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Microparticles as prognostic biomarkers in dengue virus infection
Acta Tropica 181 (2018) 21–24
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Microparticles as prognostic biomarkers in dengue virus infection a
b
c
Rucha Patil , Smrati Bajpai , Kanjaksha Ghosh , Shrimati Shetty a b c
a,⁎
T
National Institute of Immunohaematology (ICMR), 13th Floor, KEM Hospital, Parel, Mumbai 400 012, India Dept. of Medicine, Seth GS Medical College and KEM Hospital, Parel, Mumbai 400012, India Surat Raktadan Kendra & Research Centre Regional Blood Transfusion Centre, Udhana – Magdalla Road, Surat 395 002, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Dengue Cell-Derived microparticles Red blood cells Platelets Biomarkers
Promising biomarkers which may help predict the risk of developing severe dengue virus infection (DVI) are lacking and will be helpful. Thus the main aim of this study was to analyze the role of cell-derived microparticles (MP) in DVI. Sixty patients with DVI i.e. 18: dengue with warning signs (DWS); 1: DSS and 41: dengue without warning signs (DWOS); along with 15 controls (other febrile illness) were included in the study. The following MPs were assessed: annexinV, platelet (CD41a), red blood cell (RBC) (CD235a) and activated endothelial (CD62e) MPs. Patients with profound thrombocytopenia without bleeding had statistically elevated platelet MP (PMP) levels when compared to patients with profound thrombocytopenia with bleeding (p < .001). RBC MPs were found to be significantly elevated in the 2nd phase in patient with DWS which was seen earliest on day 4 of infection with a cut off of ≥2200 MPs/μl when compared to patients with DWOS (p < .0001). PMPs may prove to be a promising novel biomarker which helps discriminate patients in need of prophylactic platelet transfusion from those who do not. RBC MPs, on the other hand could be potential biomarkers capable of identifying potentially severe patients who require immediate care. Thus, MPs seem to be a promising important biomarker in many aspects of DVI.
1. Introduction Dengue is an infectious, flu- like systemic disease transmitted by Aedes mosquitoes to humans. Across the world, approximately 3.6 billion people are at risk of getting infected with dengue virus infection (DVI); 50 million being infected per year globally (Bhatt et al., 2013) with 34% being contributed by India (Chakravarti et al., 2012). From 2006 to 2012, an average of six million individuals per year in India had symptomatic illness with DVI; many cases go unreported (Shepard et al., 2014). DVI manifests as a spectrum of illnesses ranging from mild febrile illness to the severe forms, i.e. dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). According to the recent WHO guidelines, cases are now classified as dengue without warning signs (DWOS), dengue with warning signs (DWS) and severe dengue (World Health Organization, 2009). There are no vaccines or specific therapeutics currently available. Rapid diagnosis along with appropriate immediate fluid replacement is what is essential to achieve success in these cases. Promising biomarkers which may help predict the risk of developing severe DVI are lacking. Microparticles (MPs) are small (0.1–1 μm) cell-derived phospholipid membrane vesicles produced either spontaneously or in response to
⁎
various stimuli such as cell activation, apoptosis or stress (Piccin et al., 2007). There are very limited studies on the role of MPs in DVI (Hottz et al., 2013; Punyadee et al., 2015), suggesting that MPs may act as promising biomarkers for predicting the severity. In India, till date there are no studies on the role of MPs in DVI. 2. Material and methods 2.1. Ethics approval The study was approved by the Institutional Ethics Committee Review Board i.e. “Institutional Committee for Research on Human Subjects, National Institute of Immunohaematology (ICMR)”. A written informed consent was obtained from all participants and all investigations were conducted according to the principles expressed in the Declaration of Helsinki. 2.2. Patients and controls During the monsoon season from July 2016 to October 2016, patients with fever visiting the Department of Medicine, KEM Hospital are generally tested for a panel of infections by the clinicians. Being a
Corresponding author at: Department of Haemostasis and Thrombosis, National Institute of Immunohaematology (ICMR), KEM Hospital, Parel, Mumbai, India. E-mail address: [email protected] (S. Shetty).
https://doi.org/10.1016/j.actatropica.2018.01.017 Received 8 November 2017; Received in revised form 23 January 2018; Accepted 23 January 2018 0001-706X/ © 2018 Published by Elsevier B.V.
Acta Tropica 181 (2018) 21–24
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of patients with DWOS were 95500, 60000 and 46000; DWS were 92000, 44000 and 20000 and for 1 patient with DSS was 40000, 40000 and 29000 platelet/μl respectively. D Dimer test was positive in 35 patients on Day 6–7; 11 were strong positive (5 DWS, 1 DSS, and 5 DWOS). Both platelet count and D Dimer tests showed no statistical correlation to severity.
preliminary study, 60 DVI patients who tested positive for both- specific IgG and IgM capture ELISA and dengue NS1 antigen test were included in the study. Controls were 15 patients showing symptoms but tested negative for DVI, i.e. other febrile illness. The day of fever was calculated according to the onset of fever and not from the day of enrollment in the hospital. The main aim was to analyze the role played by circulating microparticles in DVI. The normal ranges of different MP levels in healthy subjects were assessed in our earlier studies (Patil et al., 2013).
3.2. Platelet MPs PMP levels in majority of our patients were found to be lower (536 ± 111 PMPs/μl) than that observed in healthy subjects (731.5 ± 377.3 PMPs/μl; cut off: > 1486 PMPs/μl) (Patil et al., 2013). Profound thrombocytopenia was seen in 13 patients out of whom 8 were without bleeding manifestations. Profound thrombocytopenia patients had a platelet count < 20000, range: 4000–20000 platelets/μl. Patients with profound thrombocytopenia with no bleeding manifestations had statistically elevated PMPs (p < .001, 95% CI: 922.53–2909.12) i.e. mean ± SD = 2270.6 ± 990.1 PMPs/μl when compared to those of the patients with profound thrombocytopenia having bleeding manifestations (mean ± SD = 354. 8 ± 88.3 PMPs/ μl) (Fig. 1). Patients with no bleeding symptoms had either elevated PMPs or those with very low PMPs had a moderate platelet levels as seen in Fig. 1.
2.3. Study design Citrate blood samples were collected at least twice as the infection progressed, either in febrile phase which is from Day 1–3, critical phase: Day 4–7 or recovery phase: ≥8 days. MP assessment by flow cytometry (standardized by participating in the International Society of Thrombosis and Haemostasis workshop) was carried out as described earlier (Patil et al., 2013). In short, Megamix beads (Biocytex, Marseille, France) which contains 0.5, 0.9 and 3 μm beads are used so as to make the microparticle gate on forward scatter (FSC) versus side scatter (SSC) graph which includes all events 0.9 μm and below and excludes all events 1 μm and above as seen in our earlier study (Patil et al., 2013). AnnexinV, platelet derived, red blood cell (RBC) and activated endothelial MPs were assessed using fluorescein isothiocyanate (FITC) labeled annexin V, phycoerythrin (PE) labeled CD 41 (CD41-PE, IgG1, K, clone HIP8), PE labeled CD235a (CD 235a-PE, IgG2b, K, clone GA-R2 (HIR2)) and PE labeled CD62e (CD 62e- PE, IgG1, K, clone 68-5H11) respectively. All the antibodies and buffers were provided by BD Biosciences, San Jose, CA. Cell blood count (CBC) and Di-dimer test by latex agglutination technique, were also performed.
3.3. Red blood cell MPs RBC MPs were statistically high in patients with DWS (Cut off: > 2200 MPs/μl) (p < .0001, 95% CI: 2946–4706) in the 2nd phase when compared to patients with DWOS (< 1100 MPs/μl) (normal range: 211–355 MPs/μl; cut off: > 427 MPs/μl) (Patil et al., 2013). This is seen earliest on Day 4 of infection. The MP profile of our controls was similar to that of DWOS. The scatter plot for the same is shown in Fig. 2. In DWS patients, no matter how high the MPs rise, they gradually decrease as infection progresses and normalize as the patient’s health improves. However, in the one case of DSS, the high levels of RBC MPs elevated even further from 5549 to 7414 RBC MPs/μl (Fig. 2). This patient expired 2 days after the last sample.
2.4. Flow cytometry analysis of MPs For absolute counts i.e. absolute numbers MPs per microliter of plasma, 30 μl of Flow CountTM fluorospheres (Beckman-Coulter, Marseille, France) is added to each tube. The total no. of MPs is calculated using the formula: Microparticles/μl = Events in microparticle gate*[C/Flow count bead events]; C = Flow count Fluospheres assayed concentrations that are provided by the manufacturer as shown in previous study (Patil et al., 2013).
3.4. Activated endothelial cell derived MPs No statistical significant difference was observed in different severities.
2.5. Statistical analysis
4. Discussion
Elevated MP levels were defined as levels > 2 standard deviations (SD) from the mean of controls. MP levels were of the different groups were also compared versus controls using the 2 tailed Student’s t-test. Statistical significance was assumed at p < .05, 95% confidence interval (CI). Data was analyzed using SPSS statistical software.
This is the first report on the role of cell-derived MPs in DVI from India and there are only two such studies worldwide. One study showed that MPs harbored a viral envelope protein, NS1 protein on their surfaces and elevated RBC MPs directly correlated with disease severity, differentiating DHF from classical DVI (Punyadee et al., 2015). Another study showed increased expression of IL-1β in platelets and PMPs which enhances endothelial permeability in vitro (Hottz et al., 2013). In our study, we had few patients with profound thrombocytopenia; generally if they have bleeding manifestations, platelet transfusion is recommended. However, for patients with thrombocytopenia without bleeding having a platelet count of < 20000 platelets/μl, the clinicians are in a dilemma whether prophylactic platelet transfusion should be given or not. We observed that patients with thrombocytopenia without bleeding had statistically elevated PMP levels. A low platelet count is known to cause bleeding manifestations. PMPs which are known to be procoagulant are derived from activated platelets, carrying all the surface antigens of platelets and thus functioning like platelets. It is possible that platelet MPs support stable hemostatic function in these patients. Thus such patients with a low platelet count but elevated PMP levels may be protected and not suffer from hemorrhagic manifestations
3. Results 3.1. Characteristics of patients and controls The age of the patients ranged from 10 to 32 years; (mean = 24 years). The patients were classified according to WHO 2009 guidelines (World Health Organization, 2009) as ■ 41 DWOS ■ 18 DWS ■ 1 DSS The characteristics of the patients and controls are given in Table 1. The platelet counts in our patients ranged from 4000 to 180,000 platelets/μl. Thrombocytopenia was seen in all DWS as well as few DWS patients. The Median platelet counts on Day 1–3, Day 4–7 and Day > 8 22
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R. Patil et al.
Table 1 Characteristics of patients and controls. Characteristics
DWOS (n = 41)
DWS (n = 18)
DSS (n = 1)
Controls (n = 15)
Sex
Male: 30 Female: 11 23.3 ± 8.1 Day 1–3: 95500 (43750–142500) Day 4–7: 60000 (37250–102500) Day > 8: 46000 (20000–105750) 7 (17%) 10 (24.4%) 5 (12.2%) 29 (70.7%) 8 (19.5%) 8 (19.5%)
Male: 13 Female: 5 26.9 ± 8.3 Day 1–3: 92000 (51000–100000) Day 4–7: 44000 (40000–49000) Day > 8: 20000 (19500–24500) 11 (61.1%) 7 (38.8%) 8 (44.4%) 16 (88.8%) 5 (27.7%) 10 (55.5%)
Male: 1 Female: 0 24 Day1–3: 40000
Male: 9 Female: 6 27 ± 6.9 Day1–3: 99800 (45500–125000)
Day 4–7: 40000
Day 4–7: 87000 (55000–100000)
Day > 8: 29000
Day > 8: 112000 (87000–155000) 0 (0)
Age (Mean ± SD) (Years) Platelet count (platelets/μl) Median (Interquatile Range)
Mucosal bleed Hypotension Liver enlargement Decreased platelet count (< 150000/μl) Profound thrombocytopenia (< 20000/μl) Organ impairment
1(100%) 1(100%) 1 (100%) 1 (100%) 0 (0) 1 (100%)
0 (0) 11 (73.3%) 0 (0) 0 (0)
Fig. 1. Relationship between platelet microparticles and platelet counts with bleeding manifestation in dengue infected patients. The scatter plot displays the PMP levels and platelet counts of dengue infected patients. These patients have been divided into those with bleeding manifestations and those without bleeding manifestations. Patients with bleeding manifestations have low PMPs and profound thrombocytopenia. The black dotted squares show that patients with profound thrombocytopenia but who do not have bleeding manifestations have highly elevated PMP levels. The green dotted squares show that patients without bleeding manifestations with very low PMP levels have moderate platelet counts. PMP: platelet microparticles.
infection. Thus patients with levels > 2200 MPs/μl may be considered at a risk of developing severe dengue. The case of DSS is interesting and novel highlighting that MPs may also be used to monitor the progress of the patient, with the main aim being that the levels should normalize. Elevated PMPs may be due to the immune-mediated peripheral platelet destruction. One of RBCs important role is trapping potentially harmful immune complexes (IC) which is accomplished by immune adherence which involves complement opsonisation followed by binding to complement receptor 1 which in expressed on the surface of
and thus will not require platelet transfusion. Punyadee et al. (2015) found a significant decrease in PMPs was associated with bleeding tendency. Thus, elevated procoagulant PMPs may act as saviours performing the necessary functions of platelets thus preventing bleeding symptoms. Thus PMPs may be used as an additional marker along with platelet count and hemorrhagic manifestations to aptly assess and discriminate patients requiring prophylactic platelet transfusion. We found elevated RBC MPs to be a potential novel biomarker capable of identifying severe patients; seen earliest by day 4 of
Fig. 2. Red blood cell microparticles in patients with different severities of dengue fever and controls. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) As seen in the scatter plots the RBC MPs in the 2nd phase, day 4–7 are significantly increased in patients with DWS when compared to DWOS and controls. In the patient with DSS (yellow), the elevated MPs exaggerate even more as infection progressed; the patient expired 2 days after the last sample was collected. DWOS: dengue without warning symptoms; DWS: dengue with warning symptoms; DSS: dengue shock syndrome; MP: microparticles.
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Acta Tropica 181 (2018) 21–24
R. Patil et al.
Funding statement
RBCs (Davies et al., 1990; Davies et al., 1992; Taylor et al., 1997). This RBC-IC complex are then transferred to liver and spleen phagocytic cells (Davies et al., 1990; Davies et al., 1992) which thus eventually leads to RBC shedding into RBC MPs. Elevated circulating ICs have been observed in acute phase of DVI. Thus, one possible reason for the increased RBC MPs may be because of the increased RBC shedding that occurs due to overburden on RBC to trap high levels of potentially harmful ICs produced in patients with DVI. There are in-vitro studies supporting this hypothesis (Punyadee et al., 2015). Though a preliminary study with a small sample size, in the search for potential biomarkers to prognosticate the severity in patients with DVI, cell-derived MPs may prove to be valuable. PMPs maybe promising novel biomarkers which may help discriminate the patients in need of prophylactic platelet transfusion. RBC MPs, on the other hand could be potential biomarkers capable of identifying potentially severe patients who require immediate care. Thus, different cell-derived MPs seem to be important biomarkers in different aspects of the infection like PMPS in depicting the seriousness of profound thrombocytopenia or RBC MPs in predicting the patients who are at a risk of developing severe DVI. In countries like India having a large load of this infection, such studies may prove to be beneficial. It would also be interesting to measure the levels of RBC MPs in paired convalescent-phase samples obtained at 2 weeks and 2 months after defervescence. This result will be useful to verify the important roles of RBC MPs in DVI. Thus larger and well-designed studies are required to confirm and validate these preliminary findings.
This research was funded by Indian Council of Medical Research (ICMR) (Reference No.: ICMR letter No. 3/1/3/PDF (12)/2015-HRD12TH Batch). References Bhatt, S., Gething, P.W., Brady, O.J., Messina, O.J., Farlow, A.W., Moyes, C.L., Drake, J.M., Brownstein, J.S., Hoen, A.G., Sankoh, O., Myers, M.F., George, D.B., Jaenisch, T., Wint, G.R., Simmons, C.P., Scott, T.W., Farrar, J.J., Hay, S.I., 2013. The global distribution and burden of dengue. Nature 496, 504–507. Chakravarti, A., Arora, R., Luxemburger, C., 2012. Fifty years of dengue in India. Trans. R. Soc. Trop. Med. Hyg. 106, 273–282. Davies, K.A., Hird, V., Stewart, S., Sivolapenko, G.B., Jose, P., Epenetos, A.A., Walport, M.J., 1990. A study of in vivo immune complex formation and clearance in man. J. Immunol. 144, 4613–4620. Davies, K.A., Peters, A.M., Beynon, H.L., Walport, M.J., 1992. Immune complex processing in patients with systemic lupus erythematosus. In vivo imaging and clearance studies. J. Clin. Invest. 90, 2075–2083. Hottz, E.D., Lopes, J.F., Freitas, C., Valls-de-Souza, R., Oliveira, M.F., Bozza, M.T., Da Poian, A.T., Weyrich, A.S., Zimmerman, G.A., Bozza, F.A., Bozza, P.T., 2013. Platelets mediate increased endothelium permeability in dengue through NLRP3-inflammasome activation. Blood 122, 3405–3414. Patil, R., Ghosh, K., Satoskar, P., Shetty, S., 2013. Elevated procoagulant endothelial and tissue factor expressing microparticles in women with recurrent pregnancy loss. PLoS One 20, e81407. Piccin, A., Murphy, W.G., Smith, O.P., 2007. Circulating microparticles: pathophysiology and clinical implications. Blood. Rev. 21, 157–171. Punyadee, N., Mairiang, D., Thiemmeca, S., Komoltri, C., Pan-Ngum, W., Chomanee, N., Charngkaew, K., Tangthawornchaikul, N., Limpitikul, W., Vasanawathana, S., Malasit, P., Avirutnan, P., 2015. Microparticles provide a novel biomarker to predict severe clinical outcomes of dengue virus infection. J. Virol. 89, 1587–1607. Shepard, D.S., Halasa, Y.A., Tyagi, B.K., Adhish, S.V., Nandan, D., Karthiga, K.S., Chellaswamy, V., Gaba, M., Arora, N.K., INCLEN. Study Group, 2014. Economic and disease burden of dengue illness in India. Am. J. Trop. Med. Hyg. 91, 1235–1242. Taylor, R.P., Ferguson, P.J., Martin, E.N., Cooke, J., Greene, K.L., Grinspun, K., Guttman, M., Kuhn, S., 1997. Immune complexes bound to the primate erythrocyte complement receptor (CR1) via anti-CR1 mAbs are cleared simultaneously with loss of CR1 in a concerted reaction in a rhesus monkey model. Clin. Immunol. Immunopathol. 82, 49–59. World Health Organization, 2009. Dengue: Guidelines for Diagnosis, Treatment, Prevention and Control. WHO, Geneva, Switzerland.
Author contribution statement Rucha Patil, Shrimati Shetty & Kanjaksha Ghosh conceived and designed the experiments. Rucha Patil performed the experiments, analyzed the data and wrote the manuscript. The clinical evaluation was done by Smrati Bajpai and Kanjaksha Ghosh. Critical revision of manuscript for important intellectual content and final approval was done by Shrimati Shetty. Conflict of interest disclosure The authors declare no competing financial interests.
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Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Microparticles as prognostic biomarkers in dengue virus infection a
b
c
Rucha Patil , Smrati Bajpai , Kanjaksha Ghosh , Shrimati Shetty a b c
a,⁎
T
National Institute of Immunohaematology (ICMR), 13th Floor, KEM Hospital, Parel, Mumbai 400 012, India Dept. of Medicine, Seth GS Medical College and KEM Hospital, Parel, Mumbai 400012, India Surat Raktadan Kendra & Research Centre Regional Blood Transfusion Centre, Udhana – Magdalla Road, Surat 395 002, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Dengue Cell-Derived microparticles Red blood cells Platelets Biomarkers
Promising biomarkers which may help predict the risk of developing severe dengue virus infection (DVI) are lacking and will be helpful. Thus the main aim of this study was to analyze the role of cell-derived microparticles (MP) in DVI. Sixty patients with DVI i.e. 18: dengue with warning signs (DWS); 1: DSS and 41: dengue without warning signs (DWOS); along with 15 controls (other febrile illness) were included in the study. The following MPs were assessed: annexinV, platelet (CD41a), red blood cell (RBC) (CD235a) and activated endothelial (CD62e) MPs. Patients with profound thrombocytopenia without bleeding had statistically elevated platelet MP (PMP) levels when compared to patients with profound thrombocytopenia with bleeding (p < .001). RBC MPs were found to be significantly elevated in the 2nd phase in patient with DWS which was seen earliest on day 4 of infection with a cut off of ≥2200 MPs/μl when compared to patients with DWOS (p < .0001). PMPs may prove to be a promising novel biomarker which helps discriminate patients in need of prophylactic platelet transfusion from those who do not. RBC MPs, on the other hand could be potential biomarkers capable of identifying potentially severe patients who require immediate care. Thus, MPs seem to be a promising important biomarker in many aspects of DVI.
1. Introduction Dengue is an infectious, flu- like systemic disease transmitted by Aedes mosquitoes to humans. Across the world, approximately 3.6 billion people are at risk of getting infected with dengue virus infection (DVI); 50 million being infected per year globally (Bhatt et al., 2013) with 34% being contributed by India (Chakravarti et al., 2012). From 2006 to 2012, an average of six million individuals per year in India had symptomatic illness with DVI; many cases go unreported (Shepard et al., 2014). DVI manifests as a spectrum of illnesses ranging from mild febrile illness to the severe forms, i.e. dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). According to the recent WHO guidelines, cases are now classified as dengue without warning signs (DWOS), dengue with warning signs (DWS) and severe dengue (World Health Organization, 2009). There are no vaccines or specific therapeutics currently available. Rapid diagnosis along with appropriate immediate fluid replacement is what is essential to achieve success in these cases. Promising biomarkers which may help predict the risk of developing severe DVI are lacking. Microparticles (MPs) are small (0.1–1 μm) cell-derived phospholipid membrane vesicles produced either spontaneously or in response to
⁎
various stimuli such as cell activation, apoptosis or stress (Piccin et al., 2007). There are very limited studies on the role of MPs in DVI (Hottz et al., 2013; Punyadee et al., 2015), suggesting that MPs may act as promising biomarkers for predicting the severity. In India, till date there are no studies on the role of MPs in DVI. 2. Material and methods 2.1. Ethics approval The study was approved by the Institutional Ethics Committee Review Board i.e. “Institutional Committee for Research on Human Subjects, National Institute of Immunohaematology (ICMR)”. A written informed consent was obtained from all participants and all investigations were conducted according to the principles expressed in the Declaration of Helsinki. 2.2. Patients and controls During the monsoon season from July 2016 to October 2016, patients with fever visiting the Department of Medicine, KEM Hospital are generally tested for a panel of infections by the clinicians. Being a
Corresponding author at: Department of Haemostasis and Thrombosis, National Institute of Immunohaematology (ICMR), KEM Hospital, Parel, Mumbai, India. E-mail address: [email protected] (S. Shetty).
https://doi.org/10.1016/j.actatropica.2018.01.017 Received 8 November 2017; Received in revised form 23 January 2018; Accepted 23 January 2018 0001-706X/ © 2018 Published by Elsevier B.V.
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R. Patil et al.
of patients with DWOS were 95500, 60000 and 46000; DWS were 92000, 44000 and 20000 and for 1 patient with DSS was 40000, 40000 and 29000 platelet/μl respectively. D Dimer test was positive in 35 patients on Day 6–7; 11 were strong positive (5 DWS, 1 DSS, and 5 DWOS). Both platelet count and D Dimer tests showed no statistical correlation to severity.
preliminary study, 60 DVI patients who tested positive for both- specific IgG and IgM capture ELISA and dengue NS1 antigen test were included in the study. Controls were 15 patients showing symptoms but tested negative for DVI, i.e. other febrile illness. The day of fever was calculated according to the onset of fever and not from the day of enrollment in the hospital. The main aim was to analyze the role played by circulating microparticles in DVI. The normal ranges of different MP levels in healthy subjects were assessed in our earlier studies (Patil et al., 2013).
3.2. Platelet MPs PMP levels in majority of our patients were found to be lower (536 ± 111 PMPs/μl) than that observed in healthy subjects (731.5 ± 377.3 PMPs/μl; cut off: > 1486 PMPs/μl) (Patil et al., 2013). Profound thrombocytopenia was seen in 13 patients out of whom 8 were without bleeding manifestations. Profound thrombocytopenia patients had a platelet count < 20000, range: 4000–20000 platelets/μl. Patients with profound thrombocytopenia with no bleeding manifestations had statistically elevated PMPs (p < .001, 95% CI: 922.53–2909.12) i.e. mean ± SD = 2270.6 ± 990.1 PMPs/μl when compared to those of the patients with profound thrombocytopenia having bleeding manifestations (mean ± SD = 354. 8 ± 88.3 PMPs/ μl) (Fig. 1). Patients with no bleeding symptoms had either elevated PMPs or those with very low PMPs had a moderate platelet levels as seen in Fig. 1.
2.3. Study design Citrate blood samples were collected at least twice as the infection progressed, either in febrile phase which is from Day 1–3, critical phase: Day 4–7 or recovery phase: ≥8 days. MP assessment by flow cytometry (standardized by participating in the International Society of Thrombosis and Haemostasis workshop) was carried out as described earlier (Patil et al., 2013). In short, Megamix beads (Biocytex, Marseille, France) which contains 0.5, 0.9 and 3 μm beads are used so as to make the microparticle gate on forward scatter (FSC) versus side scatter (SSC) graph which includes all events 0.9 μm and below and excludes all events 1 μm and above as seen in our earlier study (Patil et al., 2013). AnnexinV, platelet derived, red blood cell (RBC) and activated endothelial MPs were assessed using fluorescein isothiocyanate (FITC) labeled annexin V, phycoerythrin (PE) labeled CD 41 (CD41-PE, IgG1, K, clone HIP8), PE labeled CD235a (CD 235a-PE, IgG2b, K, clone GA-R2 (HIR2)) and PE labeled CD62e (CD 62e- PE, IgG1, K, clone 68-5H11) respectively. All the antibodies and buffers were provided by BD Biosciences, San Jose, CA. Cell blood count (CBC) and Di-dimer test by latex agglutination technique, were also performed.
3.3. Red blood cell MPs RBC MPs were statistically high in patients with DWS (Cut off: > 2200 MPs/μl) (p < .0001, 95% CI: 2946–4706) in the 2nd phase when compared to patients with DWOS (< 1100 MPs/μl) (normal range: 211–355 MPs/μl; cut off: > 427 MPs/μl) (Patil et al., 2013). This is seen earliest on Day 4 of infection. The MP profile of our controls was similar to that of DWOS. The scatter plot for the same is shown in Fig. 2. In DWS patients, no matter how high the MPs rise, they gradually decrease as infection progresses and normalize as the patient’s health improves. However, in the one case of DSS, the high levels of RBC MPs elevated even further from 5549 to 7414 RBC MPs/μl (Fig. 2). This patient expired 2 days after the last sample.
2.4. Flow cytometry analysis of MPs For absolute counts i.e. absolute numbers MPs per microliter of plasma, 30 μl of Flow CountTM fluorospheres (Beckman-Coulter, Marseille, France) is added to each tube. The total no. of MPs is calculated using the formula: Microparticles/μl = Events in microparticle gate*[C/Flow count bead events]; C = Flow count Fluospheres assayed concentrations that are provided by the manufacturer as shown in previous study (Patil et al., 2013).
3.4. Activated endothelial cell derived MPs No statistical significant difference was observed in different severities.
2.5. Statistical analysis
4. Discussion
Elevated MP levels were defined as levels > 2 standard deviations (SD) from the mean of controls. MP levels were of the different groups were also compared versus controls using the 2 tailed Student’s t-test. Statistical significance was assumed at p < .05, 95% confidence interval (CI). Data was analyzed using SPSS statistical software.
This is the first report on the role of cell-derived MPs in DVI from India and there are only two such studies worldwide. One study showed that MPs harbored a viral envelope protein, NS1 protein on their surfaces and elevated RBC MPs directly correlated with disease severity, differentiating DHF from classical DVI (Punyadee et al., 2015). Another study showed increased expression of IL-1β in platelets and PMPs which enhances endothelial permeability in vitro (Hottz et al., 2013). In our study, we had few patients with profound thrombocytopenia; generally if they have bleeding manifestations, platelet transfusion is recommended. However, for patients with thrombocytopenia without bleeding having a platelet count of < 20000 platelets/μl, the clinicians are in a dilemma whether prophylactic platelet transfusion should be given or not. We observed that patients with thrombocytopenia without bleeding had statistically elevated PMP levels. A low platelet count is known to cause bleeding manifestations. PMPs which are known to be procoagulant are derived from activated platelets, carrying all the surface antigens of platelets and thus functioning like platelets. It is possible that platelet MPs support stable hemostatic function in these patients. Thus such patients with a low platelet count but elevated PMP levels may be protected and not suffer from hemorrhagic manifestations
3. Results 3.1. Characteristics of patients and controls The age of the patients ranged from 10 to 32 years; (mean = 24 years). The patients were classified according to WHO 2009 guidelines (World Health Organization, 2009) as ■ 41 DWOS ■ 18 DWS ■ 1 DSS The characteristics of the patients and controls are given in Table 1. The platelet counts in our patients ranged from 4000 to 180,000 platelets/μl. Thrombocytopenia was seen in all DWS as well as few DWS patients. The Median platelet counts on Day 1–3, Day 4–7 and Day > 8 22
Acta Tropica 181 (2018) 21–24
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Table 1 Characteristics of patients and controls. Characteristics
DWOS (n = 41)
DWS (n = 18)
DSS (n = 1)
Controls (n = 15)
Sex
Male: 30 Female: 11 23.3 ± 8.1 Day 1–3: 95500 (43750–142500) Day 4–7: 60000 (37250–102500) Day > 8: 46000 (20000–105750) 7 (17%) 10 (24.4%) 5 (12.2%) 29 (70.7%) 8 (19.5%) 8 (19.5%)
Male: 13 Female: 5 26.9 ± 8.3 Day 1–3: 92000 (51000–100000) Day 4–7: 44000 (40000–49000) Day > 8: 20000 (19500–24500) 11 (61.1%) 7 (38.8%) 8 (44.4%) 16 (88.8%) 5 (27.7%) 10 (55.5%)
Male: 1 Female: 0 24 Day1–3: 40000
Male: 9 Female: 6 27 ± 6.9 Day1–3: 99800 (45500–125000)
Day 4–7: 40000
Day 4–7: 87000 (55000–100000)
Day > 8: 29000
Day > 8: 112000 (87000–155000) 0 (0)
Age (Mean ± SD) (Years) Platelet count (platelets/μl) Median (Interquatile Range)
Mucosal bleed Hypotension Liver enlargement Decreased platelet count (< 150000/μl) Profound thrombocytopenia (< 20000/μl) Organ impairment
1(100%) 1(100%) 1 (100%) 1 (100%) 0 (0) 1 (100%)
0 (0) 11 (73.3%) 0 (0) 0 (0)
Fig. 1. Relationship between platelet microparticles and platelet counts with bleeding manifestation in dengue infected patients. The scatter plot displays the PMP levels and platelet counts of dengue infected patients. These patients have been divided into those with bleeding manifestations and those without bleeding manifestations. Patients with bleeding manifestations have low PMPs and profound thrombocytopenia. The black dotted squares show that patients with profound thrombocytopenia but who do not have bleeding manifestations have highly elevated PMP levels. The green dotted squares show that patients without bleeding manifestations with very low PMP levels have moderate platelet counts. PMP: platelet microparticles.
infection. Thus patients with levels > 2200 MPs/μl may be considered at a risk of developing severe dengue. The case of DSS is interesting and novel highlighting that MPs may also be used to monitor the progress of the patient, with the main aim being that the levels should normalize. Elevated PMPs may be due to the immune-mediated peripheral platelet destruction. One of RBCs important role is trapping potentially harmful immune complexes (IC) which is accomplished by immune adherence which involves complement opsonisation followed by binding to complement receptor 1 which in expressed on the surface of
and thus will not require platelet transfusion. Punyadee et al. (2015) found a significant decrease in PMPs was associated with bleeding tendency. Thus, elevated procoagulant PMPs may act as saviours performing the necessary functions of platelets thus preventing bleeding symptoms. Thus PMPs may be used as an additional marker along with platelet count and hemorrhagic manifestations to aptly assess and discriminate patients requiring prophylactic platelet transfusion. We found elevated RBC MPs to be a potential novel biomarker capable of identifying severe patients; seen earliest by day 4 of
Fig. 2. Red blood cell microparticles in patients with different severities of dengue fever and controls. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) As seen in the scatter plots the RBC MPs in the 2nd phase, day 4–7 are significantly increased in patients with DWS when compared to DWOS and controls. In the patient with DSS (yellow), the elevated MPs exaggerate even more as infection progressed; the patient expired 2 days after the last sample was collected. DWOS: dengue without warning symptoms; DWS: dengue with warning symptoms; DSS: dengue shock syndrome; MP: microparticles.
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Funding statement
RBCs (Davies et al., 1990; Davies et al., 1992; Taylor et al., 1997). This RBC-IC complex are then transferred to liver and spleen phagocytic cells (Davies et al., 1990; Davies et al., 1992) which thus eventually leads to RBC shedding into RBC MPs. Elevated circulating ICs have been observed in acute phase of DVI. Thus, one possible reason for the increased RBC MPs may be because of the increased RBC shedding that occurs due to overburden on RBC to trap high levels of potentially harmful ICs produced in patients with DVI. There are in-vitro studies supporting this hypothesis (Punyadee et al., 2015). Though a preliminary study with a small sample size, in the search for potential biomarkers to prognosticate the severity in patients with DVI, cell-derived MPs may prove to be valuable. PMPs maybe promising novel biomarkers which may help discriminate the patients in need of prophylactic platelet transfusion. RBC MPs, on the other hand could be potential biomarkers capable of identifying potentially severe patients who require immediate care. Thus, different cell-derived MPs seem to be important biomarkers in different aspects of the infection like PMPS in depicting the seriousness of profound thrombocytopenia or RBC MPs in predicting the patients who are at a risk of developing severe DVI. In countries like India having a large load of this infection, such studies may prove to be beneficial. It would also be interesting to measure the levels of RBC MPs in paired convalescent-phase samples obtained at 2 weeks and 2 months after defervescence. This result will be useful to verify the important roles of RBC MPs in DVI. Thus larger and well-designed studies are required to confirm and validate these preliminary findings.
This research was funded by Indian Council of Medical Research (ICMR) (Reference No.: ICMR letter No. 3/1/3/PDF (12)/2015-HRD12TH Batch). References Bhatt, S., Gething, P.W., Brady, O.J., Messina, O.J., Farlow, A.W., Moyes, C.L., Drake, J.M., Brownstein, J.S., Hoen, A.G., Sankoh, O., Myers, M.F., George, D.B., Jaenisch, T., Wint, G.R., Simmons, C.P., Scott, T.W., Farrar, J.J., Hay, S.I., 2013. The global distribution and burden of dengue. Nature 496, 504–507. Chakravarti, A., Arora, R., Luxemburger, C., 2012. Fifty years of dengue in India. Trans. R. Soc. Trop. Med. Hyg. 106, 273–282. Davies, K.A., Hird, V., Stewart, S., Sivolapenko, G.B., Jose, P., Epenetos, A.A., Walport, M.J., 1990. A study of in vivo immune complex formation and clearance in man. J. Immunol. 144, 4613–4620. Davies, K.A., Peters, A.M., Beynon, H.L., Walport, M.J., 1992. Immune complex processing in patients with systemic lupus erythematosus. In vivo imaging and clearance studies. J. Clin. Invest. 90, 2075–2083. Hottz, E.D., Lopes, J.F., Freitas, C., Valls-de-Souza, R., Oliveira, M.F., Bozza, M.T., Da Poian, A.T., Weyrich, A.S., Zimmerman, G.A., Bozza, F.A., Bozza, P.T., 2013. Platelets mediate increased endothelium permeability in dengue through NLRP3-inflammasome activation. Blood 122, 3405–3414. Patil, R., Ghosh, K., Satoskar, P., Shetty, S., 2013. Elevated procoagulant endothelial and tissue factor expressing microparticles in women with recurrent pregnancy loss. PLoS One 20, e81407. Piccin, A., Murphy, W.G., Smith, O.P., 2007. Circulating microparticles: pathophysiology and clinical implications. Blood. Rev. 21, 157–171. Punyadee, N., Mairiang, D., Thiemmeca, S., Komoltri, C., Pan-Ngum, W., Chomanee, N., Charngkaew, K., Tangthawornchaikul, N., Limpitikul, W., Vasanawathana, S., Malasit, P., Avirutnan, P., 2015. Microparticles provide a novel biomarker to predict severe clinical outcomes of dengue virus infection. J. Virol. 89, 1587–1607. Shepard, D.S., Halasa, Y.A., Tyagi, B.K., Adhish, S.V., Nandan, D., Karthiga, K.S., Chellaswamy, V., Gaba, M., Arora, N.K., INCLEN. Study Group, 2014. Economic and disease burden of dengue illness in India. Am. J. Trop. Med. Hyg. 91, 1235–1242. Taylor, R.P., Ferguson, P.J., Martin, E.N., Cooke, J., Greene, K.L., Grinspun, K., Guttman, M., Kuhn, S., 1997. Immune complexes bound to the primate erythrocyte complement receptor (CR1) via anti-CR1 mAbs are cleared simultaneously with loss of CR1 in a concerted reaction in a rhesus monkey model. Clin. Immunol. Immunopathol. 82, 49–59. World Health Organization, 2009. Dengue: Guidelines for Diagnosis, Treatment, Prevention and Control. WHO, Geneva, Switzerland.
Author contribution statement Rucha Patil, Shrimati Shetty & Kanjaksha Ghosh conceived and designed the experiments. Rucha Patil performed the experiments, analyzed the data and wrote the manuscript. The clinical evaluation was done by Smrati Bajpai and Kanjaksha Ghosh. Critical revision of manuscript for important intellectual content and final approval was done by Shrimati Shetty. Conflict of interest disclosure The authors declare no competing financial interests.
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