Decreased Mortality Rate Among Patients with Dengue

Hemorrhagic Fever Treated with Doxycycline Associated

With Reduced Inflammatory Cytokine Levels

J. E. Z. Castro1, P. G. Martinez1, W. M. Rodriguez1, E. C. Sanchez2, M. J. Foster3, T. M.

Fredeking4

1 Centro de Investigaciones Regionales Dr. Hideyo Noguchi, Universidad Autónoma de Yucatán,

México. Avenida Itzaés # 490 x Calle 59 Colonia Centro C.P. 97000 Mérida, Yucatán, Mexico.

2 Servicios de Salud de Yucatán y Profesor de Posgrado de Facultad de Medicina de UNAM

Domicilio: Hospital Agustín O´Horan SSY, Calle Itzaes s/n. Col Centro, Mérida Yucatán 9700.

3 Global Disease Research, Inc., 1901 Norwood Drive, Hurst, TX 76054

4 Antibody Systems, Inc., 1901 Norwood Drive, Hurst, TX 76054

Abstract Patients (N=228) presenting with symptoms characteristic of dengue hemorrhagic fever (DHF) were randomized to receive either standard supportive care or supportive care in addition to oral doxycycline twice daily for 7 days. Dengue virus infection was confirmed by PCR using multiple primers. Treatment with doxycycline resulted in a 50% reduction in mortality, from 22.8% (26/114) in the control group to 11.4% (13/114) in the treatment group (p=0.025). Surviving patients in the doxycycline group were discharged significantly earlier than control subjects (mean stay of 8.2 days for controls versus 7.2 days for the treatment group; p<0.01). There was no significant difference in the mean time to death between the groups. Decreased mortality among patients receiving doxycycline was associated with lower levels of pro-inflammatory cytokines. Therefore, administration of doxycycline resulted in a significant (p<0.01) decrease in levels of TNF and IL-6 versus controls within 3 days of treatment. This trend persisted throughout the 7 day study period when blood samples were taken. Patients who died in both groups possessed significantly (p<0.01) higher levels of TNF and IL-6 compared to those who survived at all time points. The above findings indicate that doxycycline can provide a significant clinical benefit

to patients with DHF which is mediated by decreasing pro-inflammatory cytokine levels.

1. Introduction

The world wide incidence of dengue fever (DF) has increased dramatically over

the past 30 year with its reemergence in South and Central America in 1981 (1).

The World Health Organization (WHO) estimates than there are between 50-100

million cases annually occurring in over 100 countries (2, 3). Dengue is now the

most common cause of fever among tourists returning to the United States from

South or Central America and the Caribbean basin. While the mortality rate for DF

is low with supportive care (<1%), approximately 2% of patients will progress to

dengue hemorrhagic fever (DHF) characterized by high fever, severe headache,

hemorrhaging at mucosal surfaces, and hematological abnormalities. This in turn

can lead to circulatory system collapse and death in 2.5-20% of patients (4). There

is no specific treatment for DHF. No vaccines exist for dengue although several

are in late stage clinical trials (4).

Dengue virus infection is known to cause a marked elevation in pro-inflammatory

cytokine levels (5, 6, 7, 8). Elevated cytokine levels have been viewed as a

negative clinical prognostic indicator and tend to be significantly higher in

patients with DHF or dengue septic shock syndrome than those with DF (6, 7, 8).

Furthermore, increased cytokine levels may be directly responsible for the

coagulopathies characteristic of DHF (8). In vitro viral replication within human

monocytes and dendritic cells offer an increased number of targets for CD4+ and

CD8+ effector T cells (9, 10). This, in turn, causes a dramatic increase in multiple

cytokine levels including interleukin 10 (IL-10), IL-2, gamma interferon and tumor

necrosis factor (TNF) (5, 6, 7, 9, 10).

Attempts to treat both infectious and non-infectious diseases by modulation of

the cytokine response through use of drugs or antibodies, has attracted

considerable attention (11, 12, 13). While various antibiotics have been shown to

possess immune modulating activities (14), those belonging to the tetracycline

family appear to have the most promise. Patients with multiple sclerosis,

Huntington’s disease and rheumatoid arthritis have benefited clinically from

orally administered tetracyclines (15, 16, 17). Recently doxycycline or tetracycline

has been shown to down-regulate pro-inflammatory cytokine levels in patients

with tick borne encephalitis, DF or DHF (18, 19). Doxycycline was also effective at

raising serum cytokine receptor and cytokine antagonist levels in patients with DF

or DHF (19). These findings suggest that doxycycline may provide a clinical benefit

to patients with DHF. We therefore conducted a randomized, blinded, multi-

centered trail to evaluate the effect of orally administered doxycycline on the

mortality rate among patients diagnosed with DHF.

2. Material and Methods

2.1. Subjects and clinical trial design. The study protocol was approved by the

institutional review board of the Autonomous University of Yucatan, Merida,

Yucatan, Mexico. Informed consent was obtained from all adult patients and from

the parents of subject less than 18 years of age. Study subjects were recruited

while seeking treatment at two hospitals between June and November which was

shortly after the beginning of dengue season. Patients presenting with a

combination of fever ( 38C for 2 or more days), coupled with hemorrhagic

manifestations (positive tourniquet test, petechiae, ecchymosis, or mucosal or

gastrointestinal bleeding), thrombocytopenia (<100 x10 9 per liter) and evidence

of plasma leakage (hematocrit >20%, pleural effusion, or low serum albumin)

were provisionally enrolled into the study. At this time (Day 0) a blood sample

was obtained to confirm dengue virus infection by PCR (see below) and patients

were randomized to receive standard symptomatic and supportive care or

doxycycline (Vibramycin, Pfizer, N.Y., USA), 200 mg initial oral dose followed by

100 mg administered every 12 hours through day (7), in addition to standard care.

Randomized patients who tested positive for dengue virus infection were enrolled

into the study and continued to receive doxycycline through day 7. Serum

samples were obtained at days 0, 3, 5 and 7 and tested for levels of tumor

necrosis factor (TNF) and interleukin 6 (IL-6) (see below). Due to the

extraordinarily high influx of critically ill patients with dengue-like symptoms,

hospital beds were available only for the most critically ill patients. All enrolled

hospitalized patients were followed until discharged. The remaining study

subjects were visited daily by a physician associated with the study until

resolution of symptoms.

Diagnostic tests. Infection with dengue virus was confirmed by PCR using multiple primers. Tests were conducted at the Centro de Investigaciones Regionales "Dr. Hideyo Noguchi", Universidad Autónoma de Yucatán. Avenida Itzaes No. 490 x 59, Centro, CP 97000 Mérida Yucatán, México. 2.3. Cytokine Assays. Blood samples were processed to obtain serum which was stored at −70◦C. Assays were performed in a blinded manner after all samples were collected. Serum cytokine levels were quantified using commercial ELISA tests kits (R & D Systems, Minneapolis, MN, USA) per the manufacturer’s instructions. The limits of detection are as follows: IL (interleukin)-6, 0.7 pg/mL and TNF, 0.6 pg/mL. Statistical analysis. Statistical analysis comparing IL-6 and TNF levels between control (no doxycycline) and treatment (oral doxycycline) and between patients who survived and those who succumbed to infection was performed using the unpaired Student t-test. A p value <0.05 was considered significant. The significance in mortality rates between groups was by chi-square analysis. Results A total of 228 patients who met all symptomatic criteria for DHF, in addition to having a positive PCR test result, were enrolled into the study (114 per group). The demographics of the study groups were well matched. There was no statistically significant difference between the groups concerning either mean age (27.6 years for controls versus 31.2 years for treatment) or gender distribution (45 percent males in the control group versus 53 percent in the treatment group). Severity of illness at the time of enrollment was not assessed.

Levels of circulating IL-6 and TNF in patient groups are shown in Table 1. There was no significant difference (p>0.05) in IL-6 or TNF levels at the time of enrollment (Day 0) between the two groups. The administration of doxycycline resulted in a significant decline in both IL-6 and TNF by day three when levels were compared between treatment groups and within the doxycycline group. Therefore, the mean concentration of IL-6 in the treatment group had declined from 5.98 pg/ml on day 0 to 4.6 pg/ml on day 3 (p<0.05). In comparison the mean concentration of IL-6 in the control group remained virtually identical between day 0 and day 3 (5.44 pg/ml and 5.43, respectively, p>0.05). The difference between the two groups on day 3 was highly significant (p<0.01). A similar trend was observed for TNF where levels in the control group remained unchanged between day 0 and day 3 (6.37pg/ml and 6.33 pg/ml, respectively, p>0.05) compared to the treatment group where there was a significant reduction of 6.63 and 5.34, respectively (p<0.05). The difference between the control (6.33pg/ml) and treatment (5.34 pg/ml) groups on day 3 was also highly statistically significant (p<0.01). By day 5 and at day 7, both IL-6 and TNF levels had declined significantly (p<0.05) compared to day 0 in both groups reflecting an improvement in the clinical status of surviving patients (see below). However, IL-6 and TNF levels were significantly lower (p< 0.05) at both day 5 and 7 in the treatment versus control groups. The correlation between Il-6 and TNF levels in patients who died from DHF and those that survived is shown in Table 2. Elevated levels of IL-6 and TNF were associated with a fatal outcome in both the control and treatment groups. At all time points in both the treatment and control groups, IL-6 and TNF serum concentration were significantly higher (p<0.01) in subjects who died of DHF versus those who survived (Table 2). Interestingly, while IL-6 and TNF levels were significantly lower (p<0.05) in surviving patients treated with doxycycline versus survivors in the control group, this was not the case for those that died where IL-6 and TNF levels were comparable between the two groups. The ability of doxycycline to decrease circulating Il-6 and TNF was associated with a significant reduction in mortality (Table 3). Treatment with doxycycline caused a 50% reduction in mortality rate compared to untreated controls (p=0.025).

Discussion Dengue is endemic in virtually all tropical and subtropical areas (1, 2). Its recent reemergence in South and Central America and the Caribbean basin has become a major public health challenge (3, 4). In 2010, more than 1.5 million cases of dengue were reported to the Pan American Health Organization by member states (20). Of these 35,000 were consider to be severe (including dengue shock syndrome (DSS) and DHF) with 804 deaths recorded. Preliminary data from surveillance during 2011 shows no evidence that the epidemic is abating (20). Travel-associated dengue infection is becoming far more common in the US (21) with roughly 70% of cases coming from visitors to South and Central America and the Caribbean basin. Nearly a third (27%) of patients required hospitalization. There is a distinct possibility of Dengue becoming endemic in subtropical areas of the US, specifically, Florida (22). No specific treatment exists for dengue except for standard supportive care, such as treatment with anti-pyretics and fluids. Treatment of DSS and DHF require hospitalization, which creates a mayor load for the sanitary system, especially during an outbreak. For example, this study initiated shortly after the onset of dengue season, the participating rural hospitals had complete occupation due to a dramatic increase in critically ill dengue patients. The demand of care was such that some severe cases had to be treated at home or as outpatients at a regional health care clinic. Undoubtedly, this might have contributed to the high mortality rate (22.8%) observed in the control group, which is among the highest reported for severe dengue disease (4). This might be related to a vice due to the fact the patients were autoselected and were in a hospital. They already had symptoms related to hemorrhagic dengue fever and were severe cases. This could explain the high incidence of mortality seen in this study in comparison with the open population. It appears that doxycycline provides protection to the treatment group (OR = 2.2, 95% CI 1.0908 to 4.51, with a Chi2 of 5.012, p = 0.025). The total number of deaths represents the total mortality seen during the outbreak of that year in Yucatán. The above findings clearly illustrate the need for treatment improvement for dengue infections. Numerous studies have shown that infection with dengue virus

leads to increased levels of pro-inflammatory cytokines, which can lead to profound shock and death (5, 10). This opens up the possibility of treating severe dengue infections by down-regulating such mediators of systemic inflammation (11, 13). Previously, we have shown that administration of oral tetracycline or doxycycline can result in a rapid and significant decline in serum cytokine levels in patients with DF or DHF (19). Thus, the need to evaluate the ability of doxycycline to reduce mortality among patients with DHF. Doxycycline was selected due to its superior immunomodulating effect and because it has shown to inhibit dengue virus replication in vitro (23). Daily oral administration of doxycycline resulted in a 50% reduction in mortality attributed to DHF. This correlated with a significant decline in circulating levels of IL-6 and TNF, confirmed by earlier observations (19). Particularly encouraging is the fact that such effect occurred between non-hospitalized DHF patients receiving less than optimal care due to a high demand of health care services during the outbreak. The current study does have several limitations; foremost it was not conducted in a double blind, placebo-controlled manner. Additionally, all fatal outcomes in subjects were attributed to dengue infection with no post-mortem evaluation. While all enrolled subjects met the clinical criteria for DHF in addition to testing posting for dengue infection by PCR analysis, some mortality could have been either multifactorial or from an unrelated causes. The findings from the current study indicate that doxycycline could be an effective, treatment for severe dengue disease. Its low cost, long history of safety, the ability to cross the blood brain barrier and administration via the oral route appears to be an ideal candidate to be used in patients in rural areas with limited access to healthcare. We are currently exploring the feasibility of conducting a randomized, double blind, placebo-controlled trial to conclusively demonstrate the ability of doxycycline to reduce mortality associated with severe dengue disease.

Table 1. Effect of Doxycycline on Serum IL-6 and TNF Levels

Treatment Group

GMT and ranges, pg/ml

Day 0 Day 3 Day 5 Day 7

IL-6

Control 5.44 (2.41-8.88)

5.43 (1.32-9.27)

4.93* (1.89-10.4)

4.49* (1.48-9.22)

Doxycycline 5.98 (2.31-11.1)

4.60*+

(1.38-3.25)

3.52*+ (1.06-8.92)

2.99*+ (1.15-9.22)

TNF

Control 6.37 (2.41-10.1)

6.33 (1.79-10.5)

5.73* (0.89-11.5)

5.35* (0.98-10.8)

Doxycycline 6.63 (1.99-11.8)

5.34*+

(1.14-9.87) 4.21*

+

(0.67-11.2) 3.65*

+

(0.94-8.99)

* p<0.05 versus Day 0 value within the same group. + p<0.01. Control versus Doxycycline Group on same day.

Table 2. Serum IL-6 and TNF Levels in Patients Who Survived or Died from DHF

Group (Outcome)

TNF (GMT with Range, pg/ml) IL-6 (GMT with Range, pg/ml)

Day 0 Day 3 Day 5 Day 7 Day 0 Day 3 Day 5 Day 7

Control

Alive 5.90+

(1.68-9.62)

5.65+ (1.79-10.4)

5.27+ (0.89-11.3)

4.99+ (0.98-10.5)

4.96+ (2.41-8.23)

4.77+ (1.32-7.92)

4.44+ (1.43-9.47)

4.14+ (1.87-8.23)

Dead

8.06 (4.31-10.1)

8.84 (5.3-10.5)

8.62 (5.76-11.5)

8.88 (6.92-10.8)

7.11 (4.32-8.88)

7.86 (4.71-9.81)

8.14 (6.21-10.4)

8.10 (5.38-9.22)

Doxycycline

Alive 6.35+ (1.99-10.8)

5.02+ (1.14-8.88)

4.06+ (0.67-6.87)

3.53+ (0.94-7.32)

5.55+ (2.81-7.78)

4.34+ (1.38-6.91)

3.42+ (1.1-6.04)

2.82+ (1.03-4.64)

Dead

9.02 (5.89-11.8)

8.76 (7.85-9.87)

8.97 (7.75-11.2)

8.12 (7.24-8.99)

7.83 (5.49-11.1)

7.37 (4.83-9.25)

8.34 (7.78-8.92)

9.12 (9.01-5.22)

+ p<0.01 versus patients who died

Table 3. Effect of Doxycycline Treatment on Mortality Rates Associated with DHF

Group Mortality Rate (%) [Nr Dead/Total]

Mean Time to Death (days)

Control 22.8% [26/144] 5.9 (3-8)

Doxycycline 11.4%* [13/144] 4.6 (1-7)

*p=0.025

OR Control = 2.2203, 95% CI 1.0908 to 4.51.

Chi2 = 5.012, p = 0.025

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