Evaluation of Ebola Virus Inactivation Procedures for Plasmodium falciparum Malaria Diagnostics

Abstract

c Plasmodium falciparum malaria is highly endemic in the three most affected countries in the current epidemic of Ebola virus disease (EVD) in West Africa. As EVD and malaria are clinically indistinguishable, both remain part of the differential diagnosis of ill travelers from returning from areas of EVD transmission. We compared the performances of a rapid diagnostic test (Binax-NOW) and real-time PCR with P. falciparum-positive specimens before and after heat and Triton X-100 inactivation, and we documented no loss of sensitivity.T he current outbreak of Ebola virus disease (EVD) in West Africa is the largest on record (1), and guidance has emerged in hospital and outpatient settings around screening for EVD in ill travelers who have returned from areas of EVD transmission (2, 3). Plasmodium falciparum malaria is highly endemic in the three most affected countries, Sierra Leone, Liberia, and Guinea, which contribute Ͼ3 million cases annually to the global burden of malaria (4). Malaria can cause severe morbidity and mortality if diagnosis and/or treatment are delayed (5); yet, hospital laboratories are often ill-prepared to handle specimens from an individual suspected to have a filoviral infection (6). Laboratory safety must be balanced against the timely exclusion of malaria in ill travelers who have returned from EVD-affected regions in order to avoid adverse outcomes in patients. The Centers for Disease Control and Prevention (CDC) has recommended the addition of Triton X-100 and heat inactivation at 56°C prior to testing specimens from patients suspected to have filoviral infection, in addition to performing enhanced safety procedures, such as using personal protective equipment (PPE) and a certified class II biosafety cabinet (BSC) (7,8). The inactivation of blood prior to the preparation of malaria thin smears is unnecessary, as filoviruses are inherently susceptible to methanol (7, 9), the solvent in which malaria thin smears are fixed prior to staining. However, rapid diagnostic tests (RDT) for malaria are widely and routinely used in hematology and microbiology laboratories and have no corresponding fixation step. Similarly, the extraction of nucleic acid for molecular diagnostic assays is presumed to inactivate filoviruses, although data on this are lacking.The available data on the effects of filoviral inactivation procedures on the performance characteristics of malaria diagnostic assays are limited and variable (10, 11). We sought to evaluate the performance characteristics of both RDT and quantitative realtime PCR for detecting P. falciparum malaria following Triton X-100 and heat inactivation compared to those using the standard operating procedure. We documented no loss of sensitivity for either assay when performing filoviral inactivation procedures.Thirty-one P. falciparum-containing whole blood EDTA specimens, with parasitemia levels ranging from Ͻ0.1% to 6.6% and confirmed by microscopy and RDT (BinaxNOW Malaria, Alere, ME), were included, along with 10 negative-control EDTA specimens. Nin...

Buffer AVL Alone Does Not Inactivate Ebola Virus in a Representative Clinical Sample Type

“…Additionally, the dual treatment method is relatively quick, with inactivation being achievable within 30 min, which helps with throughput of samples. The heating time is reduced compared to previous methods (8)(9)(10). After inactivation, samples are then able to be manipulated outside containment, which increases sample throughput.…”

“…The CDC recommends Triton X-100 and heat treatment for 1 h for diagnostic samples containing hemorrhagic fever viruses (8), and this method has been adopted by many laboratories for handling of samples that may contain EBOV (9). Heating (alone or with acetic acid) for 1 h at 60°C has also been shown to reduce the titer of EBOV (10).…”

Rapid Bedside Inactivation of Ebola Virus for Safe Nucleic Acid Tests

“…Presently, laboratory EBOV inactivation is accomplished by gamma irradiation (8), UV radiation (9), nanoemulsion (10), and photoinducible alkylating agents (11), but these methods are not applicable in outbreak situations or as bedside inactivation methods. Other EBOV inactivation methods, such as acetic acid (12), heat (12), AVL buffer (13), TRIzol (13) or the combination of heat and Triton X-100 (14), are more applicable in outbreak situations and are currently used in field laboratories. Unfortunately, all of these methods require hands-on handling and manipulation of the sample before EBOV is inactivated.…”

Inactivating Zaire Ebolavirus in Whole-Blood Thin Smears Used for Malaria Diagnosis

“…Thus, methanol treatment alone is sufficient for EBOV inactivation and improvement of biosafety. Although not reported in this study, multiple reports have indicated that neither methanol fixation nor heat inactivation will negatively affect the quality of whole-blood thin smears or the diagnosis of malaria during the staining procedures for malaria (8,(11)(12)(13)(14).…”

“…Although it is believed that the additional Triton X-100 would inactivate EBOV particles within malaria samples and that the impact on clinical biochemical and histology assays is minimal (14,16,17), no data have been found in peer-reviewed material to support the use of Triton X-100 in inactivating EBOV.…”