Comparison of virus isolation and various polymerase chain reaction methods in the diagnosis of mucocutaneous herpesvirus infection

We compared two polymerase chain reaction (PCR) assays (simple and multiplex) and viral isolation to detect herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV) in 15 clinical specimens from 13 patients with mucocutaneous herpetic infections. HSV-1 or VZV DNA was detected in 13 specimens by simple PCRs (HSV-1 or VZV PCR) and in 12 specimens by multiplex PCR. On the other hand, viral isolation was positive for 9 specimens only. The PCR protocols used in this study are not only more sensitive and faster than the traditional viral isolation and conventional PCR protocols but also can distinguish rapidly HSV-1 from VZV. We propose the PCRs described here for rapid and precise identification of etiological agents of mucocutaneous herpetic infections.     

Detection of measles virus antigen(s) in peripheral lymphocytes from patients with subacute sclerosing panencephalitis

Summary Peripheral blood lymphocytes from 5 patients with subacute sclerosing panencephalitis (SSPE) were stimulated with phytohemagglutinin (PHA) and analyzed for the presence of the measles virus antigen(s) by immunofluorescence (IF). For detection of viral antigen fluorescein-conjugated globulins from SSPE patients or from measles convalescents both with high anti-measles titer were used. Peripheral blood lymphocytes from 5 children with measles were used as a positive control.Measles virus antigen(s) were localized in PHA-transformed lymphocytes in all SSPE patients.The present results indicate that in SSPE measles-like virus infection may be found not only in the central nervous system, but also in the circulating lymphoid cells.With 2 Figures     

Amplification of the six major human herpesviruses from cerebrospinal fluid by a single PCR

We used a novel type of primer system, a system that uses stair primers, in which the primer sequences are based on consensus sequences in the DNA polymerase gene of herpesvirus to detect herpesviruses by PCR. A single PCR in a single tube detected the six major herpesviruses that infect the central nervous system: herpes simplex virus type 1 (HSV-1), and type 2 (HSV-2), cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), and human herpesvirus 6 (HHV-6). We used the technique to analyze 142 cerebrospinal fluid (CSF) samples that had been stored at -80 degrees C and compared the results with those obtained previously for the same samples by standard, targeted PCR. Four hundred one targeted PCR tests had been run with the 142 samples to detect HSV-1, HSV-2, CMV, and VZV; screening for EBV and HHV-6 was not prescribed when the samples were initially taken. Eighteen CSF samples tested positive by classic targeted PCR. The herpesvirus consensus PCR detected herpesviruses in 37 samples, including 3 samples with coinfections and 17 viral isolates which were not targeted. Two samples identified as infected by the targeted PCR tested negative by the consensus PCR, and eight samples that tested positive by the consensus PCR were negative by the targeted PCR. One hundred three samples scored negative by both the targeted and the consensus PCRs. This preliminary study demonstrates the value of testing for six different herpesviruses simultaneously by a sensitive and straightforward technique rather than screening only for those viruses that are causing infections as suggested by clinical signs.     

Development of a multiplex nested consensus PCR for detection and identification of major human herpesviruses in CNS infections.

BACKGROUND: Rapid, sensitive and economical detection and identification of human herpesviruses as causative agents of central nervous system (CNS) infections are of clinical importance. The traditional methods for the detection of herpesviruses in CNS infections all suffer from limitations. PCR has a potential to overcome each of them. OBJECTIVES: The aims of this study were reducing the number of primers in multiplex PCR and increasing the sensitivity of the assay by nested PCR. STUDY DESIGN: A multiplex nested consensus PCR (MNC-PCR) was developed for the simultaneous detection of major human herpesviruses. A pair of conserved primers was designed for detection of HSV-1, HSV-2, CMV and EBV and another pair of conserved primers for nested PCR. For VZV, a different pair of primers was designed and another pair of primers for nested PCR. A reduction in the number of designed primer pairs (from five pairs to two in both stages of PCR) is an advantage in this assay. One hundred forty-seven cerebral spinal fluid (CSF) samples from patients that showed clinical manifestation of CNS infections were tested. Results of MNC-PCR in CSF samples were compared with those of single PCR assay for each individual DNA virus. Sensitivity of the assay was determined with a plasmid containing VZV DNA binding protein gene and another plasmid for HSV-1 DNA polymerase gene. False negative results (due to the presence of inhibitor of DNA amplification in CSF samples) were avoided by the inclusion of beta2-microglobulin primers in the MNC-PCR assay as an internal control. RESULTS: Positive results were obtained in 20 CSF samples (8 HSV-1, 2 HSV-2, 4 CMV, 3 VZV, 3 HSV-1/CMV, CMV/VZV and HSV-1/EBV coinfections). The comparison between single PCR and MNC-PCR showed a marked increase in sensitivity of MNC-PCR test, since six negative samples in single PCR proved positive in MNC-PCR (P<0.005). Sensitivity was determined 1-5 plasmid copies for VZV and 50-100 plasmid copies for HSV-1. CONCLUSIONS: The MNC-PCR assay presented in this study can provide a rapid, sensitive and economical method for detection of viral infections and is applicable to small volumes of CSF samples.     

Protective titres of measles neutralising antibody

A nation-wide measles outbreak occurred in 1988 in Taiwan. A retrospective cohort study was conducted to define the protective titre of measles neutralising (NT) antibody. Paired sera collected in 1987 and 1988 were available from 190 individuals born in 1984 who had participated in an annual hepatitis B immunisation follow-up from 1986 to 1991. Measles NT titres were quantified using a standardised neutralisation enzyme immunoassay. Measles infection was defined as a >/=4-fold rise in NT titre or seroconversion between paired sera. Symptomatic measles infection was ascribed to individuals who had measles infection and who reported measles-like symptoms between 1987 and 1988. Results demonstrated a dose-response relationship between pre-exposure NT titres and protection against measles infection. 47 of 48 individuals with measles infection in 1988 had pre-exposure NT titres 1, 000 mIU/ml may prevent measles infection and NT titres >500 mIU/ml may prevent symptomatic infection but vaccinees with undetectable or low NT titres may not necessarily be susceptible to symptomatic infection.     

Antibody-modification of measles "in vitro" infection.

While the involvement of measles-like virus with subacute sclerosing panencephalitis (SSPE) has been shown in many laboratories, the possible mechanisms by which a normal measles infection may lead to the chronic condition are unclear. The work reported here demonstrates that the growth and maintenance of Vero cells infected with the MV 30 strain of measles, in the presence of multivalent antibody, results in a redistribution of the viral antigens within the cells and an alteration in the properties of this attenuated virus which persists even after removal of the antiserum from the culture medium. It is suggested that these in vitro findings could be parallel to the situation that occurs in vivo with measles encephalitis in hamsters and human SSPE.     

Longitudinal evaluation of herpes simplex virus DNA load during episodes of herpes labialis.

BACKGROUND: Few studies have performed sequential evaluation of the herpes simplex virus (HSV) load using quantitative PCR during episodes of herpes labialis. OBJECTIVE: To determine HSV viral load kinetics during recurrences of herpes labialis. STUDY DESIGN: Twenty-two subjects were monitored by daily swabs during recurrences of herpes labialis. A real-time PCR detecting both HSV-1 and HSV-2 was used to quantify the viral load. RESULTS: Median duration of HSV-1 shedding was 60 h and 48 h by PCR and culture, respectively. No HSV-2 DNA was detected in that study. Peak viral DNA load (123.6 copies/cell) occurred at 48 h with no virus detected beyond 96 h of onset of symptoms. CONCLUSIONS: These viral load kinetics data could be used as surrogate markers of antiviral activity in future clinical trials.     

Virological diagnosis of herpes simplex virus 1 esophagitis by quantitative real-time PCR assay

Herpes simplex virus 1 (HSV-1) esophagitis diagnosis is routinely based on the endoscopic findings confirmed by histopathological examination of the esophagitis lesions. Virological diagnosis is not systematically performed and restricted to viral culture or to qualitative PCR assay from esophagitis biopsy specimens. The aim of this study was to assess the interest of quantitative real-time PCR assay in HSV-1 esophagitis diagnosis by comparing the results obtained to those of histological examination associated with immunohistochemical staining, which is considered the "gold standard." From 53 esophagitis biopsy specimens, the PCR assay detected HSV-1 in 18 of 19 histologically proven to have herpetic esophagitis and in 9 of 34 that had esophagitis related to other causes, demonstrating sensitivity, specificity, positive predictive value, and negative predictive value of 94.7%, 73%, 66.7%, and 96%, respectively. Interestingly, HSV-1 was not detected in 16 specimens without the histological aspect of esophagitis. The viral loads normalized per μg of total extracted DNA in each biopsy specimen detected positive by HSV PCR were then compared and appeared to be significantly higher in histopathologically positive herpetic esophagitis (median = 2.9 × 10(6) ± 1.1 × 10(8)) than in histopathologically negative herpetic esophagitis (median = 3.1 × 10(3) ± 6.2 × 10(3)) (P = 0.0009). Moreover, a receiver operating characteristics analysis revealed that a viral load threshold greater than 2.5 × 10(4) copies would allow an HSV-1 esophagitis diagnosis with a sensitivity and specificity of 83.3% and 100%, respectively. In conclusion, this work demonstrated that HSV quantitative PCR results for paraffin-embedded esophageal tissue was well correlated to histopathological findings for an HSV-1 esophagitis diagnosis and could be diagnostic through viral load assessment when histopathological results are missing or uncertain.     

Identification of herpes simplex virus genital infection: comparison of a multiplex PCR assay and traditional viral isolation techniques.

Genital herpes simplex virus (HSV) is of major public health importance, as indicated by the marked increase in the prevalence of genital herpes over the past two decades. Viral culture has traditionally been regarded as the gold standard for diagnosis. In this study, we compared viral culture and the amplification of HSV DNA by the polymerase chain reaction (PCR) with respect to sensitivity, cost, clinical utility, and turnaround time. Patient sample swabs from 100 individuals were inoculated onto MRC-5 cells for isolation. Positive results were confirmed via a direct fluorescent antibody technique, and serotyping, when requested, was performed using HSV-1 and -2-type-specific sera. PCR techniques employed an extraction step of the same initial swab specimen, followed by PCR amplification, using a multiplex assay for HSV-1, 2 DNA. HSV-positive results were found in 32/100 samples via culture and in 36/100 samples via PCR. PCR-positive results yielded 16 (44%) patients infected with HSV-1 and 20 (56%) patients infected with HSV-2. Turnaround time for viral culture averaged 108 hours for positive results and 154 hours for negative results; PCR turnaround time averaged 24--48 hours. Laboratory cost using viral culture was $3.22 for a negative result and $6.49 for a positive result (including direct fluorescent antibody). Serotyping added $10.88 to each culture-positive test. Although laboratory costs for PCR were higher at $8.20/sample, reimbursement levels were also higher. We propose a multiplex PCR assay for diagnosis of HSV-1 and HSV-2 from patient swabs for use in a routine clinical laboratory setting. This assay offers increased sensitivity, typing, and improved turnaround time when compared with traditional viral culture techniques. Although it appears that PCR testing in a routine clinical laboratory setting is cost prohibitive compared with the case of nonserotyped viral culture, it may be very useful when clinical utility warrants distinguishing between HSV 1 and 2 and may be cost effective when reimbursement issues are examined.     

Detection and typing of HSV-1, HSV-2, CMV and EBV by quadruplex PCR

The development of a multiplex polymerase chain reaction (PCR) method for rapid and accurate detection and typing of herpes simplex virus type 1 (HSV-1), and type-2 (HSV-2), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) is very important for clinical diagnosis to allow the deliver of therapy as early as possible. Large scale amplifications by multiplex PCR of viral DNA can lower the cost and time for viral diagnosis. In this study, therefore sensitive quadruplex PCR was achieved by optimizing parameters such as primers, and 1.5 mM magnesium and 200 uM dNTPs concentrations. The concentrations of HSV-1, HSV-2, CMV and EBV primers were 0.5, 0.3, 0.25 and 0.25 pmoles, respectively. Optimal annealing temperature was 54 degrees C. Employing these conditions, we could detect 10 copies of reconstructed template plasmid DNA, which were cloned to vectors containing target sequences of viral DNA. PCR products of 271 bp for HSV-1, 231 bp for HSV-2, 368 bp for CMV, and 326 bp for EBV were separated on 5.0% polyacrylamide gel electrophoresis and confirmed by direct sequencing. The present study showed that the quadruplex PCR assay described herein has potential application in clinical diagnosis, when rapid, accurate detection and typing of viruses HSV-1, HSV-2, CMV or EBV are necessary.     

Diagnosing genital ulcer disease in a clinic for sexually transmitted diseases in Amsterdam, The Netherlands

The most common etiologic agents of genital ulcer disease (GUD) are herpes simplex virus type 1 (HSV-1), HSV-2, Treponema pallidum, and Haemophilus ducreyi. In an outpatient clinic for sexually transmitted diseases in Amsterdam, The Netherlands, specimens from 372 patients with GUD were collected from February to November 1996. Sera were collected at the time of the symptoms and, for most patients, also during follow-up visits. Swabs in viral transport medium were used for HSV culture and for detection of DNA. The most prevalent pathogen found was HSV-2, which was detected by culture in 35% of the patients and by PCR in 48% of the patients. Also, HSV-1 infection was more often detected by PCR (7.8%) than by culture (5.6%). Evidence for an active infection with T. pallidum was found in 1.9% of the patients, using serological tests. A multiplex PCR for simultaneous T. pallidum and H. ducreyi DNA detection was positive for T. pallidum in 3.3% of the samples and for H. ducreyi in only 0.9% (3 out of 368) of the samples. The sensitivity of the PCR was superior to that of culture for HSV detection and to that of serology for T. pallidum detection. Specific H. ducreyi immunoglobulin G antibodies were detected in sera of 5.2% of the patients, with no concordance between serology and PCR. In 37% of the cases, none of the tested microorganisms was detected. Performance of PCR in addition to conventional techniques significantly improved the diagnosis of GUD.     

Characterization of virus isolates by particle-associated nucleic acid PCR

Diagnostic virus isolation is still frequently used, particularly from respiratory tract secretions. Testing positive virus cultures for all possible viruses is time-consuming, and unexpected or unknown viruses may escape detection. Therefore, a novel random PCR approach was developed that allows sequence-independent amplification of viral nucleic acids from virus isolation-positive cultures. Selectivity for viral sequences is obtained by preferential isolation of nucleic acids that are particle associated and resistant to nucleases. Using primers with a degenerated 3' end, the isolated nucleic acids are amplified and the randomly amplified PCR products are cloned and sequenced. As proof of the concept, the PAN-PCR approach was applied to supernatants of coxsackievirus B3 and murine adenovirus type 1-infected cells. Enterovirus and adenovirus sequences were obtained, demonstrating that the random PCR approach allows detection of RNA and DNA viruses. As a first application of this PAN-PCR approach, we characterized a virus isolate from mouth-washing material of a patient with chronic fatigue syndrome and high antibody titers to coxsackievirus B2. The virus isolate had tested negative for enteroviruses and respiratory viruses (influenza viruses A and B, parainfluenza virus types 1 to 3, respiratory syncytial virus, and adenovirus) by immunofluorescence and PCR. Particle-associated, nuclease-resistant RNA and DNA were prepared from the supernatant of infected cells. The DNA and the reverse-transcribed RNA were randomly amplified, and PCR products were cloned and sequenced. Of 25 sequences obtained from the DNA preparation, 24 contained herpes simplex virus type 1 (HSV-1) sequences from 14 different loci spread over the HSV-1 genome. This result was confirmed by using a standard diagnostic HSV-PCR, demonstrating that the PAN-PCR correctly identified the virus isolate. Although the identification of HSV-1 in mouth-washing material is not surprising in retrospect, it clearly demonstrates the applicability of the PAN-PCR approach. This method should be particularly useful for characterizing virus isolates that have tested negative for all expected viruses and for identifying unknown viruses.     

Laboratory Diagnosis of Common Herpesvirus Infections of the Central Nervous System by a Multiplex PCR Assay

A sensitive multiplex PCR assay for single-tube amplification that detects simultaneous herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV), human cytomegalovirus (CMV), and Epstein-Barr virus (EBV) is reported with particular emphasis on how the method was optimized and carried out and its sensitivity was compared to previously described assays. The assay has been used on a limited number of clinical samples and must be thoroughly evaluated in the clinical context. A total of 86 cerebrospinal fluid (CSF) specimens from patients which had the clinical symptoms of encephalitis, meningitis or meningoencephalitis were included in this study. The sensitivity of the multiplex PCR was determined to be 0.01 and 0.03 50% tissue culture infective doses/the reciprocal of the highest dilution positive by PCR for HSV-1 and HSV-2 respectively, whereas for VZV, CMV and EBV, 14, 18, and 160 ag of genomic DNA were detected corresponding to 48, 66, and 840 genome copies respectively. Overall, 9 (10.3%) of the CSF samples tested were positive in the multiplex PCR. HSV-1 was detected in three patients (3.5%) with encephalitis, VZV was detected in four patients (4.6%) with meningitis, HSV-2 was detected in one neonate (1.16%), and CMV was also detected in one neonate (1.16%). None of the samples tested was positive for the EBV genome. None of the nine positive CSF samples presented herpesvirus coinfection in the central nervous system. Failure of DNA extraction or failure to remove any inhibitors of DNA amplification from CSF samples was avoided by the inclusion in the present multiplex PCR assay of alpha-tubulin primers. The present multiplex PCR assay detects simultaneously five different herpesviruses and sample suitability for PCR in a single amplification round of 40 cycles with an excellent sensitivity and can, therefore, provide an early, rapid, reliable noninvasive diagnostic tool allowing the application of antiviral therapy on the basis of a specific viral diagnosis. The results of this preliminary study should prompt a more exhaustive analysis of the clinical value of the present multiplex PCR assay.     

Comparison of loop-mediated isothermal amplification, real-time PCR, and virus isolation for the detection of herpes simplex virus in genital lesions

This study compares herpes simplex virus (HSV) type-specific loop-mediated isothermal amplification (LAMP) with virus isolation and real-time PCR. Genital tract specimens were obtained from 25 patients with genital lesions; two swab samples were collected from the vulva and cervix of each patient, for a total of 50 specimens. After culturing, 10 of 50 (20%) samples were positive for HSV-1 and 12 of 50 (24%) samples were positive for HSV-2. None of the patients excreted both HSV-1 and HSV-2 virus. An original HSV type-specific LAMP assay (30 min reaction) was compared with virus isolation and HSV type-specific real-time PCR. Viral DNA was detected by LAMP in 9 of 10 HSV-1 isolated samples and 11 of 12 HSV-2 isolated samples. No viral DNA was detected in samples without virus isolation. Thus, if virus isolation was used as the standard method, the LAMP protocol was highly sensitive and specific. In comparing LAMP to real-time PCR, viral DNA was detected by the LAMP method in 9 of 12 HSV-1 DNA positive samples and 11 of 18 HSV-2 DNA positive samples. If real-time PCR was used as the standard method, then, sensitivity of the LAMP method (in particular, for HSV-2) was low. Taking this into consideration, the LAMP reaction was extended to 60 min. This led to an increase in sensitivity, resulting in an additional one and three samples testing positive for HSV-1 LAMP and HSV-2 LAMP, respectively, compared to the original LAMP protocol. Therefore, the sensitivity of the LAMP method increased to about 80%.     

Evaluation of new reagents for typing IgG to HSV-1 and HSV-2

Until recently, the lack of suitable type specific assays has hampered the serological diagnosis of herpes simplex virus (HSV) infections, due to the high crossreactivity between types 1 and 2. The aim of the present paper is the evaluation of new commercial methods for the detection of HSV-1 and HSV-2-specific IgG using glycoprotein G as antigen (BioElisa HSV-1 and BioElisa HSV-2), in their application to viral diagnosis and seroepidemiological studies. Eighty two serum samples from HSV recent infections (30 samples from 13 cases), normal children (28 samples), and patients attended in clinics for sexually transmitted diseases (STD) (24 samples from 20 patients) were studied by such methods and the results compared with those obtained by a conventional indirect ELISA, and by the complement fixation test. The methods gave a type specific identification of the antibody response in nine of the 13 HSV patients. Positive results for anti-HSV-2 IgG were obtained in four cases among the STD patients but in none among the normal children. Nineteen of the former and seven of the latter were positive for anti-HSV-1. Only one sample was reactive in the HSV-1 assay, and negative by the indirect ELISA. Type specific HSV-1 and HSV-2 assays may help the serological diagnosis of HSV infections, since they allow the correct characterization of the antibody response. Bearing in mind the high specificity of the method for HSV-2 IgG, it might be useful in screening of populations for anti-HSV-2 and especially in prevention of the neonatal HSV-2 infection.     

Increased detection rate in diagnosis of herpes simplex virus type 2 meningitis by real-time PCR using cerebrospinal fluid samples

Efficient and sensitive diagnostic methods are needed in the management of virus infections in the central nervous system. There is a demand for an evaluation of the sensitivity of PCR methods for early diagnosis of meningitis due to herpes simplex type 2 (HSV-2) and varicella-zoster virus (VZV). The objective of this study was to evaluate real-time PCR in the detection of HSV-2 and VZV DNA from cerebrospinal fluid (CSF) for etiological diagnoses in clinically well-characterized cases of primary and recurrent aseptic meningitis. Samples from 110 patients, 65 of whom were diagnosed with or were strongly suspected of having HSV-2 meningitis and 45 with acute aseptic meningitis of unknown causes, were analyzed. Results were compared with the outcome of nested PCR for HSV-2 infection. Clinical parameters were analyzed in relation to CSF viral load. With real-time PCR, HSV-2 DNA was found in CSF from 80% (52/65) of patients with clinical HSV-2 meningitis compared to 72% (47/65) found by nested PCR. The sensitivity of real-time HSV-2 PCR was found to be 87% (33/38) in primary and 70% (19/27) in recurrent meningitis. The HSV-2 viral load was significantly higher in primary than in recurrent meningitis and correlated with the degree of inflammation. VZV DNA was detected in 2 of 45 samples (4.4%). Real-time PCR for the diagnosis of HSV-2 meningitis was evaluated in a large, clinically well-characterized sample of material and found to identify more cases than nested PCR in the group of patients with recurrent meningitis. Quantification of DNA enables further research of disease prognosis and treatment.     

Assessment of the FilmArray ME panel in 4199 consecutively tested cerebrospinal fluid samples

ObjectivesIn central nervous system infections, early and correct management is of utmost importance. Rapid syndromic panel testing can potentially provide valuable guidance. The FilmArray meningitis/encephalitis (ME) panel detects 14 pathogens through multiplex PCR. Our study objectives were to assess its performance compared with established diagnostic procedures, especially real-time quantitative PCR for detection of viruses, and to determine the diagnostic and clinical significance of discrepant results.MethodsAll cerebrospinal fluid samples sent for viral diagnostics to our microbiological laboratory over 34 months were analysed with the ME panel and in-house real-time PCR for herpes simplex virus type 1 (HSV-1), HSV-2, varicella zoster virus and enteroviruses. Other pathogens detected by the panel were confirmed by routine diagnostic procedures. Discrepant results were analysed through interpretation of biological and clinical data, and performance data were calculated for individual pathogens.ResultsAltogether, 315 pathogens were detected by the ME panel in 4199 cerebrospinal fluid samples (7.5%) and an additional 21 viral targets were identified using real-time PCR. Thirty-four ME panel detections were not confirmed, totalling 55 discrepant results. After discrepancy analysis, 20 false-positive and 21 false-negative ME panel results remained. Performance varied between pathogens. Sensitivity for HSV-1 was calculated at 82.4% (59.0%–93.8%) with three false-negative results. Also noteworthy were 13 false-negative enterovirus and eight false-positive Streptococcus pneumoniae results.ConclusionsOur analysis shows good performance for the ME panel in diagnosing central nervous system infection. The risk of false-negative HSV-1 results, however, warrants additional testing when encephalitis is suspected. Uncertainties in interpretation of enterovirus and S. pneumoniae results represent other limitations.     

Acute infectious mononucleosis and coincidental measles virus infection.

BACKGROUND: Both Epstein-Barr and measles viruses (MV) cause immune suppression, and the association of the two viruses is evaluated as life threatening. The cell immune impairment caused by simultaneous Epstein-Barr and measles viral infections was responsible for the complicated course of the disease in all described previously reports and for unfavorable outcomes in most of the cases. Timely diagnosis of coincidental viral infections could be a useful predictor for the clinical course and complications. Diagnosis must be based on an accurate assessment of clinical, hematologic, serologic manifestations and supported by appropriate laboratory methods. Recognizing the infectious etiology of concomitant infections is important for both clinicians and epidemiologists. OBJECTIVE: To describe a case report of a 20-year-old woman previously vaccinated against measles infected with acute mononucleosis and coincidental measles virus infection. STUDY DESIGN: The clinical, routine laboratory, as well as serological and virologic findings of this patient were scrutinized. Special emphasis was placed on the use of RT-PCR/PCR for confirming the involvement of both measles virus and Epstein-Barr virus (EBV) in this patient's illness. RESULTS: Infectious mononucleosis was not suspected at admission to the hospital. The final diagnosis of a concomitant measles virus infection and acute infectious mononucleosis was facilitated using viral serology to detect virus-specific IgG and IgM antibodies and by RT-PCR for the detection of measles virus RNA and EBV DNA from peripheral blood monocyte cells (PBMC). CONCLUSION: The present report highlights the difficulty of diagnosing two coincidental virus infections on clinical grounds. Serological and molecular laboratory methods, specifically the PCR (RT-PCR) analysis, are found to be useful for confirming the concomitant viral infections and proper identification of the infecting pathogens.     

Development and evaluation of a real-time PCR assay for rapid identification and semi-quantitation of measles virus

A real-time PCR assay for measles virus was designed and validated using clinical samples including oral fluids, sera, urines, throat swabs, blood samples, and nasopharyngeal aspirates. The test was specific for measles virus, with a slightly higher sensitivity compared to the conventional nested PCR. Calculation of viral genome number in these samples, by comparison with a standard curve prepared from dilutions of cloned measles virus H gene, indicated that, overall, serum samples tended to have a lower viral load than oral fluid samples, and that the viral load decreased with increasing time after onset of symptoms. The real-time PCR is considered to be a sensitive and specific alternative to the conventional measles PCR, especially in situations where early and rapid diagnosis are important.