
R-CAM	2.1	0.3	0.002
MSS	11.9	1	<0.001
GCRBS	2.9	0.3	<0.001

ICU: Intensive Care Unit, MSA: Malaria Score for Adults, CAM: Coma Acidosis Malaria score, R-CAM: Respiratory rate-based CAM score, MSS: Malaria Severity Score, GCRBS Score: Glasgow coma scale, Creatinine, Respiratory rate, Bilirubin, Systolic blood pressure.

The relation between SOFA, q-SOFA and malarial severity scores was evaluated and a positive linear regression was found between SOFA and GCRBS only (p = 0.01, r2 = 0.58) (Fig 1).

Fig 1

Malarial and non-malarial severity scores.

A positive linear regression was found between Sequential Organ Failure Assessment score (SOFA) and GCRBS Score (Glasgow coma scale, Creatinine, Respiratory rate, Bilirubin, Systolic blood pressure).

ICU versus non-ICU patients

Mean age, baseline parasite density, platelet count, haemoglobin and serum creatinine levels were similar between ICU and non-ICU patients; ICU patients had a slower parasite clearance (127.2±19 vs 84.7±4.6 hours, p = 0.006) and higher total bilirubin (4.8±1.2 vs 2±0.16 mg/dl, p<0.0001), aspartate aminotransferase (AST, 159.3±34 vs 66.5±7.6 U/L, p = 0.0003) (Fig 2A) and alanine aminotransferase (ALT, 102.9±19.3 vs 61.3±7.3 U/L, p = 0.07). All ICU patients had more than 3 WHO criteria for severe malaria while the other patients met only one criterion (3.8±0.2 vs 1.3±0.11, p<0.001) (Fig 2B): specifically, the presence of neurological (p<0.001), liver (p = 0.03) or respiratory involvement (p<0.001) as single WHO criterion of severe malaria was associated to IC need.

Fig 2

Aspartate aminotransferase levels (A) and N_WHO criteria (B) between ICU and non-ICU patients. Intensive Care Unit (ICU) group showed a significantly higher aspartate aminotransferase (AST) value (A) and higher number of WHO criteria (B) compared to non-ICU group.

Univariate analysis showed that the number of WHO criteria (p<0.001), time to parasite clearance (p = 0.012), levels of serum AST (p = 0.004) and bilirubin (p = 0.003) at admission were associated with IC. Multivariate analysis showed that the number of WHO criteria (Odd Ratio [OR] = 2.58; confidence interval [CI] = 1.36–4.91; p = 0.004) and AST value (OR = 1.01; CI = 1.00–1.02; p = 0.037) were independently associated with IC (Table 3).

Table 3

Logistic regression model to evaluate risk factors associated with IC.

	Univariate		Multivariate*
	OR (95% CI)	p	OR (95% CI)	p
N_WHO criteria	4.00 (1.96–8.16)	<0.001	2.58 (1.36–4.92)	0.004
Basal parassitemia	0.97 (0.75–1.24)	0.816
Time parasitemia clearance	1.01 (1.00–1.03)	0.012
Platelet count	1.00 (0.99–1.00)	0.204
Haemoglobin	0.83 (0.65–1.07)	0.166
Creatinine	1.72 (0.39–7.51)	0.466
Bilirubine	1.74 (0.12–2.52)	0.003	1.52 (0.93–2.47)	0.089
AST	1.10 (1.00–1.01)	0.004	1.01 (1.00–1.02)	0.037
ALT	1.0 (0.99–1.01)	0.092

OR: Odds Ratio; CI: confident interval;

*with forward stepwise variable selection procedure, WHO: World Health Organization, AST: aspartate aminotransferase, ALT: alanine aminotransferase.

Discussion

Malaria remains a substantial problem in non-endemic countries. In this series of patients recruited over a 8-year period, 10 patients required IC, with a 4-day median delay of malaria diagnosis; all of them had been infected in West Africa and none of them received anti-malaria chemoprophylaxis. In a non-endemic country, any imported severe malaria case is a medical emergency, often complicated by a delayed access to care, diagnosis and initiation of specific parenteral therapy. The capacity of the different national health systems for reporting malaria cases varies substantially and depends on the capacity for a reliable diagnosis [14]. Unspecific febrile symptoms and low quality microscopy reading in non-specialised laboratories can delay diagnosis [15]. In this series of patients, P. falciparum was the main causing species although there were some P. vivax cases, with one of them requiring IC. There have been increasing reports of severe malaria caused by P. vivax [2].

Severe malaria may rapidly evolve to an unfavourable prognosis requiring IC with a case-fatality rate between 5 and 10%. Therefore, identifying severe malaria patients at higher risk of death is of paramount importance. However, not all WHO criteria have the same predictive value for the early identification of patients with unfavourable prognosis. In the 400-patient cohort study conducted in France by Bruneel et al., three baseline variables independently predicted death: older age, coma and high parasite density [16]. In our study, five prognostic malaria scores were calculated for each ICU patient (MSA, CAM, R-CAM, MSS, GCRBS score) and clinical severity at ICU admission was assessed using both SOFA and q-SOFA scores. In this study, most ICU patients had a medium-high SOFA score with a high SOFA-related mortality predictive value. In addition, there was a positive correlation between SOFA, a non-specific malaria score, and GCRBS, a specific malaria score. An early assessment of the severity status of the patient by specific score is required at admission to rapidly decide whether the patient needs to be admitted in ICU. Applying both malaria-specific (GCRBS) and general (SOFA) scores to severe malaria patients could be the best approach to assess the need for IC.

The findings of this study have few limitations. First, this is a retrospective and cross-sectional study conducted in a single centre. Second, the small number of enrolled patients, especially those requiring ICU admission, could affect the generalization of our results to a wider population. However, our results are consistent with data from previous studies. In a recent review on the clinical management of imported severe malaria in adults, the authors recommended an assessment by ICU staff for all patients with malaria even if presenting one severity criterion, only [17]. Moreover, patients with uncomplicated malaria but at risk of rapidly evolving towards severe malaria should be under continuous monitoring; a >2 q-SOFA score should be considered as an indication for ICU admission [17]. In our case series, patients requiring IC had a higher number of WHO criteria than the other group, and cerebral malaria, liver and respiratory failures were the most frequent observed complications. Indeed, at multivariate analysis, the number of WHO criteria and the AST level were independently associated with ICU admission. A prompt intravenous antimalarial regimen with adequate supportive therapy warranted a favourable outcome: the WHO guidelines for the treatment of severe malaria recommend the use of intravenous artesunate for at least the first 24 hours [2]. Such recommendation is based on the results of two large trials carried out in malaria-endemic countries that showed artesunate superiority to quinine in terms of efficacy (rapid decay of parasitaemia), safety (no major adverse events) and better survival [18,19]. All our patients requiring IC were treated with either quinine (4 patients before 2010 when parenteral artesunate was unavailable) or artesunate. All patients recovered without any complications or sequelae.

Conclusions

Considering that severe malaria may rapidly evolve to an unfavourable prognosis, a prompt and accurate evaluation for ICU admission is needed. According to our results, the assessment of both malarial and non-malarial severity scores may contribute to the proper management of severe malaria. Specifically, the number of WHO criteria and AST plasma level can predict the need of intensive care.

Strobe checklist.

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Dataset.

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4 Sep 2019

PONE-D-19-16792

Imported severe malaria and risk factors for intensive care: a single-centre retrospective analysis.

PLOS ONE

Dear Dr Iannetta,

Thank you for submitting your manuscript to PLoS ONE. After careful consideration, we felt that your manuscript requires substantial revision, following which it can possibly be reconsidered, thus governing the decision of a “major revision”. Regarding the study design, there are significant concerns raised by reviewers. According to Reviewer # 1, the criteria for IC admission is not clear and whether or not patients presented dual pathologies.  Reviewer #2 recommend to use the STROBE checklist to improve the reporting quality of results. Of further relevance, both reviewers complain that conclusion must translate the results, and authors needs to adjust that. Finally, the low number of IC patients may compromise the MS’s conclusion, and authors should include study limitation. For your guidance, a copy of the reviewers' comments was included below.   We therefore invite you to submit a revised version of the manuscript paying close attention to the specific points raised by both reviewers.

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Reviewers' comments:

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Reviewer #2: Yes

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Reviewer #1: This a retrospective single-centre review of imported severe malaria. I have the following comments:

1) What were the pre-existing admission criteria for admission to IC?

2) A total of 10 cases needing IC are reported although the reason for admission were cerebral malaria (7), shock (6) a nd ARDS (4) which totals 17, were there any patients that had dual pathologies?

3) ICU vs non-ICU patients had slower parasitemia but did the ICU patients have higher parasitemia to start off with which could explain the slower clearance?

4) 6 patients were reported as requiring vasoplressor drugs for shock but there is no mention of fluid resuscitation used. This may relevant particualry in light of the FEAST study.

5) There were no deaths or sequelae reported yet the conclusions state 'a multidisciplinary approach might be encouraged... in order to improve outcome', what outcome are they trying to improve? Furthermore what and whom should be part of a multi-disciplinary approach'?

Reviewer #2: This is a simple, concise cross-sectional study of medical record data from patients with severe malaria to assess predictive risk factors associated to the need of intensive care (IC) at a referral hospital in Italy. Authors used five prognostic malaria scores to analyse the need of IC and applyed malaria-specific and general scores to identify severe malaria patients.

The manuscript is well written, clear and intelligible. However, authors should make some changes to the manuscript in order to better interpret and conclude the results.

1) Authors are recommended to use the STROBE checklist to improve the reporting quality of results.

2) Some limitations of the study should be discussed, such as the low number of patients requiring intensive care, especially if the species was P. vivax. In addition, the cross-sectional design of the study precludes a more conclusive inference from the study results.

3) The conclusion of the study should be presented more clearly and objectively. In the Summary, for example, the conclusion presented does not translate the results found in the study.

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Reviewer #2: No

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27 Sep 2019

REVIEWER #1

This a retrospective single-centre review of imported severe malaria. I have the following comments:

1) What were the pre-existing admission criteria for admission to IC?

A: The admission criteria adopted for IC admission were:

- Glasgow Coma Scale <11;

- Presence of multiple convulsions;

- Respiratory distress with PaO2/FiO2 Ratio <100);

- Conditions requiring mechanical ventilation and/or non-invasive ventilation;

- Circulatory collapse or shock and/or MAP <70 mmHg, requiring vasopressor drugs;

- Conditions requiring close monitoring and/or prolonged sedation.

We included the IC admission criteria in the manuscript.

2) A total of 10 cases needing IC are reported although the reason for admission were cerebral malaria (7), shock (6) and ARDS (4) which totals 17, were there any patients that had dual pathologies?

A: In our study, 7 patients presented more than one clinical picture at the moment of the admission in the IC, therefore the number of the events (17) is greater than the number of patient (10). A sentence was added in the text, accordingly.

3) ICU vs non-ICU patients had slower parasitemia but did the ICU patients have higher parasitemia to start off with which could explain the slower clearance?

A: As showed in Table 1, there is no difference in basal parasitemia between ICU vs non-ICU (p= 0.811). Moreover, the data is confirmed at the univariate analysis.

4) 6 patients were reported as requiring vasopressor drugs for shock but there is no mention of fluid resuscitation used. This may relevant particuarly in light of the FEAST study.

A: According to the protocol of our Institute, the use of vasopressor drugs is secondary to the fluid resuscitation failure.

5) There were no deaths or sequelae reported yet the conclusions state 'a multidisciplinary approach might be encouraged... in order to improve outcome', what outcome are they trying to improve? Furthermore what and whom should be part of a multi-disciplinary approach'?

A: As suggested by the reviewer, we modified the conclusion of the manuscript.

REVIEWER #2

This is a simple, concise cross-sectional study of medical record data from patients with severe malaria to assess predictive risk factors associated to the need of intensive care (IC) at a referral hospital in Italy. Authors used five prognostic malaria scores to analyse the need of IC and applyed malaria-specific and general scores to identify severe malaria patients. The manuscript is well written, clear and intelligible. However, authors should make some changes to the manuscript in order to better interpret and conclude the results.

1) Authors are recommended to use the STROBE checklist to improve the reporting quality of results.

A: According to reviewer’s suggestion we modified the methods section using the STROBE checklist, which will be uploaded as a supplementary file.

2) Some limitations of the study should be discussed, such as the low number of patients requiring intensive care, especially if the species was P. vivax. In addition, the cross-sectional design of the study precludes a more conclusive inference from the study results.

A: We thank the reviewer for the suggestions. The text has been modified accordingly, introducing sentences showing some limitations of the study.

3) The conclusion of the study should be presented more clearly and objectively. In the Summary, for example, the conclusion presented does not translate the results found in the study.

A: The conclusion has been modified accordingly, introducing sentences that reflect the results showed in the text.

Submitted filename: Response to Reviewers.docx

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7 Oct 2019

PONE-D-19-16792R1

Imported severe malaria and risk factors for intensive care: a single-centre retrospective analysis.

PLOS ONE

Dear Dr Iannetta,

Thank you for submitting your manuscript for review to PLoS ONE. After careful consideration, we feel that your manuscript will likely be suitable for publication if it is revised to address major points raised now by the reviewers. Specifically, the authors should clarify a couple of topics related to study design, including why they requested written informed consent from patients’ next of kin, since all patients are adults.   A significant number of grammatical errors remained in the revised version of MS. Thus, the language needs to be properly adjusted otherwise it might compromise the publication. Finally, the reviewers complain about Data Availability that is not entirely accessible as stated by the authors.

We would appreciate receiving your revised manuscript by October 30. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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We look forward to receiving your revised manuscript.

Kind regards,

Luzia Helena Carvalho, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

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1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #2: No

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #2: (No Response)

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Although the authors stated that the study data is entirely available within the manuscript and in supplementary files, I have not had access to it completely.

Reviewer #3: This is a cross sectional and retrospective study where clinical records for patients who were admitted with severe malaria from 2007 to 2015 were reviewed and analyzed by the authors. In this revised version of the manuscript, the authors have satisfactorily addressed comments that were raised by two reviewers. The manuscript is technically sound with the data used supporting the conclusion of the paper. All statistical analyses have been done appropriately and rigorously. Nevertheless I have the following concerns with the revised manuscript which the authors need to also look into:

1. The presentation of the manuscript is intelligible and uses Standard English though it will benefit from some language usage proofreading.

2. The authors need to clearly distinguish between actual recruitment of the patients and use of records for this research study. An example is where they write that they obtained written informed consent from the patients or their next of kin before enrollment into the study without detailing how they did it considering that by the time of this research the patients were not in hospital.

3. Further the authors write that they obtained written informed consent from patients’ next of kin, since all patients are adults aged above 18 years why was consent obtained from next of kin?

4. Authors should spell out all abbreviations the first time they are used in the write up – examples are INMI, h, P. vivax etc.

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Reviewer #2: No

Reviewer #3: Yes: Peter Makaula

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Submitted filename: Reviewer notes.docx

Click here for additional data file.

25 Oct 2019

REVIEWER #2

1. Although the authors stated that the study data is entirely available within the manuscript and in supplementary files, I have not had access to it completely.

A: In the previous revision, we have already uploaded the complete database as Supplementary file (page 23, download link). We will upload again the entire database as a supplementary file. If this is not enough, please let us know what we can do further.

REVIEWER #3:

This is a cross sectional and retrospective study where clinical records for patients who were admitted with severe malaria from 2007 to 2015 were reviewed and analyzed by the authors. In this revised version of the manuscript, the authors have satisfactorily addressed comments that were raised by two reviewers. The manuscript is technically sound with the data used supporting the conclusion of the paper. All statistical analyses have been done appropriately and rigorously. Nevertheless I have the following concerns with the revised manuscript which the authors need to also look into:

1. The presentation of the manuscript is intelligible and uses Standard English though it will benefit from some language usage proofreading.

A: As suggested by the reviewer, the manuscript was widely revised for language editing.

2. The authors need to clearly distinguish between actual recruitment of the patients and use of records for this research study. An example is where they write that they obtained written informed consent from the patients or their next of kin before enrollment into the study without detailing how they did it considering that by the time of this research the patients were not in hospital.

A: As suggested by the reviewer, we modified the text. According to the protocol in use in our Institute, each patient signs an informed written consent at the time of admission for research purposes and epidemiological investigations on infectious and tropical diseases.

3. Further the authors write that they obtained written informed consent from patients’ next of kin, since all patients are adults aged above 18 years why was consent obtained from next of kin?

A: As suggested by the reviewer we modified the text. In case of person unable (e.g. coma state) to give consent, a relative/next of kin was asked to provide it.

4. Authors should spell out all abbreviations the first time they are used in the write up – examples are INMI, h, P. vivax etc.

A: As suggested by the reviewer, we modified the text.

Submitted filename: Response to Reviewers.doc.docx

Click here for additional data file.

30 Oct 2019

Imported severe malaria and risk factors for intensive care: a single-centre retrospective analysis.

PONE-D-19-16792R2

Dear Dr. Iannetta,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Luzia Helena Carvalho, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

5 Nov 2019

PONE-D-19-16792R2

Imported severe malaria and risk factors for intensive care: a single-centre retrospective analysis.

Dear Dr. Iannetta:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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With kind regards,

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on behalf of

Dr. Luzia Helena Carvalho

Academic Editor

PLOS ONE