Samples: In respiratory virus infection, samples from the affected organ system (respiratory tract and other organs) are collected for detection of virus or its components such as virus, viral antigen, or viral RNA.

• Nasal/throat swab, nasal/throat washing, nasopharyngeal swab/aspirate are the samples collected for virus isolation, antigen detection or genome detection in patients with acute upper or lower respiratory tract infection.

• Tracheal aspirate, bronchoalveolar lavage (BAL) or lung biopsy can be tested in case of lower respiratory tract infection (LRTI). However, BAL and lung biopsy involves invasive procedure, and recommended when samples of URTI comes negative.

 • Sputum can be collected for testing when produced.

• Blood or serum sample is collected for detection of antibody against the virus.

Virus isolation: Respiratory viruses can be isolated in various host systems.

Embryonated egg: Amniotic cavity of the embryonated egg is a suitable system for the primary isolation of influenza virus. Previously this system was used commonly for isolation of influenza virus from the clinical sample. However, the system suffers from low sensitivity and due to less availability of suitable pathogen free egg and development of resistance, this method is not commonly used presently.

Embryonated egg host is, however, not suitable host for primary isolation of HPIV.

Cell culture: Various cell lines are commonly used for primary isolation as well as for propagation of respiratory viruses.

Kidney cell lines from various animals such as Madin darby canine kidney (MDCK) cell lines and rhesus monkey kidney cell line LLCMK2 are commonly used for isolation of influenza, parainfluenza viruses, and other respiratory viruses. LLCMK2 and African green monkey kidney cell lines (Vero cells) are commonly used for HMPV isolation. Hep2 and HepG2 cells also support the growth of HMPV. Mixtures of two or more cell lines are available commercially for culture of respiratory viruses.

Provisional identification of virus is done by cytopathic effect which is confirmed by viral antigen detection in the infected cells by using monoclonal antibodies or virus RNA detection by RT-PCR.

Isolation is still considered as the gold standard. However, the method is time consuming and less sensitive. Because of these limitations, the method is no more used for routine diagnosis from clinical sample and has largely been replaced by molecular technique. However, it is required for virus characterization, antigen and vaccine preparation.

Shell vial technique: Also known as centrifugation enhanced technique. In this method, sample is centrifuged over the cell line during the period of incubation. This facilitates the rapid entry of virus into the cell and early expression of viral antigen on the surface of the infected cell line. The virus antigen is then detected by staining the infected cell with specific antibody by 24–48 hours. This technique is commonly used for respiratory syncytial virus (RSV).

Rapid Antigen Detection

Several tests are available for rapid antigen detection directly from respiratory sample. Immunofluorescence or enzyme immunoassay (EIA) methods are used for antigen detection. Of these, EIA format offers a point of care test system which can be done bedside without using any special equipment for testing and reading. These are mainly available for influenza virus and RSV from clinical sample.

The rapidity, simplicity and potential of point of care, makes the format useful.

For influenza virus: Sensitivity of these tests is poor and widely variable—20–90%. High sensitivity is achieved in the early part of illness due to high viral shedding.

Specificity in general is considered as good, but some recent report has shown near 40% false positivity.

Types of sample, transport media, transport temperature, processing of samples have influenced the sensitivity and specificity.

The test is based on the detection of conserved antigens like nucleoprotein and matrix protein. The method is not suitable to differentiate between types or subtypes of influenza viruses.

For RSV: Immunofluorescence assay on nasal or throat samples using monoclonal antibody demonstrates intracytoplasmic fluorescence (Fig. 1). However, this is expensive, requires fluorescence microscope and needs individual sample processing.

Fig1. RSV antigen showing cytoplasmic fluorescence in nasopharyngeal aspirate by monoclonal antibody

Molecular methods: Detection of viral RNA from clinical samples is done by various molecular tests. The advantages of molecular tests are:

• High sensitivity, specificity.

• Multiplexing to detect various types and subtypes of influenza viruses. This helps in detection of seasonal strains along with pandemic strains, also other respiratory viruses.

• Rapidity helps for short turnaround time.

 • Ability to be integrated with automation platform and high throughput system.

Polymerase chain reaction (PCR) is the most commonly used molecular method for diagnosis of influenza and other respiratory viruses and almost has achieved the place of gold standard.

Reverse transcriptase real-time PCR (rRT PCR): As most of the respiratory viruses are RNA viruses, RT-PCR is commonly used for detection of viral genome.

This method was the recommended test by CDC, Atlanta, for the detection of influenza A (H1N1) pdm09 and was the method followed for diagnosis almost worldwide including India. The test uses detection of four genes— influenza A, swine A, swine H with an in house RNAseP gene as control.

Multiplex RT-PCR: Various multiplex PCR systems are available commercially for detection of respiratory virus panel which primarily includes influenza A, influenza B, influenza A (H1N1) pdm09, HPIV 1–4, HMPV, RSV, adenovirus, human bocavirus and coronaviruses.

Loop mediated amplification (LAMP) and nucleic acid sequence based assay (NASBA) has been used for detection of influenza virus genome.

LAMP assay has reported similar sensitivity, specificity as rRT-PCR. The test uses 3 sets of primer and does not require any sophisticated equipment. Amplification occurs in isothermal temperature at 60–65°C.

Serology: Antibody starts appearing after 2 weeks of infection and takes its peak in 4–7 weeks. Antibody detection from blood is done for various purposes:

 • To look for seroconversion in acute and convalescent samples for diagnosis.

• For seroprevalence study in the community.

• To test for neutralizing antibody for immunity status.

• To check immune response to vaccine.

 • Identification of virus strain using specific antisera.

Serological Methods Used

• Hemagglutination inhibition.

• Complement fixation.

• Neutralization.

 • Enzyme immunoassay.

Demonstration of >4 -fold rise in titer of antibody between acute and convalescent sera (at 2–3 weeks interval) diagnose recent infection. The time taken and requirement of two samples is the main disadvantage of the test and because of this limitation the test is not used for routine patient diagnosis. However, it helps in diagnosis retrospectively.

Antibody detection helps in studying the seroepidemiology of a recently emerged strain. This is helpful in estimating the exposure to the new strain or immune status in the population which is important to take policy decision on vaccination.

A titer of >20 by HAI and >40 by micro neutralization (MN) test of a single convalescent sample is considered significant for seroprevalence study.

Immunogenicity of vaccine is estimated by determining the neutralizing or HAI anti body.

اخر الاخبار

اخبار العتبة العباسية المقدسة

الحزام الأخضر الجنوبي الأول يستضيف وفدًا شبابيًّا من محافظة المثنى

قسم شؤون المعارف الإسلامية يحتفي بإرث الأديب الراحل علي أنكيل ويتسلم مكتبته الشخصية

سماحة السيد الصافي يدعو إلى توثيق الأفكار والأحداث لحماية الإرث العراقي من الاندثار بغية نقله سليمًا للأجيال القادمة

قسم التطوير يقدم محاضرة ثقافية حول كتاب نهج البلاغة لملاكاته

المزيد

الآخبار الصحية

أول إرشادات غذائية قائمة على الأدلة لمعالجة الإمساك المزمن

صباح بلا إفطار؟.. صحتك في خطر!

هل يمكن للجلد أن يكشف عن صحتك العقلية؟.. دراسة حديثة تجيب

السر في مطبخك.. 6 أطعمة تمنح عظامك قوة الشباب

المزيد

الآخبار التكنلوجية

ناسا تكشف: كوكب الأرض أصبح أقل لمعانا خلال العقود الماضية

دراسة: الوقت الذي تستخدم فيه هاتفك قد يفضح حالتك النفسية

الهنغاري لاسلو كراسناهوركاي يفوز بجائزة نوبل للآداب 2025

رسميا.. إعلان الفائز بجائزة نوبل للسلام لعام 2025

المزيد

مواضيع ذات صلة

Human metapneumovirus (HMPV)

Human respiratory syncytial virus (HRSV)

Chemical Composition of Viruses

Survey of RNA-Containing Viruses

Survey of DNA-Containing Virus Families

Universal System of Virus Taxonomy

Human parainfluenza viruses (HPIV)

Complications of Influenza virus

Influenza virus

Chikungunya virus

West Nile virus

Transmission Cycle of Japanese encephalitis virus