Detection of human herpes simplex virus in clear cerebrospinal fluid by multiplex PCR at the Natiobal Reference Laboratory,Kigali,Rwanda

Abstract

Background: More than 80% of the clear cerebrospinal fluid (CSF) submitted to the National Reference Laboratory (NRL) , Centre Hospital University Kigali and King Faysal Hospital in Kigali, Rwanda, are found to be negative for Cryptococcus neoformans and Mycobacterium tuberculosis, the only microorganisms tested for at this facility. These samples are obtained from patients with clinical suspicion of meningitis and encephalitis, which may be caused by different microorganisms including herpes viruses. The purpose of this study was to test CSF obtained from different regions of Rwanda for human herpes simplex viruses (HSV) type 1 (HSV -1) and type 2 (HSV -2) using a commercial multiplex PCR kit. The objective of this study was to determine the prevalence ofHSV in clear CSF in Rwanda and to demonstrate that HSV -1 and HSV -2 can be PCR co-amplified in the same reaction tube. Methodology: Clear CSF was obtained from patients with clinical suspicion of meningitis and encephalitis from five provinces in Rwanda. All the samples were transported to the NRL for DNA extraction and then subjected to multiplex PCR for detection of HSV -1 and HSV -2. The PCR amplicons were resolved on 2% agarose gels and bands corresponding to HSV-l, HSV -2, and the internal control identified under ultra-violet (UV) light. Results: A total of 196 clear CSF samples were analyzed with this assay. Eleven out of 196 samples (5.6%) tested positive for Cryptococcus neoformans based on information supplied by medical officers where CSF originated. Of these samples, 7 (4.3%) were positive for HSV-l, 12 (6.0%) for HSV-2. 2 (1.0%) were dually infected with both HSV-l and HSV-2. Overall, 21 out of 196 samples (10.7%) were positive for HSV nationally. Conclusion: Multiplex PCR for diagnosis of HSV -1 and HSV -2 in clinical specimens (CSF) was found to be rapid and specific at the NRL. Demonstration of viral DNA detection by PCR is a major milestone as this provides a platform to the NRL to institute viral diagnostic procedures using PCR. The prevalence of HSV in CSF in this study is high enough (l 0.7%) to prompt the NRL to undertake a validation of the assay so that it can be adapted for routine HSV diagnosis in the laboratory. Once validation of the assay is done, its implementation at this referral laboratory will provide advisory leadership to physicians on whom patients can effectively benefit from available anti-HSV drugs like acyclovir, hence improving the quality oflife for all Rwandese and its neighbours.