Paul E. Klapper

Multiplex PCR: Optimization and Application in Diagnostic Virology

PCR has revolutionized the field of infectious disease diagnosis. To overcome the inherent disadvantage of cost and to improve the diagnostic capacity of the test, multiplex PCR, a variant of the test in which more than one target sequence is amplified using more than one pair of primers, has been developed. Multiplex PCRs to detect viral, bacterial, and/or other infectious agents in one reaction tube have been described. Early studies highlighted the obstacles that can jeopardize the production of sensitive and specific multiplex assays, but more recent studies have provided systematic protocols and technical improvements for simple test design. The most useful of these are the empirical choice of oligonucleotide primers and the use of hot start-based PCR methodology. These advances along with others to enhance sensitivity and specificity and to facilitate automation have resulted in the appearance of numerous publications regarding the application of multiplex PCR in the diagnosis of infectious agents, especially those which target viral nucleic acids. This article reviews the principles, optimization, and application of multiplex PCR for the detection of viruses of clinical and epidemiological importance.

New method for the extraction of viral RNA and DNA from cerebrospinal fluid for use in the polymerase chain reaction assay

A new, rapid, and simple method for the isolation of either RNA or DNA from cerebrospinal fluid samples for subsequent amplification by specific polymerase chain reaction (PCR) assays is described. The technique involves a single extraction with a guanidinium thiocyanate acid (GuSCN) buffer, and does not require the use of organic solvents. Applied to the recovery of enteroviral RNA, herpes simplex virus (HSV) and Varicella-zoster virus (VZV) DNAs the method has proved to be of equivalent or better efficiency than established methods of nucleic acid separation but is less laborious and time consuming. The simplicity of the procedure permits the processing of large numbers of samples and the use of a single preparative method for either RNA or DNA PCR makes it an attractive method for the routine laboratory.

Detection of enteroviral RNA and specific DNA of herpesviruses by multiplex genome amplification.

A reverse transcription (RT) multiplex polymerase chain reaction (PCR) assay was developed to allow rapid, sensitive and simultaneous detection of enteroviral RNA and herpesviral DNA specific sequences in a single tube. The method involves a reverse transcription step followed by a multiplex nested PCR in which the combination of primers amplifies cDNA from enteroviruses and specific herpesviruses DNA. Nested amplification utilises primers designed to anneal into the amplification product from the first reaction. Individual viruses were then detected and differentiated by the size of their PCR products determined using ethidium bromide stained agarose gels. To exclude false negatives due to sample inhibitors an internal amplification control, a cloned fragment of DNA from Pseudorabies virus (PRV DNA) was included in the reaction mixture. Detection levels between 0.01 and 0.001 TCID50 of prototype strains of Polio and Coxsackie type B viruses and between 1 and 100 molecules of cloned-DNA of herpesviruses prototype strains were achieved. The RT multiplex PCR method proved capable of detecting enteroviral RNA or herpesviral DNA in cerebro spinal fluid (CSF) samples from patients with aetiologically well characterized encephalitis or aseptic meningitis.

Detection of BK virus and JC virus DNA in urine samples from immunocompromised (HIV-infected) and immunocompetent (HIV-non-infected) patients using polymerase chain reaction and microplate hybridisation

BACKGROUND The majority of the human population is infected with two human polyomaviruses BK virus (BKV) and JC virus (JCV) during childhood. After initial infection both viruses persist within renal system. Reactivation of both viruses may be linked with immunodeficiency or immunosuppressive therapy. OBJECTIVE To evaluate the relationship between immunodeficiency and viruria, prevalence of BK and JC viruria over time was investigated in a cohort of HIV seropositive individuals at different stages of disease. The excretion in this group was compared with virus excretion in their HIV seronegative partners and in an unselected cohort of patients attending a Genito-Urinary Medicine (GUM) clinic. STUDY DESIGN The excretion of BKV and JCV DNA in multiple urine samples from HIV-infected patients at different stages of disease and their HIV-negative partners, and in single samples from a cohort of patients at a GUM clinic was investigated. A microplate hybridisation method was developed to increase both the sensitivity and specificity of detection of the PCR product. The method was also applied to estimate the DNA copy numbers of BKV and JCV in urine samples. RESULTS Within the HIV group, the level of immunosuppression (CD4+ category) was not associated with JCV viruria. By contrast, there was a modest correlation between immunodeficiency as indicated by a decline in CD4+ count and BKV viruria. Shedding of both BKV and JCV DNA together in urine samples of HIV-infected patients was much higher than in control groups (P = 0.02), indicating that HIV infection may associate with polyomavirus reactivation. The incidence of flu-like syndrome was much higher in HIV-infected asymptomatic individuals than acquired immunodeficiency syndrome (AIDS)-related complex (ARC)/AIDS patients. In general, the concentration of BKV DNA viruria (DNA copy number) was dependent to CD4+ counts (P = 0.008) while concentration of JCV DNA was independent to CD4+ cell count (P = 0.54). The prevalence of BKV and JCV DNA in patients who were infected with C. trachomatis was 9/50 (18%) and 11/50 (22%), respectively. BKV and JCV DNA was detected in 3/19 (15%) and 2/19 (10%) of patients who were infected with N. gonorrhoea. Results suggested that persons infected with C. trachomatis were more likely to show BKV and JCV viruria. CONCLUSION These results confirm that shedding of BK and JC viruses in urine is not exclusively found in immunosupression, it may also occur in healthy individuals. The frequency of virus excretion is however, apparently increased in HIV-infected patients, although no firm statistical difference could be established. One of the interesting aspects of these findings was the relatively high incidence of BKV and JCV viruria in both control groups, i.e. HIV-negative partners of HIV-infected patients and patients attending a GUM clinic.

The epidemiology of adenovirus infections in Greater Manchester, UK 1982-96

Data relating to 3313 adenovirus isolates from patients in Greater Manchester, UK between 1982 and 1996 were analysed using chi2 tests and 95% confidence intervals. Of the 3098 isolates that were typed, 18.6% were serotype 2, 14.9% serotype 3, 12.1% serotype 1 and 10.9% serotype 41. There was evidence of a seasonal occurrence of serotype 7 (March-August), serotype 2 (January-April), serotype 4 (June-August) and subgenus F (September-November). Children less than 5 years old were the most common group of patients with adenovirus infection (61.3%) compared to 24.2% for adults and only 5.6% for school children (5-15 years). Gastric symptoms were the most common amongst infants (47.6%) followed by respiratory (27.5%) and general symptoms (12.9%). In adults, the overwhelming clinical condition was conjunctivitis (88.9%). Despite the traditional association with adenoviruses, remarkably few cases of pharyngoconjunctival fever were recorded (1.7%).

Specific diagnostic methods for herpesvirus infections of the central nervous system: A consensus review by the European Union Concerted Action on Virus Meningitis and Encephalitis

BACKGROUND Herpesvirus infections of the central nervous system are often severe but are fortunately rare. The incidence of these infections has however, increased in recent years as a consequence of an increase in the number of immune-compromised individuals. New diagnostic procedures have improved our ability to diagnose these infections and herpesviruses may yet be implicated as the cause of further neurological diseases with no known aetiology. Methodological standards for selection and evaluation of patient materials are essential to the provision of reliable diagnosis, yet few studies have addressed this important issue. OBJECTIVES To describe and define methodological standards and reference methodology for diagnosis of herpesvirus infections of the CNS. STUDY DESIGN Information gathered by literature review. RESULTS Only for herpes simplex encephalitis is there sufficient data to allow the definition of reference methodology. Good methodological standards exist but few studies have adhered to these standards. As methods for the detection of specific intrathecal antibody synthesis are well established yet under-used in diagnostic virology, the principle of these measurements is reviewed in some detail. CONCLUSIONS Herpesvirus infections of the CNS are of increasing importance. High quality, multi-centre studies are needed to establish the value of the new diagnostic test procedures if further improvement in the diagnostic sensitivity and specificity of these procedures is to be achieved.

Age-related pattern of KI and WU polyomavirus infection

Abstract Background The role of two recently identified polyomaviruses, KI and WU, in the causation of respiratory disease has not been established. Objectives To determine the prevalence of KI and WU viruses (KIV and WUV) in 371 respiratory samples and evaluate their contribution to respiratory disease. Study Design Specimens were screened for KIV and WUV using single, multiplex or real time PCR; co-infection with other respiratory viruses was evaluated. Results Of the 371 samples analysed, 10 (2.70%) were positive for KIV and 4 (1.08%) were positive for WUV yielding an overall case prevalence of KIV and WUV infection of 3.77%. KIV and WUV were identified in patients aged <15 years (11 patients) with upper or lower respiratory tract infection and >45 years (3 patients) with upper respiratory tract infection. Co-infections were found in 5 (50%) and 3 (75%) of the KIV and WUV positive samples, respectively. Conclusions This study supports previous conclusions that KIV and WUV detection in the respiratory tract may be coincidental and reflect reactivation of latent or persistent infection with these viruses. The age distribution of KIV and WUV infection in this study mirrors that found for the other human polyomaviruses, BK and JC.

European Proficiency Testing Program for Molecular Detection and Quantitation of Hepatitis B Virus DNA

ABSTRACT External quality control of hepatitis B virus (HBV) DNA detection remains an important issue. This study reports and compares the results obtained from two different proficiency panels for both the qualitative and quantitative assessment of HBV DNA. The panels were designed by the European Union Quality Control Concerted Action, prepared by Boston Biomedica, Inc., and distributed in May 1999 (panel 1) and February 2000 (panel 2). Each contained two negative samples and six positive samples with 103 to 107 copies/ml (panel 1) or 103 to 2 × 106 copies of HBV DNA per ml (panel 2). For panel 1, 42 laboratories submitted 20 qualitative (all in-house PCRs) and 37 quantitative (87% commercial assays) data sets. For panel 2, 51 laboratories submitted 25 qualitative (all in-house PCRs) and 47 quantitative (94% commercial assays) data sets. Five data sets (8.8%) in panel 1 and two data sets (2.8%) in panel 2 contained totals of six and two false-positives, respectively, corresponding to false-positive result rates of 5.3% for panel 1 and 1.4% for panel 2. The false-negative result rates of 10.5% for panel 1 and 17.4% for panel 2 were dependent on the detection levels of the assays employed as well as panel composition. In the qualitative analysis of all data sets, 47.4% (panel 1) and 51.4% (panel 2) had all samples correct. An adequate or better score (all correct or only the weak-positive sample missed) was obtained with 77.2% of the panel 1 samples and 68.1% of the panel 2 samples. In the quantitative analysis, 57.1% (panel 1) and 42.6% (panel 2) of the data sets achieved an adequate or better score (positive results within the acceptable range of the geometric mean ± 0.5 log10 of all positive results). These results demonstrate that while the qualitative performance of HBV detection has considerably improved compared to that of a previously published HBV proficiency study, the detection levels of many commercial quantitative assays are still too high to allow adequate quantitation of all relevant clinical samples.

HSV‐1 and HSV‐2 in herpes simplex encephalitis: A study of sixty‐four cases in the United Kingdom

The incidence of herpes simplex virus type 1 (HSV‐1) and herpes simplex virus type 2 (HSV‐2) in herpes simplex encephalitis (HSE) was investigated using cerebrospinal fluid (CSF) samples from sixty‐four cases of HSE. A polymerase chain reaction (PCR) employing primers flanking a region of the HSV thymidine kinase gene common to both HSV‐1 and HSV‐2 was used to detect HSV in the CSF. HSV‐1 and HSV‐2 were differentiated by digestion with restriction enzymes. Two enzymes were employed; AvaI which cleaved only the HSV‐2 gene product and AvaII which cleaved only the HSV‐1 gene product. Sixty‐three cases of HSE were found to be due to HSV‐1; one case due to HSV‐2. These data confirm previous observations that HSV‐2 is a rare cause of post‐neonatal herpes encephalitis but indicates that a PCR procedure capable of detection of both viruses is essential for efficient diagnosis of HSE. J. Med. Virol. 53:1–3, 1997.

Diagnosis of viral and chlamydial keratoconjunctivitis: which laboratory test?

Conjunctivitis and keratitis are common forms of ocular morbidity seen in general practice and eye units.1 2 The aetiology of these diseases includes viral, bacterial, or parasitic infection as well as allergy, trauma, and dietary deficiency. Among the common microbial causes3-7 (Table 1) are adenovirus, herpes simplex virus (HSV), and Chlamydia trachomatis . Ocular adenovirus infections occur throughout the world in both sporadic and epidemic forms, and large scale outbreaks of epidemic keratoconjunctivitis can occur in hospitals, schools, military establishments, or factories.8 HSV type 1 ocular infection occurs in all countries with an annual incidence of up to 20.7 per 100 000 population and is the most common infective cause of blindness in developed countries.4 9 Trachoma caused by C trachomatis serovars A–C is the leading infectious cause of blindness in the world and is a major public health problem in developing countries.10 Adult chlamydial conjunctivitis, caused by C trachomatis serovars D–K, is an oculogenital infection and up to 90% of patients have concurrent genital infection.11-13 Chlamydial neonatal conjunctivitis (ophthalmia neonatorum) develops in 18%–74% of babies born to mothers with genital chlamydial infection.7 View this table: Table 1 Viral and chlamydial causes of infectious conjunctivitis This article reviews available diagnostic laboratory techniques for keratoconjunctivitis caused by adenovirus, HSV, and C trachomatis with special emphasis on modern molecular diagnostic techniques. For information on the clinical features, epidemiology, and treatment of these infections the reader is referred to a number of other reviews.8 9 14-17 Owing to the limited reliability of clinical diagnosis of adenovirus, HSV, and C trachomatis induced keratoconjunctivitis,18-23 accurate laboratory investigation for these agents in conjunctival swabs is often valuable. Failure to diagnose ocular adenoviral disease can result in outbreaks of epidemic keratoconjunctivitis. Prompt recognition of the strains of adenovirus causing this condition in patients can, …