Intracellular immunoglobulin A (icIgA) in protective immunity and vaccines.

Virus neutralization at respiratory mucosal surfaces is important in the prevention of infection. Mucosal immunity is mediated mainly by extracellular secretory immunoglobulin A (sIgA) and its role has been well studied. However, the protective role of intracellular specific IgA (icIgA) is less well defined. Initially, in vitro studies using epithelial cell lines with surface expressed polymeric immunoglobulin receptor (pIgR) in transwell culture chambers have shown that icIgA can neutralize influenza, parainfluenza, HIV, rotavirus and measles viruses. This effect appears to involve an interaction between polymeric immunoglobulin A (pIgA) and viral particles within an intracellular compartment, since IgA is transported across the polarized cell. Co-localization of specific icIgA with influenza virus in patients' (virus culture positive) respiratory epithelial cells using well-characterized antisera was initially reported in 2018. This review provides a summary of in vitro studies with icIgA on colocalization and neutralization of the above five viruses. Two other highly significant respiratory infectious agents with severe global impacts viz. SARS-2 virus (CoViD pandemic) and the intracellular bacterium-Mycobacterium tuberculosis-are discussed. Further studies will provide more detailed understanding of the mechanisms and kinetics of icIgA neutralization in relation to viral entry and early replication steps with a specific focus on mucosal infections. This will inform the design of more effective vaccines against infectious agents transmitted via the mucosal route.

Coxiella burnetii dormancy in a fatal ten-year multisystem dysfunctional illness: case report.

BACKGROUND: In a previous study of a Q fever outbreak in Birmingham, our group identified a non-infective complex of Coxiella burnetii (C.b.) antigens able to survive in the host and provoked aberrant humoral and cell-mediated immunity responses. The study led to recognition of a possible pathogenic link between C.b. infection and subsequent long-term post Q fever fatigue syndrome (QFS). This report presents an unusually severe case of C.b. antigen and DNA detection in post-mortem specimens from a patient with QFS. CASE PRESENTATION: We report a 19-year old female patient who became ill with an acute unexplained febrile encephalitis-like illness, followed by increasingly severe multisystem dysfunction and death 10 years later. During life, extensive clinical and laboratory investigations from different disciplinary stand points failed to deliver a definitive identification of a cause. Given the history of susceptibility to infection from birth, acute fever and the diagnosis of "post viral syndrome", tests for infective agents were done starting with C.b. and Legionella pneumophila. The patient had previously visited farms a number of times. Comprehensive neuropathological assessment at the time of autopsy had not revealed gross or microscopic abnormalities. The aim was to extend detailed studies with the post-mortem samples and identify possible factors driving severe disturbance of homeostasis and organ dysfunction exhibited by the course of the patient's ten-year illness. Immunohistochemistry for C.b. antigen and PCR for DNA were tested on paraffin embedded blocks of autopsy tissues from brain, spleen, liver, lymph nodes (LN), bone marrow (BM), heart and lung. Standard H&E staining of brain sections was unrevealing. Immuno-staining analysis for astrocyte cytoskeleton proteins using glial fibrillary acidic protein (GFAP) antibodies showed a reactive morphology. Coxiella antigens were demonstrated in GFAP immuno-positive grey and white matter astrocytes, spleen, liver, heart, BM and LN. PCR analysis (COM1/IS1111 genes) confirmed the presence of C.b. DNA in heart, lung, spleen, liver & LN, but not in brain or BM. CONCLUSION: The study revealed the persistence of C. b. cell components in various organs, including astrocytes of the brain, in a post-infection QFS. The possible mechanisms and molecular adaptations for this alternative C.b. life style are discussed.

Microsphere-Enabled Micropillar Array for Whispering Gallery Mode Virus Detection.

Rapid detection of pathogens and analytes at the point of care offers an opportunity for prompt patient management and public health control. This paper reports an open microfluidic platform coupled with active whispering gallery mode (WGM) microsphere resonators for the rapid detection of influenza viruses. The WGM microsphere resonators, precoated with influenza A polyclonal antibodies, are mechanically trapped in the open micropillar array, where the evaporation-driven flow continuously transports a small volume (∼µL) of sample to the resonators without auxiliaries. Selective chemical modification of the pillar array changes surface wettability and flow pattern, which enhances the detection sensitivity of the WGM resonator-based virus sensor. The optofluidic sensing platform is able to specifically detect influenza A viruses within 15 min using a few microliters of sample and displays a linear response to different virus concentrations.

Preclinical efficacy studies of influenza A haemagglutinin precursor cleavage loop peptides as a potential vaccine.

A universal influenza vaccine that does not require annual reformulation would have clear advantages over the currently approved seasonal vaccine. In this study, we combined the mucosal adjuvant alpha-galactosylceramide (αGalCer) and peptides designed across the highly conserved influenza precursor haemagglutinin (HA(0)) cleavage loop as a vaccine. Peptides designed across the HA(0) of influenza A/H3N2 viruses, delivered to mice via the intranasal route with αGalCer as an adjuvant, provided 100 % protection following H3N2 virus challenge. Similarly, intranasal inoculation of peptides across the HA(0) of influenza A/H5N1 with αGalCer completely protected mice against heterotypic challenge with H3N2 virus. Our data suggest that these peptide vaccines effectively inhibited subsequent influenza A/H3N2 virus replication. In contrast, only 20 % of mice vaccinated with αGalCer-adjuvanted peptides spanning the HA(0) of H5N1 survived homologous viral challenge, possibly because the HA(0) of this virus subtype is cleaved by intracellular furin-like enzymes. Results of these studies demonstrated that HA(0) peptides adjuvanted with αGalCer have the potential to form the basis of a synthetic, intranasal influenza vaccine.

Cytotoxic T cells are the predominant players providing cross-protective immunity induced by {gamma}-irradiated influenza A viruses.

We previously demonstrated that a single dose of nonadjuvanted intranasal gamma-irradiated influenza A virus can provide robust protection in mice against both homologous and heterosubtypic challenges, including challenge with an H5N1 avian virus strain. We investigated the mechanism behind the observed cross-protection to define which arms of the adaptive immune response are involved in mediating this protection. Studies with gene knockout mice showed the cross-protective immunity to be mediated mainly by T cells and to be dependent on the cytolytic effector molecule perforin. Adoptive transfer of memory T cells from immunized mice, but not of memory B cells, protected naïve recipients against lethal heterosubtypic influenza virus challenge. Furthermore, gamma-irradiated influenza viruses induced cross-reactive Tc-cell responses but not cross-neutralizing or cross-protective antibodies. In addition, histological analysis showed reduced lung inflammation in vaccinated mice compared to that in unvaccinated controls following heterosubtypic challenge. This reduced inflammation was associated with enhanced early recruitment of T cells, both CD4(+) and CD8(+), and with early influenza virus-specific cytotoxic T-cell responses. Therefore, cross-protective immunity induced by vaccination with gamma-irradiated influenza A virus is mediated mainly by Tc-cell responses.

Sequencing and analysis of globally obtained human parainfluenza viruses 1 and 3 genomes.

Human Parainfluenza viruses (HPIV) type 1 and 3 are important causes of respiratory tract infections in young children globally. HPIV infections do not confer complete protective immunity so reinfections occur throughout life. Since no effective vaccine is available for the two virus subtypes, comprehensive understanding of HPIV-1 and HPIV-3 genetic and epidemic features is important for diagnosis, prevention, and treatment of HPIV-1 and HPIV-3 infections. Relatively few whole genome sequences are available for both HPIV-1 and HPIV-3 viruses, so our study sought to provide whole genome sequences from multiple countries to further the understanding of the global diversity of HPIV at a whole-genome level. We collected HPIV-1 and HPIV-3 samples and isolates from Argentina, Australia, France, Mexico, South Africa, Switzerland, and USA from the years 2003-2011 and sequenced the genomes of 40 HPIV-1 and 75 HPIV-3 viruses with Sanger and next-generation sequencing with the Ion Torrent, Illumina, and 454 platforms. Phylogenetic analysis showed that the HPIV-1 genome is evolving at an estimated rate of 4.97 × 10-4 mutations/site/year (95% highest posterior density 4.55 × 10-4 to 5.38 × 10-4) and the HPIV-3 genome is evolving at a similar rate (3.59 × 10-4 mutations/site/year, 95% highest posterior density 3.26 × 10-4 to 3.94 × 10-4). There were multiple genetically distinct lineages of both HPIV-1 and 3 circulating on a global scale. Further surveillance and whole-genome sequencing are greatly needed to better understand the spatial dynamics of these important respiratory viruses in humans.

Mycoplasma pneumoniae DNA detection and specific antibody class response in patients from two tertiary care hospitals in tropical Sri Lanka.

PURPOSE: Respiratory tract infections are a major cause of global morbidity and mortality. Pneumonia is the ninth leading cause of mortality in Sri Lanka. Atypical pathogens cause about one-fifth of community-acquired pneumonia, while Mycoplasma pneumoniae accounts for about 50 %. This study aimed to determine the seroprevalence of M. pneumoniae respiratory tract infections in Sri Lanka while attempting to understand the relationships between the serology and PCR. METHODOLOGY: Paired sera from 418 adult patients (pneumonia, n=97; bronchitis, n=183; pharyngitis, n=138) and 87 healthy controls were studied. IgM, IgG and IgA antibodies were tested by M. pneumoniae enzyme-linked immunosorbent assay (ELISA). Positive IgM and or IgG seroconversion was considered to be seropositive. M. pneumoniae DNA were tested by PCR in age and gender-matched seropositives and seronegatives. RESULTS: M. pneumoniae IgG was in 14.4 % (14/97), 6.0 % (11/183) and 1.5 % (2/138) of pneumonia, bronchitis and pharyngitis patients, respectively, whilst IgM was in 6.2 % (6/97), 1.1 % (2/183) and 0 % (0/138), respectively. Amongst the pneumonia seropositives, 64.7 % (11/17) showed IgG alone, 17.5 % (3/17) showed IgM alone and 17.5 % (3/17) showed IgM and IgG. Amongst the bronchitis seropositives, 84.6 % (11/13) had IgG alone and 15.4 % (2/13) had IgM alone. In the pharyngitis seropositives, only IgG was detected 100 % (2/2). M. pneumoniae DNA was in 52.2 % (12/23) of seropositives and 15.4 % (4/26) of seronegatives. In pneumonia or bronchitis patients, specific DNA was in 77.8 % (7/10) and 50 % (6/12) of patients, respectively. M. pneumoniae DNA was not found in pharyngitis patients. Of the seropositive PCR-negative pneumonia patients, 66.7 % (2/3) showed IgG alone and 33.3 % (1/3)showed IgM alone. In bronchitis patients, 83.3 % (5/6) showed IgG alone and 16.7 % (1/6) showed IgM alone. Of the seronegative PCR-positive patients, 16.7 % (2/12) had pneumonia and 18.2 % (2/11) had bronchitis. CONCLUSION: The serological evidence for M. pneumoniae infection in Sri Lanka comprised the following prevalences: 17.5 % (17/97), 7.1 % (13/183) and 1.4 % (2/138) in adults with pneumonia, bronchitis or pharyngitis, respectively. M. pneumoniae DNA was in 52.2 % (12/23) of seropositives and 15.4 % (4/26) of seronegatives. IgG was predominant in PCR positives and negatives.

Colocalization of intracellular specific IgA (icIgA) with influenza virus in patients' nasopharyngeal aspirate cells.

Inhibition of viral replication by icIgA antibodies has only been observed with in vitro studies using epithelial cell lines in transwell cultures. This effect appears to involve an interaction between polymeric immunoglobulin A (pIgA) and viral particles within an intracellular compartment, since IgA is transported across polarized cells. Polyclonal guinea pig antisera against purified influenza A virus and mouse antisera prepared against Influenza A/H3N2 hemagglutinin (HA0) cleavage loop peptides, were used in confocal fluorescence microscopy to show specific staining of wild-type influenza H1N1 and H3N2 viruses in clinical specimens. The HA0 cleavage loop peptides used for intranasal immunization of mice were designed and synthesized from specific conserved regions of influenza A/H1N1 & A/H3N2 viruses. Anti-human secretory IgA antibodies were used to show co-localisation of influenza A virus and icIgA. The results showed specific immunofluorescent staining of influenza A/H3N2 (X31) (HA0 uncleaved)-infected MDCK cells and the presence of icIgA in respiratory exudate cells of infected patients. Both results confirm specific co-localisation and suggest interaction between influenza A virus and icIgA in patients' respiratory exudate cells. Importantly, antisera to the mouse anti-HA0 cleavage site were specific for wild-type virus in clinical specimens, indicating that the conserved region of HA0 was present in the uncleaved form. Similar staining and colocalization patterns between icIgA and virus were observed with polyclonal guinea pig antisera against influenza A virus. These are the first observations of co-localization of influenza A virus and intracellular IgA in clinical specimens. Role of icIgA: This report shows the co-localization of influenza A virus HA0 and icIgA antibodies in respiratory exudate cells of patients who were culture and viral RNA positive, suggesting that icIgA directed against the conserved HA0 site may have a privileged and unique opportunity to act on immature virus and thus prevent HA0 cleavage, maturation and subsequent cycles of viral replication. The precise mechanism by which icIgA mediates intracellular viral neutralization remains to be fully elucidated. SIGNIFICANCE: The above findings in clinical specimens would contribute strongly to our understanding of the mechanisms and kinetics of icIgA neutralization in relation to viral entry and early replication steps of mucosal viral infections. A rapid, objective and sensitive assay - by ex vivo enumeration of respiratory epithelial cells that have co-localized influenza virus and icIgA - would contribute to further mucosal immunity studies and inform the design of more effective vaccines against influenza and other viral infections transmitted via the mucosal route e.g. respiratory syncytial virus, rotavirus.

Real-time PCRs for detection of Trichomonas vaginalis beta-tubulin and 18S rRNA genes in female genital specimens.

Trichomonas vaginalis is the cause of one of the most common types of vaginitis, trichomoniasis. The incidence of trichomoniasis in developed countries has decreased substantially during the past decade, but high prevalence of this disease can still be found in rural and remote areas of Australia. Clinical manifestations of symptomatic women are generally non-specific, but include vaginal discharge, vaginitis and irritation. T. vaginalis infection has also been linked to the increased risk of human immunodeficiency virus transmission. Current diagnosis of T. vaginalis relies on the visualization of motile organisms in a wet-mount preparation. Culture is used mainly in reference laboratories. The latter two methods require viable organisms and would not be suitable for use where transportation of specimens can be delayed. Two real-time fluorescence resonance energy transfer (FRET) hybridization probe PCR assays were used in this study to test for T. vaginalis DNA, targeting the beta-tubulin and 18S rRNA genes. We tested 500 randomly selected female patients, in an STD setting, for T. vaginalis DNA. The FRET PCRs targeting the beta-tubulin gene and the 18S rRNA gene detected 96 % (85/89) and 100 % (89/89) , respectively, of the positive specimens (first-void urine sample or genital swabs). Wet-mount microscopy was performed on 76 of these PCR-positive specimens and showed a sensitivity of 38 % (29/76). The prevalence, by PCR, of trichomoniasis was 18 % in this study. The two real-time PCRs developed in this study, targeting different genetic regions of the organism, provide a rapid, sensitive and specific diagnosis of T. vaginalis infection.

Sequencing and analysis of globally obtained human respiratory syncytial virus A and B genomes.

BACKGROUND: Human respiratory syncytial virus (RSV) is the leading cause of respiratory tract infections in children globally, with nearly all children experiencing at least one infection by the age of two. Partial sequencing of the attachment glycoprotein gene is conducted routinely for genotyping, but relatively few whole genome sequences are available for RSV. The goal of our study was to sequence the genomes of RSV strains collected from multiple countries to further understand the global diversity of RSV at a whole-genome level. METHODS: We collected RSV samples and isolates from Mexico, Argentina, Belgium, Italy, Germany, Australia, South Africa, and the USA from the years 1998-2010. Both Sanger and next-generation sequencing with the Illumina and 454 platforms were used to sequence the whole genomes of RSV A and B. Phylogenetic analyses were performed using the Bayesian and maximum likelihood methods of phylogenetic inference. RESULTS: We sequenced the genomes of 34 RSVA and 23 RSVB viruses. Phylogenetic analysis showed that the RSVA genome evolves at an estimated rate of 6.72 × 10(-4) substitutions/site/year (95% HPD 5.61 × 10(-4) to 7.6 × 10(-4)) and for RSVB the evolutionary rate was 7.69 × 10(-4) substitutions/site/year (95% HPD 6.81 × 10(-4) to 8.62 × 10(-4)). We found multiple clades co-circulating globally for both RSV A and B. The predominant clades were GA2 and GA5 for RSVA and BA for RSVB. CONCLUSIONS: Our analyses showed that RSV circulates on a global scale with the same predominant clades of viruses being found in countries around the world. However, the distribution of clades can change rapidly as new strains emerge. We did not observe a strong spatial structure in our trees, with the same three main clades of RSV co-circulating globally, suggesting that the evolution of RSV is not strongly regionalized.

Detection and subtyping of Herpes simplex virus in clinical samples by LightCycler PCR, enzyme immunoassay and cell culture.

BACKGROUND: Prompt laboratory diagnosis of Herpes simplex virus (HSV) infection facilitates patient management and possible initiation of antiviral therapy. In our laboratory, which receives various specimen types for detection of HSV, we use enzyme immunoassay (EIA) for rapid detection and culture of this virus. The culture of HSV has traditionally been accepted as the diagnostic 'gold standard'. In this study, we compared the use of real time PCR (LightCycler) for amplification, detection and subtyping of specific DNA with our in-house developed rapid and culture tests for HSV. RESULTS: The LightCycler PCR (LC-PCR) detected and subtyped HSV in 99% (66/67) of HSV positive specimens, compared to 81% (54/67) by rapid antigen EIA or 57% (36/63) by culture. A specimen was considered positive when two or more tests yielded HSV identifications or was culture positive. Discordant results were confirmed with an in-house developed PCR-ELISA or DNA sequence analysis. The typing results obtained with the LC-PCR and by culture amplified test were completely concordant. CONCLUSIONS: This study showed that the LC-PCR provided a highly sensitive test for simultaneous detection and subtyping of HSV in a single reaction tube. In addition to increased sensitivity, the LightCycler PCR provided reduced turn-around-times (2 hours) when compared to enzyme immunoassay (4 hours) or culture (4 days).

Rapid detection and simultaneous subtype differentiation of influenza A viruses by real time PCR.

A real time RT-PCR, using the LightCycler, was developed and compared with rapid antigen enzyme immunoassay (AgEIA) and enhanced virus culture for rapid detection of influenza A viruses in stored and prospectively collected respiratory specimens. Specific hybridization probes were used for simultaneous detection and differentiation between H1N1 and H3N2 subtypes. The sensitivity of the RT-PCR for influenza A H1N1 was 120 copies and H3N2 350 copies of in vitro transcribed RNA. A specimen was considered positive for influenza A when it was culture positive or at least two methods yielded a positive test result. Using these criteria, with stored samples, the RT-PCR sensitivity, specificity, positive and negative predictive values were 82.9, 95.5, 98.9 and 52.5%, respectively. In specimens collected prospectively the RT-PCR sensitivity, specificity, positive and negative predictive values were 100, 87.9, 82.8 and 100%, respectively. There was complete concordance with subtype differentiation by hybridization probe melting temperature analysis and haemagglutination inhibition assay.

Designer antigens for elicitation of broadly neutralizing antibodies against HIV.

Broadly neutralizing antibodies (bNAbs) are a consistent protective immune correlate in human immunodeficiency virus (HIV) patients as well as in passive immunotherapy studies. The inability to elicit bNAbs is the core reason underlining the repeated failures in traditional HIV vaccine research. Rare monoclonal bNAbs against HIV, however, have been produced. The significance of producing and studying more monoclonal bNAbs against HIV is underlined by its capability of defining critical epitopes for antigen designs aimed at the development of a serum-neutralizing HIV vaccine. In this regard, traditional antigen preparations have failed. There is a need to clearly advocate the concept, and systematic study, of more sophisticated 'designer antigens' (DAGs), which carry epitopes that can lead to the elicitation of bNAbs. Using an extremely efficient cell-to-cell HIV infection model for the preparation of HIV prefusion intermediates, we have investigated a novel and systematic approach to produce (not screen for) potential bNAbs against HIV. We have established the concept and the experimental system for producing formaldehyde-fixed HIV DAGs that carry temperature-arrested prefusion intermediates. These prefusion intermediates are structures on the cell surface after viral attachment and receptor engagement but before fully functional viral entry. Using defined HIV prefusion DAGs, we have produced monoclonal antibodies (mAbs) specific to novel epitopes on HIV prefusion intermediates. These mAbs do not react with the static/native surface HIV or cellular antigens, but react with the DAGs. This is a paradigm shift from the current mainstream approach of screening elite patients' bNAbs.

The virus inoculum volume influences outcome of influenza A infection in mice.

When establishing animal models of viral respiratory infection, the optimal dose and route of delivery are critical to ensure reproducible outcomes. The mouse model for influenza infection is widely used due to the small animal size and simplicity of viral inoculation. During establishment of a mouse model of influenza A infection we observed a marked shift in morbidity when identical influenza A inoculum doses were delivered in less than 35 µL. We show for the first time that mice challenged with a 25 µL inoculum volume readily recovered following infection with an infectious dose of influenza A virus that was fatal when inoculated in 35 or 50 µL volumes.

Influenza antiviral resistance in the Asia-Pacific region during 2011.

Despite greater than 99% of influenza A viruses circulating in the Asia-Pacific region being resistant to the adamantane antiviral drugs in 2011, the large majority of influenza A (>97%) and B strains (∼99%) remained susceptible to the neuraminidase inhibitors oseltamivir and zanamivir. However, compared to the first year of the 2009 pandemic, cases of oseltamivir-resistant A(H1N1)pdm09 viruses with the H275Y neuraminidase mutation increased in 2011, primarily due to an outbreak of oseltamivir-resistant viruses that occurred in Newcastle, as reported in Hurt et al. (2011c, 2012a), where the majority of the resistant viruses were from community patients not being treated with oseltamivir. A small number of influenza B viruses with reduced oseltamivir or zanamivir susceptibility were also detected. The increased detection of neuraminidase inhibitor resistant strains circulating in the community and the detection of novel variants with reduced susceptibility are reminders that monitoring of influenza viruses is important to ensure that antiviral treatment guidelines remain appropriate.

Neutralizing monoclonal antibodies to different clades of Influenza A H5N1 viruses.

Four IgG(1kappa) monoclonal antibodies (mAbs) against Influenza A/Chicken/Vietnam/8/2004 (H5N1) virus are described. Three of these showed neutralizing activities against H5N1 strains from clades 1, 2 and 3 using a retroviral pseudotype or live virus microneutralization assay. In the pseudotype assay, the IC(90) neutralizing titre range was >1600-51,200, and with the microneutralization was 80> or =10,240. MAb 1C1 showed strong neutralizing activities in both assays. All four mAbs reacted specifically to the immunogen by immunohistochemical staining and to A/Hong Kong/483/1997 (H5N1) and A/Thailand/1(KAN-1)/2004 (H5N1)-infected MDCK cells by immunofluorescence. ELISA titrations of the mAbs showed specificity for H5N1 haemagglutinin (HA) and no cross-reactivity to 15 other Influenza A subtypes. Only mAbs 1C1 and the non-neutralizing 1F7 reacted with HA(1), the cleaved subunit of HA, by Western blot. These results suggest that the mAbs recognize distinct or overlapping epitopes and will be useful reagents for construction of specific rapid point-of-care assays or for therapeutic use.

Emergence and spread of oseltamivir-resistant A(H1N1) influenza viruses in Oceania, South East Asia and South Africa.

The neuraminidase inhibitors (NAIs) are an effective class of antiviral drugs for the treatment of influenza A and B infections. Until recently, only a low prevalence of NAI resistance (<1%) had been detected in circulating viruses. However, surveillance in Europe in late 2007 revealed significant numbers of A(H1N1) influenza strains with a H274Y neuraminidase mutation that were highly resistant to the NAI oseltamivir. We examined 264 A(H1N1) viruses collected in 2008 from South Africa, Oceania and SE Asia for their susceptibility to NAIs oseltamivir, zanamivir and peramivir in a fluorescence-based neuraminidase inhibition assay. Viruses with reduced oseltamivir susceptibility were further analysed by pyrosequencing assay. The frequency of the oseltamivir-resistant H274Y mutant increased significantly after May 2008, resulting in an overall proportion of 64% (168/264) resistance among A(H1N1) strains, although this subtype represented only 11.6% of all isolates received during 2008. H274Y mutant viruses demonstrated on average a 1466-fold reduction in oseltamivir susceptibility and 527-fold reduction in peramivir sensitivity compared to wild-type A(H1N1) viruses. The mutation had no impact on zanamivir susceptibility. Ongoing surveillance is essential to monitor how these strains may spread or persist in the future and to evaluate the effectiveness of treatments against them.

Molecular diagnosis of medical viruses.

The diagnosis of infectious diseases has been revolutionized by the development of molecular techniques, foremost with the applications of the polymerase chain reaction (PCR). The achievable high sensitivity and ease with which the method can be used to detect any known genetic sequence have led to its wide application in the life sciences. More recently, real-time PCR assays have provided additional major contributions, with the inclusion of an additional fluorescent probe detection system resulting in an increase in sensitivity over conventional PCR, the ability to confirm the amplification product and to quantitate the target concentration. Further, nucleotide sequence analysis of the amplification products has facilitated epidemiological studies of infectious disease outbreaks, and the monitoring of treatment outcomes for infections, in particular with viruses which mutate at high frequency. This review discusses the applications of qualitative and quantitative real-time PCR, nested PCR, multiplex PCR, nucleotide sequence analysis of amplified products and quality assurance with nucleic acid testing (NAT) in diagnostic laboratories.