In signal amplification assays, the signal is directly proportional to the amount of the target sequence present in the clinical specimen, reducing false-positive results due to cross contamination; also, the development of quantitative assays is more reliable.

These techniques use enzyme-mediated processes, in which the enzymes synthesize several copies of target nucleic acid. The amplification products are detected by two oligonucleotide primers that bind to complementary sequences. The final result is the production of millions of copies of the targeted sequence. There is possibility of contamination, so false positive results must be reduced through special laboratory design, practices and workflow.

These methods differ from those that use target amplification in which the amplification products contain only a sequence present in the initial probes. Some examples of probe amplification methods are ligase chain reaction [23Wu DY, Wallace RB. The ligation amplification reaction (LAR) - amplification of specific DNA sequences using sequential rounds of template-dependent ligation Genomics 1989; 4: 560-9.], cycling probe technology [24Suzuki Y, Saito R, Sato I, et al. Identification of oseltamivir resistance among pandemic and seasonal influenza A (H1N1) viruses by a His 275Tyr genotyping assay using the cycling probe method J Clin Microbiol 2011; 49: 125-30.] and cleavase-invader technology [25Lyamichev V, Mast A, Hall JG, et al. Polymorphism identification and quantitative detection from genomic DNA by invasive cleavage of oligonucleotide probes Nat Biotechnol 1999; 17: 292-6.].

A DNA array (or DNA chip) is a collection of spots attached to a solid support where each spot contains one or more single-stranded DNA oligonucleotide fragment [26Clinical and Laboratory Standards Institute. D iagnostic nucleic acid microarrays; proposed guideline CLSI document MMP12-P 2005.]. High density arrays, that permit attaching hundred or thousands of oligonucleotides, are referred to as microarrays. A labeled amplification product is hybridized to the probes, and hybridization signals are mapped to several positions within the array. The pattern of hybridization can identify the sequence of PCR, if the number of probes is sufficiently large. The results of hybridization between the bound probe and labeled sequences in the sample applied and tested are revealed by scanning or imaging the array surface. Confocal microscopy is used to scan the chip, detecting fluorescent signals that reveal hybridization at precise locations on the chip. As many DNA sequences can be present on a slide, it is possible for microarray analysis to test for multiple viruses simultaneously.

The first application in diagnostic virology has been for rapid sequencing to detect HIV mutations associated with resistance to antiretroviral drugs [27Kozal MJ, Shah N, Shen N, et al. Extensive polymorphisms observed in HIV-1 clade B protease gene using high-density oligonucleotide arrays Nat Med 1996; 2: 753-9.]. Since then, some research groups have developed microarrays that detect several viruses such as respiratory viruses [28Coiras MT, López-Huertas MR, López-Campos G, Aguilar JC, Pérez-Breña P. Oligonucleotide array for simultaneous detection of respiratory viruses using a reverse-line blot hybridization assay J Med Virol 2005; 76: 256-64.], hepatitis C virus [29Xu X, Li YM, Ji H, Hou CZ, Cheng YB, Ma FP. Changes of ECM and CAM gene expression profile in the cirrhotic liver after HCV infection: analysis by cDNA expression array World J Gastroenterol 2005; 14: 2184-7.] and virus causing CNS infection [30Leveque N, Van Haecke A, Renois F, Boutolleau D, Talmud D, Andreoletti L. Rapid virological diagnosis of central nervous system infectionsby use of a multiplex reverse transcription-PCR DNA microarray J Clin Microbiol 2011; 49: 3874-9.].

Microsphere-based suspension array technologies, such as the Luminex ^® xMAP^TM system, offer a platform for nucleic acid detection that have some advantages including rapid data acquisition, excellent sensitivity and specificity and multiplexed analysis capability [31Dunbar SA. Applications of Luminex ® xMAPTM technology for rapid, high-throughput multiplexed nucleic acid detection Clin Chim Acta 2006; 363: 71-82.]. As compared to planar microarrays, suspension arrays have the advantages of ease of use, low cost, statistical superiority, faster hybridization kinetics and more flexibility in array preparation [31Dunbar SA. Applications of Luminex ® xMAPTM technology for rapid, high-throughput multiplexed nucleic acid detection Clin Chim Acta 2006; 363: 71-82.].

This system incorporate microspheres dyed with two spectrally different fluorochromes. An array is created consisting of 100 distinct microsphere sets with specific spectrum. A third fluorochrome quantifies the biomolecular interaction that has occurred at the microsphere surface. Microspheres pass through separate lasers in the Luminex analyzer. High speed digital signal processing classifies the microsphere based on its spectral signal and quantifies the reaction on the surface. Thousands of microspheres are analyzed per second resulting in an analysis system capable of analyzing and reporting a lot of different reactions. The assay can be used as direct DNA hybridization, competitive DNA hybridization and solution-based chemistries with microsphere capture.

A multiplexed assay for detection and quantitation of viral nucleic acids using this system has been developed for HIV, HCV and HSV with high specific results [32Smith PL, WalkerPeach CR, Fulton RJ, DuBois DB. A rapid, sensitive, multiplexed assay for detection of viral nucleic acids using the FlowMetrix system Clin Chem 1998; 44: 2054-60.].

This method has improved classical PCR in its reaction simplicity, accuracy and higher amplification efficiency. The procedure is very rapid, and the amplification can be completed in less than 1 hour. The main advantage of LAMP is that it does not require thermal cyclers, and the amplification can be carried out with a water bath or heating block.

LAMP is a one-step amplification reaction that proceeds at isothermal conditions. The chemistry of LAMP amplification is based on the principle of strand displacement reaction, described previously [34Notomi T, Okayama H, Masubuchi H, et al. Loop-mediated isothermal amplification of DNA Nucleic Acids Res 2000; 28: E63.]. The mechanism consists in three steps: an initial non-cycling step, a cyclic amplification step and an elongation step. The addition of reverse transcriptase makes it possible to amplify cDNA from RNA (RT-LAMP).

LAMP method has been already used for emerging human viral pathogens such as Dengue and SARS viruses [35Parida M, Horioke K, Ishida H, et al. Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplificaton assay J Clin Microbiol 2005; 43: 2895-903., 36Hong TC, Mai QL, Cuong DV, et al. Development and evaluation of a novel loop mediated isothermal amplification (LAMP) method for rapid detection of SARS Corona virus J Clin Microbiol 2004; 42: 1956-61.]. Also, RT-LAMP assays have been developed for influenza A and B viruses’ detection [37Ito M, Watanabe M, Nakagawa N, Ihara T, Okuno Y. Rapid detection and typing of influenza A and B by loop-mediated isothermal amplification: comparison with immunochromatography and virus isolation J Virol Methods 2006; 135: 272-5.], as well as for CMV, HSV, VZV, BK virus and HPV [38Okamoto S, Yoshikawa T, Ihira M, et al. Rapid detection of Varicella-zoster virus infection by a loop-mediated isothermal amplification method J Med Virol 2004; 74: 677-82.-42Hagiwara M, Sasaki H, Matsuo K, Honda M, Kawase M, Nakagawa H. Loop-mediated isothermal amplification method for detection of human papillomavirus type 6, 11, 16 and 18 J Med Virol 2007; 79: 605-15.].

HDA is an isothermal amplification method similar to DNA replication in vivo by using a DNA helicase to separate two complementary DNA strands (dsDNA) and further extension by a DNA polymerase. The initial heat denaturation and subsequent thermocycling are not necessary, and the entire HDA reaction can be performed at a single uniform temperature. This technique provides a useful tool to amplify DNA in vitro under isothermal conditions.

HDA technique has been developed to detect several viruses in different clinical samples such as HIV-1 in human plasma [43Tang W, Chow WHA, Li Y, Kong H, Tang Y, Lemieux B. Nucleic acid assay system for tier II labs and moderately complex clinics to detect HIV in low-resource settings J Infect Dis 2010; 201: S46-51.] and HSV types 1 and 2 from genital lesions [44Kim HJ, Tong Y, Tang W, et al. A rapid and single isothermal nucleic acid amplification test for detection of herpes simplex virus types 1 and 2 J Clin Virol 2011; 50: 26-30.].

Laboratory workers must be trained in both the pre-analytical (specimen extraction and processing) and the analytical procedures. Professionals that work in this kind of laboratory should have a correct training or experience in molecular methods and also should have theoretical knowledge of molecular virology. The majority of manufacturers provide overview presentations on molecular biology as well as technical information on their specific testing platform. The Director of the Virology Laboratory should provide individualized training in molecular virology for all laboratory workers for success in performing correct molecular testing. It is very important to keep special attention to maintain strict adherence to standard operating procedures (SOP) and avoid the samples contamination using aseptic techniques.

The laboratory must have available detailed SOP, training materials and checklists for each technique performed in the laboratory. Respect to this fact, it would be very important to have a technical expert to provide a reference person in order to apply this methodology in the clinical laboratory. Laboratory-developed tests require that the technical resources to resolve problems related to the assay are available within the laboratory.

In order to minimize or decrease the risk of specimen contamination, it is necessary a physical separation of processes as well as to have reagents and equipment for use only in the molecular laboratory. Each laboratory should define their work areas but, in general, four different work areas are recommended: a reagent preparation area to prepare PCR master mix, a sample processing area where different procedures are performed (like nucleic acid extraction), a target loading area where the specimen is added to the PCR master mix and an amplification area where thermocycling and probe detection is performed.

The reagent preparation area should be kept free of all specimens and DNA/RNA extracts. The number of tubes that should be simultaneously opened must be minimizing in order to avoid cross-contamination between different samples. An important issue is that the different devices used in PCR such as pipettes, tubes, reagents should be dedicated exclusively to each working area. Reagents should be prepared and aliquoted into single use or small volume.

All working surfaces should be cleaned before and after each use with a reagent that eliminates nucleic acid. The manufacturer´s recommendations must be followed for cleaning of instruments, processing blocks and other instrument surfaces and parts.

Gloves should be changed frequently at least before beginning each procedure and must always be changed if moving from one to another work area.

After selection and successful introduction of a molecular testing platform into the virology laboratory, work flow should be implemented at the same time that this technology is being introduced. The main factors to establish an effective work flow are determined by the arrival times of specimens, number of samples of each test, clinical urgency for the results and laboratory functionality. Each laboratory should establish its own work flow depending of each technique and each viral determination, so this fact should be individualized.

CMV infection can have several clinical presentations such as non-specific viral syndrome, ocular and congenital disease. Infection can also occur in the CNS, and in the majority of them the clinical presentation is in the form of encephalitis, but also as myelitis, radiculomyelopathy and mononeuritis multiplex. Studies suggest that the detection of CMV DNA in the cerebrospinal fluid (CSF) is highly sensitive and specific for CMV neurologic disease [45Flood J, Drew WL, Miner R, et al. Diagnosis of cytomegalovirus (CMV) polyradiculopathy and documentation of in vivo anti-CMV activity in cerebrospinal fluid by using branched DNA signal amplification and antigen assays J Infect Dis 1997; 176: 348-52.]. CMV DNA could be detected by conventional PCR in the CSF of HIV patients [46Minjolle S, Arvieux C, Gautier AL, et al. Detection of herpesvirus genomes by polymerase chain reaction in cerebrospinal fluid and clinical findings J Clin Virol 2002; 25(Suppl 1 ): S59-70.] but is rarely detected in HIV-infected patients without clinical neurological disease. CMV viral load testing using PCR techniques (including real-time PCR) or hybrid capture assays can detect and quantify CMV DNA or DNA-RNA hybrids in clinical specimens, including the CSF [47Aberle SW, Puchhammer-Stöckl E. Diagnosis of herpesvirus infections of the central nervous system J Clin Virol 2002; 25(Suppl 1): S79-85.]. CMV viral load assays can be performed quickly, but it is important to use the same assay while monitoring an individual patient. Interpretation of the results of the viral load is sometimes problematic and unclear because CMV viral load have not been standardized, so it is not possible to define cutoff values.

Herpes simplex virus (HSV) is a cause of a wide spectrum of clinical manifestations such as CNS, genital and dermal diseases. HSV is the most common cause of non-epidemic sporadic acute focal CNS disease (mainly encephalitis). HSV can also cause aseptic meningitis, usually a self-limited disease that resolves without specific therapy. Because of this, there is a need for a rapid and accurate diagnostic test for HSV CNS diseases, so CSF PCR testing has been evaluated as a diagnostic test for HSV encephalitis and meningitis, being a rapid and a very high sensitivity and specificity diagnostic tool [48Lakeman FD, Whitley RJ. Diagnosis of herpes simplex encephalitis: application of polymerase chain reaction to cerebrospinal fluid from brain-biopsied patients and correlation with disease J Infect Dis 1995; 171: 857-63.]. However, because neither the sensitivity nor specificity is 100%, HSV PCR results should be interpreted with caution. False-negative HSV PCR results could be possible and false-positive HSV PCR results can be also possible due to eventual contamination. Even so, currently it has been established that molecular amplification of HSV DNA is the new gold standard for the laboratory diagnosis of these infections.

PCR is positive early in the course of the illness (within the first 24 hours) and remains positive during the first week of therapy [49Wildemann B, Ehrhart K, Storch-Hagenlocher B, et al. Quantitation of herpes simplex virus type 1 DNA in cells of cerebrospinal fluid of patients with herpes simplex virus encephalitis Neurology 1997; 48: 1341-46.]. In some cases, viral genomes persist in the CSF for two weeks or longer after the onset of antiviral therapy [49Wildemann B, Ehrhart K, Storch-Hagenlocher B, et al. Quantitation of herpes simplex virus type 1 DNA in cells of cerebrospinal fluid of patients with herpes simplex virus encephalitis Neurology 1997; 48: 1341-46.].

A real-time PCR assay is being used for the diagnosis of CMV, HSV-1 and HSV-2, EBV and VZV from CSF specimens [50Stöcher M, Holzl G, Stekel H, Berg J. Automated detection of five human herpes virus DNAs by a set of Light Cycler PCRs complemented with a single multiple internal control J Clin Virol 2004; 29: 171-8.]. Compared to conventional PCR, these real-time assays are rapid, simple and convenient for testing for herpesviruses DNA in the routine laboratory. The similarity of clinical features of these viruses is a reason for include several targets for CSF testing rather than for a single unique sequence of one virus.

With respect to dermal and genital disease caused by HSV, real-time HSV PCR assays have emerged as a more sensitive method to confirm HSV infection in clinical specimen obtained from genital ulcers and muco-cutaneous lesions [51Smith TF, Uhl JR, Espy MJ, et al. Development, implementation, and trend analysis of real-time PCR tests for the clinical microbiology laboratory Clin Microbiol Newsl 2004; 26: 145-54.]. Conventional PCR was not adapted for the detection of HSV in dermal or genital sources, because cell culture or direct staining techniques were relatively more sensitive for detecting HSV in these specimens. The main limiting factor in introducing real-time HSV PCR as the primary diagnostic tool in the laboratory is the cost of this assay. Moreover, the lack of uniform validation of the PCR assay as a diagnostic method for detecting HSV in clinical specimens other than cerebrospinal fluid has limited its availability in some laboratories.

VZV is a cause of CNS disease such as encephalitis, myelitis and acute meningitis. In a retrospective study, VZV DNA was detected from 5% of CSF specimens [47Aberle SW, Puchhammer-Stöckl E. Diagnosis of herpesvirus infections of the central nervous system J Clin Virol 2002; 25(Suppl 1): S79-85.]. Real-time PCR assay provide rapid and sensitive confirmation of VZV from clinical specimens obtained from several samples such as exudate from skin lesions, broncho-alveolar lavage and CSF [52Stránská R, Schuurman R, de Vos M, van Loon AM. Routine use of a highly automated and internally controlled real-time PCR assay for the diagnosis of herpes simplex and varicella-zoster virus infections J Clin Virol 2004; 30: 39-44., 53Schmutzhard J, Merete Riedel H, Zweygberg WB, Grillner L. Detection of herpes simplex virus type 1, herpes simplex virus type 2 and varicella-zoster virus in skin lesions. Comparison of real-time PCR, nested PCR and virus isolation J Clin Virol 2004; 29: 120-6.]. Several studies comparing different assays against real-time PCR have demonstrated that VZV DNA was detected in more samples that the rest of techniques [53Schmutzhard J, Merete Riedel H, Zweygberg WB, Grillner L. Detection of herpes simplex virus type 1, herpes simplex virus type 2 and varicella-zoster virus in skin lesions. Comparison of real-time PCR, nested PCR and virus isolation J Clin Virol 2004; 29: 120-6., 54Harbecke R, Oxman MN, Arnold BA, et al. A real-time PCR assay to identify and discriminate among wild-type and vaccine strains of varicella-zoster virus and herpes simplex virus in clinical specimens, and comparison with the clinical diagnoses J Med Virol 2009; 81: 1310-22.]. Moreover, PCR-based testing was highly specific because no cross-reactivity was identified when tested against several other viruses. PCR testing is also useful for other indications such as the diagnosis of VZV infection in a patient with vaccine-modified infection [55Leung J, Harpaz R, Baughman AL, et al. Evaluation of laboratory methods for diagnosis of varicella Clin Infect Dis 2010; 51: 23-32.], as well as testing of serum or blood might also be helpful in the transplant patient who has visceral disease prior to the appearance of cutaneous lesions [56de Jong MD, Weel JF, van Oers MH, Boom R, Wertheim-van Dillen PM. Molecular diagnosis of visceral herpes zoster Lancet 2001; 357: 2101-.].

EBV has been implicated in the development of lymphomas, above all in immunosupressed patients. Detection of EBV DNA by molecular techniques in CSF could be useful for the diagnosis of this infection. This technique has a sensitivity of 80-90% and a specificity that approaches 100% [57Yarchoan R, Jaffe ES, Little R. Diagnosing central nervous system lymphoma in the setting of AIDS: a step forward J Natl Cancer Inst 1998; 90: 346-7.]. Detection of EBV DNA might also be useful in patients in whom a brain biopsy is not possible to do. On the other hand, detection of EBV DNA in CSF also provides a marker to monitor the response to treatment for CNS lymphomas. EBV DNA can be detected by conventional PCR [58Broccolo F, Iuliano R, Careddu AM, et al. Detection of lymphotropic herpesvirus DNA by polymerase chain reaction in cerebrospinal fluid of AIDS patients with neurological disease Acta Virol 2000; 44: 137-43.], by in situ hybridization [59Phan TG, O’Neill BP, Kurtin PJ. Posttransplant primary CNS lymphoma Neurooncology 2000; 2: 229-38.] and also more recently has been introduced real-time PCR assays to detect EBV DNA from CNS lymphomas [60Bossolasco S, Cinque P, Ponzoni M, et al. Epstein-Barr virus DNA load in cerebrospinal fluid and plasma of patients with AIDS-related lymphoma J Neurovirol 2002; 8: 432-8.].

Infection with parvovirus B19 might cause asymptomatic infection or a wide spectrum of disease (erythema infectiosum in children with arthropathy, severe anemia and systemic affectation) as well as hydrops fetalis, congenital anemia and abortion if the infection is produced in pregnant women.

Although parvovirus B19 infection is diagnosed by serologically detecting IgM and IgG class antibodies with ELISA assays, the main application of DNA detection by PCR is the control of transmission of the virus present in blood [61Weimer T, Streichert S, Watson C, Gröner A. High-titer screening PCR: a successful strategy for reducing the parvovirus B19 load in plasma pools for fractionation Transfusion 2001; 41: 1500-4.]. Detecting B19 DNA using molecular tests is now being used in many clinical laboratories, and these techniques are much more sensitive than antigen-based detection systems. Most of these techniques detect genotype 1 DNA but not genotypes 2 or 3.

Some real-time PCR assays such as LightCycler parvovirus B19 quantitative assay (Roche Diagnostics, Indianapolis, IN) and ABI TaqMan (Applied Biosystems) have been developed for detecting B19 nucleic acids in association with infection during pregnancy or assessing the prevalence of the virus DNA in blood products [62Harder TC, Hufnagel M, Zhan K, et al. New Light Cycler PCR for rapid and sensitive quantification of parvovirus B19 DNA guides therapeutic decision-making in relapsing infections J Clin Microbiol 2001; 39: 4413-9., 63Knöll A, Louwen F, Kochanowski B, Plentz, et al. Parvovirus B19 infection in pregnancy: quantitative viral DNA analysis using a kinetic fluorescence detection system (TaqMan PCR) J Med Virol 2002; 67: 259-66.]. The LightCycler parvovirus B19 quantitative assay is highly sensitive for genotype 1 but is not suitable for detecting genotypes 2 or 3. With respect to RealArt Parvo B19 LC PCR (Qiagen, Hamburg), this assay can detect all three genotypes according some researchers, but no by others [64Hokynar K, Norja P, Laitinen H, Palomäki, et al. Detection and diferentiation of human parvovirus variants by commercial quantitative real-time PCR tests J Clin Microbiol 2004; 42: 2013-9.]. Appropriate clinical specimens for nucleic acid analysis include plasma, serum, bone marrow, placental and fetal tissues and amniotic fluid.

Both viruses were recovered in cell cultures in 1971. BK virus is associated with nephropathy, above all in renal transplant patients as well as in patients with ureteral stenosis and hematuria [65Li RM, Mannon RB, Kleiner D, et al. BK virus and SV40 co-infection in polyomavirus nephropathy Transplantation 2002; 74: 1497-504.]. On the other hand, it is well knowing the association of JC virus infection with progressive multifocal leukoencephalopathy in immunocompromised patients (such as those with AIDS) [66Du Pasquier RA, Kuroda MJ, Zheng Y, Jean-Jacques J, Letvin NL, Koralnik IJ. A prospective study demonstrates an association between JC virus specific cytotoxic T lymphocytes and the early control of progressive multifocal leukoencephalopathy Brain 2004; 127: 1970-78.].

Conventional PCR for detection of JC virus in the cerebrospinal fluid has replaced the brain biopsy for the diagnosis of presence of this virus in patients with leukoencephalopathy [67Telenti A, Aksamit AJ, Proper J Jr, Smith TF. Detection of JC virus DNA by polymerase chain reaction in patients with progressive multifocal leukoencephalopathy J Infect Dis 1990; 162: 858-61.]. This technique had sensitivity that range 70-90% and a specificity that range 90-100% before the highly active antiretroviral therapy [68Cinque P, Scarpellini P, Vago L, Linde A, Lazzarin A. Diagnosis of central nervous system complications in HIV-infected patients: cerebrospinal fluid analysis by the polymerase chain reaction AIDS 1997; 11: 1-17.]. However, at the moment, the application of this therapy recovery the immune system, so the viral replication becomes to be decreased, being JC virus PCR negative in CSF. Currently, it has been developed a qualitative real-time PCR for JC virus detection in CSF.

BK virus can be detected in urine samples by conventional PCR; also, a quantitative real-time PCR technique has been developed to monitoring BK virus DNA in renal transplant recipients [69Randhawa P, Ho A, Shapiro R, et al. Correlates of quantitative measurement of BK polyomavirus (BKV) DNA with clinical course of BKV infection in renal transplant patients J Clin Microbiol 2004; 42: 1176-80.]. Active BK virus nephropathy is associated with high quantitative levels of BK virus DNA, and resolution of nephropathy was correlated with decreased DNA virus level in urine. However, although it is a sensitive test, the presence of BK virus DNA in this kind of samples does not necessarily means a true infection because there is asymptomatic reactived infection in 10-45% of renal transplant patients. Therefore, the result should be confirmed using blood samples [70Randhawa P, Vats A, Shapiro R. Monitoring for polyomavirus BK and JC in urine: comparison of quantitative polymerase chain reaction with urine cytology Transplantation 2005; 79: 984-6.]. In this sense, research has demonstrated that renal transplant recipients with higher urine DNA levels are more likely to show detectable DNA in blood. On the other hand, a negative result does mean no association of BK virus with nephritis.

Finally, PCR from kidney biopsy specimens is not an appropriate test for primary diagnosis of BK nephropathy, because persistent but low-level target DNA in biopsy specimens can be detected in asymptomatic patients [71Schmid H, Nitschko H, Gerth J, et al. Polyomavirus DNA and RNA detection in renal allograft biopsies: results from a European multicenter study Transplantation 2005; 80: 600-4.].

Rapid laboratory diagnosis is critical for infection control, so several diagnostic tests have been developed and are available for the detection of influenza viruses. Rapid influenza diagnostic tests are less sensitive and specific than fluorescent antibody assays and RT-PCR [72Ginocchio CC, Zhang F, Manji R, et al. Evaluation of multiple test methods for the detection of the novel 2009 influenza A (H1N1) during the New York City outbreak J Clin Virol 2009; 45: 191-5.]. RT-PCR is the preferred diagnostic assay for influenza virus. These tests are the most sensitive and specific and can differentiate between influenza types (A or B) and subtypes [72Ginocchio CC, Zhang F, Manji R, et al. Evaluation of multiple test methods for the detection of the novel 2009 influenza A (H1N1) during the New York City outbreak J Clin Virol 2009; 45: 191-5.]. The main problem of this technique is that it could not be available in all laboratories, so there is a need of other tests in these settings.

Real-time PCR is much more sensitive than other methods of detection and is available for detecting influenza virus [73Boivin G, Côté S, Déry P De, Serres G, Bergeron MG. Multiplex real-time PCR assay for detection of influenza and human respiratory syncytial viruses J Clin Microbiol 2004; 42: 45-51.] but is more expensive.

With respect to parainfluenza viruses, types 1 to 4 have been associated with bronchiolitis, croup and pneumonia in children, but also in elderly and immunocompromised patients. PCR is an adequate technique for the detection of parainfluenza virus, above all in immunocompromised patients [74Mahony JB. Detection of respiratory viruses by molecular methods Clin Microbiol Rev 2008; 21: 716-47.]. PCR has a sensitivity of 100% and specificity that range 95-98% if compared with culture method [75Liolios L, Jenney A, Spelman D, Kotsimbos T, Catton M, Wesselingh S. Comparison of a multiplex reverse transcription-PCR-enzyme hybridization assay with conventional viral culture and immunofluorescence techniques for the detection of seven viral respiratory pathogens J Clin Microbiol 2001; 39: 2779-83.]. Multiplex PCR assays can differentiate between a wide variety of respiratory pathogens [75Liolios L, Jenney A, Spelman D, Kotsimbos T, Catton M, Wesselingh S. Comparison of a multiplex reverse transcription-PCR-enzyme hybridization assay with conventional viral culture and immunofluorescence techniques for the detection of seven viral respiratory pathogens J Clin Microbiol 2001; 39: 2779-83.]. Also, a rapid and sensitive multiplex real-time PCR assay for detection of four serotypes of parainfluenza viruses has been developed [76Templeton KE, Scheltinga SA, Beersma MF, Kroes AC, Claas EC. Rapid and sensitive method using multiplex real-time PCR for diagnosis of infections by influenza A and influenza B viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3 and 4 J Clin Microbiol 2004; 42: 1564-69.].

For more information, it can see the article entitled “Laboratory detection of respiratory viruses by automated techniques”.

PCR is a specific and sensitive assay for detecting adenovirus DNA from a wide variety of clinical specimens, but results must be always interpreted in the context of the clinical findings of adenovirus disease. Quantitative real-time PCR is being now used for the evaluation of adenovirus infections in immunocompromised patients [77Claas EC, Schilham MW, de Brouwer CS, et al. Internally controlled real-time PCR monitoring of adenovirus DNA load in serum or plasma of transplant recipients J Clin Microbiol 2005; 43: 1738-44.]. Moreover, quantification of adenovirus DNA could be useful for assessing response to antiviral therapy [78Lankester AC, van Tol MJ, Claas EC, Vossen JM, Kroes AC. Quantification of adenovirus DNA in plasma for management of infection in stem cell graft recipients Clin Infect Dis 2002; 34: 864-7.]. Real-time PCR assays mainly are used to detect adenovirus type 4 (subgroup E) that can cause respiratory, ocular and other infections. The sensitivity of real time PCR against conventional PCR is greater for the detection of adenovirus DNA [79Heim A, Ebnet C, Harste G, Pring-Akerblom P. Rapid and quantitative detection of human adenovirus DNA by real-time PCR J Med Virol 2003; 70: 228-39.].

Enteroviruses such as echoviruses, parechoviruses (echoviruses 22 and 23) and coxsackieviruses A and B could produce several diseases such as respiratory tract infections, aseptic meningitis, myocarditis and neonatal systemic enteroviral disease. Cell culture has a low sensitivity, so molecular techniques like RT-PCR have been developed for the diagnosis of this kind of viruses [80Rotbart HA, Robero JR. Laboratory diagnosis of enteroviral infections In: Rotbart HA, Ed. Human enterovirus infections. Washington DC: ASM Press 1995; pp. 401-18.]. Moreover, RT-PCR has higher sensitivity than cell culture for detecting enteroviruses in the CSF [81Pozo F, Casas I, Tenorio A, Trallero G, Echevarria JM. Evaluation of a commercially available reverse transcription-PCR assay for diagnosis of enteroviral infection in archival and prospectively collected cerebrospinal fluid specimens J Clin Microbiol 1998; 36: 1741-5.].

A rapid and sensitive detection of enterovirus in CSF could be performed by the introduction of real-time PCR techniques in the laboratory. Real-time PCR assays amplify conserved target nucleic acid sequences of the virus. Sensitivity for detecting enterovirus is similar between conventional PCR and real-time PCR but the last one is less labor intensive and easier to implement in the clinical laboratory [82Rabenau HF, Clarici AM, Muhlbauer G, et al. Rapid detection of enterovirus infection by automated RNA extraction and real-time fluorescence PCR J Clin Virol 2002; 25: 155-64.].

Molecular techniques can be very useful for the diagnosis of viral hepatitis infections such as hepatitis A, B, C, D and E in cases in which serological assays are no conclusive. However, currently the main application of nucleic acid test in the management of these viruses, above all hepatitis B and C, is the detection of serum or plasma viral load of these viruses for monitoring therapeutic responses of infected patients. Assays to quantify hepatitis B and C virus load in liver tissue have also been described [83White PA, Pan Y, Freeman AJ, Ffrench RA, Lloyd AR, Rawlinson WD. Quantification of hepatitis C virus in human liver and serum samples by using LightCycler reverse transcriptase PCR J Clin Microbiol 2002; 40: 4346-8., 84Zanella I, Rossini A, Domenighini D, Albertini A, Cariani E. Real-time quantitation of hepatitis B virus (HBV) DNA in tumorous and surrounding tissue from patients with hepatocellular carcinoma J Med Virol 2002; 68: 494-9.]. HBV DNA detection and HBV DNA viral load are also essential to explore a viral reactivation. Real-time PCR quantification assays are widely used because of their sensitivity, specificity, accuracy, broad dynamic range and positive predictive values. The World Health Organization (WHO) has defined international standards [85Saldanha J, Gerlich W, Lelie N, Dawson P, Heermann K, Heath A. An international collaborative study to establish a World Health Organization international standard for hepatitis B virus DNA nucleic acid amplification techniques Vox Sang 2001; 80: 63-71.]. However, because of these techniques do not use the same HBV primers, follow up of patients should be carried out with the same technical assay in order to compare viral DNA load evolution. With respect to the HBV genotypes, there is currently a need to perform it due to several factors such as the prediction of clinical outcomes and the association with response to interferon treatment [86Lin CL, Kao JH. The clinical implications of hepatitis B virus genotype: recent advances J Gastroenterol Hepatol 2011; 26(Suppl 1 ): 123-30.]. Genotyping of chronic HBV infections can help practicing physicians identify those at risk of disease progression and determine optimal anti-viral therapy. Methods to determine the viral genotypes are based on hybridization and sequencing, and genotype affiliation rely on phylogenetic analyses.

With respect to HCV, molecular diagnostic assays represent an essential approach in the management of HCV patients. Qualitative and quantitative HCV molecular assays are used for the diagnosis of acute and chronic HCV infections, viral genotyping, viral-load determination, treatment, monitoring and prognosis. RT-PCR, transcription-mediated amplification and branched DNA amplification are commonly employed for detection of HCV RNA. Recently, new HCV molecular assays that employ nanostructures have emerged and have been proposed as suitable, without loss specificity and sensitivity [87Al Olaby RR, Azzazy HM. Hepatitis C virus RNA assays: current and emerging technologies and their clinical applications Expert Rev Mol Diagn 2011; 11: 53-64.].

For more information, it can see the article entitled “Introduction of automated systems for the diagnosis and quantification of Hepatitis B and Hepatitis C viruses”.

HIV-1 and HIV-2 RNA levels in the plasma of infected patients could be detected by quantitative rapid real-time PCR assays. There are some techniques for this detection that have similar sensitivity and specificity, but differ in the probe and in the amplification-detection systems used.

Quantitative assays for measurement of HIV-1 and HIV-2 proviral DNA have also been developed, as well as qualitative real-time PCR assay for detection of proviral DNA.

For more information, it can see the article entitled “Improving clinical laboratory efficiency: introduction of systems for the diagnosis and monitoring of HIV infection”.

Molecular methods to detect HPV DNA in clinical specimens have been introduced in clinical practice, and the majority of protocols for detection of this infection are currently based in the study of HPV DNA. HPV DNA detection could be carried out by several methods such as type-specific PCR, PCR with general primers and liquid hybridization (hybrid capture). Several studies have shown the utility of HPV DNA testing for management of women with abnormal Pap smears [88Manos MM, Kinney WK, Hurley IB, et al. Identifying women with cervical neoplasia: Using human papillomavirus DNA testing for equivocal Papanicolaou results JAMA 1999; 281: 1605-0.]. Identifying women at high risk by testing for HPV DNA could avoid unnecessary colposcopy procedures.

For more information, it can see the article entitled “Human papillomavirus (HPV) genotyping: automation and application in routine laboratory testing”.