Association of thrombocytopenia with splenomegaly in malaria patients in East Kalimantan: A cross-sectional, retrospective study

Background: Malaria still presents as a major health problem in Indonesia and specifically in East Kalimantan. One common sign found in malaria patient is thrombocytopenia, the mechanism of which is still unclear. Several studies have suggested some mechanisms, one of which is splenomegaly. This study aimed to discover the association between thrombocytopenia and splenomegaly of malaria patients in East Kalimantan. Methods: This study was a descriptive retrospective study with clinical and laboratory data obtained from the medical records of malaria patients in four major public hospitals from January 2015 to July 2018. The association between thrombocytopenia with splenomegaly was analysed using Chi-Square test. Results: A total of 215 patients were included; 189 male (87.9%) and 26 female (12.1%). The etiologic agents found in these patients were Plasmodium vivax (43.2%), Plasmodium falciparum (42.8%), and mixed infection ( Plasmodium falciparum and Plasmodium vivax) (4.6%). The thrombocyte count was normal in 28 patients (13%) and decreased in 187 patients (87%). Among patients with thrombocytopenia, the percentage of mild, moderate and severe thrombocytopenia was 18.2%, 43.8% and 33%, respectively. Splenomegaly was found in only 11 patients (5.1%). We found no association between thrombocytopenia with splenomegaly (p=0.61). Conclusions: We conclude that splenomegaly, which was a rare clinical finding of these malaria patients, was not associated with thrombocytopenia.

Data from the East Kalimantan Provincial Health Office indicated that there were 7,045 cases of malaria in 2010. This number fluctuated in the following years, with 3,021 cases in 2011, 9,966 cases in 2012 and 2,603 cases in 2013. In terms of Annual Parasitic Incidence (API), in 2014 East Kalimantan was still above the national average with an API of 2,04 per 1000 population, leading to it being categorized as a low cumulative incidence area 2 .
One common finding in malaria is decreased platelet count or so-called thrombocytopenia. This laboratory finding is often confused with other infectious diseases, especially dengue infection in which thrombocytopenia is a major diagnostic parameter. There are several studies that demonstrate thrombocytopenia in malaria patients [3][4][5] , and this is found in both infection with Plasmodium falciparum and Plasmodium vivax. The results from our previous study demonstrated that from 1041 malaria cases in East Kalimantan, 85% presented with thrombocytopenia of varying degrees 4 . Therefore, thrombocytopenia has been suggested as one important diagnostic parameter in malaria. The mechanism of thrombocytopenia in malaria is still unclear. Several theories, such as mechanical trapping of thrombocytes inside the spleen and immune response that attacks thrombocytes, has been proposed 5 . A study by Coelho et al. found that platelet phagocytosis may contribute to thrombocytopenia in vivax malaria 6 .
Splenomegaly is also one common clinical finding in malaria patients. The spleen is part of the reticuloendothelial system, which becomes active in order to get rid of plasmodium-infected erythrocytes. Splenomegaly can also contribute to increased destruction of thrombocytes due to mechanical trapping. Therefore, the aim of the present study was to determine the association of thrombocytopenia with splenomegaly in malaria patients in East Kalimantan.

Study design and participants
This study was a cross-sectional retrospective study. This study was approved by the Ethical Committee for Health Research at Abdul Wahab Syahranie Public Hospital Samarinda, East Kalimantan (approval number 124/KEPK-AWS/V/2018). Patient consent for the use of their data records was waived by the ethical committee due to the retrospective nature of the study.

Data collection
Data were collected between June and August 2018 from the medical records of patients with malaria during the period of January 2015 to July 2018. We collected clinical and laboratory data of both outpatients and inpatients diagnosed with malaria from four major hospitals in East Kalimantan: Abdul Wahab Sjahranie Hospital in Samarinda, Aji Putri Botung Hospital in Penajam Paser Utara, Abdul Rivai Hospital in Tanjung Redeb, and Panglima Sebaya Hospital in Tanah Grogot.
All patients with malaria, both paediatric and adult patients, were included in the study. Patients were excluded from the study if the necessary data were incomplete or patients were discharged upon own's request during treatment.
In order to collect data, first, the hospital's database was searched for patients diagnosed with malaria. Second, after identifying these patients, the relevant medical records were retrieved which contained age, gender, type of Plasmodium, thrombocyte count, and presence of splenomegaly. Type of Plasmodium was determined using microscopic method by obtaining thick blood smear and staining by Giemsa 3%. The thrombocyte count included in this study was from laboratory data at the time of hospital visit or admission before receiving any treatment. The thrombocyte count was determined using automated haematology analyser of different types and manufacturers available in each hospital.

Data analysis
For descriptive data, we described patients' characteristics that include age, sex, type of Plasmodium, and thrombocyte count on the first day admission. The association between thrombocytopenia and splenomegaly was analyzed by Chisquare test using SPSS 23.0 software. Results were considered statistically significant if p<0.05.

Results
Our study identified a total of 215 malaria patients from January 2015 to July 2018 from four hospitals in East Kalimantan. There were 87.9% (189/215) male and 12.1% (26/215) female patients ( Table 1).
The association of thrombocytopenia with splenomegaly in malaria patients shown in Table 2. There were 11 patients with

Amendments from Version 2
We revised the wording in our conclusion (on both abstracts and conclusion). We added some information about the method of determining thrombocyte count.
Any further responses from the reviewers can be found at the end of the article In our study, we describe the thrombocyte count in malaria patients with splenomegaly. We found that there was no association between thrombocytopenia with splenomegaly (p=0.611). Therefore, we propose that thrombocytopenia is not caused by the mechanical trapping of thrombocytes in the spleen. This result suggests another mechanism of thrombocytopenia that involves immune process, as proposed by Coelhoe et al. in 2013 6 . Until now, definitive mechanism of thrombocytopenia in malaria is still unclear. However, some factors that contribute to thrombocytopenia have been reported, such as decreased thrombopoiesis, peripheral destruction induced by P. falciparum and disseminated intravascular coagulation 11 .

REVISED
This study has its limitations. Different types of automatic haematology analysers were used in the hospitals and this might contribute to variations in thrombocyte count. Therefore, we suggest the use of a single haematology analyser across hospitals to improve accuracy. In addition, we did not exclude other causes of thrombocytopenia that could coexist with malaria infection, such as viral infection and autoimmune diseases, which would be required to further validate our findings. Finally, the examination of splenomegaly was conducted by different physicians, which might contribute to subjective factors in determining splenomegaly, such as physician's expertise and thoroughness, especially in cases with minor or subclinical splenomegaly.
Overall, we conclude that splenomegaly, which was a rare clinical finding in this set of malaria patients, was not associated with thrombocytopenia. presence of splenomegaly. This is an important question to ask in relation to thrombocytopenia in malaria, since the mechanisms involved are not well-defined -splenic trapping is certainly a valid mechanism to consider. Their platelet data support many previous studies of other patient cohorts showing thrombocytopenia is a common clinical feature in malaria. However, the relatively small number of patients exhibiting splenomegaly (11/204 or 5.1%) precludes making a definitive conclusion about the lack of association with thrombocytopenia. Therefore, I recommend this limitation be included in the abstract and final conclusions of the paper.

Data availability
Eg. Suggested wording: Splenomegaly, which was a rare clinical feature of this malaria patient cohort, was not associated with thrombocytopenia.
The methods should include notes about how platelet counts were determined, and the fact that different hematological analyzers were used (this is currently mentioned in the Discussion).

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Partly
No competing interests were disclosed. Competing Interests: Reviewer Expertise: Immunology and genetics (basic research).
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. , which permits unrestricted use, distribution, and reproduction in any medium, provided the original Attribution License work is properly cited. 1.

Jontari Hutagalung
Biomedical and Basic Health Technology, National Institute of Health Research and Development, Ministry of Health (Indonesia), Jakarta, Indonesia Is the work clearly and accurately presented and does it cite the current literature? Partly

Are sufficient details of methods and analysis provided to allow replication by others? Partly
If applicable, is the statistical analysis and its interpretation appropriate? Partly