**5. Viral hepatitis**

Viral hepatitis is a necroinflammatory liver disease of variable severity. Persistent infection by HBV is often associated with chronic liver disease that can lead to the development of cirrosis and hepatocellular carcinoma being a global public health as chronic diseases, cause of infectious disease mortality globally, each year causing approximately 1.4 million deaths. In addition, viral hepatitis are cause of liver cancer and the most common reason for liver transplantation [119]. The viruses more common are Hepatitis A, Hepatitis B, and Hepatitis C. Approximately 400 million persons living with chronic hepatitis B virus (HBV) or hepatitis C virus infection who die from cirrhosis or liver cancer, years and decades after of their infection [120]. Hepatitis B is caused by infection with HBV, which may lead to acute or chronic hepatitis. HBV is the 9th leading cause of death worldwide. It causes cirrhosis, liver failure and hepatocellular carcinoma. HBV is a small nonenveloped DNA virus that is a member of the Hepadnaviridae family, HBV contains a 3.2-kb partially double-stranded DNA genome with 4 open reading frames encoding 7 proteins (P/viral polymerase, S/Surface antigen proteins/HBsAg, C/core protein, HBeAg y X/HBx protein) [121]. Eight genotypes of HBV (designated-H) have been identified by sequence divergence of >8% over the entire genome of HBV DNA [122]. HBV is efficiently transmitted by percutaneous or mucous membrane exposure to infectious blood or body fluids that contain blood. Acute HBV infection can be either asymptomatic or symptomatic. Symptoms in acute HBV infection are clinically indis‐ tinguishable from those in other acute viral hepatitis infections [123]. Diagnosis of hepatic viral infection is carried out by studying biochemical as liver function tests, serologic assays and histological parameters. Serological HBV diagnosis identifies virally-encoded antigens and their corresponding antibodies in serum. Three clinical useful antigen-antibody systems have been identified for hepatitis B: hepatitis B surface antigen (HBsAg) and antibody to HBsAg (anti-HBs), antibody (anti-HBc IgM and anti-HBc IgG) to hepatitis B core antigen (HBcAg), hepatitis B e antigen (HBeAg) and antibody to HBeAg (anti-HBe) [124]. B surface antigen (HBsAg) is the first serological marker to appear during the course of HBV infection, is present in both acute and chronic infection. The immunoassays to detect HBsAg are highly spectific and show a sensitivity, allowing the detection of <0.15 ng/ml of HbsAg [125]. Detection of HBsAg allowed for the first time screening of inapparently infected blood donors for a dangerous pathogen. The simultaneous detection of antibodies against HBsAg (anti-HBs) and HBcAg (anti-HBc) can be useful to know the evolution of disease. The presence of IgM antibody to hepatitis B core antigen (IgM anti-HBc) is diagnostic of acute or recently acquired HBV infection. Antibody to HBsAg (anti-HBs) is produced after a resolved infection and is the only HBV antibody marker present after immunization. The presence of HBsAg and total anti-HBc, with a negative test for IgM anti-HBc, indicates chronic HBV infection. The presence of anti-HBc alone might indicate a false-positive result or acute, resolved, or chronic infection [126, 127]. Chronic HBV infection is defined by the persistence of serum HBsAg for more than 6 months [128]. Serologic assays for HBV are the mainstay diagnostic tools for HBV infection. The clinically silent HBV infections are a strong driving force in the development of modern virus diagnostics to analyze the HBV replication profile, HBV DNA levels and the viral protein expression [129]. When serological testing could be inconclusive for the diagnosis of a HBV infection (due to the presence of genetic variants of HBV), the molecular detection of HBV DNA may help to resolve the uncertainties. The NAT assays in plasma or serum can detect to 10 copies/mL [130]. The viral genomes can be detected and quantified by polymerase chain reaction (PCR), transcription-mediated amplification (TMA), and with signal amplification

**MOLECULAR DIAGNOSIS METHOD**

104 Trends in Infectious Diseases

NASBA amplification and real-time detection using molecular beacon probes

**5. Viral hepatitis**

PCR-microarray PapilloCheck HPV-Screening Test

(Greiner Bio-One)

Infiniti HPV Genotyping assay

Clart HPV 2 (Genomics)

(AutoGenomics)

Next-Generation assay NextGen HPV Screening Assay

**Table 3.** Principal methods of molecular diagnosis of HPV.

NucliSENS EasyQ HPV (BioMérieux)

and Platform (Qiagen)

transcription such as ST3Gal III, ST3Gal IV and ST6Gal I in CIN [118].

Information based on Kroupis C, et al., 2011 [90]; Arney A, et al., 2010 [100].

**NAME TEST GENOTYPES AND TARGET DETECTED**

and CP6108.

L1 gene.

using E1 gene.

genotypes using L1 gene.

73, 82 and 85 using E1 gene.

16, 18, 31, 33 and 45.

Invader chemistry Cervista HPV HR Test (Hologic) Detection of 14-type High Risk HPV genotypes: 16,

Analysis of the host factor p16INK4a (p16), a cyclin-dependent kinase inhibitor could be a molecular marker to HPV infection. The increase of p16 indicates removal of the negative feedback control supplied by the retinoblastoma gene, pRB. When oncogenic HPV E7 proteins bind to pRB, p16 is overexpressed and elevated, representing active expression of HPV oncogenes [117]. The sialylation modification observed during oncogenic transformation, tumor metastases and invasion, has been associated with enhanced sialyltransferases (STs)

Viral hepatitis is a necroinflammatory liver disease of variable severity. Persistent infection by HBV is often associated with chronic liver disease that can lead to the development of cirrosis

67, 68, 69, 70, 71, 72, 73 (MM9), 81, 82, 83, 84, IS39

18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 using

Type-specific identification of 18 high-risk(16,18, 31,33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82) and 6 low-risk types of HPV (6, 11, 40, 42, 43, 44)

Identification of HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 68, 70, 71, 72, 73, 81, 82, 83, 84, 85 and 89

Genotyping of HPV 6, 11, 16, 18, 26, 30, 31, 33, 34, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 67, 68, 69, 70,

Discriminates the E6/E7 mRNA between genotypes

15 high risk subtypes (+ types 66 and 82)

methods such as hybrid-capture and the branched DNA assay. Quantification of genome of HBV can be used for diagnosing HBV infection and monitoring the effect of antiviral therapy. HBV DNA is the earliest detectable marker in acute HBV infection and the gold standard for the diagnosis of occult HBV infection. HBV DNA testing is particularly useful in the detection of the early phase of acute HBV infection prior to the appearance of serum HBsAg as well as in occult HBV infection [131, 132]. Several commercial molecular assays have been developed for quantitation of HBV DNA. Such as COBAS Amplicor HBV Monitor, which is based on the amplification of DNA targets by PCR with HBV-specific primers. Between the hybridizations methods are Hybrid capture Ultrasensitive hybrid capture II and Branched DNA VERSANT hepatitis B virus DNA 3.0. Recently developed real-time target amplification methods have improved viral genome detection and quantification for clinical and research purposes. Real time PCR for HBV DNA has reached an excellent level of performance with a detection limit close to the theoretical minimum of 1 DNA molecule per reaction mix and a huge dynamic range up to 107 or more. In 1991 the WHO introduced International Standard preparations and an arbitrary International Unit (IU) of HBV DNA. The number of molecules per IU depends on the assay; but typically 5 molecules correspond to one IU HBV DNA [133]. Real-Time PCR (Real Time Abbott PCR, Smart HBVTM, Real Art HBV, COBAS AmPliprep, Cobas TaqMan HBV, Aptima HBV Quantitative assay) [134-136]. Fosun real-time PCR HBV kit is a comercial assay for quantitation of serum HBV DNA based on TaqMan PCR technology, which is useful for monitoring HBV DNA levels in patients with chronic hepatitis B. The limit of the duplex real-time PCR assay was 29.5 IU/ml, whereas the specificity was 100% for the detection of HBV DNA [137]. A trial has been tested, a TaqMan locked nucleic acid (LNA) real-time polymerase chain reaction (PCR) probe for the accurate quantification and detection of hepatitis B virus (HBV) DNA in serum (plasma) [138]. The genotyping analysis of HBV can performed by realtime PCR using (GQ-PCR) method or the direct sequencing and reverse hybridization with INNO-LiPA HBV genotyping assay [139]. Other option to diagnostics of hepatotrophic viruses is Real-time PCR array, useful in the rapid detection of multiple viral pathogens, between them hepatitis B virus (HBV), hepatitis C virus (HCV) using the SYBR Green chemistry. The array detected: 10 genome equivalents (geq)/ml of HCV, 50 geq of HBV (genotype A) [140]. Micro‐ RNAs (miRNAs) are noncoding RNAs that regulate gene expression primarily at the posttranscriptional level by binding to mRNAs. The circulating miRNA in serum or plasma might be a very useful biomarker for the diagnosis and prognosis of HBV-related diseases, indicating a promising future in the treatment of HBV-related diseases [141]. Hepatitis C virus (HCV) is a major public health problem and a leading cause of chronic liver disease. An estimated 180 million people are infected worldwide, several of these patients go on to develop chronic HCV infection, often developing into liver cirrhosis, hepatic failure and hepatocellular carcinoma [142]. Hepatitis C virus is a single stranded RNA, enveloped virus, belongs to the Flaviviridae family and is the only member of the Hepacivirus genus [143]. The commercially-available diagnostic tests are based on enzyme immunosorbent assays (EIA) for the detection of HCVspecific antibodies and recombinant immunoblot assays (RIBA). Although, in the diagnosis of influenza is applied the algorithm showed in figure 5 [144-146]. Testing for circulating HCV by genomic sequence amplification (PCR and branched DNA assay) has been successfully utilized for confirmation of serological results and the effectiveness of antiviral therapy [134,

147]. An alternative to HCV diagnosis is Loop-mediated isothermal amplification (LAMP) assay for rapid detection of HCV genomic RNA [148]. The molecular HCV assays includes to RT-PCR (AmpliScreen 2.0, Amplicor HCV 2.0, Cobas Amplicor Monitor HCV 2.0, Versant HCV RNA, Procleix HIV/HCV assay, Procleix Ultrio assay). The methods for accurate quantification of HCV RNA levels are key tools in the clinical management of patients. The HCV RNA Assay by RT-PCR includes to Amplicor HCV Monitor 2.0, Cobas Amplicor Monitor HCV 2.0, Cobas AmliPrep/Cobas TaqMan HCV, Versant HCV RNA 3.0 Quantitative assay, LCx HCV RNA Quantitative assay, SuperQuant, Abbott RealTime [135]. To evaluate the response to antiviral therapies is possible analyze the absence or alteration of genetic material in clinical specimens from successfully treated patients. In situ hybridization (ISH) enables visualization of specific nucleic acid in morphologically preserved cells and tissue sections. The anti-sense probe detected HCV RNA, with a sensitivity and specificity of 95% and 100%, respectively. HCV genomic RNA can be variably distributed in tissue sections and was located primarily in the perinuclear regions in hepatocytes [149]. Viral hepatitis is one of the major health problems worldwide. Hepatitis delta virus (HDV) is also not uncommon world-wide. Hepatitis D virus (HDV) or delta virus is a defective virus. It requires the help of another virus that is hepatitis B virus for its multiplication. It always occur with HBV either in the form of co-infection or super-infection [150]. HCV, HBV, and HDV share parallel routes of transmis‐ sion due to which dual or triple viral infection can occur in a proportion of patients at the same time. HBV and HCV are important factors in the development of liver cirrhosis (LC) and hepatocellular carcinoma [151]. In the diagnosis of co-infections, individually each infection (HBV, HCV and HDV) is confirmed by the presence of the serum surface antigen, hepatitis B, C and D envelope antigen and specific antibodies to the hepatitis B, C and D core [152]. By Deep Sequencing were readily detected at high coverage in plasma of patients with chronic viral hepatitis B and C. Although, this protocol also is adapted to other samples such as urine, bile, saliva and other body fluids by viral metagenomic survey [153]. For other side, other molecular technique as the protein micro-array gives a way to diagnosis multiple viral infections; using two viral antigens (HBsAg, HBeAg) and seven viral antibodies (HBsAb, HBcAb, HBeAb, HCVAb, HDVAb, HEVAb, HGVAb) of human hepatitis viruses [154]. Finally, the successful simultaneous detection of HAV, HBV and HCV was performed with the Magicplex HepaTrio Real-time Detection test, whose sensitivity and specificity of the Hepa‐ Trio test were 93.8% and 98.2%, respectively, for detecting HBV infection, and 99.1% and

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Mumps is an acute viral infection caused by a member of the *Rubulavirus* genus in the *Paramyxoviridae* family. Mumps virus (MuV) is pleomorphic, enveloped, comprising a nucleocapsid core with helical structure composed of the 15384 nucleotide. Nonsegmented negative-sense RNA genome has the organization: 3′-NP-P-M-F-SH-HN-L-5′ [156]. The molecular epidemiology of MuV is characterized by the co-existence of 10 (or more) distinct genotypes named A-J based on the nucleotide sequence of the SH gene. MuV genotypes (C,

100.0%, respectively, for HCV infection [155].

**6. Mumps virus**

147]. An alternative to HCV diagnosis is Loop-mediated isothermal amplification (LAMP) assay for rapid detection of HCV genomic RNA [148]. The molecular HCV assays includes to RT-PCR (AmpliScreen 2.0, Amplicor HCV 2.0, Cobas Amplicor Monitor HCV 2.0, Versant HCV RNA, Procleix HIV/HCV assay, Procleix Ultrio assay). The methods for accurate quantification of HCV RNA levels are key tools in the clinical management of patients. The HCV RNA Assay by RT-PCR includes to Amplicor HCV Monitor 2.0, Cobas Amplicor Monitor HCV 2.0, Cobas AmliPrep/Cobas TaqMan HCV, Versant HCV RNA 3.0 Quantitative assay, LCx HCV RNA Quantitative assay, SuperQuant, Abbott RealTime [135]. To evaluate the response to antiviral therapies is possible analyze the absence or alteration of genetic material in clinical specimens from successfully treated patients. In situ hybridization (ISH) enables visualization of specific nucleic acid in morphologically preserved cells and tissue sections. The anti-sense probe detected HCV RNA, with a sensitivity and specificity of 95% and 100%, respectively. HCV genomic RNA can be variably distributed in tissue sections and was located primarily in the perinuclear regions in hepatocytes [149]. Viral hepatitis is one of the major health problems worldwide. Hepatitis delta virus (HDV) is also not uncommon world-wide. Hepatitis D virus (HDV) or delta virus is a defective virus. It requires the help of another virus that is hepatitis B virus for its multiplication. It always occur with HBV either in the form of co-infection or super-infection [150]. HCV, HBV, and HDV share parallel routes of transmis‐ sion due to which dual or triple viral infection can occur in a proportion of patients at the same time. HBV and HCV are important factors in the development of liver cirrhosis (LC) and hepatocellular carcinoma [151]. In the diagnosis of co-infections, individually each infection (HBV, HCV and HDV) is confirmed by the presence of the serum surface antigen, hepatitis B, C and D envelope antigen and specific antibodies to the hepatitis B, C and D core [152]. By Deep Sequencing were readily detected at high coverage in plasma of patients with chronic viral hepatitis B and C. Although, this protocol also is adapted to other samples such as urine, bile, saliva and other body fluids by viral metagenomic survey [153]. For other side, other molecular technique as the protein micro-array gives a way to diagnosis multiple viral infections; using two viral antigens (HBsAg, HBeAg) and seven viral antibodies (HBsAb, HBcAb, HBeAb, HCVAb, HDVAb, HEVAb, HGVAb) of human hepatitis viruses [154]. Finally, the successful simultaneous detection of HAV, HBV and HCV was performed with the Magicplex HepaTrio Real-time Detection test, whose sensitivity and specificity of the Hepa‐ Trio test were 93.8% and 98.2%, respectively, for detecting HBV infection, and 99.1% and 100.0%, respectively, for HCV infection [155].

## **6. Mumps virus**

methods such as hybrid-capture and the branched DNA assay. Quantification of genome of HBV can be used for diagnosing HBV infection and monitoring the effect of antiviral therapy. HBV DNA is the earliest detectable marker in acute HBV infection and the gold standard for the diagnosis of occult HBV infection. HBV DNA testing is particularly useful in the detection of the early phase of acute HBV infection prior to the appearance of serum HBsAg as well as in occult HBV infection [131, 132]. Several commercial molecular assays have been developed for quantitation of HBV DNA. Such as COBAS Amplicor HBV Monitor, which is based on the amplification of DNA targets by PCR with HBV-specific primers. Between the hybridizations methods are Hybrid capture Ultrasensitive hybrid capture II and Branched DNA VERSANT hepatitis B virus DNA 3.0. Recently developed real-time target amplification methods have improved viral genome detection and quantification for clinical and research purposes. Real time PCR for HBV DNA has reached an excellent level of performance with a detection limit close to the theoretical minimum of 1 DNA molecule per reaction mix and a huge dynamic

or more. In 1991 the WHO introduced International Standard preparations and

an arbitrary International Unit (IU) of HBV DNA. The number of molecules per IU depends on the assay; but typically 5 molecules correspond to one IU HBV DNA [133]. Real-Time PCR (Real Time Abbott PCR, Smart HBVTM, Real Art HBV, COBAS AmPliprep, Cobas TaqMan HBV, Aptima HBV Quantitative assay) [134-136]. Fosun real-time PCR HBV kit is a comercial assay for quantitation of serum HBV DNA based on TaqMan PCR technology, which is useful for monitoring HBV DNA levels in patients with chronic hepatitis B. The limit of the duplex real-time PCR assay was 29.5 IU/ml, whereas the specificity was 100% for the detection of HBV DNA [137]. A trial has been tested, a TaqMan locked nucleic acid (LNA) real-time polymerase chain reaction (PCR) probe for the accurate quantification and detection of hepatitis B virus (HBV) DNA in serum (plasma) [138]. The genotyping analysis of HBV can performed by realtime PCR using (GQ-PCR) method or the direct sequencing and reverse hybridization with INNO-LiPA HBV genotyping assay [139]. Other option to diagnostics of hepatotrophic viruses is Real-time PCR array, useful in the rapid detection of multiple viral pathogens, between them hepatitis B virus (HBV), hepatitis C virus (HCV) using the SYBR Green chemistry. The array detected: 10 genome equivalents (geq)/ml of HCV, 50 geq of HBV (genotype A) [140]. Micro‐ RNAs (miRNAs) are noncoding RNAs that regulate gene expression primarily at the posttranscriptional level by binding to mRNAs. The circulating miRNA in serum or plasma might be a very useful biomarker for the diagnosis and prognosis of HBV-related diseases, indicating a promising future in the treatment of HBV-related diseases [141]. Hepatitis C virus (HCV) is a major public health problem and a leading cause of chronic liver disease. An estimated 180 million people are infected worldwide, several of these patients go on to develop chronic HCV infection, often developing into liver cirrhosis, hepatic failure and hepatocellular carcinoma [142]. Hepatitis C virus is a single stranded RNA, enveloped virus, belongs to the Flaviviridae family and is the only member of the Hepacivirus genus [143]. The commercially-available diagnostic tests are based on enzyme immunosorbent assays (EIA) for the detection of HCVspecific antibodies and recombinant immunoblot assays (RIBA). Although, in the diagnosis of influenza is applied the algorithm showed in figure 5 [144-146]. Testing for circulating HCV by genomic sequence amplification (PCR and branched DNA assay) has been successfully utilized for confirmation of serological results and the effectiveness of antiviral therapy [134,

range up to 107

106 Trends in Infectious Diseases

Mumps is an acute viral infection caused by a member of the *Rubulavirus* genus in the *Paramyxoviridae* family. Mumps virus (MuV) is pleomorphic, enveloped, comprising a nucleocapsid core with helical structure composed of the 15384 nucleotide. Nonsegmented negative-sense RNA genome has the organization: 3′-NP-P-M-F-SH-HN-L-5′ [156]. The molecular epidemiology of MuV is characterized by the co-existence of 10 (or more) distinct genotypes named A-J based on the nucleotide sequence of the SH gene. MuV genotypes (C,

specific IgG, detected using a serologic assay (EIA or IFA), is considered evidence of mumps immunity but does not predict the presence of neutralizing antibodies or protection from mumps disease [163]. A near patient test (NPT) for the detection of mumps-specific IgM in oral fluid specimens was developed and evaluated using 196 oral fluid specimens from suspected cases of mumps and measles. Compared to EIA, the sensitivity, specificity, positive and negative predictive value of the mumps IgM NPT were 79.5%, 100%, 100%, and 72.6%, respectively. Mumps IgM NPT is rapid and simple to perform for confirmation of a clinical diagnosis. The NPT strip is also a suitable matrix for preserving nucleic acid, enabling virusspecific RT-PCR to be performed [164]. Standard diagnostics that detect virus or virus-specific antibody are dependable for confirming primary mumps infection in immunologically naïve persons, but these methods perform inconsistently for individuals with prior immune exposure. Detection of activated mumps-specific antibody-secreting B cells (ASCs) by an enzyme-linked immunospot (ELISPOT) assay has the potential for use as an alternative method of diagnosis when suspect cases cannot be confirmed by detection of IgM or virus. The mumps-specific memory B cells are detected at a much lower frequency than measles-or rubella-specific cells, suggesting that mumps infection may not generate robust B-cell memory [165]. Successful virus isolation by cell culture should always be confirmed by immunofluor‐ escence with a mumps-specific monoclonal antibody or by molecular techniques such as RT-PCR, nested-PCR or real-time RT-PCR [166-168]. In patients vaccinated, pre-existing vaccineinduced antibodies. Moreover, acute infection has to be demonstrated by direct detection of the virus by viral isolation or genomic amplification. RT-PCR allows a diagnosis and also forms the basis for genotype characterization by sequencing the SH gene, useful tool for mumps surveillance, management and control, according to WHO recommendations. Virus genotyp‐ ing allows the building of a sequence database that will help to know transmission pathways of mumps strains circulating in the world and to distinguish wild-type mumps virus from vaccine virus [169]. Standard RT-PCR to detect the SH gene of mumps virus can be used to detect mumps RNA in clinical samples or in infected cell culture [170]. The sequence of the PCR product containing the coding region of the SH gene can be used to determine the viral genotype [171]. A multiplex real-time RT-PCR test for the detection of RNA from mumps virus in patient specimens, using primers and probes that target the mumps SH gene [172]. Several test of real-time RT-PCR are performed with TaqMan Assay [173]. A multiplex real-time RT-PCR assay, for rapid mumps diagnosis in a clinical setting. The assay used oligonucleotide primers and a TaqMan probe targeting the mumps SH gene. This test showed 100% correlation with results from viral culture. [172]. Other qRT-PCR assay is directed to the mumps virus F gene [174]. Recently, by RT-PCR it was detect a case of corneal endothelitis following the

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Measles produces a highly contagious respiratory infection and may cause extensive epidem‐ ics. Measles is one of the most important causes of child morbidity and mortality. [176]. A safe and efficacious live attenuated virus vaccine is available toward global measles control [177,

mumps parotitis [175].

**7. Measles virus**

**Figure 5.** HCV Testing Algorithm and molecular assays.

D, H, J) and vaccine strains (Urabe Am9) have been associated with enhanced neurovirulence [157]. MuV is known to affect the salivary glands causing parotid swelling lasting at least two days, but may persist longer than ten days [158]. The mumps incubation period ranges from 12–25 days, but parotitis typically develops 16 to 18 days after exposure to mumps virus. The MuV also produce an acute systemic infection involving glandular, lymphoid and nervous tissues, leading to some important complications, the main central nervous system (CNS) complication of mumps virus infection is aseptic meningitis (in up to 15% of cases) [159, 160, 161]. Mumps epidemics are usually caused by airborne transmission of mumps virus (MuV) and have high morbidity in non-immunized children. Massive vaccination programs have decreased the incidence of MuV infection worldwide. The annual incidence of mumps in the absence of immunization was in the range of 100–1000 cases/100 000 people, outbreaks have not been completely eliminated even in populations with high vaccination coverage [162]. Laboratory confirmation of mumps infection can be made by the detection of immunoglobulin M (IgM) antibodies specific to mumps virus in acute-phase serum samples (gold standard for mumps diagnosis). Mumps virus can be detected from fluid collected from the parotid duct, other affected salivary gland ducts, the throat, from urine, and from cerebrospinal fluid (CSF). Parotid duct swabs yield the best viral sample. With previous contact with mumps virus either through vaccination or natural infection, serum mumps IgM test results may be negative; IgG test results may be positive at the initial blood draw. The serologic tests available for laboratory confirmation of mumps acute infection and confirmation of previous exposure to mumps vary among laboratories. Tests for IgM antibody includes Enzyme Immunoassay (EIA): a highly specific test for diagnosing acute mumps infection and Immunofluorescence assay (IFA) a test that is relatively inexpensive and simple, but the test is particularly susceptible to interference by high levels of mumps-specific IgG. A significant rise in IgG antibody titer, in acute-and convalescent-phase serum specimens is a positive result of infection. The presence of mumpsspecific IgG, detected using a serologic assay (EIA or IFA), is considered evidence of mumps immunity but does not predict the presence of neutralizing antibodies or protection from mumps disease [163]. A near patient test (NPT) for the detection of mumps-specific IgM in oral fluid specimens was developed and evaluated using 196 oral fluid specimens from suspected cases of mumps and measles. Compared to EIA, the sensitivity, specificity, positive and negative predictive value of the mumps IgM NPT were 79.5%, 100%, 100%, and 72.6%, respectively. Mumps IgM NPT is rapid and simple to perform for confirmation of a clinical diagnosis. The NPT strip is also a suitable matrix for preserving nucleic acid, enabling virusspecific RT-PCR to be performed [164]. Standard diagnostics that detect virus or virus-specific antibody are dependable for confirming primary mumps infection in immunologically naïve persons, but these methods perform inconsistently for individuals with prior immune exposure. Detection of activated mumps-specific antibody-secreting B cells (ASCs) by an enzyme-linked immunospot (ELISPOT) assay has the potential for use as an alternative method of diagnosis when suspect cases cannot be confirmed by detection of IgM or virus. The mumps-specific memory B cells are detected at a much lower frequency than measles-or rubella-specific cells, suggesting that mumps infection may not generate robust B-cell memory [165]. Successful virus isolation by cell culture should always be confirmed by immunofluor‐ escence with a mumps-specific monoclonal antibody or by molecular techniques such as RT-PCR, nested-PCR or real-time RT-PCR [166-168]. In patients vaccinated, pre-existing vaccineinduced antibodies. Moreover, acute infection has to be demonstrated by direct detection of the virus by viral isolation or genomic amplification. RT-PCR allows a diagnosis and also forms the basis for genotype characterization by sequencing the SH gene, useful tool for mumps surveillance, management and control, according to WHO recommendations. Virus genotyp‐ ing allows the building of a sequence database that will help to know transmission pathways of mumps strains circulating in the world and to distinguish wild-type mumps virus from vaccine virus [169]. Standard RT-PCR to detect the SH gene of mumps virus can be used to detect mumps RNA in clinical samples or in infected cell culture [170]. The sequence of the PCR product containing the coding region of the SH gene can be used to determine the viral genotype [171]. A multiplex real-time RT-PCR test for the detection of RNA from mumps virus in patient specimens, using primers and probes that target the mumps SH gene [172]. Several test of real-time RT-PCR are performed with TaqMan Assay [173]. A multiplex real-time RT-PCR assay, for rapid mumps diagnosis in a clinical setting. The assay used oligonucleotide primers and a TaqMan probe targeting the mumps SH gene. This test showed 100% correlation with results from viral culture. [172]. Other qRT-PCR assay is directed to the mumps virus F gene [174]. Recently, by RT-PCR it was detect a case of corneal endothelitis following the mumps parotitis [175].

#### **7. Measles virus**

D, H, J) and vaccine strains (Urabe Am9) have been associated with enhanced neurovirulence [157]. MuV is known to affect the salivary glands causing parotid swelling lasting at least two days, but may persist longer than ten days [158]. The mumps incubation period ranges from 12–25 days, but parotitis typically develops 16 to 18 days after exposure to mumps virus. The MuV also produce an acute systemic infection involving glandular, lymphoid and nervous tissues, leading to some important complications, the main central nervous system (CNS) complication of mumps virus infection is aseptic meningitis (in up to 15% of cases) [159, 160, 161]. Mumps epidemics are usually caused by airborne transmission of mumps virus (MuV) and have high morbidity in non-immunized children. Massive vaccination programs have decreased the incidence of MuV infection worldwide. The annual incidence of mumps in the absence of immunization was in the range of 100–1000 cases/100 000 people, outbreaks have not been completely eliminated even in populations with high vaccination coverage [162]. Laboratory confirmation of mumps infection can be made by the detection of immunoglobulin M (IgM) antibodies specific to mumps virus in acute-phase serum samples (gold standard for mumps diagnosis). Mumps virus can be detected from fluid collected from the parotid duct, other affected salivary gland ducts, the throat, from urine, and from cerebrospinal fluid (CSF). Parotid duct swabs yield the best viral sample. With previous contact with mumps virus either through vaccination or natural infection, serum mumps IgM test results may be negative; IgG test results may be positive at the initial blood draw. The serologic tests available for laboratory confirmation of mumps acute infection and confirmation of previous exposure to mumps vary among laboratories. Tests for IgM antibody includes Enzyme Immunoassay (EIA): a highly specific test for diagnosing acute mumps infection and Immunofluorescence assay (IFA) a test that is relatively inexpensive and simple, but the test is particularly susceptible to interference by high levels of mumps-specific IgG. A significant rise in IgG antibody titer, in acute-and convalescent-phase serum specimens is a positive result of infection. The presence of mumps-

**Figure 5.** HCV Testing Algorithm and molecular assays.

108 Trends in Infectious Diseases

Measles produces a highly contagious respiratory infection and may cause extensive epidem‐ ics. Measles is one of the most important causes of child morbidity and mortality. [176]. A safe and efficacious live attenuated virus vaccine is available toward global measles control [177,

178]. Measles vaccination is highly effective, safe and relatively cost-effective and has inter‐ rupted measles transmission in most parts of the world [179]. Despite tremendous achieve‐ ments towards global measles mortality reduction and measles elimination goals, globally, in 2010, there were 327305 measles cases reported and an estimated 139300 measles deaths) [180, 181]. Mortality is highest in children and most acute measles deaths are due to secondary infections that result from a poorly understood measles-induced suppression of immune responses [182]. In addition to the risks of acute infection, children under the age of 2 years are also vulnerable to development of subacute sclerosing panencephalitis (SSPE), a progres‐ sive, uniformly fatal neurologic disease associated with persistent measles virus infection of the nervous system, have documented high levels of antibody to measles virus [183]. Other diseases related to measles are systemic lupus erythematosus [184] and multiple sclerosis [185]. The measles virus is a member of the Morbillivirus genus of the family Paramyxoviridae. The virions are pleomorphic and range in size from 100 to 300 nm. The measles virus is antigenically stable and genetic differences are few among vaccine strains. Wild-type viruses are more variable. Several different genotypes of wild measles virus are currently circulating worldwide and this genetic variation provides the basis for the application of molecular epidemiological techniques to study the transmission of measles virus [186]. Laboratory confirmation of clinically diagnosed measles was traditionally based on methods such as immunofluorescence antibody for detection of viral antigen and haemagglutination inhibition (HI), haemolysin inhibition, complement fixation, and plaque-reduction neutralization (PRN) for detection of measles antibody in serum (Table 4) [187]. Currently, detection of measles-specific IgM antibody and measles RNA by real-time RT-PCR are the most common methods for confir‐ mation of measles infection [168]. Initially laboratory confirmation of cases of measles infection is performed by detection of measles-specific immunoglobulin M (IgM) antibodies, the test of choice for rapid diagnosis of measles cases in a single serum specimen collected within the first few days of rash onset, usually become detectable in serum after four days post onset of rash (90-100% sensitivity) and decline rapidly after one month. The virus can be detected in serum samples, blood spots, throat swab (or nasopharyngeal swab), urine and or collection of both respiratory and urine samples that can increase the likelihood of detecting virus. [188]. Oral fluids can also be used to detect viral ribonucleic acid (RNA) [189]. The enzyme immu‐ noassay is the most commonly used method for detecting measles-specific IgM or IgG antibodies. Both capture and indirect formats for IgM detection are available commercially. Some tests available are LIAISON IgM measles (DiaSorin, Saluggia, Italy) a new automated chemiluminescence immunoassay and the enzyme immunoassay (EIA) Enzygnost (Siemens, Marburg, Germany), which have a a sensitivity of 93.7% and 98.8%, whereas the specificity was 96.8% and 97.9%, respectively [190]. DiaSorin Liaison (Saluggia, Italy) is other option for measles diagnosis, with a sensitivity and specificity for measles IgM of 92% and 100% respectively [191]. Commercially, there are some test directs to simultaneous detection of measles and rubella as Enzygnost (Siemens) and Platelia (Bio-Rad), useful for detecting IgM against measles and rubella [192].

does not ensure protection from infection or re-infection. Screening the young adult population about to enter college or the military, pregnant women, and other individuals at risk, for

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**LABORATORY TEST**

Enzyme-linked immunosorbent assay (ELISA) is normally used to quantify the amount of serum IgG antibodies against measles (measles in addition to mumps, rubella, and varicellazoster virus, MMRV). However, a multiplex immunoassay for the simultaneous detection of antibodies against MMRV showed be a good alternative to conventional ELISAs and suitable for use in serosurveillance and vaccine studies [193]. An enzyme linked Immunosorbent commercial assay is Captia Measles IgG based (Trinity, Biotech, USA). In countries where disease prevalence is low, intensified surveillance typically implemented during and after an importation will result in some false positive IgM results since no assay is 100% specific. So, it is necessary to introduce other techniques of diagnosis related to ARN genome. In the Fifth Hands-on Training on the Laboratory Diagnosis of Measles and Rubella focusing on Molecular Detection and Sequence Analysis, in Hong Kong, China. It was exposed the need of introduce the molecular detection of measles and rubella viruses using new real-time polymerase chain reaction (PCR) as well as conventional PCR, quantitative real-time PCR and others molecular techniques such as sequencing. Dr Paul Rota (Chief, Measles Virus Section, Centers for Disease Control and Prevention USA), presented on the use of real-time and conventional RT-PCR for case classification and molecular surveillance of measles virus. These tests can detect 10-100 copies of RNA/sample in a high throughput format and produce results within two hours. It can help to confirm a case when serologic results are inconclusive but negative results do not

seropositivity, is a valuable tool for determining their immune status.

Detection of IgM antibody by indirect IgM ELISA, IgM-capture

measles RNA can be detected from nasopharyngeal swab, urine or peripheral blood lymphocytes (oral fluid/ throat swabs) up to 5 days

RT-PCR, RT-nested PCR, Real-Time RT-PCR and sequencing

**Table 4.** Laboratory diagnosis for measles in clinical materials.

Serological assays Using serum sample

ELISA or EIA for IgM

measles antibody NAT analysis

post disease manifestation.

HI

Virus neutralization test

Detection of IgG antibody by IgG ELISA

Cell culture and immunofluorescence microscopy Using urine, nasopharyngeal swabs or blood lymphocytes and detect to virus using specific

In acute, uncomplicated measles, there is a significant rise in measles-specific IgG antibodies between acute-and convalescent-phase serum specimens. A positive test result for specific IgG antibodies in a serum indicates past infection with measles virus or measles vaccination, but does not ensure protection from infection or re-infection. Screening the young adult population about to enter college or the military, pregnant women, and other individuals at risk, for seropositivity, is a valuable tool for determining their immune status.


**Table 4.** Laboratory diagnosis for measles in clinical materials.

178]. Measles vaccination is highly effective, safe and relatively cost-effective and has inter‐ rupted measles transmission in most parts of the world [179]. Despite tremendous achieve‐ ments towards global measles mortality reduction and measles elimination goals, globally, in 2010, there were 327305 measles cases reported and an estimated 139300 measles deaths) [180, 181]. Mortality is highest in children and most acute measles deaths are due to secondary infections that result from a poorly understood measles-induced suppression of immune responses [182]. In addition to the risks of acute infection, children under the age of 2 years are also vulnerable to development of subacute sclerosing panencephalitis (SSPE), a progres‐ sive, uniformly fatal neurologic disease associated with persistent measles virus infection of the nervous system, have documented high levels of antibody to measles virus [183]. Other diseases related to measles are systemic lupus erythematosus [184] and multiple sclerosis [185]. The measles virus is a member of the Morbillivirus genus of the family Paramyxoviridae. The virions are pleomorphic and range in size from 100 to 300 nm. The measles virus is antigenically stable and genetic differences are few among vaccine strains. Wild-type viruses are more variable. Several different genotypes of wild measles virus are currently circulating worldwide and this genetic variation provides the basis for the application of molecular epidemiological techniques to study the transmission of measles virus [186]. Laboratory confirmation of clinically diagnosed measles was traditionally based on methods such as immunofluorescence antibody for detection of viral antigen and haemagglutination inhibition (HI), haemolysin inhibition, complement fixation, and plaque-reduction neutralization (PRN) for detection of measles antibody in serum (Table 4) [187]. Currently, detection of measles-specific IgM antibody and measles RNA by real-time RT-PCR are the most common methods for confir‐ mation of measles infection [168]. Initially laboratory confirmation of cases of measles infection is performed by detection of measles-specific immunoglobulin M (IgM) antibodies, the test of choice for rapid diagnosis of measles cases in a single serum specimen collected within the first few days of rash onset, usually become detectable in serum after four days post onset of rash (90-100% sensitivity) and decline rapidly after one month. The virus can be detected in serum samples, blood spots, throat swab (or nasopharyngeal swab), urine and or collection of both respiratory and urine samples that can increase the likelihood of detecting virus. [188]. Oral fluids can also be used to detect viral ribonucleic acid (RNA) [189]. The enzyme immu‐ noassay is the most commonly used method for detecting measles-specific IgM or IgG antibodies. Both capture and indirect formats for IgM detection are available commercially. Some tests available are LIAISON IgM measles (DiaSorin, Saluggia, Italy) a new automated chemiluminescence immunoassay and the enzyme immunoassay (EIA) Enzygnost (Siemens, Marburg, Germany), which have a a sensitivity of 93.7% and 98.8%, whereas the specificity was 96.8% and 97.9%, respectively [190]. DiaSorin Liaison (Saluggia, Italy) is other option for measles diagnosis, with a sensitivity and specificity for measles IgM of 92% and 100% respectively [191]. Commercially, there are some test directs to simultaneous detection of measles and rubella as Enzygnost (Siemens) and Platelia (Bio-Rad), useful for detecting IgM

In acute, uncomplicated measles, there is a significant rise in measles-specific IgG antibodies between acute-and convalescent-phase serum specimens. A positive test result for specific IgG antibodies in a serum indicates past infection with measles virus or measles vaccination, but

against measles and rubella [192].

110 Trends in Infectious Diseases

Enzyme-linked immunosorbent assay (ELISA) is normally used to quantify the amount of serum IgG antibodies against measles (measles in addition to mumps, rubella, and varicellazoster virus, MMRV). However, a multiplex immunoassay for the simultaneous detection of antibodies against MMRV showed be a good alternative to conventional ELISAs and suitable for use in serosurveillance and vaccine studies [193]. An enzyme linked Immunosorbent commercial assay is Captia Measles IgG based (Trinity, Biotech, USA). In countries where disease prevalence is low, intensified surveillance typically implemented during and after an importation will result in some false positive IgM results since no assay is 100% specific. So, it is necessary to introduce other techniques of diagnosis related to ARN genome. In the Fifth Hands-on Training on the Laboratory Diagnosis of Measles and Rubella focusing on Molecular Detection and Sequence Analysis, in Hong Kong, China. It was exposed the need of introduce the molecular detection of measles and rubella viruses using new real-time polymerase chain reaction (PCR) as well as conventional PCR, quantitative real-time PCR and others molecular techniques such as sequencing. Dr Paul Rota (Chief, Measles Virus Section, Centers for Disease Control and Prevention USA), presented on the use of real-time and conventional RT-PCR for case classification and molecular surveillance of measles virus. These tests can detect 10-100 copies of RNA/sample in a high throughput format and produce results within two hours. It can help to confirm a case when serologic results are inconclusive but negative results do not rule out a case. These molecular tests are more sensitive than conventional (endpoint) RT-PCR. Sequence information from the conventional PCR is required for genotype assignment and confirmation of vaccine reactions [194].

several weeks. The samples should undergo virus isolation on sensitive cell lines, such as B95a cells. Measles virus can be isolated from clinical specimens, including; throat swab, conjuncti‐

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Acquired Immunodeficiency Syndrome (AIDS) is one of the most critically acclaimed endemic diseases, caused by two lentiviruses HIV-1 and 2. Human immunodeficiency virus (HIV) is the pathogen causing the acquired immunodeficiency syndrome (AIDS) [209, 210]. HIV is an enveloped virus with tropism for CD4+lymphocytes and monocytes. HIV is classified in the family *Retroviridae*, subfamily *Lentivirinae*, and genus *Lentivirus.* HIV comprising a single stranded, positive-sense ribonucleic acid (RNA) genome of about 9.7 kilobases. From the 5' end of genome are located the three genes that characterize retroviruses: *gag-pol-env.* The *gag* gene codes for the internal structural proteins, the *pol* gene for the three viral enzymes, and the *env* gene for the envelope glycoproteins. LTR (Long Terminal Repeat) sequences are found at each extremity of the genome, containing the signals for the regulation of expression of the viral genes. The genome also has six additional genes called "accessory" genes: *vif, nef, vpr, tat, rev* and *vpu* (HIV-1) or *vpx* (HIV-2) [211]. There are two strands of HIV RNA and each strand has a copy with nine genes, which encode 15 proteins. The RNA is surrounding by a cone shaped capsid which consists of approximately 2000 copies of the p24 viral protein. Surround‐ ing the capsid is the viral envelope. Each envelope subunit consists of two non-covalently linked membrane proteins: glycoprotein (gp) 120, the outer envelope protein, and gp41, the transmembrane protein that anchors the glycoprotein complex to the surface of the virion. The envelope protein is the most variable component of HIV, although gp120 itself is structurally divided into highly variable (V) and more constant (C) regions. The variability of the HIV envelope also confers a uniquely complex antigenic diversity. The virion contains three enzymes necessary for multiplication: reverse transcriptase (enables the viral RNA to be transcribed into DNA), and endonuclease (enables the DNA to be integrated into the host cell, the viral genome then becomes proviral DNA) and the protease (enables the virus to mature at a late stage in the cycle of intracellular multiplication). The presence of two copies of the retroviral genome in each particle promotes genetic recombination between the RNAs. This and the high error rate of the viral reverse transcriptase leads to considerable genetic variation in the viral progeny. AIDS is characterized by the selective targeting of the CD4+/CD8+T cells by HIV which fatally impairs the immune system. The window period for this retrovirus is from several weeks to few months altogether before detection of earliest antibodies in blood serum raised against HIV [210]. HIV infection is one of the major threats to human health due to the lack of relevant vaccine and drugs to cure AIDS. Its early diagnosis is thus important in controlling HIV transmission. With acute HIV infection, high levels of infectious virus are detectable in serum and genital secretions. The rate of transmission during acute HIV infection is higher than the established HIV infection, for this reason, new HIV testing strategies need to focus on sensitivity, especially for this highly contagious phase immediately after infection. There are two types of virus, HIV-1 and HIV-2, which are further divided into groups and/or

val swabs, nasopharyngeal aspirates or urine [208].

**8. HIV**

Measles RNA can be detected (oral fluid/ throat swabs) up to 5 days post disease manifesta‐ tion. Molecular assays to measles virus includes measles H (haemagglutinin) gene real-time PCR and hybridization [195] and nucleic acid sequence analysis of the nested N-gene PCR amplicons (nucleocapsid)[196, 197].Anestedreverse transcriptasePCR(RT-PCR)thatdetected measles virus (MV)from dried filter papers was set up using MV infected cells diluted in sterile phosphate-buffered saline. Although, the nested RT-PCR results of low titer viruses dried onto filter papers are not reproducible and reliable [198, 199]. Detection of RNA in PBMC by RT-PCRis the most effective methodfordiagnosis of measles.Astudyperformedin 2010, analyzed sixty-three throat swabs, 84peripheral blood mononuclear cell(PBMC) samples, and85plasma samples were collected from 85 cases of suspected measles. The percentage of positive results from PBMC by RT-PCR and virus isolation was 100 and 91.7%, respectively. The percentage of positive results from throat swabs by RT-PCR and virus isolation was 91.2 and 52.8%, respec‐ tively [200]. Measles can produce congenital infections with a risk of neurological complica‐ tions in the newborn. Mother-to-child transmission of the measles virus, it has been widely documented in the newborns either by RT-PCR in saliva or by IgM detection in blood. An early viral RT-PCR detection allows successful immunoglobulin prophylaxis in one newborn avoiding the development classical or neurological clinical signs of measles infection [201]. Molecular detection of measles virus has been optimized by amplification of nucleocapsid (N) and human RNase P mRNA for a one-step quantitative reverse transcription (qRT)-PCR [202]. The qRT-PCR for measles diagnosis can use SYBR Green or TaqMan (ABI) in real-time reverse transcription-polymerase chain reaction (RT-PCR) assays. For the real-time RT-PCR, primer sets are design from a region of the MV H gene of the Edmonston strain (genotype A) and a TaqMan probe specific for the H gene of genotype D MV [203]. Currently, is possible estimate the titer of measles, mumps and rubella (MMR) viruses by a TaqMan-based real-time reverse transcription-polymerase chain reaction (qPCR-RT) assay optimized in infected cell culture supernatants [204]. For genetic typing of measles virus in clinical samples is xMAP technolo‐ gy that employs specific oligonucleotide probes of genotypes D4, D6 and D7 of virus [205]. Other alternative employed for the genotype analysis of measles virus is sequencing of the 450 nucleotide ofnucleoproteingene (N450)that contributes to the genetic characterizationof wildtype measles viruses and offers data in the study of viral transmission pathways. N450 is amplifying with the primer pair, MeV216/MeV214. It is clear that the molecular tools improve‐ ment the molecular characterization of circulating measles viruses globally and provides enhanced quality control measures [206]. Demonstration of the usefulness of molecular tests in the diagnosis of measles during outbreaks or epidemic peaks was reported in an outbreak in Paris (France)in 2011. 171 oralfluid samples and 235 serum samples collected from 270 patients weretestedusinganovelone-stepreal-timeRT-PCRassay.This studyshowedthatthedetection rateofMV-RNAbyRT-PCRwas98%(100/102)fororalfluidand95%(97/102)for serumsamples. The detection rate of MV-IgM was 85% (87/102). In addition, it was found than during the early stage ofinfection, the MV-RNA viral load in serum was lowerin patient's positive than in those negative for MV-IgG [207]. The detection and identification of the virus in cell culture may take several weeks. The samples should undergo virus isolation on sensitive cell lines, such as B95a cells. Measles virus can be isolated from clinical specimens, including; throat swab, conjuncti‐ val swabs, nasopharyngeal aspirates or urine [208].

### **8. HIV**

rule out a case. These molecular tests are more sensitive than conventional (endpoint) RT-PCR. Sequence information from the conventional PCR is required for genotype assignment and

Measles RNA can be detected (oral fluid/ throat swabs) up to 5 days post disease manifesta‐ tion. Molecular assays to measles virus includes measles H (haemagglutinin) gene real-time PCR and hybridization [195] and nucleic acid sequence analysis of the nested N-gene PCR amplicons (nucleocapsid)[196, 197].Anestedreverse transcriptasePCR(RT-PCR)thatdetected measles virus (MV)from dried filter papers was set up using MV infected cells diluted in sterile phosphate-buffered saline. Although, the nested RT-PCR results of low titer viruses dried onto filter papers are not reproducible and reliable [198, 199]. Detection of RNA in PBMC by RT-PCRis the most effective methodfordiagnosis of measles.Astudyperformedin 2010, analyzed sixty-three throat swabs, 84peripheral blood mononuclear cell(PBMC) samples, and85plasma samples were collected from 85 cases of suspected measles. The percentage of positive results from PBMC by RT-PCR and virus isolation was 100 and 91.7%, respectively. The percentage of positive results from throat swabs by RT-PCR and virus isolation was 91.2 and 52.8%, respec‐ tively [200]. Measles can produce congenital infections with a risk of neurological complica‐ tions in the newborn. Mother-to-child transmission of the measles virus, it has been widely documented in the newborns either by RT-PCR in saliva or by IgM detection in blood. An early viral RT-PCR detection allows successful immunoglobulin prophylaxis in one newborn avoiding the development classical or neurological clinical signs of measles infection [201]. Molecular detection of measles virus has been optimized by amplification of nucleocapsid (N) and human RNase P mRNA for a one-step quantitative reverse transcription (qRT)-PCR [202]. The qRT-PCR for measles diagnosis can use SYBR Green or TaqMan (ABI) in real-time reverse transcription-polymerase chain reaction (RT-PCR) assays. For the real-time RT-PCR, primer sets are design from a region of the MV H gene of the Edmonston strain (genotype A) and a TaqMan probe specific for the H gene of genotype D MV [203]. Currently, is possible estimate the titer of measles, mumps and rubella (MMR) viruses by a TaqMan-based real-time reverse transcription-polymerase chain reaction (qPCR-RT) assay optimized in infected cell culture supernatants [204]. For genetic typing of measles virus in clinical samples is xMAP technolo‐ gy that employs specific oligonucleotide probes of genotypes D4, D6 and D7 of virus [205]. Other alternative employed for the genotype analysis of measles virus is sequencing of the 450 nucleotide ofnucleoproteingene (N450)that contributes to the genetic characterizationof wildtype measles viruses and offers data in the study of viral transmission pathways. N450 is amplifying with the primer pair, MeV216/MeV214. It is clear that the molecular tools improve‐ ment the molecular characterization of circulating measles viruses globally and provides enhanced quality control measures [206]. Demonstration of the usefulness of molecular tests in the diagnosis of measles during outbreaks or epidemic peaks was reported in an outbreak in Paris (France)in 2011. 171 oralfluid samples and 235 serum samples collected from 270 patients weretestedusinganovelone-stepreal-timeRT-PCRassay.This studyshowedthatthedetection rateofMV-RNAbyRT-PCRwas98%(100/102)fororalfluidand95%(97/102)for serumsamples. The detection rate of MV-IgM was 85% (87/102). In addition, it was found than during the early stage ofinfection, the MV-RNA viral load in serum was lowerin patient's positive than in those negative for MV-IgG [207]. The detection and identification of the virus in cell culture may take

confirmation of vaccine reactions [194].

112 Trends in Infectious Diseases

Acquired Immunodeficiency Syndrome (AIDS) is one of the most critically acclaimed endemic diseases, caused by two lentiviruses HIV-1 and 2. Human immunodeficiency virus (HIV) is the pathogen causing the acquired immunodeficiency syndrome (AIDS) [209, 210]. HIV is an enveloped virus with tropism for CD4+lymphocytes and monocytes. HIV is classified in the family *Retroviridae*, subfamily *Lentivirinae*, and genus *Lentivirus.* HIV comprising a single stranded, positive-sense ribonucleic acid (RNA) genome of about 9.7 kilobases. From the 5' end of genome are located the three genes that characterize retroviruses: *gag-pol-env.* The *gag* gene codes for the internal structural proteins, the *pol* gene for the three viral enzymes, and the *env* gene for the envelope glycoproteins. LTR (Long Terminal Repeat) sequences are found at each extremity of the genome, containing the signals for the regulation of expression of the viral genes. The genome also has six additional genes called "accessory" genes: *vif, nef, vpr, tat, rev* and *vpu* (HIV-1) or *vpx* (HIV-2) [211]. There are two strands of HIV RNA and each strand has a copy with nine genes, which encode 15 proteins. The RNA is surrounding by a cone shaped capsid which consists of approximately 2000 copies of the p24 viral protein. Surround‐ ing the capsid is the viral envelope. Each envelope subunit consists of two non-covalently linked membrane proteins: glycoprotein (gp) 120, the outer envelope protein, and gp41, the transmembrane protein that anchors the glycoprotein complex to the surface of the virion. The envelope protein is the most variable component of HIV, although gp120 itself is structurally divided into highly variable (V) and more constant (C) regions. The variability of the HIV envelope also confers a uniquely complex antigenic diversity. The virion contains three enzymes necessary for multiplication: reverse transcriptase (enables the viral RNA to be transcribed into DNA), and endonuclease (enables the DNA to be integrated into the host cell, the viral genome then becomes proviral DNA) and the protease (enables the virus to mature at a late stage in the cycle of intracellular multiplication). The presence of two copies of the retroviral genome in each particle promotes genetic recombination between the RNAs. This and the high error rate of the viral reverse transcriptase leads to considerable genetic variation in the viral progeny. AIDS is characterized by the selective targeting of the CD4+/CD8+T cells by HIV which fatally impairs the immune system. The window period for this retrovirus is from several weeks to few months altogether before detection of earliest antibodies in blood serum raised against HIV [210]. HIV infection is one of the major threats to human health due to the lack of relevant vaccine and drugs to cure AIDS. Its early diagnosis is thus important in controlling HIV transmission. With acute HIV infection, high levels of infectious virus are detectable in serum and genital secretions. The rate of transmission during acute HIV infection is higher than the established HIV infection, for this reason, new HIV testing strategies need to focus on sensitivity, especially for this highly contagious phase immediately after infection. There are two types of virus, HIV-1 and HIV-2, which are further divided into groups and/or subtypes. The pandemic is caused by HIV-1 group M. HIV-1 and HIV-2 have differences as clinical progression of the disease is slower and mother-to-child transmission is less likely with HIV-2 than with HIV-1 (maternal-fetal transmission < 2% in the absence of treatment). Recombinant HIV strains are known as CRF (Circulating Recombinant Forms). Although HIV-2 is also associated with AIDS, it is not transmitted as readily and, generally speaking, progression toward immunodeficiency is much slower in individuals with an HIV-2 infection. HIV-1 is responsible for a chronic infection that gradually develops and causes the destruction of the body's CD4+T lymphocytes. HIV-1 is responsible for a chronic infection that gradually causes the destruction of CD4+ T lymphocytes [212]. To detect a HIV infection several tests are used to analyze the HIV infection status of a patient, evaluate the progression of disease, and monitor the effectiveness of antiretroviral therapy (ART). HIV infection can be diagnosed by direct visualization of virions or electron microscopy; cultivation by lymphocyte culture; measurement of HIV-specific serologic responses; detection of viral antigens; and detection of viral nucleic acids [213]. For many years, laboratory diagnosis of HIV is based on the identi‐ fication of HIV antibodies using immuno-enzymatic (ELISA) tests or other immunological techniques of equivalent sensitivity. Still considering the limitation of this approach in the known 'window period' between the time of infection and the initial instance of detectable antibody, this may last for several weeks. Primary infection is asymptomatic in more than 50% of cases. In the remaining cases, symptoms appear two to three weeks after infection and clinical signs usually resemble those of flu-like or mononucleosis syndromes. Plasma viraemia levels are generally high (≥10<sup>6</sup> copies of viral genome/ml) during primary infection.

diagnostics, Delkenheim, Germany), Elecsys 2010 HIV Combi (Roche Diagnostics GmbH, Mannheim, Germany) and Vidas HIV Duo Quick (Biomerieux, France). All of the assays had sensitivities of 100% on clinical samples. The specificities of the AxSYM, ARCHITECT, Elecsys 2010 HIV Combi, and Elecsys HIV Combi PT were 99.6, 99.6, 99.0, and 99.5%, respectively [218]. Genscreen Ultra HIV Ag-Ab is other new version of the HIV p24 antigen and antibody combination assays [218]. The commercial ELISAs such as Vironostika HIV Ag/Ab, Enzygnost Anti-HIV 1/2 Plus Genscreen HIV-1/2 Version 2 and INNO-LIA HIV I/II are suitable tools for making HIV test performance accessible to people [219]. The accurate diagnosis of HIV infection demands that to consider a positive result, at least three assays with different antigenic base should be used, one of them, Western-Blot being mandatory for confirmation. Confirmatory techniques which are used most frequently they are the Western Blot (WB) and the recombinant immunoblot or immunoassay online (LIA) who have at least the same sensitivity than ELISA and a higher specificity. Both techniques they can incorporate antigens of HIV-2. It detects antibodies against the glycoprotein gp160 envelope, gp120 and gp41, p55, p24 and p17 encoded-gag and p66, p51 and p311. The interpretation of the results is crucial; a negative test is the total absence of reactivity. To assess, the positivity numerous criteria applied, according to the Center for Disease Control (CDC) a positive result occurs with at least 2 bands of p24, gp41, and gp160gp120 are detected. WHO recognizes a positive test with 2 bands. The ARC (American Red Cross) indicates three bands, one of each structural gene, and the Consortium for Retrovirus Serology Standardization indicates at least one of gp120 or gp160 and one of p24 or p31 [220, 221]. It is interpreted as an undetermined result, any reactivity that does not meet the minimum criteria of positivity. Since, the causes of WB indeterminate are diverse and they may correspond to early phases or advanced stages of infection associated to severe immune impairment, or to the presence of immune complexes than can reduce the antibodies circulating, between other causes. Detection of some band of envelope with or without bands of gag gene, may be due to HIV infection. In these cases, is necessary performed others confirmatory tests as LIA and sometimes complement them with the determination of proviral DNA or viral load or p24 antigen to assess a possible primoinfeccion. In any case on an indeterminate WB is required a new sample [222-224]. The comparison between the Ag/Ab combo assay and RNA viral load showed that in an acute HIV infection in human gave a similar result. HIV Combo detected 97% of infections acute. The ARCHITECT HIV Combo assay can detect p24 Ag when RNA is above approximately 58,000 copies/mL [225]. However, the comparison between the results of HIV RNA nucleic acid test (NAT) and 4th-generation Ag/Ab assay (ARCHITEC HIV Ag/Ab Combo [HIV Combo] assay, Abbott Diagnostics) in 2744 HIV antibody-negative samples were identified fourteen people with acute HIV infection (HIV antibody negative/NAT positive). The HIV Combo assay detected nine of these individuals [226 delete these rows, from the stage word to the HIV Word [226In the 2012 HIV Diagnostics Conference: the molecular diagnostics perspective, gives in Atlanta, GA, USA, 12–14 December 2012. The forum was foccusing in the evaluation of molecular diagnostics and their role in HIV diagnosis. Many scientific presentations exposed the role played by RNA testing and new developments in molecular diagnostics, including detection of total and integrated HIV-1 DNA, detection and quantification of HIV-2 RNA, and rapid formats for detection of HIV-1

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RNA [227]. HIV infection monitoring is based on counting the number of CD4+

lymphocytes

An assay useful in Multispot HIV-1/HIV-2 Rapid Test (BIO-RAD) directed to a rapid test for detection and differentiation of HIV-1 and HIV-2 antibodies in human serum and plasma. This test have a time of results of 10 minute and shows HIV-1 sensitivity: 100%, HIV-2 sensitivity: 100% and specificity: 99.9% [214]. BIO-RAD also has an assay know as HIV-1/HIV-2 PLUS O, an ELISA-immuno assay utilizing recombinant proteins and synthetic peptides for the detection of antibodies to HIV-1 (groups M and O) and/or HIV-2.

New HIV screening tests approved by the US FDA in 2010-2011 include immunoassays capable of detecting p24 antigen and HIV antibody simultaneously. The fourth generation combo assays could reduction the window period due to their ability to detect viral protein s in addition to IgG and IgM class antibodies against both HIV-1 and HIV-2 [215], but the moni‐ toring of HIV disease progression is mostly accomplished by the quantitation of CD4 T cells and viral RNA [216]. The use of combined ELISA tests called "4th generation" tests, enables for more effective early detection of infections which are very often asymptomatic. Also, these assays can detect acute and chronic infections. An example is the ARCHITECT HIV Ag/Ab Combo assay (Abbott Diagnostics), which uses anti-HIV-1 p24 antibodies as reagents to detect HIV-1 p24 antigen, thereby decreasing the window period and improving early detection of HIV infection. The assay is useful to determine the presence of HIV-1 p24 antigen, antibodies to HIV-1 (group M and group O), and antibodies to HIV-2 in human serum or plasma using chemiluminescent microparticle immunoassay A technology. The test has an analytical sensitivity of <50 pg/mL for HIV-1 p24 antigen [217]. There are others fourth-generation human immunodeficiency virus-1 (HIV-1) screening assays as the AxSYM HIV Ag/Ab Combo (Abbott diagnostics, Delkenheim, Germany), Elecsys 2010 HIV Combi (Roche Diagnostics GmbH, Mannheim, Germany) and Vidas HIV Duo Quick (Biomerieux, France). All of the assays had sensitivities of 100% on clinical samples. The specificities of the AxSYM, ARCHITECT, Elecsys 2010 HIV Combi, and Elecsys HIV Combi PT were 99.6, 99.6, 99.0, and 99.5%, respectively [218]. Genscreen Ultra HIV Ag-Ab is other new version of the HIV p24 antigen and antibody combination assays [218]. The commercial ELISAs such as Vironostika HIV Ag/Ab, Enzygnost Anti-HIV 1/2 Plus Genscreen HIV-1/2 Version 2 and INNO-LIA HIV I/II are suitable tools for making HIV test performance accessible to people [219]. The accurate diagnosis of HIV infection demands that to consider a positive result, at least three assays with different antigenic base should be used, one of them, Western-Blot being mandatory for confirmation. Confirmatory techniques which are used most frequently they are the Western Blot (WB) and the recombinant immunoblot or immunoassay online (LIA) who have at least the same sensitivity than ELISA and a higher specificity. Both techniques they can incorporate antigens of HIV-2. It detects antibodies against the glycoprotein gp160 envelope, gp120 and gp41, p55, p24 and p17 encoded-gag and p66, p51 and p311. The interpretation of the results is crucial; a negative test is the total absence of reactivity. To assess, the positivity numerous criteria applied, according to the Center for Disease Control (CDC) a positive result occurs with at least 2 bands of p24, gp41, and gp160gp120 are detected. WHO recognizes a positive test with 2 bands. The ARC (American Red Cross) indicates three bands, one of each structural gene, and the Consortium for Retrovirus Serology Standardization indicates at least one of gp120 or gp160 and one of p24 or p31 [220, 221]. It is interpreted as an undetermined result, any reactivity that does not meet the minimum criteria of positivity. Since, the causes of WB indeterminate are diverse and they may correspond to early phases or advanced stages of infection associated to severe immune impairment, or to the presence of immune complexes than can reduce the antibodies circulating, between other causes. Detection of some band of envelope with or without bands of gag gene, may be due to HIV infection. In these cases, is necessary performed others confirmatory tests as LIA and sometimes complement them with the determination of proviral DNA or viral load or p24 antigen to assess a possible primoinfeccion. In any case on an indeterminate WB is required a new sample [222-224]. The comparison between the Ag/Ab combo assay and RNA viral load showed that in an acute HIV infection in human gave a similar result. HIV Combo detected 97% of infections acute. The ARCHITECT HIV Combo assay can detect p24 Ag when RNA is above approximately 58,000 copies/mL [225]. However, the comparison between the results of HIV RNA nucleic acid test (NAT) and 4th-generation Ag/Ab assay (ARCHITEC HIV Ag/Ab Combo [HIV Combo] assay, Abbott Diagnostics) in 2744 HIV antibody-negative samples were identified fourteen people with acute HIV infection (HIV antibody negative/NAT positive). The HIV Combo assay detected nine of these individuals [226 delete these rows, from the stage word to the HIV Word [226In the 2012 HIV Diagnostics Conference: the molecular diagnostics perspective, gives in Atlanta, GA, USA, 12–14 December 2012. The forum was foccusing in the evaluation of molecular diagnostics and their role in HIV diagnosis. Many scientific presentations exposed the role played by RNA testing and new developments in molecular diagnostics, including detection of total and integrated HIV-1 DNA, detection and quantification of HIV-2 RNA, and rapid formats for detection of HIV-1 RNA [227]. HIV infection monitoring is based on counting the number of CD4+ lymphocytes

subtypes. The pandemic is caused by HIV-1 group M. HIV-1 and HIV-2 have differences as clinical progression of the disease is slower and mother-to-child transmission is less likely with HIV-2 than with HIV-1 (maternal-fetal transmission < 2% in the absence of treatment). Recombinant HIV strains are known as CRF (Circulating Recombinant Forms). Although HIV-2 is also associated with AIDS, it is not transmitted as readily and, generally speaking, progression toward immunodeficiency is much slower in individuals with an HIV-2 infection. HIV-1 is responsible for a chronic infection that gradually develops and causes the destruction of the body's CD4+T lymphocytes. HIV-1 is responsible for a chronic infection that gradually

used to analyze the HIV infection status of a patient, evaluate the progression of disease, and monitor the effectiveness of antiretroviral therapy (ART). HIV infection can be diagnosed by direct visualization of virions or electron microscopy; cultivation by lymphocyte culture; measurement of HIV-specific serologic responses; detection of viral antigens; and detection of viral nucleic acids [213]. For many years, laboratory diagnosis of HIV is based on the identi‐ fication of HIV antibodies using immuno-enzymatic (ELISA) tests or other immunological techniques of equivalent sensitivity. Still considering the limitation of this approach in the known 'window period' between the time of infection and the initial instance of detectable antibody, this may last for several weeks. Primary infection is asymptomatic in more than 50% of cases. In the remaining cases, symptoms appear two to three weeks after infection and clinical signs usually resemble those of flu-like or mononucleosis syndromes. Plasma viraemia

An assay useful in Multispot HIV-1/HIV-2 Rapid Test (BIO-RAD) directed to a rapid test for detection and differentiation of HIV-1 and HIV-2 antibodies in human serum and plasma. This test have a time of results of 10 minute and shows HIV-1 sensitivity: 100%, HIV-2 sensitivity: 100% and specificity: 99.9% [214]. BIO-RAD also has an assay know as HIV-1/HIV-2 PLUS O, an ELISA-immuno assay utilizing recombinant proteins and synthetic peptides for the

New HIV screening tests approved by the US FDA in 2010-2011 include immunoassays capable of detecting p24 antigen and HIV antibody simultaneously. The fourth generation combo assays could reduction the window period due to their ability to detect viral protein s in addition to IgG and IgM class antibodies against both HIV-1 and HIV-2 [215], but the moni‐ toring of HIV disease progression is mostly accomplished by the quantitation of CD4 T cells and viral RNA [216]. The use of combined ELISA tests called "4th generation" tests, enables for more effective early detection of infections which are very often asymptomatic. Also, these assays can detect acute and chronic infections. An example is the ARCHITECT HIV Ag/Ab Combo assay (Abbott Diagnostics), which uses anti-HIV-1 p24 antibodies as reagents to detect HIV-1 p24 antigen, thereby decreasing the window period and improving early detection of HIV infection. The assay is useful to determine the presence of HIV-1 p24 antigen, antibodies to HIV-1 (group M and group O), and antibodies to HIV-2 in human serum or plasma using chemiluminescent microparticle immunoassay A technology. The test has an analytical sensitivity of <50 pg/mL for HIV-1 p24 antigen [217]. There are others fourth-generation human immunodeficiency virus-1 (HIV-1) screening assays as the AxSYM HIV Ag/Ab Combo (Abbott

detection of antibodies to HIV-1 (groups M and O) and/or HIV-2.

T lymphocytes [212]. To detect a HIV infection several tests are

copies of viral genome/ml) during primary infection.

causes the destruction of CD4+

114 Trends in Infectious Diseases

levels are generally high (≥10<sup>6</sup>

and quantification of plasma viral RNA. These tests are performed every 6 months if the CD4 count is > 500/mm3 and every 3 to 4 months if the CD4 count is between 200 and 500/mm3 . Plasma viral load is measured using quantitative tests based on molecular tools: gene ampli‐ fication (PCR-polymerase chain reaction, LCR-ligase chain reaction, TMA-transcription mediated amplification, NASBA-nucleic acid sequence based amplification) or hybridization followed by signal amplification (bDNA-branched DNA). Most tests have sensitivity of the order of 50-100 copies/ml. Although the new HIV diagnostic algorithm relies on RNA assays as a supplemental test, it is not clear how accessible these assays will be for clinical laboratories. Currently, only one HIV RNA test is approved by the US FDA for HIV diagnosis (Hologic Gen-Probe APTIMA HIV-1 RNA Qualitative Assay) and some clinical laboratories may need to send specimens out for RNA testing. Currently, molecular diagnosis of HIV infection is only used as a complementary diagnosis although viral load test is used to monitor disease progression and responsiveness to antiviral therapy. Recently, it was proposed to the first-line HIV molecular techniques performed on a routine basis routed to the use of HIV molecular tools for the screening of blood products, organs and tissue from human origin. Directed to medically assisted procreation and in neonates from HIV-infected mothers [228]. In 2012 HIV Diagnostics Conference was presented the design of a new HIV-1 proviral DNA assay capable of detecting two copies of HIV-1 DNA in a qualitative format and quantitatively of three to 30,000 copies per ml. Since the lack a nucleic acid test for HIV-2, in this meeting was described a novel HIV-2 RNA viral load assay based on the 5´ long terminal repeat of HIV-2, with a lower quantification limit of 29 infectious units per ml. In addition, an assay capable of detecting HIV-2 proviral DNA, which combines three separate amplification reactions from three regions of the proviral genome, detected both A and B HIV-2 subtypes at between five and ten copies of the HIV-2 proviral genome [228]. For other side, HIV-1 detection in plasma samples with a molecular beacon-based multiplex NASBA assay of a region in the HIV-1 pol gene showed a limit of quantification of the assay was <1000 copies/ml for HIV-1 with 98% sensi‐ tivity and 100% specificity [229]. Also, it was reported the detection and quantification of HIV-1 group O RNA in plasma by an RT-qPCR assay [230]. In addition, there are several HIV molecular assays showed in the table 5 [231].

Routine follow-up of HIV-infected individuals includes measurement of CD4+ T cell count to evaluate the immune status, of viral load to assess virus replication directed to events of therapeutic failure (therapeutic escape), and of changes in the viral genome to characterize resistance to drugs and tropism. Genotype resistance testing can detect a potential viral escape due to poor compliance with the treatment regimen, metabolic problems or the selection of resistant mutants. In summary, the diagnosis of HIV infection is established by one of the following methods: detecting antibodies to the virus; detecting the viral p24 antigen; detecting viral nucleic acid; or culturing HIV.

access to immediate HIV test results could improve the application of prophylactic regimens

**METHOD AND NAME TEST**

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**DNA/RNA qualitative assays** used to diagnosis HIV

COBAS AmpliPrep/TaqMan HIV-1 (Roche Molecular)

**Genotyping drug** used to Antiretroviral drug resistance

**Phenotyping drug** used to Antiretroviral drug resistance

Information based on Tang YW, et al. 2012 [231].

**Table 5.** Main molecular diagnostics methods for HIV-1.

Versant HIV-1RNA (Siemens) NucliSens HIV RNA QT (bioMérieux) RealTime m2000 HIV-1 (Abbott Molecular)

TruGene HIV-1 genotyping (Siemens) ViroSeq HIV-1 genotyping (Abbott Molecular) HIV PRT GeneChip assay (Affymetrix) HIV RT Line Probe assay (Innogenetics)

AntiVirogram assay (Virco Lab) Trofile (MonoGram BioSciences) SensiTrop II HIV (Pathway Diagnostics)

APTIMA HIV-1 RNA Qualitative assay or Procleix HIV-1/HCV Assay (Gen Probe) **RNAviral load testing** used for clinical diagnosis and/or monitoring of HIV-1

The analysis of the cost-effectiveness of initial diagnosis with a rapid HIV test, to screen out HIV-uninfected infants shows that in the comparison of DNA-PCR and rapid HIV test approaches, the first assay identified 94.3% (91.8–94.7%) of HIV-infected infants, as compared with 87.8% (79.4–90.5%) for the latter. Moreover, the total cost of the POC testing program was about 40% less than that of DNA-PCR (\$59 vs. \$38 per infant aged 6–9 months). Assessing the cost-effectiveness of several HIV testing as well as establishing a specific threshold of positivity for routine testing of HIV dignosis will be critical in AIDS control [235, 236]. Despite the global effort to control the AIDS pandemic, human immunodeficiency virus (HIV) infection continues to spread relatively unabated in many parts of the world. As the AIDS epidemic continues is necessary establish new strategies of prevention, treatment and molecular diagnostics assays to discriminate "window phase" infections from those that are serologically positive. There is opportunity to implement and evaluate the incremental diagnostic usefulness of new test modalities that are based on sophisticated molecular diagnostic technologies and that can be performed in settings where laboratory infrastructure is minimal [237]. Human immunodefi‐ ciency virus type 1 (HIV-1) is a highly diverse virus, a global scale, and within individual HIV-1 infected subjects [238]. The genetic variants constituting the viral population are called haplotypes, and these haplotypes form a viral quasispecies [239]. This viral diversity is highly

to reduce vertical transmission when used intrapartum or postpartum [234].

The most widely used test is the detection of antibodies to HIV [232]. Rapid serological tests take only 15 minutes with acceptable specificity and sensibility. But, there is the possibility of have a negative false result with them. NATs are assays more sensitive in HIV diagnosis, but more expensive. The expanded use of point-of-care (POC) tests to HIV testing plays an important role in HIV prevention, both in developed and in developing countries [233]. The


**Table 5.** Main molecular diagnostics methods for HIV-1.

and quantification of plasma viral RNA. These tests are performed every 6 months if the CD4

Plasma viral load is measured using quantitative tests based on molecular tools: gene ampli‐ fication (PCR-polymerase chain reaction, LCR-ligase chain reaction, TMA-transcription mediated amplification, NASBA-nucleic acid sequence based amplification) or hybridization followed by signal amplification (bDNA-branched DNA). Most tests have sensitivity of the order of 50-100 copies/ml. Although the new HIV diagnostic algorithm relies on RNA assays as a supplemental test, it is not clear how accessible these assays will be for clinical laboratories. Currently, only one HIV RNA test is approved by the US FDA for HIV diagnosis (Hologic Gen-Probe APTIMA HIV-1 RNA Qualitative Assay) and some clinical laboratories may need to send specimens out for RNA testing. Currently, molecular diagnosis of HIV infection is only used as a complementary diagnosis although viral load test is used to monitor disease progression and responsiveness to antiviral therapy. Recently, it was proposed to the first-line HIV molecular techniques performed on a routine basis routed to the use of HIV molecular tools for the screening of blood products, organs and tissue from human origin. Directed to medically assisted procreation and in neonates from HIV-infected mothers [228]. In 2012 HIV Diagnostics Conference was presented the design of a new HIV-1 proviral DNA assay capable of detecting two copies of HIV-1 DNA in a qualitative format and quantitatively of three to 30,000 copies per ml. Since the lack a nucleic acid test for HIV-2, in this meeting was described a novel HIV-2 RNA viral load assay based on the 5´ long terminal repeat of HIV-2, with a lower quantification limit of 29 infectious units per ml. In addition, an assay capable of detecting HIV-2 proviral DNA, which combines three separate amplification reactions from three regions of the proviral genome, detected both A and B HIV-2 subtypes at between five and ten copies of the HIV-2 proviral genome [228]. For other side, HIV-1 detection in plasma samples with a molecular beacon-based multiplex NASBA assay of a region in the HIV-1 pol gene showed a limit of quantification of the assay was <1000 copies/ml for HIV-1 with 98% sensi‐ tivity and 100% specificity [229]. Also, it was reported the detection and quantification of HIV-1 group O RNA in plasma by an RT-qPCR assay [230]. In addition, there are several HIV

and every 3 to 4 months if the CD4 count is between 200 and 500/mm3

.

T cell count to

count is > 500/mm3

116 Trends in Infectious Diseases

molecular assays showed in the table 5 [231].

viral nucleic acid; or culturing HIV.

Routine follow-up of HIV-infected individuals includes measurement of CD4+

evaluate the immune status, of viral load to assess virus replication directed to events of therapeutic failure (therapeutic escape), and of changes in the viral genome to characterize resistance to drugs and tropism. Genotype resistance testing can detect a potential viral escape due to poor compliance with the treatment regimen, metabolic problems or the selection of resistant mutants. In summary, the diagnosis of HIV infection is established by one of the following methods: detecting antibodies to the virus; detecting the viral p24 antigen; detecting

The most widely used test is the detection of antibodies to HIV [232]. Rapid serological tests take only 15 minutes with acceptable specificity and sensibility. But, there is the possibility of have a negative false result with them. NATs are assays more sensitive in HIV diagnosis, but more expensive. The expanded use of point-of-care (POC) tests to HIV testing plays an important role in HIV prevention, both in developed and in developing countries [233]. The

access to immediate HIV test results could improve the application of prophylactic regimens to reduce vertical transmission when used intrapartum or postpartum [234].

The analysis of the cost-effectiveness of initial diagnosis with a rapid HIV test, to screen out HIV-uninfected infants shows that in the comparison of DNA-PCR and rapid HIV test approaches, the first assay identified 94.3% (91.8–94.7%) of HIV-infected infants, as compared with 87.8% (79.4–90.5%) for the latter. Moreover, the total cost of the POC testing program was about 40% less than that of DNA-PCR (\$59 vs. \$38 per infant aged 6–9 months). Assessing the cost-effectiveness of several HIV testing as well as establishing a specific threshold of positivity for routine testing of HIV dignosis will be critical in AIDS control [235, 236]. Despite the global effort to control the AIDS pandemic, human immunodeficiency virus (HIV) infection continues to spread relatively unabated in many parts of the world. As the AIDS epidemic continues is necessary establish new strategies of prevention, treatment and molecular diagnostics assays to discriminate "window phase" infections from those that are serologically positive. There is opportunity to implement and evaluate the incremental diagnostic usefulness of new test modalities that are based on sophisticated molecular diagnostic technologies and that can be performed in settings where laboratory infrastructure is minimal [237]. Human immunodefi‐ ciency virus type 1 (HIV-1) is a highly diverse virus, a global scale, and within individual HIV-1 infected subjects [238]. The genetic variants constituting the viral population are called haplotypes, and these haplotypes form a viral quasispecies [239]. This viral diversity is highly relevant on pathogenesis, drug resistance, and vaccine development. Currently, virus popu‐ lations can be studied much faster using next-generation sequencing (NGS) platforms. NGS is a valuable tool for the detection and quantification of HIV-1 variants *in vivo* [240, 241]. As Venet exhibited in 2004, a major evolution in the near future will be the generalization of NAT for the diagnosis of viral etiology in patients, mostly with respiratory, CNS or hepatic diseases. Major technical improvements have been made to avoid obstacles that still limit this general‐ ization, related to genetic variability of viruses, multiplex detection or contamination risk [242]. Real-time amplification has allowed the development of new NAT platforms and the intro‐ duction of others techniques as NGS than contributes with data that support to global diagnostic [243].

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In this chapter was presented the some methods applied to diagnosis and monitoring of an infectious disease of viral etiology with global incidence, such as hepatitis, mumps or influenza that have been authorized by WHO and CDC. In addition, new applications of molecular techniques that facilitate fast identification of the etiology of an infectious disease were presented.
