Niki Spyrou

Detection and typing of HSV-1, HSV-2, and VZV by a multiplex polymerase chain reaction.

The development of a multiplex polymerase chain reaction method for the rapid and accurate detection and typing of HSV‐1, HSV‐2, and VZV from clinical specimens is described. A sensitive multiplex polymerase chain reaction was achieved by optimization of parameters such as the primers, magnesium, and dNTPs concentrations. False‐negative results that sometimes arise due to inhibitors of DNA amplification or failure of DNA extraction procedure used may be avoided by assaying each specimen with α‐tubulin primers. Multiplex PCR amplified viral sequences from all 55 specimens obtained from patients with clinical evidence of HSV or VZV infection indicated 100% sensitivity. From 55 patients who were investigated by multiplex PCR, HSV‐1 was detected in 28, HSV‐2 in 20, and VZV in 7 specimens. The reported results indicate that the present multiplex PCR assay has a potential application in clinical diagnosis when a rapid and accurate detection and typing of involved viruses HSV‐1, HSV‐2, or VZV is needed. J. Clin. Lab. Anal. 14:214–219, 2000.

Isolation of polioviruses and other enteroviruses in south Greece between 1994 and 1998.

During the five‐year period between 1994 and 1998, a total of 217 clinical samples were assessed for the isolation of enteroviruses at the Enterovirus Reference Centre for South Greece. Fourteen enterovirus strains belonging to different serotypes were isolated. These field strains were detected by cell culture in appropriate cell lines. They were subsequently identified by neutralizing antibodies with the LBM (Lim‐Benyesh Melnick) mixed antisera pools up to 1995 and RIVM (National Institute of Public Health and the Environment, Bilthoven, The Netherlands) pools from 1996 onwards. The isolated viruses included two strains of poliovirus type 2 Sabin‐like, three strains of poliovirus type 1 non‐Sabin‐like, one Coxsackie B2 (CBV2) strain, one Coxsackie B5 (CBV5) strain, one Echo 5 (ECV5) strain, one Echo 7 (ECV7) strain, three Coxsackie A16 (CAV16) strains, and two currently enteroviral strains unidentified by RIVM pools. Reverse transcription‐polymerase chain reaction (RT‐PCR) using poliovirus‐specific primers or poliovirus type‐specific primers and enterovirus specific primers from the highly conserved 5′‐UTR, the latter followed by RFLP, was also applied in 6 clinical isolates (3 strains of poliovirus type 1 non‐Sabin‐like, 1 polio type 2 Sabin‐like, and 2 non‐identified by RIVM pools enteroviruses). The advantages and the drawbacks of these assays against the conventional ones are discussed here. The isolations and the subsequent identification of the strains were carried out from fecal samples of clinical cases that included hand‐foot‐and‐mouth disease, meningitis, and acute flaccid paralysis. The reappearance of non‐Sabin‐like poliovirus strains in Greece in 1996 after 14 years is considered to have an important medical and clinical value. J. Clin. Lab. Anal. 14:157–163, 2000.

Development of a quadriplex polymerase chain reaction for human cytomegalovirus detection.

The development of a quadriplex PCR method with amplification of HCMV in a single‐step procedure using primers taken from four different regions of the viral genome is described. Different concentrations of dNTPs and MgCl2 were assayed in order to optimize the constitution of the buffer for the multiplex PCR. The specificity of the PCR was tested with 100ng, 10ng, and 1ng of genomic MRC‐5 cell DNA infected with CMV in the presence of 10μg of uninfected MRC‐5 cell DNA. The sensitivity of the PCR was evaluated by the amplification of various amounts (100ng, 10ng, 1ng, and 0.1ng) of genomic MRC‐5 cell DNA infected with CMV. The specificity and sensitivity assays were performed for each pair of primers and for the combined four primer pairs in the multiplex PCR. CMV was consistently detected from 10ng of genomic MRC‐5 cell DNA with each primer pair. When all four sets of primers were combined in a single reaction tube, the sensitivity of the assay was equivalent to 10ng of genomic MRC‐5 cell DNA, whereas amplification from 1ng genomic MRC‐5 cell DNA produced only a subset of the amplimers. By amplifying four target‐sequences of HCMV simultaneously with minimum incubation time at each temperature, a quadriplex, highly sensitive PCR assay was performed. The use of four primer sets designed in different genomic regions of HCMV allowed the detection of variants and achieved maximal sensitivity and specificity which are essential for a diagnostic utilization. J. Clin. Lab. Anal. 13:99–105, 1999.

Clear detection and typing of herpes simplex virus types 1 and 2 by an indirect ELISA assay: comparison with three different combined methods--capture ELISA, restriction enzymes, and polymerase chain reaction.

The severity and recurrences of Herpes Simplex Virus (HSV) infection depend on the type of the infectious agent (HSV‐1 or HSV‐2), which induces the necessity of a nonambiguous detecting typing. The commonly used capture ELISA technique has to be often supported by DNA analysis to confirm the detection and the typing of HSV viruses in exposed patients. In this report, we describe a rapid and cheap indirect ELISA method using anti‐HSV monospecific polyclonal antibodies prepared in the laboratory. The typing of the studied samples was clear, did not need series of dilution, and allowed the immediate classification of viruses without further control examination. We tested 51 specimens, which were typed 25 HSV‐1 and 26 HSV‐2 strains. The comparison with capture ELISA, restriction enzyme and polymerase chain reaction analysis definitely allowed our method to be assessed as a useful tool for a routine diagnostic. J. Clin. Lab. Anal. 11:146–153, 1997.

Molecular and phylogenetic analysis of haemagglutinin and neuraminidase sequences from recent human influenza type A (H3N2) viral isolates in Southern Greece

Summary. Eighteen haemagglutinin (HA1) gene segments and eleven neuraminidase (NA) genes of human influenza type A (H3N2) viruses isolated from non-vaccinated individuals presenting severe influenza-like illness at peak influenza activity in Southern Greece during the surveillance period 1996–1999, were subjected to sequence and phylogenetic analyses following propagation in embryonated hen’s eggs. The HA1 gene segment of the clinical isolates differed from the recent reference influenza type A (H3N2) vaccine strains in an Ile at residue 186, a Val at residue 194 and a Val at residue 226 for one, two and thirteen isolates of the 1996–1997 and 1996–1999 periods, respectively. The analogous differences in the NA gene were confined in an Asp to Asn substitution at residue 198 in one A/Wuhan/359/95 (H3N2)-like isolate of the 1996–1997 period, primarily. In addition, phylogenetic analysis revealed that an isolate of the 1997–1998 period was a recombinant with its HA1 gene segment being closely related to that of A/Wuhan/359/95-like viruses and its NA to viruses of the A/Sydney/5/97 (H3N2) lineage. These findings confirmed the profound genetic instability of influenza type A (H3N2) viruses and underscored the importance for periodic molecular surveys of HA and NA in the effective prevention and management of viral outbreaks. Most importantly, however, they contributed the first complete epidemiological material for influenza in Southern Greece, the archival nature of which constitutes valuable reference for future surveys.