**3. Influenza virus**

*aegypti*, as well as to study virus replication in different cell lines using TaqMan probes [38]. A real time RT-PCR (qRT-PCR) for DENV using TaqMan Minor Groove Binding (MGB) probe approach was development for detection and quantitation of all four serotypes using a single probe primer set targeted against the 3'UTR of DENV. In this assay, the limit of detection was DENV-1 (0.98 UFP/ml), DENV-2 (0.99 UFP/ml), DENV-3 (0.99 UFP/ml) and DENV-4 (0.99 UFP/ml) [39]. Other option to diagnosis to provide serotype specificity is a multiplex rRT-PCR assay targeting the 5' untranslated region and capsid gene of the DENV genome using molecular beacons. This assay was linear from 7.0 to 1.0 log10 cDNA equivalents/mL for each serotype. The limit of detection was calculated to be 0.3 cDNA equivalents/mL for DENV-1, 13.8 for DENV-2, 0.8 for DENV-3, and 12.4 for DENV-4 [40]. A technique for the multiplex detection and typing of all DENV serotypes from clinical samples is PCR-ligase detection reaction (LDR). In this technique, a serotype-specific PCR amplifies the regions of genes C and E simultaneously. Then, two amplicons are targeted in a multiplex LDR, and the resultant fluorescently labeled ligation products are detected on a universal array. The sensitivity of the assay was 98.7%, and its specificity was 98.4%, relative to the results of real-time PCR. The detection threshold was 0.017 PFU for DENV-1, 0.004 PFU for DENV-2, 0.8 PFU for DENV-3, and 0.7 PFU for DENV-4. The assay is specific; it does not cross-react with the other flaviviruses tested (West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Kunjin virus, Murray Valley virus, Powassan virus, and yellow fever virus). The detection limit of the assay ranged from 0.004 to 0.7 equivalent PFU/reaction. The assay was 100 times more sensitive for DENV-2 and DENV-1 (LOD, 0.004 and 0.017 equivalent PFU, respectively) than for the others serotypes [41]. Detection of DENV in urine is other diagnostic method, a study by real-time RT-PCR, reported the detection of viral genome in urine between days 6 to 16[42]. Recently, CDC development a CDC DENV-1-4 Real-Time RT-PCR Assay, the first molecular test

WHO, Geneva, 2009 [10].

94 Trends in Infectious Diseases

**Figure 2.** Immune response to virus dengue. In the infection course of dengue virus, the host's B cells produce IgM and IgG antibodies, which are released for recognize and neutralize the dengue virus and molecules such as the den‐ gue NS1 protein with the purpose of eliminates the virus. The figure shows the viremia can detect by the amplification of the NS1 gene by RT-PCR and the antibodies titers by ELISA. Information based on Guzman MG, et al., 2010 [25] and

Influenza is a highly contagious respiratory disease of humans, with propensity for seasonal epidemics and occasional pandemics. This disease constitutes a global health issue, leading to morbidity, mortality, and economic losses. During influenza season, influenza viruses circulate ubiquitously in the population. Global influenza surveillance forms the primary line of defense against the occurrence of influenza pandemics by identifying emerging influenza virus strains that pose a potential threat [45]. Influenza affects all age groups that result from its pulmonary complications. The virus initially infects the upper airways but can directly extend to the lower airways in severe cases, resulting in a viral pneumonia with significant morbidity and mortality [46, 47]. Influenza viruses belong to the Orthomyxoviridae family and are organize into types A, B and C. Influenza types A and B are responsible for epidemics of respiratory illness in humans and animals. The etiologic agent of influenza is the influenza virus with negative-strand, segmented RNA genome. Influenza type A and B viruses have 8 genes that code for 10 proteins. The virion has two surface glycoproteins (hemagglutinin (HA) and neuraminidase (NA) and the M2 protein protrude through envelop (Figure 3). Influenza virus binds to its sialic (neuraminic) acid receptor on respiratory epithelial cells by means of the HA protein [48]. The influenza type A viruses are sub classified into different subtypes according to HA and NA proteins, there are 16 HA subtypes and 9 NA subtypes, all of them have been identified and isolated from birds, humans and can affect a range of mammal species. The influenza A subtypes that circulated extensively in humans are A(H1N1); A(H1N2); A(H2N2); and seasonal influenza A (H3N2) [49]. Influenza A viruses (IAV) are the cause of pandemics, which are generated by the rearrangement (reassortment) of viral RNA segments in cells infected with two different viral strains [50].

Pandemic viruses of influenza A virus including "Spanish influenza" (H1N1 in 1918) and A/ H1N1 or A(H1N1)pdm09 (H1N1 in 2009) [51] or rarely, a novel influenza A virus infection. For the diagnosis, the influenza tests that provide accurate and timely results are the most recommended. The appropriate respiratory samples for influenza testing are upper respiratory

suggestive of influenza, follow-up testing with RT-PCR and/or viral culture should be considered to confirm negative test results [55]. Besides, of its low sensitivity RIDTs may be used to help with diagnostic and treatment decisions for patients in clinical settings, such as whether to prescribe antiviral medications [45]. These tools are continuously improved, a double-sandwich ELISA (pH1N1 ELISA), based on two monoclonal antibodies against haemagglutinin (HA) of the pH1N1 virus has a sensitivity of 92.3% (84/91, 95% CI 84.8–96.9%), being significantly higher than that of the BD Directigen EZ Flu A+B test (70.3%, p <0.01). In addition, this assay can directly differentiate pandemic (H1N1) 2009 (pH1N1) virus from other respiratory pathogens, including seasonal influenza virus [56]. A hemagglutination inhibition assay is an extremely reliable tool for typing, subtyping and analyze the antigenic character‐ istics of influenza viral isolates if the reference antisera used contain antibodies to currently circulating viruses [53]. An HAI test showed that the patients with influenza A H1N1 have effective immune response [57]. The Directigen EZ Flu A+B test is a rapid chromatographic immunoassay for the qualitative detection of influenza A and B viral antigens, it has an accuracy of 95.5% and 96.8%, respectively. This kit has the ability to detect H5N1 isolates and of the A/California/07/2004 strain [BD Diagnostics, Becton Dickinson and Company, 2005]. Viral culture is considering one "gold standard" for detection of infection with human influenza viruses. Although, viral isolation is not a screening routine test in outbreaks, during periods of low influenza activity, could performed on respiratory specimens collected from persons with suspected influenza. Viral culture does not provide timely results (1-14 days) [58, 59]. In the influenza seasonal and in outbreaks, the viral culture is essential as a source of virologic data about strain characteristics, such as antigenic comparison to influenza vaccine strains and antiviral susceptibility that are important for clinicians and public health. Shell vial centrifugation cultures have been used to shorten the time to results to 1 to 5 days, to detect viral antigens [60]. Immunofluorescence using direct fluorescent antibody or indirect fluores‐ cent antibody staining for influenza antigen detection are used as screening tests. This test exhibits slightly lower sensitivity and specificity than viral isolation in cell culture, but results are obtained within hours [45]. An example is the Respiratory Screen direct immunofluores‐ cence antibody (DFA) (direct fluorescent antibody) staining of cells collected using nasophar‐ yngeal (NP) swabs or NP aspirates can detects not only influenza, but RSV, parainfluenza types 1,2,3, and adenovirus. DFA is reported within 2-3 hrs [61]. However, for detection of respira‐ tory viruses in clinical specimens, the nucleic acid tests (NATs) are fast, accurate and sensitive test. Several nucleic acid based amplification approaches have been applied for the detection of individual respiratory viruses including PCR, nucleic acid sequence-based amplification (NASBA), loop mediated isothermal amplification (LAMP), and multiplex ligation-dependent probe amplification (MLPA) [62-65]. For NATs, the nucleic acid is extracted from the samples by using commercial kits, like QIAamp viral RNA extraction kit (Qiagen, Hilden, Germany) using silica gel membrane technology. KingFisher mL (ThermoFisher Scientific Inc., Worces‐ ter, MA, USA) and easyMAG (bioMérieux, Marcy l'Etoile, France) kits using magnetic beadsbased technology [66, 67]. TruTip (Akonni Biosystems, Inc., Frederick, MD, USA) use a porous nucleic acid binding matrix embedded within a pipette tip [68]. Reverse transcriptase-PCR (RT-PCR) or Real-time RT-PCR can be considered the other "gold standard" for detection of influenza viruses due to its high sensitivity and specificity for detection of influenza A and B

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**Figure 3.** Schematic representation of influenza virus A (not drawn to scale). On the envelope viral are anchored: HA, M2 (ion channel protein) and NA protein. Inside the virion the negative-RNA single-stranded and segmented viral ge‐ nome with ARN polymerase formed for PB1, P2 and PA. Also, there is a NEP protein.

tract specimen such as deep nostrils (nasal swab), throat (throat swab) and nasopharynx (nasopharyngeal swab). Nasopharyngeal aspirate and bronchial aspirate are also useful. Samples should be collected within the first 4 days of illness [52,53]. According to WHO the diagnosis of influenza is based in enzyme-linked immunosorbent assay (ELISA), haemagglu‐ tination inhibition (HI) and neuraminidase inhibition (NI) tests, egg and cell culture inocula‐ tion, and conventional reverse transcriptase PCR (RT-PCR) and Real-Time PCR (Table 2). The reference standards for laboratory confirmation of influenza virus infection are reverse transcription-polymerase chain reaction (RT-PCR) or viral culture [54]. Serologic testing is usually not recommended to detect evidence of human influenza virus infection for manage‐ ment of acute illness. However, a variety of serological tests, including the hemagglutination inhibition (HAI) test, complement fixation and EIA are used for testing paired acute and convalescent phase sera to detect antibody titers. The Rapid Influenza Diagnostic Tests (RIDTs) can identify the presence of influenza A and B viral nucleoprotein antigens, the result only is positive or negative. The RIDTs are immunoassays than provide results in 10–30 min but exhibit decreased sensitivity (70%–90% in children and 40% to 60% in adults), compared with RT-PCR and with viral culture. There is a potential for false negative results. Negative results of RIDTs do not exclude influenza virus infection in patients with signs and symptoms suggestive of influenza, follow-up testing with RT-PCR and/or viral culture should be considered to confirm negative test results [55]. Besides, of its low sensitivity RIDTs may be used to help with diagnostic and treatment decisions for patients in clinical settings, such as whether to prescribe antiviral medications [45]. These tools are continuously improved, a double-sandwich ELISA (pH1N1 ELISA), based on two monoclonal antibodies against haemagglutinin (HA) of the pH1N1 virus has a sensitivity of 92.3% (84/91, 95% CI 84.8–96.9%), being significantly higher than that of the BD Directigen EZ Flu A+B test (70.3%, p <0.01). In addition, this assay can directly differentiate pandemic (H1N1) 2009 (pH1N1) virus from other respiratory pathogens, including seasonal influenza virus [56]. A hemagglutination inhibition assay is an extremely reliable tool for typing, subtyping and analyze the antigenic character‐ istics of influenza viral isolates if the reference antisera used contain antibodies to currently circulating viruses [53]. An HAI test showed that the patients with influenza A H1N1 have effective immune response [57]. The Directigen EZ Flu A+B test is a rapid chromatographic immunoassay for the qualitative detection of influenza A and B viral antigens, it has an accuracy of 95.5% and 96.8%, respectively. This kit has the ability to detect H5N1 isolates and of the A/California/07/2004 strain [BD Diagnostics, Becton Dickinson and Company, 2005]. Viral culture is considering one "gold standard" for detection of infection with human influenza viruses. Although, viral isolation is not a screening routine test in outbreaks, during periods of low influenza activity, could performed on respiratory specimens collected from persons with suspected influenza. Viral culture does not provide timely results (1-14 days) [58, 59]. In the influenza seasonal and in outbreaks, the viral culture is essential as a source of virologic data about strain characteristics, such as antigenic comparison to influenza vaccine strains and antiviral susceptibility that are important for clinicians and public health. Shell vial centrifugation cultures have been used to shorten the time to results to 1 to 5 days, to detect viral antigens [60]. Immunofluorescence using direct fluorescent antibody or indirect fluores‐ cent antibody staining for influenza antigen detection are used as screening tests. This test exhibits slightly lower sensitivity and specificity than viral isolation in cell culture, but results are obtained within hours [45]. An example is the Respiratory Screen direct immunofluores‐ cence antibody (DFA) (direct fluorescent antibody) staining of cells collected using nasophar‐ yngeal (NP) swabs or NP aspirates can detects not only influenza, but RSV, parainfluenza types 1,2,3, and adenovirus. DFA is reported within 2-3 hrs [61]. However, for detection of respira‐ tory viruses in clinical specimens, the nucleic acid tests (NATs) are fast, accurate and sensitive test. Several nucleic acid based amplification approaches have been applied for the detection of individual respiratory viruses including PCR, nucleic acid sequence-based amplification (NASBA), loop mediated isothermal amplification (LAMP), and multiplex ligation-dependent probe amplification (MLPA) [62-65]. For NATs, the nucleic acid is extracted from the samples by using commercial kits, like QIAamp viral RNA extraction kit (Qiagen, Hilden, Germany) using silica gel membrane technology. KingFisher mL (ThermoFisher Scientific Inc., Worces‐ ter, MA, USA) and easyMAG (bioMérieux, Marcy l'Etoile, France) kits using magnetic beadsbased technology [66, 67]. TruTip (Akonni Biosystems, Inc., Frederick, MD, USA) use a porous nucleic acid binding matrix embedded within a pipette tip [68]. Reverse transcriptase-PCR (RT-PCR) or Real-time RT-PCR can be considered the other "gold standard" for detection of influenza viruses due to its high sensitivity and specificity for detection of influenza A and B

tract specimen such as deep nostrils (nasal swab), throat (throat swab) and nasopharynx (nasopharyngeal swab). Nasopharyngeal aspirate and bronchial aspirate are also useful. Samples should be collected within the first 4 days of illness [52,53]. According to WHO the diagnosis of influenza is based in enzyme-linked immunosorbent assay (ELISA), haemagglu‐ tination inhibition (HI) and neuraminidase inhibition (NI) tests, egg and cell culture inocula‐ tion, and conventional reverse transcriptase PCR (RT-PCR) and Real-Time PCR (Table 2). The reference standards for laboratory confirmation of influenza virus infection are reverse transcription-polymerase chain reaction (RT-PCR) or viral culture [54]. Serologic testing is usually not recommended to detect evidence of human influenza virus infection for manage‐ ment of acute illness. However, a variety of serological tests, including the hemagglutination inhibition (HAI) test, complement fixation and EIA are used for testing paired acute and convalescent phase sera to detect antibody titers. The Rapid Influenza Diagnostic Tests (RIDTs) can identify the presence of influenza A and B viral nucleoprotein antigens, the result only is positive or negative. The RIDTs are immunoassays than provide results in 10–30 min but exhibit decreased sensitivity (70%–90% in children and 40% to 60% in adults), compared with RT-PCR and with viral culture. There is a potential for false negative results. Negative results of RIDTs do not exclude influenza virus infection in patients with signs and symptoms

**Figure 3.** Schematic representation of influenza virus A (not drawn to scale). On the envelope viral are anchored: HA, M2 (ion channel protein) and NA protein. Inside the virion the negative-RNA single-stranded and segmented viral ge‐

nome with ARN polymerase formed for PB1, P2 and PA. Also, there is a NEP protein.

96 Trends in Infectious Diseases

viruses. The results are available within 4–6 h after specimen submission. These molecular tools are used as a confirmatory test. Several different gene targets have been used for amplification such as the matrix (*m*) to detect all influenza A subtypes, *ha* to distinguish between influenza A, B and C or between influenza A subtypes and non-structural protein genes (*nsp*) [69-71]. In all eight segments of influenza A virus, the first 12 nucleotides of the 3′ terminus (Uni12) and the first 13 nucleotides of 5' terminus (Uni13) are conservative and are the target to designed a primers pair. Detection of influenza A virus in human nasal swabs can performed by RT-PCR with Uni12 and Uni13 primers. The coupling of RT-PCR with sequenc‐ ing analysis provides information about viral genotype [72]. Multiplex RT-PCR assays have been widely used for detection and differentiation of a panel of respiratory viral pathogens. FluPlex is a multiplex RT-PCR enzyme hybridization assay, capable of typing influenza viruses and subtyping HA (H1, H2, H3, H5, H7, and H9) and NA (human N1, animal N1, N2, and N7) with high sensitivity (10–100 copies/reaction) [73]. As the Qiagen ResPlex II multiplex RT-PCR kit, an test with high specificity for detection of 17 viral pathogens in nasopharyngeal swab samples such as influenza A virus (FluA), FluB, FluA 2009 pandemic A(H1N1)pdm09 and others pathogens as parainfluenza virus 1 (PIV1), PIV2, PIV3, PIV4, respiratory syncytial virus (RSV), human metapneumovirus (hMPV), rhinoviruses (RhV), adenoviruses (AdV), four coronaviruses (229E, OC43, NL63 and HKU1) [74]. The TrueScience RespiFinder Identification Panels (Applied Biosystems) is other multiplex PCR test to detect and differentiate 15 respi‐ ratory pathogens, using the multiplex ligation-dependent probe amplification (MLPA) technology, which starts by a preamplification step, which ensures the detection of both RNA and DNA viruses.

Follow is performed a reverse transcription, a PCR reaction, the hybridization probes to their target regions to the PCR product. A ligation probes and the final PCR exponentially amplifies the ligated probes with only two primers. The targets are detected through capillary electro‐ phoresis. The pathogens potentially identified are Influenza A, Influenza B, Influenza A H5N1, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, Parainfluenza 4, RSV-A, RSV-B, Corona 229E, Corona NL63, Corona OC43, Adenovirus, Rhinovirus, Bordetella pertussis, Chlamydo‐ phila pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae. MPLA shows specificities and sensitivities of 98.2% and 100%, respectively, for influenza A virus [75]. A successful test to detect a H5N1 virus was based on Loop-Mediated Isothermal Amplification (LAMP) method. For other side, detection, quantification and subtyping of influenza viruses can be performed by real-time RT-PCR (qRT-PCR). There are many papers of this technique applied to influenza A diagnosis such as TaqMan qRT-PCR method, which detects HA and NA genes of HPAI H5N1 virus [76]. A new generation qRT-PCR approach designated as Super high-speed qRT-PCR (SHRT-PCR) is a version of qRT-PCR with an extremely short reaction time (less than 20 min per run for 40 cycles) capable to detects viral RNA segments of influenza A [77]. RealTime ready Influenza A/H1N1 Detection Set (Roche) is an assay to detect the M2 gene (M2 PCR) of a generic influenza virus A and a specific PCR targeting the HA of A/H1N1 pdm09 (HA PCR, 2009 H1N1), to detect and quantify the 2009 H1N1 virus in clinical samples [78]. CDC developed the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel-Influenza A/H7 (Eurasian Lineage) assay for patients with signs and symptoms of respiratory infection. The kit contains a dual-labeled hydrolysis probe (TaqMan). The limit of detection of

this kit was 103.4 [79]. On April 1, 2013, an outbreak of human infections with a new avian influenza A (H7N9) virus was first reported in China by the World Health Organization. The diagnostics of this virus used the real time RT-PCR by TaqMan assay. Other molecular technique to diagnosis is DNA microarray such as FluChip-55 [Sakurai & Shibasaki, 2012] used

Information based on Centers for Disease Control and Prevention, National Center for Immunization and Respiratory Diseases (NCIRD). Guidance for Clinicians on the Use of Rapid Influenza Diagnostic.2011. Petric M, et al. 2006 [64];

**METHOD TYPE INFLUENZA VIRUS TYPE SPECIMENS**

A and B

A and B

Identification of subtypes of A

A and B Serum

Identification of subtypes of A

A and B NP swab, throat swab, NP or bronchial

Molecular Diagnostics as an Indispensable Tool for the Diagnosis of Infectious Diseases of…

sputum

A and B NP swab, throat swab, NP or bronchial

sputum

A and B NP swab, nasal wash, NP aspirate, throat swab

wash, nasal or endotracheal aspirate,

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wash, nasal or endotracheal aspirate,

NP swab, throat swab, NP or bronchial wash, NP or endotracheal aspirate, sputum

NP swab, thrat swab, NP or bronchial wash, NP or endotracheal aspirate, sputum

Cell culture (Madin-Darby canine kidney (MDCK), mink lung epithelial cell line (Mv1Lu), rhesus monkey kidney (LLC MK2), and buffalo green monkey kidney

Rapid cell culture (commercial shell vials,

Immunofluorescence microscopy by direct fluorescent antibody (DFA) test or an immunofluorescent antibody (IFA) test, using commercial monoclonal antibody for influenza A and B viruses; or non commercial monoclonal antibody specific for H1, H3 H5, H7 and H9

Immunospecific assay for viral antigen

Rapid influenza diagnostic test (RIDT's)

Nucleic acid testing (Conventional RT-PCR singleplex and multiplex, Multiplex PCR, Degenerate PCR, Probe-based realtime PCR (TaqMan), SBRY Green I-based real-time PCR, Microarrays, NABA, LAMP,

Wang&Taubenberger, 2010 [65].

**Table 2.** Methods of diagnosis of influenza virus.

Viral antibody detection Virus neutralization test Haemmaglutination inhibition ELISA using commercially anti-human

antibody conjugates Complement fixation

Pyrosequencing

single or mixed cell lines)

(BGMK)

detection


Information based on Centers for Disease Control and Prevention, National Center for Immunization and Respiratory Diseases (NCIRD). Guidance for Clinicians on the Use of Rapid Influenza Diagnostic.2011. Petric M, et al. 2006 [64]; Wang&Taubenberger, 2010 [65].

**Table 2.** Methods of diagnosis of influenza virus.

viruses. The results are available within 4–6 h after specimen submission. These molecular tools are used as a confirmatory test. Several different gene targets have been used for amplification such as the matrix (*m*) to detect all influenza A subtypes, *ha* to distinguish between influenza A, B and C or between influenza A subtypes and non-structural protein genes (*nsp*) [69-71]. In all eight segments of influenza A virus, the first 12 nucleotides of the 3′ terminus (Uni12) and the first 13 nucleotides of 5' terminus (Uni13) are conservative and are the target to designed a primers pair. Detection of influenza A virus in human nasal swabs can performed by RT-PCR with Uni12 and Uni13 primers. The coupling of RT-PCR with sequenc‐ ing analysis provides information about viral genotype [72]. Multiplex RT-PCR assays have been widely used for detection and differentiation of a panel of respiratory viral pathogens. FluPlex is a multiplex RT-PCR enzyme hybridization assay, capable of typing influenza viruses and subtyping HA (H1, H2, H3, H5, H7, and H9) and NA (human N1, animal N1, N2, and N7) with high sensitivity (10–100 copies/reaction) [73]. As the Qiagen ResPlex II multiplex RT-PCR kit, an test with high specificity for detection of 17 viral pathogens in nasopharyngeal swab samples such as influenza A virus (FluA), FluB, FluA 2009 pandemic A(H1N1)pdm09 and others pathogens as parainfluenza virus 1 (PIV1), PIV2, PIV3, PIV4, respiratory syncytial virus (RSV), human metapneumovirus (hMPV), rhinoviruses (RhV), adenoviruses (AdV), four coronaviruses (229E, OC43, NL63 and HKU1) [74]. The TrueScience RespiFinder Identification Panels (Applied Biosystems) is other multiplex PCR test to detect and differentiate 15 respi‐ ratory pathogens, using the multiplex ligation-dependent probe amplification (MLPA) technology, which starts by a preamplification step, which ensures the detection of both RNA

Follow is performed a reverse transcription, a PCR reaction, the hybridization probes to their target regions to the PCR product. A ligation probes and the final PCR exponentially amplifies the ligated probes with only two primers. The targets are detected through capillary electro‐ phoresis. The pathogens potentially identified are Influenza A, Influenza B, Influenza A H5N1, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, Parainfluenza 4, RSV-A, RSV-B, Corona 229E, Corona NL63, Corona OC43, Adenovirus, Rhinovirus, Bordetella pertussis, Chlamydo‐ phila pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae. MPLA shows specificities and sensitivities of 98.2% and 100%, respectively, for influenza A virus [75]. A successful test to detect a H5N1 virus was based on Loop-Mediated Isothermal Amplification (LAMP) method. For other side, detection, quantification and subtyping of influenza viruses can be performed by real-time RT-PCR (qRT-PCR). There are many papers of this technique applied to influenza A diagnosis such as TaqMan qRT-PCR method, which detects HA and NA genes of HPAI H5N1 virus [76]. A new generation qRT-PCR approach designated as Super high-speed qRT-PCR (SHRT-PCR) is a version of qRT-PCR with an extremely short reaction time (less than 20 min per run for 40 cycles) capable to detects viral RNA segments of influenza A [77]. RealTime ready Influenza A/H1N1 Detection Set (Roche) is an assay to detect the M2 gene (M2 PCR) of a generic influenza virus A and a specific PCR targeting the HA of A/H1N1 pdm09 (HA PCR, 2009 H1N1), to detect and quantify the 2009 H1N1 virus in clinical samples [78]. CDC developed the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel-Influenza A/H7 (Eurasian Lineage) assay for patients with signs and symptoms of respiratory infection. The kit contains a dual-labeled hydrolysis probe (TaqMan). The limit of detection of

and DNA viruses.

98 Trends in Infectious Diseases

this kit was 103.4 [79]. On April 1, 2013, an outbreak of human infections with a new avian influenza A (H7N9) virus was first reported in China by the World Health Organization. The diagnostics of this virus used the real time RT-PCR by TaqMan assay. Other molecular technique to diagnosis is DNA microarray such as FluChip-55 [Sakurai & Shibasaki, 2012] used in influenza diagnosis and others viruses such as parainfluenza, respiratory syncytial virus, human metapneumovirus, adenovirus, rhrinoviruses, coronaviruses causes pneumonia, which can be analyzed by multiplex PCR that assist in the discrimination of the etiologic agent [Pavia, 2011; Mahony, 2008]. The NGEN respiratory virus analyte-specific assay (Nanogen, San Diego, CA) detects influenza A, influenza B and others pathogens such as PIV type 1 (PIV-1), PIV-2, PIV-3, and RSV on a NanoChip 400 electronic microarray. The FilmArray Respiratory Panel (RP) is a PCR array test (Idaho Technology, Inc., Salt Lake City, UT) that can detect up to 21 viral and bacterial respiratory pathogens within about an hour as Adenovirus, Bocavirus, Coronavirus 229E, Coronavirus HKU1, Coronavirus NL63, Coronavirus OC43, Human Metapneumovirus, Parainfluenza virus 1, Parainfluenza virus 2, Parainfluenza virus 3, Parainfluenza virus 4, Rhinovirus/Enterovirus, Respiratory Syncytial Virus, Bordetella pertussis, Chlamydophila pneumoniae, and Mycoplasma pneumonia and Influenza A, Influenza A subtype H1, Influenza A subtype H3, Influenza A subtype H1 2009, Influenza B. This assay is a feasible alternative to viral culture in an acute clinical setting [80]. Recently, it was reported the method for the detection of genetic markers associated with high pathoge‐ nicity of influenza virus [81].

> less than 35 years old is particularly significant. The possible causes that result in cervical cancer to tend to occur in young include remarkable increase of HPV infection rate, especially the high-risk HPV 16 and 18 infection closely related to cervical cancer [91]. The primary screening and diagnostic methods have been cytology and histology. Papanicolaou (Pap) staining is the gold standard for detecting abnormal cervical epithelial cells, using microscopic analyses of conventional cervical smears or cell suspensions from liquid cytology medium. The limitation of Pap smear are low specificity, is need repeat the screening at short intervals [92]. Morphological findings from a cytology analysis determine the level of risk for develop‐ ing cervical malignancies. Cervical epithelial cells atypical or abnormal are known as "atypical squamous cells of undetermined significance" (ASCUS). Some ASCUS signals the presence of low-grade squamous intraepithelial lesions (LSIL). However, some ASCUS are associated with underlying high-grade disease, including cervical intraepithelial neoplasia (CIN) [93-95]. HPV serology is not used diagnostically. Detection of the humoral antibody response is type-specific and first detected 6–18 months after infection. The response is weak and only between 50–60% of patients positive to HPV DNA-positive mount a measurable antibody response [96]. Although, the role played by the humoral immune response during the HPV infection is not very well understood; it was observed that this response is generated all throughout the malignant process; 54%-69% of women with incident HPV 16, 6, or 18 infections had antibod‐ ies. Serum antibodies against many different viral products of HPV have been detected by ELISA. The best characterized and most type-specific antibodies are those directed against conformational epitopes of the L1 capsid protein assembled as VLPs (virus-like particles) such as HPV-16 L1 VLP (virus-like particle)-based ELISA [97]. There is some high-throughput single-serum-dilution enzyme-linked immunoassay (ELISA) system for determining anti-

> **Figure 4.** Linear representation of Human Papillomavirus (HPV) genome. The circular HPV genome has approximately 7900 base pairs, in the schematic with eight overlapping reading frames. L1 is signaled as the most used genomic tar‐

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At present there is no "gold standard" for HPV detection. Guidelines for the management of women with cervical neoplasia or abnormal cervical cancer screening tests indicates the immediate colposcopy (cervical exam), cytologic follow-up, and triage by HPV DNA testing

HPV antibody titers following vaccination against HPV [98].

get for detection of HPV.
