Viral Culture & Immunofluorescence (IF) Assay – strategies used in diagnosis of human Infectious diseases

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View Knowledge Base – Diagnostic antibodies and antigens for infectious disease testing including HIV/AIDS, HCV, Influenza A/B, Syphilis, Helicobacter pylori, HSV-1/2, CMV, rubella virus, toxoplasma.>>

Abstract

Infectious diseases are a significant burden on public health and economic stability of societies all over the world. They have been among the leading causes of death and disability and presented growing challenges to health security and human progress for centuries. Infectious diseases are generally caused by microorganisms. The routes of them entry into host is mostly by the mouth, eyes, genital openings, nose, and the skin. Damage to tissues mainly results from the growth and metabolic processes of infectious agents intracellular or within body fluids, with the production and release of toxins or enzymes that interfere with the normal functions of organs and/or systems [1]. Advances in basic science research and development of molecular technology and diagnostics have enhanced understanding of disease etiology, pathogenesis, and molecular epidemiology, which provide basis for appropriate detection, prevention, and control measures as well as rational design of vaccine [2]. The diagnosis of infectious diseases is particularly critical for the prevention and control of the epidemic. Here we introduce the insights and detection methods of infectious disease, aiming to provide some helps for clinical diagnosis as well as epidemic prevention and control of infectious diseases.

Viral Culture & Immunofluorescence (IF) Assay - strategies used in diagnosis of human Infectious diseases

Virus culture, isolation and identification are the gold standards for laboratory identification of pathogens. However, viral culture results do not yield timely results to inform clinical management. Shell-vial tissue culture results may take 1-3 days, while traditional tissue-cell viral culture results may take 3-10 days. Due to the long incubation time, high technical requirements, and must be carried out in a level III safe biological laboratory, it is not suitable for rapid virus diagnosis during the epidemic period [28].

 

Immunofluorescence (IF) technique is widely used for rapid detection of virus infections by identifying virus antigens in clinical specimens. IF staining is usually considered very rapid (about 1 to 2 hr) and overall gives a sensitive and specific viral identification [29-32]. Unfortunately, IF technique may not able to confirm the identity of all virus strains, for instance viruses of the “enterovirus” group; since most monoclonal antibodies (MAbs) for enteroviral identification have been shown to lack sensitivity, while cross-reactivity with rhinoviruses is extremely common [33]. In contrast, IF has been successfully used for better management of influenza virus infection and surveillance of influenza virus activity [30, 31]. As recommended by CDC, when influenza activity is low, positive results should be confirmed by direct immunofluorescence assay (DFA), viral culture, or RT-PCR, as false positive test results are more likely; while during peak influenza activity confirmatory testing using DFA, viral culture, or PCR must always be considered because a negative test may not rule out influenza viral infection. Interestingly, although IF is generally considered less sensitive then ELISA and PCR, a recent publication reports DFA as an optimal method for rapid identification of varicella-zoster virus (VZV), when compared with conventional cell culture [34]. In contrast, the Herpes simplex virus (HSV) DFA test accuracy was found very low (sensitivity 61%, specificity 99%), when tested to identify mucocutaneous HSV infection in children [35]. Furthermore, a monoclonal antibody designated CHA 437 was developed against HSV showed no cross-reactivity against the varicella-zoster virus, cytomegalovirus, or Epstein-Barr virus, however direct specimen testing resulted in overall low sensitivity (84.6%) and specificity (95.7%) [36]. On the other hand, an antigen detection assay for severe acute respiratory syndrome (SARS) coronavirus (CoV) could detect SARS-CoV in 11 out of 17 (65%) samples from SARS patients. As such IF technique is well-accepted laboratory diagnostics test, however, sometime these assays could be quite expensive, due to the cost of antibodies used. Additional variability may also be introduced due to non-specific binding, or cross-reactivity of commercially available antibodies [37].

As such IF technique is well-accepted laboratory diagnostics test, however, sometime these assays could be quite expensive, due to the cost of antibodies used. Additional variability may also be introduced due to non-specific binding, or cross-reactivity of commercially available antibodies.

Figure 4. Immunofluorescence staining of vaccinia virus infected cell [38]. Areas of virus assembly within the cell are pink. Host and viral DNA (deoxyribonucleic acid) is blue. The host cell's DNA is contained within its nucleus (large oval). Actin protein filaments, which make up part of the cytoskeleton, are green.

Summary

Infectious diseases are a real public health threat, outbreaks can have serious social, political, and economic effects. A complex number of factors relating to human behavior and activities, pathogen evolution, poverty, and changes in the environment as well as dynamic human interactions with animals have been found to contribute to infectious disease emergence and transmission. Aggressive research is warranted to unravel important characteristics of pathogens necessary for diagnostics, therapeutics, and vaccine development. Here we describe some strategies for the diagnosis of human infectious diseases, hoping to be helpful for clinical diagnosis and epidemic prevention and control of infectious diseases. To date, multiple diagnostic techniques have been developed. Various diagnostic tools show both significances and limitations. Conventional approaches to quantify infective viral particles are labor-intensive, time-consuming, and often associated with poor reproducibility. Immunological tests generally provide quick results, however, is quite expensive due to the requirement of antigen-specific antibody. While RT-PCR may be able to provide results within a matter of hours, it is laborious, requires a skilled operator, and is sensitive to contamination. TEM-based quantification, although highly accurate in determining the shape and the total number of viral particles, often considered time-consuming, extremely expensive and impractical for high sample numbers. Moreover, TEM sample preparation is tedious, and the technique requires sophisticated instrument and a skilled operator. To alleviate these limitations, there is still a need to develop new cost-effective analytical methods that can allow users to quickly and easily determine virus concentrations and reduce constrictions coupled with current assays. Nevertheless, any such emerging methods must be carefully evaluated in terms of their efficiency, precision and linear range. The evaluation of each diagnostic technique and approval from the FDA are necessary before practical application.

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Cytokine release syndrome (CRS) is an acute systemic inflammatory syndrome characterized by fever and multiple organ dysfunction.

Genemedi produces core diagnostic ingredients for test of infectious diseases and related syndrome.

GeneMedi offers paired diagnostics grade antibodies (monoclonal antibody,mab)and antigens for infectious disease rapid test kit including infection of HIV/AIDS,Hepatitis C virus(HCV), Influenza A/B,Treponema Pallidum caused Syphilis,Helicobacter pylori (H. pylori) Bacteria, Herpes simplex virus 1/2(HSV-1/HSV-2),Cytomegalovirus (CMV),rubella virus,toxoplasma gondii and so on.

All the antibodies of inflammation and CRS test are suitable for in functional ELISA, and other immunoassays in diagnostics. The antibody can act as a capture antibody and detection antibody.

Reference

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