Zika Virus (ZIKV) (Flavivirus, Flaviviridae) (Arbovirus) - Molecular diagnosis by real - time PCR (rt-RT-PCR) and detection of specific antibodies (serology) by neutralization tests

 

Information 01-02-2016.

 

Zika virus (ZIKV) is an arbovirus of the Flaviviridae (genus flavivirus) family, transmitted by mosquitoes, causing Zika fever, typhus self - limiting febrile syndrome, similar to dengue. Signs and common symptoms are mild fever, maculopapular rash, arthralgia, myalgia, headache and conjunctivitis. Less often they can manifest edema, pharyngitis, cough and vomiting. There are cases that have been associated with Guillain-Barré syndrome and hearing loss. Currently, there is evidence, which continue to research, linking this virus with increasing incidence rates of microcephaly in the affected regions.

 

This virus was first described in 1947 in Uganda in rhesus monkey chimpanzee sentinel for detecting yellow fever Zika forest detected after several mosquito species Aedes spp. It could be shown infectivity to mice with serum sentinel mono and transmission to experimental mice infected mosquitoes. Between 1951 and 1981, have been reported serological evidence of human infection in several African countries (Uganda, Tanzania, Egypt, Central African Republic, Sierra Leone, Gabon, ...), in parts of Asia (India, Malaysia, Philippines, Thailand, Vietnam, Indonesia, ...), where there have been documented outbreaks. The finding for the first time on the island of Yap (2007), the Federated States of Micronesia, showed that he could be out of Africa and Asia. Outbreaks have occurred in parts of the region (-2007 French Polynesia, New Caledonia, Easter Island, Cook Islands, peninsular Malaysia and Borneo, Thailand, Cambodia, Indonesia, ...), affecting travelers in the area. Its reservoir is supported non - human primates, among which is transmitted by mosquitoes of sylvatic habits, not urban.

 

The first cases of infection detected in Brazil occurred in 2015, and in January 2016, according to the PHO (Pan Asmerican Health Organization). Most Brazilian states have reported locally acquired cases is estimated that there have been 1.5 million cases. In addition, we detected indigenous cases in Barbados, Colombia, Ecuador, El Salvador, Guatemala, Guyana, French Guyana, Haiti, Honduras, Martinique, Mexico, Panama, Paraguay, Puerto Rico, St. Martin, Guadeloupe, Surinam, Venezuela and Bolivia.

 

Zika virus disease

 

Infection with this virus usually produces mild symptoms or even asymptomatic infection in 75% of cases. Its symptoms can be confused with that caused by infection with dengue virus chikungunya virus.

 

The incubation period may vary from 3 to 12 days, symptoms and maintained for 2 to 7 days.

 

The main symptoms are:

 

• • Moderate fever above 37.2 ° C.
  • Maculopapular rash.
  • Arthritis or transient, mainly in joints of hands and feet arthralgia.
  • Conjunctival hyperemia.
  • Myalgia, fatigue, headaches.

Complications of virus infection ZIKA

 

Zika virus infection in most cases is asymptomatic (75%). In 25% of cases the symptoms are mild.

 

However, there are cases of complications. These complications are:

 

Neurological complications as Guillen-Barré syndrome (GBS). These complications have been linked to the presence of antibodies to this virus in patients with this syndrome and temporomandibular spatial association with the incidence of GBS.

 

Neurological disorders in newborns: increased incidence of malformations central nervous system, which have been linked to the presence of antibodies in mothers.

 

Microcephaly: increase in cases in regions with outbreaks of infection with this virus. There is ongoing research to establish the relationship between the virus in amniotic fluid and congenital malformations and some cases of the virus to the fetus or term newborns.

 

Macular eye injury and cerebral calcifications in infants with microcephaly and intrauterine infection Zika virus.

 

Other: meningitis, meningoencephalitis or myelitis.

 

Drivetrain - arthropod vectors

 

Several species of Aedes mosquitoes: Aedes aegypti, Aedes albopictus (present in several regions of the Spanish Levante, including the Balearic Islands) and Aedes polynesiensis. The presence of the species Aedes albopictus has been found in 7 regions of Spain: Catalonia, Valencia, Murcia, the Balearic Islands, Andalusia, the Basque Country and Aragon.

 

It has not yet demonstrated its transmission by transfusion, but can not be excluded that can occur.

 

It could also be transmitted via sexual contact, as has been demonstrated in a case in the US

 

Zika risk of introduction of virus in Spain

 

This disease has not been previously known in Spain and the European Union, and there is difficulty in diagnosis because in most cases (75%) infection is asymptomatic. In addition, the symptoms are similar to other infections triggered by Arbovirus infection as Dengue or Chikungunya virus, among others, also present with fever, more or less intensity, and rash. Therefore, there is a risk for travelers from areas where there is currently incidence (number of countries in Latin America, including Brazil, Haiti, etc. - Thailand and Southeast Asia, Polynesia, - ...), and that our country can move to regions where the mosquito species Aedes albopictus is present, they pose a risk to establish a focus. Must be regarded as the Mediterranean coast, where the vector is present, it is one of the preferred destinations for foreigners from countries where outbreaks.

 

Zika risk of transmission of virus in Spain

 

The risk of transmission depends on the following factors:

 

1. 1. Presence of the vector. It exists in several areas of peninsular Levante and the Balearic Islands.
   2. Introduction of the virus by an infected traveler from endemic areas. This is possible, because little or no symptoms resulting in most infected.
   3. Presence of susceptible population in Spain.
   4. Coincidence in space and time of an imported case in areas where there are mosquitoes, made possible because infected individuals may move to the Spanish Levante.
   5. Possibility that the virus finds favorable conditions for transmission.

All these circumstances can be found in the geographical areas where the vector is present, especially during the months of May to October, so there is a real risk that it could be a source of infection by Zika virus in our country.

 

Diagnosis of Zika virus infections

 

The diagnosis of Zika virus infection can be performed by:

 

• • Virus isolation in cell culture (only during the first week after exposure) ..

• • Molecular detection by RT-PCR or real - time PCR (only during the first week after contact) ..

• • Detection of antibodies from the 3rd to 5th day of the onset of symptoms. We used several methods as ELISA or indirect immunofluorescence. These methods have the problem of existing antibodies generated by other viruses that cause similar symptoms, as with other Flavivirus (Dengue, Chikungunya) cross reactions, so to overcome this problem, and to detect specific antibodies Zika virus, it is necessary neutralization method. In IVAMI, we have this method to detect antibodies specific to the virus.

Note: To exclude contact in the absence of symptoms, it is recommended to test for specific antibodies after at least 15 days from the contagion. In these cases it is not recommended, or virus isolation test, or the test molecular detection by RT-PCR or real time PCR.

 

Tests in IVAMI:

 

• • Real - time PCR (Real-Time PCR) with primers (primers) and probe specific Zika VRU. This test is only indicated in the first days (up to 10 days) after infection, when the infected person can have the virus in blood.

• • Zika virus specific antibodies, by viral neutralization. This method does not cross - react with antibodies due to other infections Arbovirus with such symptoms as in virus infections or Dengue virus Chikungunya. This test is not indicated in the first days after infection because it has not yet had time to antibodies against the virus are formed. This test is indicated after infection and an elapsed time (the recommended at least 2 to 3 weeks) to be given time to form antibodies. This test can be useful when infected by the virus (eg after a few bites of mosquitoes), although they have not had symptoms because the immune system recognizes the virus and creates antibodies against it.

Detection limit of the method of real - time PCR:

           

• • 50 copies / mL

Sample types

 

• • Tests Real - time PCR: peripheral blood or plasma sample in the acute phase, and taken within the first 10 days of the disease.

• • Antibody tests: sample separated blood serum obtained from the 5th or 7th day of onset of clinical symptoms, or after 15 days of possible infection. If you have not had symptoms and the test is performed only as a control to see if has been infected, we recommend to wait 3 weeks before blood extraction. The extraction of the blood sample and separation of blood serum, can be performed in a clinical laboratory, having deposited serum (minimum 1 mL) in a sterile vial and this in turn into another tube to protect during transport.

Preservation and shipment of sample:

           

• • Whole blood or separated blood serum: refrigerated (preferred) for less than 2 days (white box cork -frigolín- pack frozen). While the shipment is not performed the sample must be kept in a refrigerator.

• • Frozen (only for separate sample blood serum): if you will keep more than 2 days before shipment.

  Delivery of results:

 

• • Real - time PCR: 24 to 48 hours.

• • Zika specific virus neutralizing antibodies: 7 to 15 working days.

 

Cost of testing

Consult ivami@ivami.com