Preliminary study of dengue virus infection in Iran
Introduction
Dengue is one of the most significant viral haemorrhagic fever infections worldwide. In the past 50 years the incidence of dengue fever has increased 30-fold and has expanded to areas which were previously free from the virus. An estimated 2.5 billion people live in dengue-endemic countries, including Malaysia, India and Pakistan and 50–100 million people contract dengue each year.1 Dengue virus (DENV) belongs to the family of Flaviviridae and genus Flavivirus with a single stranded positive-sense, RNA. The virus consists of four serotypes (DEN-1 to -4).2 Humans and the main route of transmission to humans is by the bites of infected Aedes mosquitoes, principally A. aegypti.3
Dengue has a wide spectrum of clinical symptoms, often with unpredictable clinical outcomes. After 4–10 days incubation period, infection by any of the four virus serotypes can produce a wide spectrum of illnesses, although most infections are asymptomatic or subclinical. While the infection is self-limiting in the majority of cases and patients fully recover, in a small proportion of patients the infection progresses to severe disease, mostly characterized by plasma leakage with or without haemorrhage. In its haemorrhagic form, it can be easily misdiagnosed as other haemorrhagic diseases.4 Crimean-Congo Haemorrhagic Fever (CCHF) is the main type of viral haemorrhagic fever in Iran, and suspected cases with haemorrhagic signs are initially considered to be CCHF and are tested in the Arboviruses and Viral Haemorrhagic Fevers Laboratory (National Reference Laboratory) at the Pasteur Institute of Iran.5
In 2008, the first case of dengue fever was reported in Iran, where the patient had previously travelled to Malaysia.6 Thereafter, no other cases were officially reported in the Iranian population. Furthermore, since Iran is in close proximity to dengue-endemic countries such as Pakistan,7 it has led us to investigate the possibility of dengue virus infection.
The principal aim of this retrospective study was to evaluate the presence of dengue infection in Iranian patients who had previously tested negative for CCHF.
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Materials and methods
Serum samples from throughout Iran were collected at random from suspected cases of CCHF with haemorrhagic symptoms and were submitted by the Iranian Centre for Disease Control (CDC) of the Ministry of Health to the Arboviruses and Viral Haemorrhagic Fevers Laboratory (National Reference Laboratory) of the Pasteur Institute of Iran. All samples were serologically and PCR negative for CCHF.
The serum samples had been collected and stored in the serum bank of Arboviruses and Viral Haemorrhagic
Serological assay
IgM; Serum samples were diluted (1/20) by adding 5 μl serum samples to 95 μl of sample dilution, after which 80 μl of serum solution was added into wells except those assigned to controls. Subsequently, 20 μl of the 1/20 dilutions of serum samples, 100 μl of positive control, 100 μl of cut off and 100 μl of negative control were added into the corresponding wells and incubated at 37 °C for 60 min. The following steps were undertaken according to the manufacturer's instructions of the Dengue
Molecular assay
Viral RNA was extracted from serum using QIAamp Viral RNA extraction, according to the manufacturers' instructions (QIAGEN, Germany). The extracted RNA was amplified by RT-PCR using a one-step RT-PCR kit (QIAGEN, Germany).11 DENV serotyping was performed genetically using a nested PCR mix. The reaction mixture was prepared using diluted first-round PCR products, dNTPs, taq DNA Polymerase and primer D1 and dengue virus type-specific primers: TS1, TS2, TS3and TS4. The PCR products were analysed
Results
In this study, serum samples from 300 patients, mostly from the Sistan and Baluchistan province in southeast Iran, were tested. Of these samples, 15 (5%) were serologically positive, of which 12 (4%) were only serologically positive and 3 (1%) were both serologically and PCR positive. The mean (±SD) age of the positive cases was 49.6 (±16.7).
Of 15 sera which were positive serologically for Dengue, 5 (33.3%) were positive for both IgM and IgG, 5 (33.3%) were negative for IgM but positive for IgG
Conclusions
Dengue is a serious public health concern in the Western Pacific Region.13 Between 2001 and 2008, more than 1 million cases were reported in Cambodia, Malaysia, Philippines, and Vietnam, the four countries in the Western Pacific Region with the highest numbers of cases and deaths.14 In addition, it should not be overlooked that other countries with dengue cases such as Saudi Arabia which lies in close proximity to Iran, and Pakistan which has a common border with Iran, pose a potential risk for
Funding
None.
Conflict of interest
None declared.
Acknowledgement
We express our gratitude to Mr Hooman Goharriz (Animal Health and Veterinary Laboratories Agency, Wildlife Zoonoses and Vector-Borne Diseases Research Group, UK) for assistance in editing the manuscript and also from acknowledge assistant the European Virus Archive.
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