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Clinical Response in Patients with Dengue Fever to Oral Calcium Plus Vitamin D
Administration: Study of 5 Cases

Article  in  Proceedings of the Western Pharmacology Society · January 2009

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Proc. West. Pharmacol. Soc. 52: 14-17 (2009) CASE REPORT

Clinical Response in Patients With Dengue Fever to Oral Calcium Plus Vitamin D Administration:
Study of 5 Cases
Emilio Sánchez-Valdéz1*, Melissa Delgado-Aradillas2, José Angel Torres-Martínez3 and
José Martín Torres-Benítez4
1,2
Community Center “Las Américas” Tampico, Tamaulipas, 3Biomedical Engineering, 4Faculty of Medicine,
Tec de Monterrey, Autonomous University of Tamaulipas, México
Email: dr.emiliosanchez@productosmoka.com
Abstract
A dengue epidemic is one of the most important public health problems in the tropical and subtropical areas of the
World. In 2005, 7,062 dengue cases were reported in Tamaulipas on Mexico's eastern coast, including 1,832 (26%)
cases classified as Dengue Hemorrhagic Fever (DHF). Dengue fever (DF) is characterized by fever, intense headache,
myalgias, arthralgias, rash, nausea and vomiting. A proportion of infected persons may develop DHF characterized
by prominent hemorrhagic manifestations associated with thrombocytopenia. An immune mechanism of
thrombocytopenia due to increased platelet destruction appears to be operative in patients with DHF. Excessive
capillary permeability may lead to Dengue Shock Syndrome (DSS). Patients with DHF/DSS who also have prolonged
fever (> 5 days) are at high risk for concurrent bacteremia. Standard treatment is limited to electrolytic solutions,
rest, measurements of body temperature, blood pressure, hematocrit, platelet count, and administration of
antipyretics like paracetamol when fever is too high. Extracellular calcium plays a key role in platelet aggregation
and for the regulation of the immune response in persons infected with Dengue Virus (DV), and dihydroxy-vitamin
D has recently been found to alter IL-12 expression and dendritic cell maturation. We report the cases of five
patients who received oral calcium carbonate plus Vitamin D3, who improved overall clinical condition and reduced
the duration of signs and symptoms of DF.

Introduction
Dengue transmission requires the simultaneous
presence of three factors; susceptible humans, a
competent mosquito vector, and DV [1]. Studies of DF
endemic zones have shown that low income groups
have a higher incidence of disease. Two and one-half
billion people in more than one hundred countries are
at risk of infection, with an estimated 50 million cases
per year.[2] DV is a mosquito-borne flavivirus and the
most prevalent arbovirus in tropical and subtropical
regions of the globe [3]. DF may result from infection
with any of the four antigenically related dengue virus
serotypes (DEN-1-4)[4] of the genus Flavivirus [5].
DENV-2 was the predominant serotype in the
Tamaulipas outbreak that occurred in 2005 [6]. An
Immunoglobulin M (IgM)-capture enzyme-linked
immunosorbent assay (ELISA) confirms recent dengue
infection.[7]
Among both DF and DHF cases, more than 98 percent of
the patients had serologic evidence for a secondary
dengue virus infection from another serotype of dengue
virus or a non-dengue flavivirus [8]. Epidemiological
studies indicate that homologous immunity provides
nearly permanent protection against re-infection with a
previously experienced serotype; in contrast,
heterologous immunity does not provide protection
against re-infection in the long term [9]. The antibody-
dependent enhancement (ADE) theory states that cross-
reactive, yet non-neutralizing, antibody from a previous
flavivirus infection enhances infections in Fc receptor-
bearing cells such as monocytes and macrophages and
possibly dendritic cells [8]. The theory elevates the
relative importance of immune enhancement as a risk
factor for DHF, but these findings do not reduce the
importance of other risk factors like genotype.
The vitamin D receptor (VDR) mediates the immuno-
regulatory effects of 1,25-dihydroxy-vitamin D3 (1,25-
D3), which include activating monocytes, stimulating
cellular immune responses, and suppressing immuno-
globulin production and lymphocyte proliferation. A
study of genetic susceptibility to DHF, showed that the
tt genotype of a single nucleotide polymorphism (SNP)
at position 352 of the VDR gene may be associated with
altered risk for severe dengue, since activated B and T
lymphocytes express VDR, and 1,25-D3 affects
monocytes, the main sites of dengue virus infection and
replication [10].
Immune response mechanisms modulated by calcium:
Langerhans Cells (LC) are the sentinels of the skin
immune system. LC and dermal dendritic cell's (DC) role
is to launch and modulate the innate and acquired

immune responses to virus infections. Following the
mosquito bite, LC capture antigens encountered in the
epidermis, and migrate toward local draining lymph
nodes. Activation and migration of LC relies on calcium
dependent processes, recruiting the participation of
Langerin and E-cadherine. Possible stimulus for
migration via the activation of LC through engagement
of class II MHC antigens on the cell surface has been
suggested.[11] LC efficiently leads to an ordered series
of events which stimulate a lymphocyte to divide and
produce descendants, some of them convert into
effector cells. The complete immune response includes
both the induction of cellular proliferation and the
expression of the immune functions. The lymphocytes
are activated when specific ligands bind to receptors on
the surface. The required ligands are different for T and
B cells, but the response is similar for both.[12]
Figure 1. Lymphocyte replication and the importance of
calcium. Schematic representation of the immune cell
stimulatory events described for the importance of
calcium in the propagation of immune cells.
The increase in protein tyrosine kinase (PTK) activity is
an early event when the T or B cell binds to ligand [12].
The PTK activates phospholipase C-γ1 enzyme (PLC γ1),
which hydrolyzes phosphatidylinositol. As a result,
diacylglycerol (DAG) and 1,4,5-inositol triphosphate
(IP3) are produced, both are second messengers. DAG
remains inside the membrane where it activates protein
kinase C (PKC) which phosphorylates other proteins on
serine and threonine residues. IP3 is released to the
cytoplasm, binds to specific receptors on calcium
storage organelles and triggers an increase in
intracellular calcium concentration [Ca2+]i, which
diffuses into the cytosol reaching its highest
concentration in the first minute following the contact
with the activator stimuli (Fig. 1).
The activation of the PKC and the rapid increase in
[Ca2+]i are critical in the activation of subsequent
events, not all of which are known.
Calcium shares an important quality with other
cytoplasmic messengers, namely that the cytoplasmic
concentration of calcium is determined by events that
occur inside the membrane as well as calcium entry.
Proc. West. Pharmacol. Soc. 52: 14-17 (2009)
Intracellular calcium concentrations fluctuate to induce
different responses in cells (Table 1) [13]. For instance,
when a phagocytic cells comes in contact with a foreign
particle and migrates to devour it, the increased [Ca2+]i
may reflect its role in the regulation of changes in
cytoskeletal activity [14].
Table 1. Calcium effect in different cell types [15].
Cells Response
Vascular Smooth Muscle Contraction
Gastric Smooth Muscle Contraction
Skeletal Muscle Contraction
Blood Platelets Change in shape, clotting
Lacrimal gland Increase in potassium
Calcium Function in Platelets The adhesion of platelets
to one another occurs under both normal and
pathological conditions. Platelets that come in contact
with hemorrhagic tissue are activated by agonists such
as adenosine diphosphate (ADP) [16]. The activation
involves a change in their shape, and glycoprotein
IIb/IIIa receptor sites become exposed. Afterwards, new
platelets stick to those adhering to collagen and a white
thrombus forms.
Aggregation takes place in two phases [17]. In the
primary phase platelets attach loosely to each other
and if the stimulation process is weak the aggregation is
reversible. The secondary phase requires a longer
period of time, it begins when platelets release their
own ADP and calcium from storage granules and
synthesize thromboxane A2 (TXA2). If platelets are
unable to release ADP or synthesize TXA2, secondary
phase aggregation cannot occur and wound bleeding
will take longer to stop. [16]
In addition, fibrinogen and calcium ions (Ca2+) are
needed for platelet aggregation. Fibrinogen's role is to
form bridges between platelets. Ca2+ is released by
platelets from storage granules to provide high
concentrations in the affected area. Ca2+ is essential for
platelet clotting, but the exact role remains unknown
[12].
Case Studies
Five patients that met study criteria were selected, they
had fever over 38°C of 2 to 4 days of duration,
headache, muscle and joint pain, nausea and/or
vomiting. All the patients included in the study had at
least 4 of the clinical symptoms for case definition of DF
and were positive for tourniquet test (Rumpel-Leede
Proc. West. Pharmacol. Soc. 52: 14-17 (2009)
Capillary-Fragility Test). They were invited to participate
in the study and received standard treatment, in
addition to an oral dosage of calcium carbonate plus
vitamin D3 (Caltrate 600 + D, Division Wyeth Mexico).
Individual explanations were provided for patients and
their relatives; written consent to participate in the
study was obtained from all participants.
Standard treatment consisted on electrolytic solutions
and 500 mg of paracetamol every 12 hr. Patients
received one tablet of calcium carbonate + vitamin D3
every 8 hr the first 3 days, totaling 1,800 mg of calcium
and 600 UI of Vitamin D3. The following day’s doses
were reduced gradually to one tablet every 12 hours for
three days, totaling 1,200 mg of calcium and 400 UI of
vitamin D3. A maintenance dose was provided,
consisting on one tablet per day, totaling 600 mg of
calcium and 200 UI of vitamin D3 for three more days,
and until patients were asymptomatic.
We report three men and two women between 18 and
59 years old, with an average of 8.8 in the visual analog
pain score, and average platelet counts of 136,000
cells/mm3 ± 69,508 at the beginning of the treatment,
and 179,600 cells/mm3 ± 56,584 at the end. The clinical
profile evolved with a gradually decrease of clinical
symptoms and an average duration of 4.8 days ± 2.5. All
patients were discharged on the fifth day. Among
patients, the most striking case was a 29 year-old male
patient who had thrombocytopenia with a 113,000
cells/mm3 on the platelet count at the beginning of the
treatment which increased to 257,000 cells/mm3 on the
fourth day. A 45 year old female with a 116,000
cells/mm3 on the initial platelet count increased to
186,000 cells/mm3 on the third day of treatment.
Patients were followed for up two weeks, and all were
asymptomatic for DF and had normal platelet counts.
Discussion
When calcium levels in the human being are not enough
to accomplish the body necessities, the human is
exposed to numerous health problems. Lowered
calcium levels occur most of the time due to an
inadequate diet. The majority of the population fails to
meet daily calcium requirements irrespective of
infection. The traditional Mesoamerican diet of a low
income groups is based on corn tortillas and black
beans, is rich in complex carbohydrates [18] and has a
decreased intake of dairy products, which are the main
source of the dietary calcium [19]. In addition, the low
socioeconomic groups are the most likely to consume
milk formula, instead of whole milk, because of the lack
of resources. Milk formula is an industrialized dairy
product made with vegetal fat, and may not provide the
same amounts of proteins, calcium and Vitamin D3 as
whole milk.
We cannot completely discard the possibility of the
importance of infectious disease interacting with poor
nutrition,[20] therefore we suggest that patients in this
study are likely to have a subclinical hypocalcemic state
due to a decreased intake of calcium on the diary diet.
Since calcium is essential for platelet clotting and
modulates the immune response, an oral administration
of calcium plus Vitamin D3 may thus restore free Ca2+
more quickly and could explain the earlier clinical
improvement and the reduction of thrombocytopenia
observed on the five patients.
There were some limitations in this study. First, the
small number of study cases with DF makes statistical
power too small to make conclusions. Second, the
selection of patients for the study was based on the
clinical symptoms for case definition of DF. No control
population was prospectively used for comparison.
Finally, further studies to elucidate more information
regarding clinical response and effect of oral calcium
plus vitamin D3 administration on thrombocytopenia in
patients with DF are warranted.
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