Tropical Parasitology

ORIGINAL ARTICLE
Year
: 2020  |  Volume : 10  |  Issue : 2  |  Page : 95--101

Thrombocytopenia in malaria patients from an arid region of Western Rajasthan (India)


Yashik Bansal1, Vinod Maurya1, Nidhima Aggarwal1, Vibhor Tak1, Vijaya Lakshmi Nag1, Abhishek Purohit2, Akhil Dhanesh Goel3, Gopal Krishna Bohra4, Kuldeep Singh5,  
1 Department of Microbiology, All India Institute of Medical Sciences, Jodhpur, Rajasthan, India
2 Department of Pathology, All India Institute of Medical Sciences, Jodhpur, Rajasthan, India
3 Department of Community Medicine and Family Medicine, All India Institute of Medical Sciences, Jodhpur, Rajasthan, India
4 Department of General Medicine, All India Institute of Medical Sciences, Jodhpur, Rajasthan, India
5 Department of Pediatrics, All India Institute of Medical Sciences, Jodhpur, Rajasthan, India

Correspondence Address:
Vibhor Tak
Department of Microbiology, All India Institute of Medical Sciences, Basni Phase-2, Jodhpur - 342 005, Rajasthan
India

Abstract

Context: The arid climate of Western Rajasthan is challenging for malaria transmission, with the number of cases correlating directly with the annual rainfall pattern. Moreover, >90% of the cases in this region are caused by Plasmodium vivax, which has recently been shown to cause a similar degree of thrombocytopenia as Plasmodium falciparum. Aims and Objectives: The aim of the study was to determine the degree of thrombocytopenia in malaria patients and its association with different species of malaria in this region with an unstable malaria epidemiology. Materials and Methods: This retrospective study was conducted on all microbiologically confirmed malaria patients with documented platelet counts from August 2017 to October 2018. Microbiological diagnosis was established by rapid diagnostic tests and peripheral blood film examination. Platelet counts were used to assess the degree of thrombocytopenia. Results: A total of 130 cases were included in the study, of which 118 (91%) were caused by P. vivax, while the rest 12 (9%) were caused by P. falciparum. Thrombocytopenia was present in 108 (83%) cases, and the mean values of platelets in thrombocytopenic patients with P. vivax and P. falciparum infection were 72600/μL and 48500/μL, respectively. Although P. falciparum infection was significantly associated with severe thrombocytopenia (odds ratio: 4.7, [95% confidence interval 1.3–16.1]), extremely low platelet counts (n = 5) warranting platelet transfusions (n = 1) were seen only in P. vivax cases. Only one patient required platelet transfusions in these patients suggesting good tolerance to thrombocytopenia. Conclusions: Avoiding unnecessary transfusions in febrile thrombocytopenic patients with an established malaria diagnosis can help in reducing transfusion-transmitted infections.



How to cite this article:
Bansal Y, Maurya V, Aggarwal N, Tak V, Nag VL, Purohit A, Goel AD, Bohra GK, Singh K. Thrombocytopenia in malaria patients from an arid region of Western Rajasthan (India).Trop Parasitol 2020;10:95-101


How to cite this URL:
Bansal Y, Maurya V, Aggarwal N, Tak V, Nag VL, Purohit A, Goel AD, Bohra GK, Singh K. Thrombocytopenia in malaria patients from an arid region of Western Rajasthan (India). Trop Parasitol [serial online] 2020 [cited 2021 Apr 13 ];10:95-101
Available from: https://www.tropicalparasitology.org/text.asp?2020/10/2/95/307798


Full Text



 Introduction



Malaria is a major public health problem for a tropical country like India for centuries with India contributing majority of the cases in the South-East Asian region.[1] A total of 4 Plasmodium species have been known to cause the disease in humans, and a fifth species, i.e., Plasmodium knowlesi, has been reported from South-East Asia as a cause of malaria in the past two decades or so.[2] Among these, Plasmodium falciparum is known to cause the most severe form of malaria[3] until recently when it was established that Plasmodium vivax is also capable of causing the similar magnitude of severe malaria with similar clinical manifestations.[4]

The city of Jodhpur is located in Western Rajasthan at the edge of the Thar desert and has an arid climate with an unstable pattern of rainfall.[5],[6] Classically, the arid regions are considered areas with low malaria outbreak potential. However, the malaria outbreaks in this region in the past three decades have shown that rainfall is directly linked with the malaria incidence.[7] As per the available data, majority of the cases in this region are attributable to P. vivax in contrast to the national data where 66% of the malaria cases are caused by P. falciparum, largely contributed by the North Eastern and Southern states.[1]

A few studies have now shown that thrombocytopenia (platelet count in blood less than 1,50,000/μL) which was earlier seen in association with P. falciparum malaria is also seen in P. vivax malaria.[8] While some studies claim that thrombocytopenia should be viewed as a complication of severe malaria,[9] other studies are in disagreement to this aspect.[10] The manifestations of severe malaria are also showing a changing trend in the past decade.[11] Apart from malaria, Rajasthan is also showing an increasing burden of dengue in the past decade,[12] an arboviral, mosquito-borne disease notorious for severe thrombocytopenia to the degree warranting multiple platelet transfusions.

The emerging threat of dengue in India has renewed the interest of thrombocytopenia in malaria patients, as avoiding unnecessary platelet transfusions in these patients is of huge benefit[13] since it reduces transfusion-transmitted infections (TTI) and helps better use of blood products in a resource-poor country like ours where demand for blood and blood components exceeds the supply.[14] There is a lack of evidence in this region having an unstable malaria epidemiology with a much lower incidence of P. falciparum malaria as compared to the national average.

Keeping in view of the above facts, the overlapping season of dengue and malaria, similar clinical presentations of these two diseases,[15] the conflicting role of thrombocytopenia in malaria pathogenesis, and the huge public health importance, this study was conducted in malaria patients from Western Rajasthan to study: (i) the prevalence of thrombocytopenia in malaria patients from this region, (ii) the degree of thrombocytopenia, (iii) the pattern and severity of thrombocytopenia in association with different Plasmodium species, and (iv) need for platelet transfusions in malaria patients.

 Materials and Methods



The present study was a retrospective observational study conducted on patients presenting to our tertiary care hospital in Western Rajasthan. Ethical clearance was obtained from the institutional ethics committee (vide AIIMS/IEC/2020-21/2026 dated May 27, 2020). The study was timed in such a manner that it covered two periods of excess rainfall in the region,[16] i.e., between August 2017 and October 2018. All the patients with a microbiologically confirmed diagnosis of malaria were selected and analyzed for inclusion and exclusion criteria. Patients with microbiologically confirmed malaria and a documented platelet count report at the time of presentation were included in the study. Patients for whom platelet count data were not available or patients with the presence of other coinfections were excluded from the study.

For microbiological diagnosis of malaria, 3 ml of whole blood samples of the patients was collected in ethylenediaminetetraacetic acid vacutainers. Two tests available for the diagnosis of malaria in our institute are immunochromatography-based rapid diagnostic tests (RDTs) for malaria (Alere Trueline™ Rapid test kit for Malaria Ag Pf/PAN, Massachusetts, USA) and microscopy/peripheral blood film (PBF) examination.

The RDT detected parasitic lactate dehydrogenase, which is genus specific and histidine-rich protein-II which is specific for P. falciparum parasite. For microbiological confirmation of malaria, all samples received in the laboratory were subjected to RDT followed by PBF examination. Giemsa stain was used to stain the PBF slides for microscopy. Hemogram testing was performed using Sysmex XN-1000 hematology analyzer (Sysmex Corporation, Kobe, Japan). If thrombocytopenia was detected by the analyzer, its verification was done using a manual method by counting platelets in a peripheral blood smear to verify the accuracy of the platelet count in such patients. Hematological parameters such as platelet count, hemoglobin levels besides reports of other available investigations such as ultrasound abdomen, renal function tests, and liver function tests were retrieved from the hospital information system of the hospital.

The first platelet count done on the initial visit of the patient was considered for documenting thrombocytopenia in the patients. The trends of subsequent platelet counts in the patients were also recorded in the data. Finally, the clinical data of the patients were accessed to see for any documented evidence of bleeding and the type of management given to the patients with thrombocytopenia.

Thrombocytopenia in the patients was graded as per the National Cancer Institute common terminology criteria for adverse events version 3.0, i.e., Grade I (75,001–149,999/μL), Grade II (50,001–75,000/μL), Grade III (25,001–50,000/μL), and Grade IV (≤25,000/μL).[17] For the statistical analysis, Grade III and IV thrombocytopenia was categorized as severe thrombocytopenia and the statistical analysis was performed using SPSS software version 23 (IBM corp., Armonk, New York, USA).

If a patient had a documented evidence of bleeding, the severity was graded as per the WHO bleeding scale, i.e., Grade 0 bleeding (no bleeding), Grade 1 bleeding (minimal, e.g., petechiae), Grade 2 bleeding (mild, e.g., visible blood in stools or urine), Grade 3 bleeding (gross, e.g., profuse bleeding in gastrointestinal tract and minor intracranial hemorrhage), and Grade 4 bleeding (debilitating or life-threatening).[18]

 Results



A total of 4152 samples were received in the laboratory, of which 153 samples were microbiologically confirmed for malaria parasite during the study period. Among these, 130 (3.7%) cases were included in the study after removing 13 cases as per the inclusion and exclusion criteria. Majority of the cases were seen in the month of September (n = 24) in the year 2017, while in the year 2018, almost equal number of cases were seen from June to September. The month-wise rainfall and number of malaria cases seen in that month are shown in [Figure 1].{Figure 1}

Of 130 cases, 94 (72%) were male and 36 (28%) were female. The mean age of the patients was 28.7 years (median: 24 years, range: 0–90 years). The majority (n = 38, 29.3%) were from the age group of 21–30 years, followed by 11–20 years (n = 35, 26.9%) [Table 1]. Of these 130 cases, 118 (91%) had P. vivax infection, whereas the rest 12 (9%) had P. falciparum infection. No case of mixed infection was diagnosed during the study period.{Table 1}

In these patients, platelet count data revealed that the mean platelet count in the patients was 92,000/μL (range: 5000–413,000/μL, median: 77,000/μL). The mean platelet count in P. vivax patients was 93,700/μL (range: 5000–413,000/μL), whereas in patients with P. falciparum infection, the mean platelet count was 75,000/μL (range: 25,000–251,000/μL). One hundred and eight (83%) patients had thrombocytopenia and these were categorized into four grades of thrombocytopenia depending on the platelet counts. Among thrombocytopenic patients, the mean values in P. vivax and P. falciparum cases were 72,600/μL and 48,500/μL, respectively. The age-wise platelet count is given in [Table 1]. The gender-wise distribution along with platelet counts in P. vivax and P. falciparum cases with the severity of thrombocytopenia is given in [Table 2].{Table 2}

The risk of severity of thrombocytopenia was assessed for the infecting species, and we found that patients with P. falciparum infection were at a significantly higher risk of developing severe thrombocytopenia (Grade 3 and 4) as compared to patients with P. vivax infection in this study (odds ratio: 4.7, [95% confidence interval (CI): 1.3–16.1]) after adjusting for age and gender. The unadjusted risk for the above parameter was also found to be significantly higher in patients with P. falciparum infection (odds ratio: 4.3 [95% CI: 1.3–14.6]).

Although the severity of thrombocytopenia was significantly higher in P. falciparum cases, the number of patients with an extremely low platelet count warranting platelet transfusions was only seen in P. vivax cases. The lowest platelet count in a P. falciparum case was 25,000/μL, while the lowest count in a P. vivax case was a mere 5000/μL. A total of 5 patients with P. vivax malaria had a platelet count of ≤20,000/μL. Platelet transfusions were given to only one of these five patients who had evidence of Grade 1 bleeding (petechiae). No other patient had a documented evidence of bleeding.

As the follow-up platelet count data were not available for all the patients, the resolution of thrombocytopenia was analyzed only for the patients with severe thrombocytopenia. Follow-up of platelet count data in this group was available for 25 of 36 patients. Among these 25 patients, eight had a platelet count between 50,000 and 100,000/μL after a median duration of 3 days, nine patients had a platelet count of 100,000–149,000/μL after a median duration of 5 days, while the rest eight patients had a platelet count of >150,000/μL after a median duration of 5 days [Table 3] and [Table 4].{Table 3}{Table 4}

 Discussion



This retrospective study conducted on patients over a period of 13 months saw a spike in number of cases following a large excess rainfall in the premonsoon season of 2017 as compared to long-term usual trends with a deficient rainfall thereafter. The year 2018 saw 79% excess rainfall in the month of June 2018 with a deficient rainfall in the rest of the months.[19] A similar spike in the number of malaria cases was seen in the subsequent months of July to September 2018. The situation is not much different from about a decade back when Lingala also found similar malaria epidemiology in this arid part of Western Rajasthan from 2009 to 2012.[1]

It is difficult to gauge the exact problem of malaria in Jodhpur based on this single-center study. Of 4152 samples that were examined for malaria by RDT and microscopy, 130 were positive, suggesting malaria prevalence of 3.7% in febrile patients. In India, an extensive malaria control program brought the number of malaria cases to a very low number by the end of the 1950s, before a re-emergence in the late 1960s that was largely attributed to the insecticide resistance in the vector, i.e., Anopheles sp. and shortage of Dichlorodiphenyltrichloroethane (DDT).[20] In 2017, there were less than 1 million reported cases of malaria in India.[21] The higher prevalence of malaria in males could be attributable to a variety of factors such as greater outdoor activity in males, custom of more clothing in females, and higher proportions of males seeking treatment in a tertiary care center.

The number of P. falciparum cases was observed to be substantially lower than the national average with not even a single case of mixed/dual infection diagnosed in our patients during the study period. P. vivax is found to be the prevalent species in this part of Rajasthan, as also suggested by Anand et al.[5] Nationwide, the proportion of P. falciparum malaria cases has increased to almost 66% of the total cases.[1]

Malaria parasite affects all components of the blood, and there are various mechanisms through which the malaria parasite affects the platelets resulting in varying degrees of thrombocytopenia in the patient. Some of these mechanisms documented in the literature include lysis of the platelets by the parasite, structural abnormalities in the platelets, or an invasion of the platelets by the parasite.[22] Recently, Punnath et al. studied the effects of inflammatory cytokines on the development of thrombocytopenia in malaria patients.[23] They found that cytokines such as interleukin (IL)-10, tumor necrosis factor (TNF)-α, and IL-6 influence the development of severe thrombocytopenia, as the patients with severe thrombocytopenia had decreased levels of IL-6 (IL-6 promotes platelet formation) and increased levels of IL-10 and TNF-α (both are known to reduce the platelet counts).

The platelets also undergo dysfunction in two stages: the first stage of platelet hyperactivity followed by platelet hypoactivity. This platelet dysfunction is transient and the two phases last from 1 to 2 weeks after which the platelet function returns to normal, along with the platelet count.[22] In a study by Moulin et al., the platelet counts in malaria patients returned to normal maximum by 28 days.[24] In our patients, subsequent platelet counts were done during follow-ups only in cases where there was a higher degree of thrombocytopenia, as discussed in the study limitations below.

In this study, the evidence of bleeding was seen in only one patient who was given platelet transfusions. No other case of malaria patients with thrombocytopenia in this study required platelet transfusions. This suggests a good tolerance to thrombocytopenia in the patients. The transfusion of platelets can be either prophylactic or therapeutic. Prophylactic transfusion is recommended below a certain platelet cutoff value to avoid the risk of bleeding or in patients with bleeding score 0 to 1[25] as per the modified WHO bleeding score, while therapeutic transfusion is given to patients with evidence of bleeding (score 2 or higher) during thrombocytopenia regardless of the platelet count, although the need of platelets is rare in a thrombocytopenic patient with a platelet count above 100,000/μL.[26]

There is no single exhaustive guideline on platelet transfusion and no guideline dealing specifically with thrombocytopenia and malaria patients. Instead, many guidelines have been published from time to time that address the need for transfusion in a specific category of patients.[25],[27],[28],[29] The WHO and NVBDCP guidelines for the management of dengue patients with thrombocytopenia advice prophylactic platelet transfusion only in patients with a platelet count of ≤10,000/μL,[30] while some recommend that the cutoff can be further lowered to 5000/μL in capable centers with good monitoring support, although a major concern reported for such low threshold is the accuracy of the automated counters used in hematology laboratories.[25]

It is pertinent to highlight that two guidelines address the transfusion needs of a large variety of clinical scenarios. A 21-member panel undertook a systematic review based on which a clinical practice guideline on prophylactic platelet transfusion was formulated by formerly American Association of Blood Banks in 2014 [Table 5].[27] This guideline also recommends prophylactic platelet transfusion in patients with hypoproliferative thrombocytopenia (Punnath et al.[23] demonstrated decreased IL-6 in malaria patients with thrombocytopenia as already described above) with a platelet count < 10,000/μL. In 2016, the British Committee for Standards in Hematology published its guidelines for the use of platelet transfusions in various clinical settings.[25]{Table 5}

There are a few limitations in the study. First, being a retrospective study, the recovery from thrombocytopenia could not be studied in all the patients in a systematic manner. A daily follow-up of platelet count is not practical in routine practice in the outpatient department setup, and moreover, the cost factor is to be taken into consideration, so follow-up platelet counts are usually done only for those patients who are at risk of bleeding due to severe thrombocytopenia. Second, the method for verification of thrombocytopenia that was followed (manual counting on peripheral blood smear) is not the accurate reference method but rather gives an approximate platelet count for the purpose of correlation with the machine count. Third, the number of P. falciparum cases included in the study was low, owing to the rainfall linked epidemic nature of malaria in this region resulting in a lesser number of cases.

This study shows that P. vivax malaria is capable of causing severe thrombocytopenia requiring platelet transfusions. However, bleeding episodes in malaria patients are very uncommon, and platelet transfusions are rarely needed. With the recent rise in dengue cases in India along with its spread to a greater geographical area, the platelet count and thrombocytopenia have received the greater focus of clinicians in the management of dengue cases, and thus, there is a need to impart similar focus on thrombocytopenia in malaria.

 Conclusions



Malaria continues to remain endemic in Jodhpur despite the relatively hot climate suggesting vector survival in a hidden niche which will require a sustained entomological and epidemiological control strategy. In times when dengue is an emerging challenge to the Indian subcontinent, with an overlapping dengue and malaria season, thrombocytopenia in a febrile patient should be regarded as an important feature of both P. falciparum and P. vivax malaria. Prompt initiation of the antimalarial therapy can help avoid unnecessary transfusions, thereby preventing serious transfusion-related infections and complications in these patients.

Acknowledgments

We express our sincere gratitude to Dr. Naveen Bansal (MD, Transfusion Medicine), ESI Hospital, Baddi, Himachal Pradesh, for providing his inputs.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest

References

1Lingala MAL. Effect of meteorological variables on Plasmodium vivax and Plasmodium falciparum malaria in outbreak prone districts of Rajasthan, India. J Infect Public Health 2017;10:875-80.
2Barber BE, Rajahram GS, Grigg MJ, William T, Anstey NM. World Malaria Report: Time to acknowledge Plasmodium knowlesi malaria. Malar J 2017;16:135.
3Trampuz A, Jereb M, Muzlovic I, Prabhu RM. Clinical review: Severe malaria. Crit Care 2003;7:315-23.
4WHO. Management of Severe Malaria – A Practical Handbook. 3rd ed. Geneva, Switzerland: WHO; 2013.
5Anand PK, Swarn L, Yadav SP, Singh H. Disease dynamics, distribution and surveillance of malaria in arid ecology of Jodhpur, Rajasthan, India during 2002 to 2006. J Public Health Epidemiol 2011;3:301-7.
6Joshi V, Adha S, Singh H, Singhi M, Dam PK. Introduction, transmission and aggravation of malaria in desert ecosystem of Rajasthan, India. J Vector Borne Dis 2006;43:179-85.
7Akhtar R, McMichael AJ. Rainfall and malaria outbreaks in Western Rajasthan. Lancet 1996;348:1457-8.
8Muley A, Lakhani J, Bhirud S, Patel A. Thrombocytopenia in Plasmodium vivax malaria: How significant? J Trop Med 2014;2014:567469.
9Saravu K, Rishikesh K, Kamath A. Determinants of mortality, intensive care requirement and prolonged hospitalization in malaria – A tertiary care hospital based cohort study from South-Western India. Malar J 2014;13:370.
10Hanson J, Phu NH, Hasan MU, Charunwatthana P, Plewes K, Maude RJ, et al. The clinical implications of thrombocytopenia in adults with severe falciparum malaria: A retrospective analysis. BMC Med 2015;13:97.
11Gahlot NK, Rozh D, Aswal VK, Sirshath KS, Agarwal M, Kochar SK. Clinicopathological manifestation of falciparum malaria in Bikaner (Northwest Rajasthan) population and proteomic investigation for identification of potential serum marker proteins. Int J Mosq Res 2017;4:01-04.
12Mutheneni SR, Morse AP, Caminade C, Upadhyayula SM. Dengue burden in India: Recent trends and importance of climatic parameters. Emerg Microbes Infect 2017;6:e70.
13Gupta P, Guddattu V, Saravu K. Characterization of platelet count and platelet indices and their potential role to predict severity in malaria. Pathog Glob Health 2019;113:86-93.
14Jenny HE, Saluja S, Sood R, Raykar N, Kataria R, Tongaonkar R, et al. Access to safe blood in low-income and middle-income countries: Lessons from India. BMJ Glob Health 2017;2:e000167.
15Epelboin L, Boullé C, Ouar-Epelboin S, Hanf M, Dussart P, Djossou F, et al. Discriminating malaria from dengue fever in endemic areas: Clinical and biological criteria, prognostic score and utility of the C-reactive protein: A retrospective matched-pair study in French Guiana. PLoS Negl Trop Dis 2013;7:e2420.
16Indian Meteorological Department of Ministry of Earth Sciences. India: Government of India; cMinistry of Earth Sciences, Customized Rainfall Information System(CRIS). Available from: http://hydro.imd.gov.in/hydrometweb/(S(cilgd545fwsnmb55kblo22id))/DistrictRaifall.aspx. [Last accessed on 2019 Sep 21.]
17National Cancer Institute Criteria for Adverse Events Version 3. Bethesda: US Department of Health and Human Services; 2006. p. 4.
18Nagrebetsky A, Al-Samkari H, Davis NM, Kuter DJ, Wiener-Kronish JP. Perioperative thrombocytopenia: Evidence, evaluation, and emerging therapies. Br J Anaesth 2019;122:19-31.
19Indian Meteorological Department of Ministry of Earth Sciences. India: Government of India; cMinistry of Earth Sciences, CRIS Rainfall Statistics of India; 2017. Available from: http://hydro.imd.gov.in/hydrometweb/(S (qwtoq0el02xlif45hvyrhijk))/PRODUCTS/Publications/Rainfall%20Statistics%20of%20India%20-%202017/Rainfall%20Statistics%20of%20India%20-%202017.pdf. [Last accessed on 2019 Sep 21].
20Sharma VP, Mehrotra KN. Malaria resurgence in India: A critical study. Soc Sci Med 1986;22:835-45.
21Ghosh SK, Rahi M. Malaria elimination in India-The way forward. J Vector Borne Dis 2019;56:32-40.
22Gill MK, Makkar M, Bhat S, Kaur T, Jain K, Dhir G. Thrombocytopenia in malaria and its correlation with different types of malaria. Ann Trop Med Public Health 2013;6:197-200.
23Punnath K, Dayanand KK, Chandrashekar VN, Achur RN, Kakkilaya SB, Ghosh SK, et al. Association between inflammatory cytokine levels and thrombocytopenia during Plasmodium falciparum and P. vivax infections in South-Western coastal region of India. Malar Res Treat 2019;2019:4296523.
24Moulin F, Lesage F, Legros AH, Maroga C, Moussavou A, Guyon P, et al. Thrombocytopenia and Plasmodium falciparum malaria in children with different exposures. Arch Dis Child 2003;88:540-1.
25Estcourt LJ, Birchall J, Allard S, Bassey SJ, Hersey P, Kerr JP, et al. Guidelines for the use of platelet transfusions. Br J Haematol 2017;176:365-94.
26Yaddanapudi S, Yaddanapudi L. Indications for blood and blood product transfusion. Indian J Anaesth 2014;58:538-42.
27Arya RC, Wander G, Gupta P. Blood component therapy: Which, when and how much. J Anaesthesiol Clin Pharmacol 2011;27:278-84.
28Kaufman RM, Djulbegovic B, Gernsheimer T, Kleinman S, Tinmouth AT, Capocelli KE, et al. Platelet transfusion: A clinical practice guideline from the AABB. Ann Intern Med 2015;162:205-13.
29National Blood Authority. Australia: The Authority; cCommon Wealth of Australia. The Patient Blood Management Guidelines Companions. Available from: https://www.blood.gov.au/patient-blood-management-guidelines-companions. [Last accessed on 2020 Feb 02].
30World Health Organization. Country Office for India: The Organization; cWHO 2020. National Guidelines for Clinical Management of Dengue Fever. World Health Organization; 2015. Available from: https://apps.who.int/iris/handle/10665/208893. [Last accessed on 2020 Feb 02].