Robert J. Cooper

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.

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%).

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, …

Polymerase chain reaction for rapid diagnosis of respiratory adenovirus infection.

Endemic (type 1, 2, 5 and 6) and epidemic (type 3, 4 and 7) respiratory adenovirus infections are associated with upper respiratory tract symptoms, pharyngoconjunctival fever, and pneumonia. Improved methods of diagnosis are needed, particularly in immunocompromized patients. We examined 93 throat swabs or nasopharyngeal aspirates from patients with acute respiratory disease using virus isolation and an adenovirus-specific polymerase chain reaction (PCR) based on consensus primers H1 and H2 derived from the hexon region DNA sequences of serotypes 2 and 5. Specimens which yielded viruses other than adenovirus in cell culture (n = 23) or which were negative for infectious viruses (n = 25) were negative in the PCR. The sensitivity of DNA amplification was 76% (34/45) in comparison with virus culture, being markedly lower with subgenus B (types 3 and 7) strains than with subgenus C (type 1, 2, 5 and 6) isolates (8/16 (50%)) vs. 26/28 (93%). P = 0.004) despite the use of a low annealing temperature to maximize detection of adenoviruses belonging to subgenera other than C. Of the 11 samples falsely negative in a single-round PCR but yielding adenovirus type 1 (n = 1), type 2 (n = 1). type 3 (n = 7), type 7 (n = 1), or untyped isolates (n = 1) in cell culture, nine (82%) gave positive results after nested DNA amplification. Possible approaches to further improving the performance of adenovirus PCR with respiratory specimens are discussed.

Infection and temporal arteritis: a PCR-based study to detect pathogens in temporal artery biopsy specimens.

The possibility of infectious triggers stimulating the development of inflammatory vascular diseases has generated much recent interest. This study uses PCR to detect the presence of Chlamydia pneumoniae, parvovirus B19 and all the human herpes viruses except HHV8 in temporal artery biopsy specimens. Samples from 37 temporal artery biopsies with histological evidence of arteritis and 66 samples from histologically negative temporal artery biopsies, all from different patients, were negative for C. pneumoniae, HSV, VZV, EBV, and HHV7 DNA. Two of the 37 histologically positive specimens were positive for HHV6, another two for CMV and a further two for parvovirus B19 DNA. Parvovirus B19 DNA was also detected in five histologically negative biopsies, one positive for HCMV DNA and a further one was positive for HHV6 DNA. There is no statistically significant difference to the presence of virus DNA in the two types of specimens (P = 0.538). This study does not support a role for C. pneumoniae, parvovirus B19 or human herpes viruses in the pathogenesis of temporal arteritis. J. Med. Virol. 80:501–505, 2008.

Detection of human herpes virus 6 DNA in fetal hydrops

Human herpes virus 6 (HHV6) DNA was detected in two of eight fetuses with hydrops and none of ten non-hydropic dead fetuses. Both cases with HHV6 DNA had chromosomal abnormalities. Positive results were confirmed with a second PCR specific for an alternate region of the HHV6 genome. Restriction endonuclease analysis confirmed that the viral DNA was representative of HHV6 type A.

The polymerase chain reaction for detecting adenovirus DNA in formalin-fixed, paraffin-embedded tissue obtained post mortem

The polymerase chain reaction (PCR) was used to detect adenovirus DNA in formalin-fixed, paraffin-embedded tissue obtained post mortem. Adenovirus DNA was successfully amplified from specimens of lung and liver from two patients with disseminated adenovirus infection confirmed by virus isolation, electron microscopy and/or immunohistochemistry. Negative results were obtained for specimens of lung from two patients with cytomegalovirus pneumonia. The specificity of the adenovirus PCR was confirmed by means of a digoxigenin-labelled probe generated in a separate PCR. Detection of viral nucleic acid by PCR in tissues obtained post mortem has considerable diagnostic potential.

Multiplex polymerase chain reaction for human herpesvirus-6, human cytomegalovirus, and human β-globin DNA

Human cytomegalovirus and human herpesvirus-6 are closely related viruses which cause similar diseases, have similar cellular repositories of latent infection, and may be detected largely in the same types of clinical specimens. DNA amplification appears likely to play an increasing role in the diagnosis of recent and remote infection with these agents. A sensitive multiplex polymerase chain reaction was therefore developed for the two viruses and for human β-globin DNA. Optimization of parameters such as the primers, primer concentrations, magnesium concentration, and buffer constituents was crucial in achieving a sensitive assay. Preliminary results indicated that the assay could simultaneously monitor DNA extraction from clinical specimens and allow detection of HCMV or HHV-6 in patients with diseases possibly caused by either pathogen.

Prevalence and quantitation of adenovirus DNA from human tonsil and adenoid tissues

In this study, real‐time PCR was used to quantify adenovirus DNA in cell suspensions prepared from 106 right and left tonsils and 10 adenoids obtained from 57 patients who underwent routine tonsillectomies and/or adenoidectomies. Eighty‐four (72.4%) tonsils and adenoids samples were positive for HAdV by real‐time PCR. The viral load ranged from 2.8 × 102 to 2.6 × 106 copies/107 cells and varied up to sixfold between the right and left tonsils. In some cases, only one tonsil was positive and the viral load was lower in older children. Seventy‐eight of 84 positive samples could be typed by sequencing of the hexon L1 region. Species C (types 1, 2, and 5) were detected in 84.1% of the patients followed by types 3 and 7 of species B (6.8%), HAdV‐E4 (6.8%), and HAdV‐F41 (2.3%). In one patient adenovirus C2 was found in the left tonsil and adenovirus C5 in the right tonsil. No DNA methylation was detected in either the E1A promoter or the major late promoter region of adenovirus DNA from six tonsils and adenoids samples and two clinical isolates. J Med. Virol. 85:1947–1954, 2013..

Detection of human cytomegalovirus, human herpesvirus type 6 and human herpesvirus type 7 in urine specimens by multiplex PCR

OBJECTIVES To develop a sensitive multiplex PCR to detect HCMV, HHV6 and HHV7, to test this PCR on urine specimens sent to the virus diagnostic laboratory and on stored urine samples from HIV-positive patients and their HIV-negative partners and to compare the sensitivity of the multiplex PCR with the diagnostic laboratorys routine service for the detection of HCMV. STUDY DESIGN Primers specific for each of the three viruses were combined in a multiplex PCR that was then optimised for sensitivity. This PCR was applied prospectively to 413 unselected routine urine specimens over a 1 year period and retrospectively to 258 urine specimens from 63 HIV-positive patients and 10 HIV-negative partners. METHODS In the prospective study, the multiplex PCR detected 40 specimens positive for HCMV alone, 10 for HHV6, 3 for HHV7 and 3 with a dual infection of HCMV and HHV6. The sensitivity for HCMV was 93.5% by multiplex PCR compared to 28.3% by culture. HHV6 DNA was detected in 6 neonates (2-21 days) and HHV7 DNA in 2 neonates (4 and 20 days). In the retrospective study of HIV patients, HCMV was the most commonly detected virus (55.6%) compared to HHV6 (7.9%) and HHV7 (4.8%). CONCLUSIONS . The multiplex PCR was significantly more sensitive than non-DNA based procedures for the detection of HCMV. Urine may be a useful non-invasive specimen for the detection of HHV6 and HHV7 and their presence in neonates suggest perinatal transmission or the possibility of in utero infection.