Monday, August 15, 2011

Hemorrhagic fever viruses Diagnosis

The diagnosis of VHF can be confirmed by Ab testing or by detection of viral RNA in blood or other body fluids. In the U.K., virus isolation is usually also attempted (Aitken & Jeffries, 2001).
Ebola and Marburg laboratory diagnosis
Patients in the acute stages of disease have high case fatality rates, and most of those who die do so without developing detectable antibodies. This emphasizes the diagnostic need for isolation of the virus or, antigens or RNA detection. The specimens must be obtained and sent to a laboratory with the appropriate expertise. Because outbreaks have continued to include care givers who have direct contact with patients, those who collect samples need to take adequate barrier nursing precautions while obtaining the samples (Richards et al., 2000).
Additionally, special care should be taken to avoid needle sticks and to dispose of the sharps in an appropriate manner. Collection of specimens should be done with a mind toward sterility and prevention of cross-contamination of specimens; this has become particularly important in the day of sensitive techniques such as RT-PCR amplification for diagnosis and characterization of the virus genome (Kiley and Lloyd, 1998).
• Electron microscope
EM has been particularly useful in the diagnosis of Filovirus infections. Negative-contrast electron microscopic examination of initial-passage cell culture can be performed. Although the fine structure of viruses is distorted, resolution of nucleocapsid structures within viruses can establish the diagnosis (Yang et al., 2000).

Electron micrograph of Filovirus (B├╝chen-Osmond, 2006)
• Virus isolation and cell culture
Although high-level containment is warranted because of the extreme hazard associated with handling the viruses, virus isolation remains relatively simple and sensitive. The virus grows well in a large variety of cell lines, although Vero cells have been most used. CPE can be recognized by microscopy and the virus identified by immunologic or EM means (Ksiazek et al., 1999).
• Antigen detection
Detection of either Ebola or Marburg virus Ag by ELISA has often been the most rapid means of diagnosis. The assay time is approximately 3 - 4 hours, and detection of antigen in the blood or serum of patients in the acute course of the disease was very successful when compared to virus isolation or to RT-PCR (Ksiazek et al., 1999).
Filoviruses Ag can also be detected by IHC examination of formalin-fixed tissues by using specific polyclonal and MAbs. These methods have role in cases in which archival tissues are the only specimens available for diagnosis. During the epidemic in Kikwit, Zaire, a novel sensitive IHC test was developed at CDC in which skin biopsy specimens were used for the diagnosis of Ebola virus (Lloyd et al., 1999).
Formalin-fixed biopsy specimens are not infectious, they may be taken in the most basic field conditions, and they may be sent without special precautions or refrigeration. This technique was useful for the diagnosis of fatal cases in subsequent Ebola outbreaks in Gabon (Zaki and Goldsmith, 1998); lesser sensitivity was noted in the recent Marburg outbreak in the DRC (WHO, 1999).
• Serology
IgM Ab ELISA proved useful in the diagnosis of recent infections in surviving patients. IgG response of the patients was somewhat delayed and realistically could be expected only in the early convalescent sera of those patients who were destined to recover from their infections. IgM Abs detected by this test have been found to persist for only 2 - 3 months in monkey that was experimentally infected and similar relatively short period in surviving humans (Ksiazek et al., 1999).
• Molecular detection
RNA amplification by RT-PCR has added another rapid and sensitive tool for the diagnosis of Marburg or Ebola viruses. The technique was first utilized during the 1989-1990 outbreak of the newly discovered Ebola Reston virus, but it has been used much more commonly in the recent human outbreaks in DRC in 1995 and in Gabon from 1994 - 1997 (Sanchez et al.,1999). The RT-PCR should be combined with direct sequencing of the amplified product. This provides virus sequence information that enables phylogenetic comparison of the virus in clinical specimen to other, previously recognized strains of the virus (Georges-Courbot et al., 1997).

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