**5.1. Viral culturing**

Viral isolation in cell culture remains the "gold standard" for the diagnosis of Lassa fever given the challenges in diagnosing the virus due to mutations [38], although RT-PCR and immunoassays are commonly used assays for a clinically actionable diagnosis in recent time [7, 40]. Viral culturing is carried out by the inoculation of suspected samples containing the virus in Vero E6 cells in incubator at 37°C. A positive result may yield a cell cytopathic effect (CPE) [41], while, a second method of detection, such as viral antigen detection, RT-PCR, or electron microscopy, should be used as confirmatory for the presence of the virus. There is always viremia presence at the time of presentation to medical care and reduces after 6 days of illness in subjects, who survived the infection and there may be persistence presence until death in fatal cases [42]. The viral agent can also be cultured from throat swabs, blood, urine, and cerebrospinal fluid specimens from subjects [4]. The detection of virus in urine and throat swabs can be inconsistent in subjects with serum viremia. Positivity of the viral culture may exists in organ specimens such as spleen, liver, lung, heart, kidney, and placenta at post mortem in fatal infections cases [4].

usage allows improved access and assay development. A lateral passage test for the virus nucleoprotein (ReLASV) is another type of diagnostic test, which may be used for point-ofcare diagnosis [45]. Following initial development and testing efforts, this test received the CE mark in 2013, although approval by the US FDA has not been granted for the test. According to the product insert, the assay generates results in 15–25 minutes and has 85 and 99% sensitivity and specificity, respectively using confirmed Lassa virus-positive blood specimens [4]. In real sense, a diagnostic test would not only identify Lassa virus infection but would also check for other pathogens with similar clinical symptoms endemic in West Africa at the same time [39]. A transitioning Lassa and Ebola virus antigen- and IgM-based ELISAs onto a MAGPIX system has been described that uses individually labeled magnetic beads to identify multiple targets in a single test. This test has lower limits of detection for Lassa virus nucleoprotein and IgM than conventional ELISAs. Further development of multiplex MAGPIX assays, including testing for Lassa virus antigen and common endemic infections such as malarial infection, will assist with the diagnosis and clinical management of suspected cases of Lassa fever, especially in cases of coinfection with other pathogens where multiple therapeutic modalities may be indicated [38]. Patients on medical care dalliance following onset of the disease could affect negatively on the virus identification in a nucleoprotein detection test, and detection by viral-specific IgM might be more appropriate and reliable [37, 42]. IgG of the virus levels may increase later than IgM levels, with a mean time to detection of 25.6 days after symptom onset, although positive IgG titers have occasionally been detected presence in subjects with acute fever within the first few days of sickness onset [38]. IgM of the virus usually becomes noticeable and detectable in the second week of infections onset, although it could be detectable within 4 days of onset of illness in some subjects. Lack of an antibody

A Reemerging Lassa Virus: Aspects of Its Structure, Replication, Pathogenicity and Diagnosis

http://dx.doi.org/10.5772/intechopen.79072

159

Real-time RT-PCR is a commonly used diagnostic technique for infectious agents due to the high specificity and sensitivity and has become a gold standard clinically for Lassa fever identification [37, 46, 47]. Automated coupled specimen processing and 96-well plate thermocyclers, large samples can be evaluated fast and cheaply. The methods could detect viral agent for early illness and a longer time when compared to culturing of virus and might be carried out on samples that are inactivated chemically [4, 48]. Cycle threshold figures usage with quantitative rt-PCR can help with estimates of viremia using the right positive-control equipment for generation of standard curve [49]. Based on the virus strain and primers used, the 95% probability limit of detection estimates with RT-PCR vary from 1237 to 4290 RNA copies/ml [49]. With highly diverse viral agent such as Lassa virus, molecular diversity can be a problem for such assay, as even a single nucleotide variant in one of the primers can have a significant negative effect on the sensitivity of the assay depending on the location of the nucleotide variant [50–52]. The availability of additional sample testing and sequencing data has made mismatches identified using established assays, necessitating assay redesign to enhance performance. Multiplex panels to simultaneously detect a multitude of viruses that could produce hemorrhagic fever syndromes, including Lassa and Ebola viruses, using RT-PCR alone or in synergy with either

enzyme hybridization or ligase detection reactions have also been produced [53, 54].

response has been found in some fatal cases of Lassa fever.

**5.3. Polymerase chain reaction (PCR) methods**

Culturing of viruses allows for the identification, which is genetically independent of variabilities between types and further typing of the viral agent, if desired is achieved [38]. This technique also guarantee the quantification of viremia that might provide further viral typing data, as viremia with 10<sup>3</sup> 50% tissue culture infective doses (TCID50)/ml has a fatality odds ratio of 3.7 as regards to viremia with less than 10<sup>3</sup> TCID50/ml. The method is neither fast, taking at least several days to yield results, and it is not widely available due to the need for BSL-4 precautions to handle live viral samples that limits its utility for the early diagnosis of the viral infection [31].

#### **5.2. Rapid immunogenic tests**

Rapid immunogenic tests are attractive alternatives to the technical requirements and high specificity of the PCR methods especially in LASV endemic areas [14, 37]. The antibody/antigen binding is usually less specific than primer/probe hybridization, leaving for greater flexibility in identifying diverse viral agents. Detection of antigens relies on specific antibodies usage against Lassa virus components to detect viral antigens in blood samples [14]. Nonspecific Lassa virus antigens with polyclonal antibodies are detected with initial assays, whereas more current ELISAs target the Lassa virus nucleoprotein antigen [31, 38]. A diagnosis based on the detection of the relatively conserved Lassa nucleoprotein antigen could reduce the differences in test efficacy between genetically diverse virus types and comparison to DNA-based techniques [43]. Antigenemia-increased levels have been identified in fatal cases of the fever when compared with nonfatal cases. The short time of antigenemia enhances the detection of the virus antigen more specific to acute infection with the virus when compared to detection with antibody tests. The antigen of the virus nucleoprotein can be detected in subjects with the virus in the first week of illness and wanes during the second week in temporal association with the increase in detectable immunoglobulins [42]. Antigen detection tests could identify Lassa fever earlier during illness than antibody tests, as antibodies often may not be detectable until the second week of illness [43, 44]. The virus antigen levels might become undetectable despite viremia persistence. It should be noted that, negative antigen test during an acute illness does not mean that the patient is free from the fever [44].

A lot of IgM and antigen capture ELISA methods have been invented with inactivated viral agent; but the protocol is restricted to BSL-4 capable machine [41]. Recombinant antigens usage allows improved access and assay development. A lateral passage test for the virus nucleoprotein (ReLASV) is another type of diagnostic test, which may be used for point-ofcare diagnosis [45]. Following initial development and testing efforts, this test received the CE mark in 2013, although approval by the US FDA has not been granted for the test. According to the product insert, the assay generates results in 15–25 minutes and has 85 and 99% sensitivity and specificity, respectively using confirmed Lassa virus-positive blood specimens [4].

In real sense, a diagnostic test would not only identify Lassa virus infection but would also check for other pathogens with similar clinical symptoms endemic in West Africa at the same time [39]. A transitioning Lassa and Ebola virus antigen- and IgM-based ELISAs onto a MAGPIX system has been described that uses individually labeled magnetic beads to identify multiple targets in a single test. This test has lower limits of detection for Lassa virus nucleoprotein and IgM than conventional ELISAs. Further development of multiplex MAGPIX assays, including testing for Lassa virus antigen and common endemic infections such as malarial infection, will assist with the diagnosis and clinical management of suspected cases of Lassa fever, especially in cases of coinfection with other pathogens where multiple therapeutic modalities may be indicated [38]. Patients on medical care dalliance following onset of the disease could affect negatively on the virus identification in a nucleoprotein detection test, and detection by viral-specific IgM might be more appropriate and reliable [37, 42]. IgG of the virus levels may increase later than IgM levels, with a mean time to detection of 25.6 days after symptom onset, although positive IgG titers have occasionally been detected presence in subjects with acute fever within the first few days of sickness onset [38]. IgM of the virus usually becomes noticeable and detectable in the second week of infections onset, although it could be detectable within 4 days of onset of illness in some subjects. Lack of an antibody response has been found in some fatal cases of Lassa fever.

#### **5.3. Polymerase chain reaction (PCR) methods**

immunoassays are commonly used assays for a clinically actionable diagnosis in recent time [7, 40]. Viral culturing is carried out by the inoculation of suspected samples containing the virus in Vero E6 cells in incubator at 37°C. A positive result may yield a cell cytopathic effect (CPE) [41], while, a second method of detection, such as viral antigen detection, RT-PCR, or electron microscopy, should be used as confirmatory for the presence of the virus. There is always viremia presence at the time of presentation to medical care and reduces after 6 days of illness in subjects, who survived the infection and there may be persistence presence until death in fatal cases [42]. The viral agent can also be cultured from throat swabs, blood, urine, and cerebrospinal fluid specimens from subjects [4]. The detection of virus in urine and throat swabs can be inconsistent in subjects with serum viremia. Positivity of the viral culture may exists in organ specimens such as spleen, liver, lung, heart, kidney, and placenta at post mor-

Culturing of viruses allows for the identification, which is genetically independent of variabilities between types and further typing of the viral agent, if desired is achieved [38]. This technique also guarantee the quantification of viremia that might provide further viral typing

taking at least several days to yield results, and it is not widely available due to the need for BSL-4 precautions to handle live viral samples that limits its utility for the early diagnosis of

Rapid immunogenic tests are attractive alternatives to the technical requirements and high specificity of the PCR methods especially in LASV endemic areas [14, 37]. The antibody/antigen binding is usually less specific than primer/probe hybridization, leaving for greater flexibility in identifying diverse viral agents. Detection of antigens relies on specific antibodies usage against Lassa virus components to detect viral antigens in blood samples [14]. Nonspecific Lassa virus antigens with polyclonal antibodies are detected with initial assays, whereas more current ELISAs target the Lassa virus nucleoprotein antigen [31, 38]. A diagnosis based on the detection of the relatively conserved Lassa nucleoprotein antigen could reduce the differences in test efficacy between genetically diverse virus types and comparison to DNA-based techniques [43]. Antigenemia-increased levels have been identified in fatal cases of the fever when compared with nonfatal cases. The short time of antigenemia enhances the detection of the virus antigen more specific to acute infection with the virus when compared to detection with antibody tests. The antigen of the virus nucleoprotein can be detected in subjects with the virus in the first week of illness and wanes during the second week in temporal association with the increase in detectable immunoglobulins [42]. Antigen detection tests could identify Lassa fever earlier during illness than antibody tests, as antibodies often may not be detectable until the second week of illness [43, 44]. The virus antigen levels might become undetectable despite viremia persistence. It should be noted that, negative antigen test during an acute illness does not mean that the patient is free from the fever [44]. A lot of IgM and antigen capture ELISA methods have been invented with inactivated viral agent; but the protocol is restricted to BSL-4 capable machine [41]. Recombinant antigens

50% tissue culture infective doses (TCID50)/ml has a fatality odds

TCID50/ml. The method is neither fast,

tem in fatal infections cases [4].

ratio of 3.7 as regards to viremia with less than 10<sup>3</sup>

158 Current Topics in Tropical Emerging Diseases and Travel Medicine

data, as viremia with 10<sup>3</sup>

the viral infection [31].

**5.2. Rapid immunogenic tests**

Real-time RT-PCR is a commonly used diagnostic technique for infectious agents due to the high specificity and sensitivity and has become a gold standard clinically for Lassa fever identification [37, 46, 47]. Automated coupled specimen processing and 96-well plate thermocyclers, large samples can be evaluated fast and cheaply. The methods could detect viral agent for early illness and a longer time when compared to culturing of virus and might be carried out on samples that are inactivated chemically [4, 48]. Cycle threshold figures usage with quantitative rt-PCR can help with estimates of viremia using the right positive-control equipment for generation of standard curve [49]. Based on the virus strain and primers used, the 95% probability limit of detection estimates with RT-PCR vary from 1237 to 4290 RNA copies/ml [49]. With highly diverse viral agent such as Lassa virus, molecular diversity can be a problem for such assay, as even a single nucleotide variant in one of the primers can have a significant negative effect on the sensitivity of the assay depending on the location of the nucleotide variant [50–52].

The availability of additional sample testing and sequencing data has made mismatches identified using established assays, necessitating assay redesign to enhance performance. Multiplex panels to simultaneously detect a multitude of viruses that could produce hemorrhagic fever syndromes, including Lassa and Ebola viruses, using RT-PCR alone or in synergy with either enzyme hybridization or ligase detection reactions have also been produced [53, 54].
