Global travel is an efficient route of transmission for highly infectious pathogens and increases the chances of such pathogens moving from high disease-endemic areas to new regions. We describe the rapid and safe identification of the first imported case of Ebola virus disease in a traveler to Lagos, Nigeria, using conventional reverse transcription polymerase chain reaction (RT-PCR) in a biosafety level (BSL)-2 facility.
On 20 July 2014, a traveler arrived from Liberia at Lagos International Airport and was admitted to a private hospital in Lagos, with clinical suspicion of Ebola virus disease.
Blood and urine specimens were collected, transported to the Virology Unit Laboratory at the College of Medicine, University of Lagos, and processed under stringent biosafety conditions for viral RNA extraction. RT-PCR was set-up to query the Ebola, Lassa and Dengue fever viruses. Amplicons for pan-filoviruses were detected as 300 bp bands on a 1.5% agarose gel image; there were no detectable bands for Lassa and Dengue viral RNA. Nucleotide BLAST and phylogenetic analysis of sequence data of the RNA-dependent RNA polymerase (L) gene confirmed the sequence to be
Our BSL-2 facility in Lagos, Nigeria, was able to safely detect Ebola virus disease using molecular techniques, supporting the reliability of molecular detection of highly infectious viral pathogens under stringent safety guidelines in BSL-2 laboratories. This is a significant lesson for the many under-facilitated laboratories in resource-limited settings, as is predominantly found in sub-Saharan Africa.
Ebola virus is the causative agent of Ebola virus disease (EVD), previously known as Ebola haemorrhagic fever.
On 17 July 2014, the Ministries of Health in Liberia, Guinea and Sierra Leone, in collaboration with the World Health Organization, announced a cumulative total of 1048 suspected and 745 laboratory-confirmed cases of EVD, with 632 (60.3%) deaths
A 40-year-old Liberian man, who had been living in Elwa, Monrovia, travelled by air through Accra (Ghana) and Lomé (Togo) to Lagos, Nigeria. Upon arrival at Lagos International Airport on 20 July 2014, he collapsed and was taken to a private hospital in the Obalende area of Lagos, Nigeria (
Travel route of index case with Ebola virus disease from Liberia to Lagos, Nigeria. Lower inset shows the closest health care facility to the airport (LASUTH), the private health care facility where the index case was admitted (Private Hospital), the Laboratory where the diagnosis was made (Virology Lab, CMUL/LUTH) and the Genomics Centre where the corroborative diagnosis and nucleotide sequencing was done (ACEGID). Map was plotted with GPS coordinates using Arc GIS 10.1.
No ethics approval was required for the diagnosis of an infection of great public health importance and which was part of the normal course of management of any infected patient.
Due to the fact that no BSL-3 or BSL-4 facilities were available to handle the patient’s specimens, all samples received were handled in a BSL-2 facility with extreme care and in line with US Centers for Disease Control and Prevention (CDC) safety guidelines.
For viral RNA extraction, neat (undiluted), 1:10 and 1:100 dilutions in sterile phosphate-buffered saline specimens of both blood and urine were processed using a Qiagen RNA extraction kit (Qiagen, Germantown, Maryland, United States). Segments of the L-gene of the Ebola virus, the S-gene of the Lassa virus and the 3’ non-coding region of the Dengue fever virus were amplified in singleplex PCRs using the primers listed in
Primers used in the laboratory investigation of viral haemorrhagic diseases.
Virus | Primer | Sequence | Amplicon size (bp) |
---|---|---|---|
Lassa fever virus | 36E2 |
5’-GTT CTT TGT GCA GGA MAG GGG CAT KGT CAT-3’ |
~300 |
Dengue fever virus | DenS |
5’-GGA TAG ACC AGA GAT CCT GCT GT-3’ |
97 |
Filovirus strains | FiloA2.2 |
5’-AAG CCT TTC CTA GCA ACA TGA TGG T-3’ |
298 |
PCR amplicons on the agarose gel were purified using an agarose gel extraction kit (Jena Bioscience, Jena, Germany). Purified non-infectious PCR products, packaged and transported using triple-level packaging, were sequenced using the Filo A2.3 primer on the Sanger dideoxy sequencing technology platform with an Applied Biosystems 3130xl Genetic Analyser at Jena Bioscience in Jena, Germany. A sequence trace file was used for BLAST analysis in GenBank.
Agarose gel image showing PCR amplicon bands of the specimens. The ~300 bp band is the target band size for Filoviruses.
Investigations carried out on viral RNA purified from the blood and urine specimens of the patient revealed the presence of the expected amplicon size (300 bp) for Filoviruses (
Phylogenetic analysis of the L-gene partial CDS indicated that the virus evolved from the Guinea and Mano River isolates of the 2014 outbreak with a bootstraping value of 87% (
Molecular phylogenetic analysis of the L-gene segment of EBOV/Hsap/NGA/2014/LIB-NIG 01072014 in comparison with selected filoviruses sequences by the maximum likelihood method. Sequences are labelled using the ICTV consensus nomenclature for variants of the
By using molecular techniques and following CDC safety guidelines, our BSL-2 laboratory was able to safely identify the Ebola virus in blood and urine samples. The clinical symptoms of the index case were initially linked to malaria and typhoid fever, until the patient began to haemorrhage, which led to a differential diagnosis of Lassa, Ebola or Dengue fever. The time lapse between hospital admission, malaria/typhoid treatment and the clinical suspicion of EVD was two days and led to the exposure of 72 persons at the airport and the hospital.
Partial genomic sequence analysis data identified
Many African countries, governments and research institutions are inadequately equipped or trained in diagnostics, active surveillance and reporting of highly infectious diseases.
Molecular detection using PCR is the most sensitive method for viral diagnostics. The use of chaotropic lysis buffer for the isolation of viral nucleic acids from the highly contagious specimens was a key step, because it rendered specimens non-infectious and thus safe for processing in a BSL-2 laboratory. However, despite our success, it should be noted that manipulations of highly pathogenic viruses such as Ebola cannot be attempted in a BSL-2 laboratory. Manipulations, such as culturing live Ebola viruses, must be restricted to BSL-4 and BSL-3-enhanced laboratories, because of the higher risk of contracting Ebola virus when incidents occur.
One of the major lessons learnt from this outbreak is that that there is an urgent need to build capability for rapid detection of and response to disease outbreaks in resource-limited countries, especially in West Africa where the health systems are very weak. This should serve as a wake-up call, not only for African governments but also to the world, that investment in laboratory infrastructure and improvements in laboratory capabilities, as well as building capacity for disease surveillance, infection control and biosafety, is critical so that these countries do not constitute the weak links in the ongoing fight against infectious diseases.
The authors are grateful to and salute the courage of the late Dr Ameyo Stella Adadevoh (First Consultant Hospital). Her courage in restraining the index patient and promptly contacting the authorities led to the prompt diagnosis of the virus and initiated the chain of events that ended the outbreak in Nigeria with very few casualties. We are also grateful to the management and staff of First Consultant Hospital, Obalende, Lagos, Lagos State Ministry of Health, Nigerian Centre for Disease Control / Federal Ministry of Health and all those that assisted to curtail the spread of the Ebola virus in Nigeria.
The authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article.
Our Laboratory is a National Reference Laboratory for Viral Haemorraghic Fevers and a World Health Organization’s Regional Office for Africa Reference Laboratory for EVD. This project was supported by the Federal Ministry of Health and the World Health Organization’s Regional Office for Africa.
S.A.O. was the laboratory director and team lead. O.B.S., A.B.J. and B.O.O. were responsible for experimental and project design, analysis of data and writing the manuscript. M.R.O., R.A.A. and M.A.A. performed most of the experimental analysis. C.H. made conceptual contributions, performed some experimental analysis and assisted in preparing the manuscript. J.I., I.A.A-S., A-S.N., F.T.O. and O.T. all made conceptual contributions and assisted in preparing the manuscript.