Viral Infections Lab Diagnosis: Introduction, List of Common Virus, Types of Methods and Keynotes

Introduction of Viral Infections Lab Diagnosis

A virus is the smallest acellular organism having either DNA or RNA. It is non-pathogenic to deadly causing common cold (Rhinovirus) to hemorrhagic fever (Ebola- deadly virus). Marburg virus and Ebola come under risk group 4 microorganisms with a high fatality rate. The common viruses that are encountered in the clinical laboratory are HIV, HBV, HCV, HAV, HEV, Japanese encephalitis, Poliovirus, Meseals viruses, Cytomegalovirus, and Herpes simplex type I and II viruses, etc. CoVID-19 pandemic causing virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was the most common that was diagnosed laboratory so far. Viral Infections Lab Diagnosis is the only tool that helps to confirm the etiological agents of viral infections/diseases. The general approaches are serology, microscopy, cultivation, molecular assay.

List of Common Viral Infections Lab Diagnosis

The viruses of the principal interest in the countries of  Eastern South Asia are hepatitis, HIV/AIDS,  Japanese encephalitis, Poliovirus, Meseals viruses, Cytomegalovirus, and Herpes simplex type I and II viruses, SARS-CoV-2, etc.

Methods of Viral Infections Lab Diagnosis

Viral Infections Lab Diagnosis by Serological methods

The following different Serological testing methods are available varying upon the type of the virus infection and site of infection.

• RPHA ( Reversed Passive Haemagglution) Assay: The technique utilizes stabilized red cells coated with specific viral antibodies. e.g. HBV-HBsAg detection can be done by RPHA.

• Latex/gelatin particles Agglutination Tests – Commerical kits are available. e.g Adenovirus, HIV/AIDS, etc.
• HAI (Haemagglutination Inhibition) Tests  – e.g. HAI tests for JE.
• ELISA – Enzymes Linked Immunosorbent Assay – e.g. TORCH (toxoplasmosis, rubella cytomegalovirus, herpes simplex) range and HIV infection. The commercial test kits are available with positive and negative controls including different buffers required for the washing etc.
• WB (Western Blot) Assay – The WB test is a little more sophisticated test and it is used for the confirmation of HIV-positive cases.
• IFA (Immunofluorescence Assay) Immunofluorescence also called cell imaging technique is a combination of Immuno and fluorescence and this assay is used primarily on biological samples and is classically defined as a procedure to detect antigens in cells using antibodies. The specificity of the antibody to its antigen is the base for immunofluorescence.  This is a useful test in virology. The commercial kits are expensive. A fluorescence microscope is required and the facilities.

For Routine Use the following Tests are Recommended in Viral Infections Lab Diagnosis

• Stool samples – In children with diarrhea the stool samples are tested for Rotaviruses by ELISA techniques.

The Stool samples for Poliovirus Culture – The stool sample from all the cases of Acute Flaccid Paralysis (AFP) are preserved in the refrigerator and transported in cold packs to the sophisticated laboratory for Polio Virus Culture.

• Nasopharyngeal aspirates/Throat swabs- The tissue culture is recommended for the investigation of Influenzae Viruses. These samples are collected by aseptic techniques in special containers and transported in dry ice.
• Blood Samples- The blood samples are collected for the serodiagnosis of many of the viral infections. The sensitive third-generation test in which the low levels of antibodies ( up to 10 ng/ml serum) can be detected in blood/serum samples. e.g Japanese Encephalitis, Dengue Viruses infection. herpes simplex virus type I and II etc. This test is called ELISA.

Techniques of sample collection for Viral Infections Lab Diagnosis

• The blood samples are collected aseptically in a sterile test tube.
• The blood is allowed to clot at room temperature (RT).
• The serum is separated aseptically into a sterile screw-capped tube.
• The sample was labeled properly and transported in the cold pack to the referral center.
• Cerebrospinal fluid (CSF) – The CSF sample is collected by Lumar puncture aseptically before the antibiotic is given, CSF being preserved in a sterile container for the JE test.

Virus Transport Medium (VTM)

VTM is a buffered medium used to maintain the viability of viruses during their transport to a Virology Laboratory.

To make about 25 bottles:

Hank’s balanced salt solution: 43 ml

Bovine albumin, 100 g/l (10% wlv): 5 ml

Phenol red, 4 g/l (0.4% w/v): 0.25 ml

Nystatin, 2500 IU/ml in sterile phosphate-buffered saline (PBS): 0.5 ml

Penicillin, 104 IU/ml and streptomycin 10 mg/ml in sterile: 0.5 ml

Commercially this medium is available from Oxoid or it can be made in the lab

Specimens for virus culture –

• Respiratory viruses

Rhinoviruses  – Throat swabs

Influenza  – Nasal secretion

Parainfluenza – Nasal secretion

Adenoviruses   – Throat swabs/Stool specimens

Enterovirus

Rheovirus

• Skin/mucous membrane infection

Herpes simplex I, II  – Throat fluid

Varicella-Zoster virus and Scrapings

Measles Virus – Throat swab

Rubells Virus –  Blood

• CNS infections

Enterovirus – Stool/CSF

Encephalitis virus -CSF /Blood

Lymphocytic Chorio

Meningitis virus (LCM) – CSF

Herpes simplex -Throat swab/CSF

Rabies virus – Saliva

• Parotitis

Mumps virus – Throat swab

• Hepatitis A virus

A blood sample is tested for HAV antibody

Hepatitis B virus – HBV

There are many types of serological tests available for the detection of HBs Ag antigens in the blood samples such as:-

1.  Latex agglutination test- Virutex (Tulip labs)
2. RPHA tests
3. ELISA tests – Wellcome Ltd, Organon Teknika etc.
4. Membrane blot tests

• Most of these tests are screening methods and are very sensitive 3rd generation assays.
• It is important to screen the Blood for HBs Ag before transfusion and is now compulsory to test HBs  Ag in Blood Bank.

Hepatitis c virus HCV- It is necessary to screen the blood before donation. The test kits are available commercially.

Diagnosis of HIV/AIDS

1. Diagnosis – Provisional diagnosis can be done by detailed clinical history and presence of the risk behavior such as IVDU, sex promiscuity, multiply sex partners, Health Care Workers  (HCWs ) are all the time exposed to the infected blood. and unscreened blood transfusion also poses a great risk of HIV infections.
2. Clinical diagnosis – Physicians can elicit the clinical signs/symptoms that give clues towards HIV/AIDS infection such as

• Extreme emaciation
• Oral candidiasis
• Lymphadenopathy
• Kaposi sarcoma
• Lymphoma
• Dementia and CNS problems.

Laboratory diagnosis – These techniques detect the antibodies only.

Screening Test

PA( Particles agglutination) test for HIV 1 and 2

The test pack is available from Serodia, Fujire Bio Inc. Japan. It is a self-contained pack containing HIV 1 and 2 coated gelatine particles as antigens and the other necessary buffers., positive and negative controls, etc. one has to follow the instruction leaflet in the test pack.

HIV Spot HIV 1 and 2

This test is available from Gene Lab. The test kit is a self-contained pack with necessary buffers, positive and negative controls, and gold conjugate, etc. The test cartridges contain HIV 1 and 2 antigens as the spot in the center. The instruction leaflet accompanies the positive results is indicated by the development of a purple spot at the center. The test is easy to perform with little training.

Capsule Test 

This is the agglutination test. It detects HIV 1 and 2 by agglutinations. This is also a self-contained pack with positive and negative control. The test is easy to perform.

ELISA

This technique involves the use of an enzymes system and an immunosorbent ( an absorbing material specific for one component of reaction, antibody, or antigen). The absorbing material could be either agarose or a solid matrix ( microwell, membrane). The test kits are available commercially from Welcome Ltd, Organon Teknika. interpretation by the naked eye and also by using a Sepctrophotomer if available. In ELISA after every step washing by suitable buffer must be done properly.

• Confirmation tests

Western blot WB – Commonly done

This technique detects antibodies directed to the Gag, Pol, and ENV components of HIV virus type 1 and 2

Gag   – p24. p55, p18

Pol- p66, p51, p31

ENV- gp160, gp120, gp41

Positive – positive should have 3 bands of

Anti – gp 160/120

Anti -gp 41

Anti- p 24

Indeterminate – When one or more bands are seen but not specific to any of the above bands are interpreted as indeterminate. The tests should be re-tested after 2-3 months.

Negative – The test strip should show no bands if only one band develops it is no significance.

Japanese Encephalitis Virus – JE virus

JE virus is transmitted by mosquito bites.

Viral Infections Lab Diagnosis

In an area to JE, the patients with rigor, headache, general malaise, and convulsions with abnormal sensorium are suspected of having JE infection.

• Blood sample collected immediately for base-level JE antibody.
• CSF sample is also collected by Lumbar puncture CSF can be tested for IgM antibody.
• The second blood sample was collected 2 weeks after the onset of the clinical illness. Both blood samples are tested for the Anti JE IgM antibody.
• A four-fold rise in antibody titer indicates JE infection.
• The presence of JE IgM in the CSF indicates the local antibody production suggesting encephalitis.

There are many conventional surgical tests for JE antibody

• HIA- haemo- agglutination inhibition tets.
• CFT – Complement Fixation test
• If = Immunofluorescemce tets
• Neutralization test (NT)- Plaque reduction NT
• ELISA – Enzyme-linked ImmunoSorbent Assay. Mac ELISA is the most important test for JE
• PCR – Now the latest techniques for JE in which JE viral genomic sequences are detected by PCR (Polymerase Chain Reaction)

Keynotes on Viral Infections Lab Diagnosis

• The specimens have to be collected by sterile swabs and transported in the virus transported medium. In the case of stool specimen, 1gm of stool is added to virus transport media. The specimens are then transported to the referral for the virus culture.
• The confirmatory test of HIV is Western blot.
• Some antigen assays have explored the use of viral transport medium (VTM) during sample collection, but the use of VTM may dilute the specimen and may decrease the sensitivity of the assay (possibly causing false test results).
• Regarding SARS-CoV-2 Antigen Test-Decontaminate work surfaces and equipment with appropriate disinfectants by using an EPA (emergency use authorization ) -approved disinfectant for SARS-CoV-2
• Introduction of SARS-CoV-2 Antigen Test: SARS-CoV-2 antigen test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is used as screening tests on asymptomatic persons to detect or exclude Corona Virus Disease 2019 (COVID-19) or to determine whether a person who previously was diagnosed with COVID-19 remains infectious. It is an immunoassay for that nasopharyngeal or nasal swab specimen is used. The test is simple, rapid, and easy to perform even in a low level of laboratory setup. The report may be dispatched within half an hour.
• The antibody is the immunoglobulin and detectable antibodies in antibody-detecting rapid diagnostic tests (Ab-RDTs) are IgM and IgG but rarely IgA too. SARS-CoV-2 structural components like nucleocapsid (N), spike (S) protein, and receptor-binding domain (RBD) has been used as antigens for antibody assay. Among them, nucleocapsid (N) is the most common antigen for the SRAS-CoV-2 antibody test. IgM could be detected as early as 1-day post- symptom onset (PSO) and was detectable in 85% of COVID-19 confirmed patients 7 days PSO.  As for IgG, over 90% of COVID-19 confirmed patients produced this type of antibody 14 days after illness but in rare cases yet fails to produce IgM/ IgG 40 days after symptom onset. Blood is taken as a specimen.
• PCR stands for a polymerase chain reaction and PCR test applies to detect genetic material from a specific organism but here for a virus, SARS-Cov-2. The test detects the presence of genes of a virus if you have been infected at the time of the test. The assay is also applicable to detect fragments of the virus even after you have no longer been infected. Test specimens are nasopharyngeal swab,  oropharyngeal swab, bronchoalveolar lavage, sputum, etc.

Further Readings on Viral Infections Lab Diagnosis

1. https://www.sciencedirect.com/topics/medicine-and-dentistry/viral-diseases-diagnosis
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7149825/#:~:text=The%20traditional%20approaches%20to%20laboratory,measurement%20of%20antibodies%20in%20the
3. https://academic.oup.com/cid/article/31/3/739/297506
4. https://en.wikipedia.org/wiki/Laboratory_diagnosis_of_viral_infections
5. https://dermnetnz.org/topics/laboratory-tests-for-viral-infections
6. https://www.msdmanuals.com/home/infections/overview-of-viral-infections/overview-of-viral-infections
7. https://nios.ac.in/media/documents/dmlt/Microbiology/Lesson-54.pdf
8. https://www.uofmhealth.org/health-library/hw235580