Viral sample collection/isolation and identification of viruses

 

Steps

  • Sampling/sample collection
  • Labeling of the sample
  • Transportation
  • Isolation
  • Identification

1.Sampling


                Viral samples should be collected within 4 days after the onset of viral infection. If we collect a sample after 7 days, then there will be the chance of no viruses in the sample. We collect the sample from CSF, urine, blood, and broncho-alveolar lavage (BAL) etc.

2.Labeling


                Label sample properly. Write the date of illness, onset, date and time of collection of samples, a tentative diagnosis of the physician, and source of the specimen.

3.Transportation


                Most viruses remain stable at 4o C so store sample at 4oC.

General categories of sample


1.Blood


                We clean the vein area with 70 % isopropyl alcohol. After this, we apply 20 % iodine tincture. Then we allow iodine tincture to dry. After this collect 8-10 ml of blood in a tube containing anticoagulant. We do not need to store samples at 4oC. We collect blood for virus isolation

E.g., Cytomegaly virus

2.CSF


                Disinfect the site with 2 % tincture iodine. Insert the needle with stylet at inter-space between L3-L4 vertebrae, L4-L5, or L5-S1.

Stylet: Cylindrical medical probe used for tracheal intubation.

                When stylet reaches the subarachnoid space, remove stylet, and collect the 2-5 ml of CSF. Store at 4oC immediately after collection.

                CSF is mostly isolated to detect mumps virus, enterovirus, echovirus, and less frequently it is collected to detect the LCMB virus (Lymphocytic choriomeningitis virus), rabies virus, and HSV (Herpes simplex virus).

3.Vaginal swab or cervical swab


                If lesions are present in the vaginal or cervical region, swab lesion vigorously, and store swab. If lesions are not present, remove the mucous from the cervix with a swab and collect this mucous in the screw-capped tube and discard the swab.  UTM (Chlamydia mycoplasma urea plasm transportation medium) mostly used to detect cytomegaly, papilloma, and HSV.

4.Conjunctiva swab


                Collect sample from lower conjunctiva with flexible fine shifted swab which is already moistened with sterilized saline. After the collection, the swab should be stored in UTM. It is mostly for adenoviruses, entero-viruses, HSV, CMV and cox-sickie virus.

5.Feces


                Collect feces 2-4 grams in a clean dry container. There is no need for a transportation medium. After collection, it should be stored at 4oC. It is for adeno, entero, and rotaviruses.

6.Nasal swab


                Pass a flexible, fine shifted swab into the nostril and rotate slowly for 5 seconds so that it will absorb the secretion. Remove the swab and place it in UTM. Repeat the same procedure to the other side of the nostril and store swab at 4oC. It is mostly for Influenza, parainfluenza, and rhino-respiratory syncytial viruses (RSV).

7.Nasopharynx aspirate or wash


                Pass approximate sized tubing or catheter into the nasopharynx. Aspirate the material with a small syringe and store it into a sterilized container. If we do not get material, then tilt the patient head back at 70o and instill 4 ml of sterilized PBS (Phosphate buffer saline). Re-aspirate the material after a few seconds. It is for influenza, parainfluenza and rhinoviruses. If we take nasopharynx swab, then store it in UTM at 4oC.

8.Oral swab

       If lesions are present in the mouth, take a sample from the base of the lesions with a swab. Store swab in UTM at 4oC. It is mostly for enterovirus and HSV.

9.Vesicular sampling

      Collect the vesicular samples carefully. Collect samples from the fresh vesicle because older vesicles are dehydrated and they may not contain viruses. Burst the vesicle with scalpal or needle. When fluid comes out, absorb fluid with a swab. Store vesicular swab in UTM at 4oC. It is mostly for the isolation of enterovirus, echovirus, poxvirus, HSV, and VZV (Varicella-zoster virus).

10.Throat swab

       Use a tongue depressor to depress the tongue and collect the sample from the throat with the help of a swab from posterior pharynx, tonsils and inflamed area. The tongue is depressed to prevent contamination with saliva. Store swab in UTM at 4oC. IT is mostly for adenovirus, enterovirus, HSV, measle virus, mumps virus, and parainfluenza viruses.

11.Tissue sampling

        Take a sample from the area directly adjacent to the affected tissue. After collection, store it in UTM at 4oC. It is for lots of viruses.

12.Urethral swab

         Before the collection of the sample, make sure that the patient has not urinated for about 1 hour. Insert the flexible fine shifted swab up to 4 cm into the urethra of the patient. Then rotate the swab 2-3 times to obtain maximum secretion. Store swab in UTM at 4oC. It is mostly for CMV and HSV.

13.Urine

          Collect 5 ml midstream clean voided urine in a sterilized container. It gives a clear picture of blood glucose conc. Store at 4oC. No UTM is required. It is for adenovirus, CMV, HSV, mumps virus, and less frequently rubella virus.

4.Isolation of Viruses

   Viruses require a living host. Host may be an animal, bacteria, or plant.

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