DEVELOPMENT OF THE TOOLS FOR PCR-DETECTION OF HEPATITIS A AND C VIRUSES IN INTRAHOSPITAL VIRAL CONTAMINATION

RESEARCH.

1D. I. Ivanovsky Virology Institute, Ministry of Health Russian Federation. 2Chumakov Institute of Poliomyelitis and Viruses Encephalitis, Moscow, Russia;

3Karolinska Institutet, Stockholm, Sweden; 4Riga Stradins University, Riga, Lativa.

S. A. Esper1, T. V. Grebennikova 1, K. K. Kyuregyan 2 ,M. G. Issagouliantis 1, 3, 4

- Previous studies have shown the importance of surfaces and objects in the transmission of viruses, and that many viruses can remain infections outside the body for long period of time.

- This suggests that viruses can potentially serve as a biomarker for hospital-acquired infections. Thus, monitoring of hospital surfaces is crucial to limit the spreading of these viruses in the environment, which could contribute to more effective evaluation of microbiological condition in hospital settings, effective measures for the disease spread prevention, and treatment of patients.

The Aim of Work:

-Choosing the most sensitive PCR kit for detection of the hepatitis A and C viruses (HAV and HCV).

-Understanding for how long time the viruses of hepatitis A and C can be stored on the surface.

•Methadology and ResultsPCR kits: -Amplicence HAV-FL-Amplicence HCV-Eph PCR-nested PCR using laboratory developed

primers

Viruses: - Positive controls of viruses HAV Cultured

virus genotype A in inactivated bovine serum, concentration 105 RNA copies/ml.

- HCV Serum from patients with chronic hepatitis C, concentration 107 copies/ml.

Kit Comparison Experiment In order to select the most sensitive

method. Comparison was produced between “Amplicence HAV-FL” kit based on Real-time PCR, and nested PCR using laboratory developed primers were used for detection of HAV genome.

- It was found that Real-time PCR and nested PCR were able to detect 1-10 RNA copies of HAV.

- For HCV, Comparision between “Аmplicence HCV-Eph” kit and nested PCR using laboratory developed primers.

- “Аmplicence HCV-Eph” has ability to determine 103 RNA copies/ml, while nested PCR can detect 10 RNA copies/ml.

- Comparison of these analyses was shown, that nested PCR using home-made primers was more sensitive than PCR using “Аmplicence HCV-Eph” kit.

nested PCR

-Experiment to understand for how long the viruses of hepatitis A and C can be stored on the surface.

- Series of HAV dilutions was prepared: from 105 to 103 RNA copies/ml and series of HCV dilutions: from 107 to 105 RNA copies/ml.

Isolation of RNA

PCR Detection for HCV and HAV

Collection occurred after 8 hr, 24hr, 32hr, 48hr, 56hr.

Surfaces with different HAV, HCV Dilutions

-The dilutions were spread on surface. Samples were collected after 8 hr, 24hr, 32hr, 48hr, 56hr.

- It was found that HAV in concentration 105 RNA copies/ml remain detectable 56 hours later, after spreading on the surface, while HAV in dilution 104 RNA copies/ml was detected after 24 hours, and HAV in dilution 103 RNA copies/ml was detected 12 hours later, after spreading on the surface

Hours after spreading HAV RNA copies/ml

56 105

24 104

12 103

- HCV concentrations 107 RNA copies/ml was discovered 32 hours later. While HCV concentration 106 RNA copies/ml was discovered 24 hours later, and concentration 105 RNA copies/ml was discovered 8 hours later, after spreading on the surface.

Hours after spreading HCV RNA copies/ml

32 107

24 106

8 105

Conclusion:- Viruses of Hepatitis A and Hepatitis C

depending on concentration remain stable on surfaces in environment from several hours to two days. So they could be used for environmental monitoring and identification the level of intrahospital contamination.

- For HAV detection could be used both Real-time PCR and nested PCR, for HCV detection should be used nested PCR.

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