Comparative evaluation of measles virus-specific RT-PCR methods through an international collaborative study

Comparison of RT-PCR assays established in house at various places revealed that laboratories could differ in sensitivity by as much as 1,000-fold in terms of the ability to detect measles virus sequences in clinical samples. The study indicates that PCR findings, positive or negative, are questionable if they are not supported by the associated data demonstrating the overall sensitivity of the assay applied. Measles virus-specific RT-PCR-based assays need to be validated using standard virus preparation or nucleic acid-based target templates. A correlation between real-time quantitative PCR and the conventional PCR for measles virus is highly desirable.     

Comparative evaluation of measles virus specific TaqMan PCR and conventional PCR using synthetic and natural RNA templates

Comparative evaluation of TaqMan RT-polymerase chain reaction (PCR) methodology developed during this study with the conventional RT-PCR-nested PCR methodology developed earlier, using measles virus RNA templates derived from synthetic and natural sources against a number of primer sets belonging to various regions of the genome, revealed the existence of similar assay thresholds for both methods. An exception to this finding was, however, noted using primer sets of the N and M genes regions with RNA templates extracted from the wild type measles virus strain where the nested PCR method proved to be 10- to 100-fold more sensitive than the end points established with the N gene specific TaqMan RT-PCR method with synthetic RNA templates. These differences were not evident when the same primer sets were evaluated with RNA templates extracted from a brain sample of SSPE patient. These findings indicate that the genetic make up of measles virus strain in any given clinical specimen, in relation to the amplifying primers/probe sequences, can have impact on the overall sensitivity and specificity of the methodology applied. Both methods are equally suitable for the molecular detection of measles virus sequences in clinical specimens, although the TaqMan RT-PCR method may be preferred due to its advantages of contamination control, automation, and real-time product quantitation.     

Co‐circulation of multiple measles virus genotypes during an epidemic in France in 2008

In 2008, measles reappeared in France in a series of outbreaks. During this period, 604 measles cases were reported to a routine surveillance system and 305 (50%) of these cases were then confirmed in the laboratory. To understand better the current epidemiological situation and the circulation of different measles strains, a phylogenetic characterization of 113 (19%) of the measles cases from these outbreaks was performed. All measles cases met the WHO clinical criteria and were confirmed either by laboratory detection of measles‐specific IgM and/or by detection of the virus genome by polymerase chain reaction (PCR) and viral isolation. PCR products generated from blood, oral fluid, urine, or nasopharyngeal‐swab samples were sequenced for molecular epidemiology studies. Phylogenetic analysis showed a co‐circulation of genotypes D4 and D5 during the first measles outbreak in the city of Reims in early 2008. Over the course of the year, the A, B3.2, D8, and D9 genotypes also appeared. The data from this study show the simultaneous circulation of several measles genotypes in France and describe genotypes D8 and D9 for the first time in this country. The data also suggest that there are still many pockets of unvaccinated individuals helping to maintain the circulation of measles virus in the population. Phylogenetic studies allowed the corroboration of epidemiologic links and showed that nosocomial transmission can create significant risk for measles dissemination. Finally, the pattern of changes in viral genotypes during 2008 suggests a regular introduction of measles strains from abroad. J. Med. Virol. 82:1033–1043, 2010. © 2010 Wiley‐Liss, Inc.     

Simultaneous infection of measles and varicella-zoster virus in a child in India

Simultaneous occurrence of measles and chickenpox in a single individual is a rare event despite the fact that each of these infections alone is very common. The clinical presentation and molecular characterization of a dual infection caused by measles and Varicella-Zoster virus (VZV) in a 3-year female child is reported for the first time from India. The child presented with high fever, cough, cervical lymphadenopathy, and maculopapular rash followed by vesicular skin rash. The child was not immunized against measles and chickenpox. The viral nucleic acids extracted from the clinical specimen were subjected to PCR-Sequencing for confirmation of a dual infection with measles and VZV. The PCR and sequence analysis from the throat swab samples confirmed the coinfection of wild-type measles (genotype D4) and Varicella-Zoster virus (PstI(+) BglI(+)). The measles virus RNA and VZV DNA could be detected successfully from a single specimen of a throat swab. The case recovered uneventfully. Dual infection with measles and VZV does occur but may be underreported in the literature.     

Rapid diversification of measles virus genotypes circulating in Morocco during 2004-2005 epidemics

Measles virus strains circulating in six different regions in Morocco during 2004-2005 were analysed. They were genotyped using two different methods: the recently developed method based on real-time PCR amplification and melting curve analyses, and the conventional method based on nucleic acid sequencing and phylogenetic analysis of 456 nucleotides of the 3'-region of the nucleoprotein (N) gene sequence. Five genotypes (A, B3.2, C2, D7 and D8) were shown to be circulating during this period. Previous studies on measles virus genotypes in Morocco (1998-2003) showed that only the genotype C2 was present and was considered to be endemic. Sequence comparison of the 2004-2005 viruses with other measles strains suggests that measles strains belonging to genotype B3.2 were probably imported from West Africa, whereas those belonging to genotypes D7 and D8 were imported from Europe. These studies which identify the route of importation of measles are important for developing strategies for measles elimination in Morocco.     

Measles virus strains circulating in Ethiopia in 1998-1999: molecular characterisation using oral fluid samples and identification of a new genotype

A measles outbreak in December 1998 in Bedelle (vaccine coverage <40%) and two sporadic cases in Addis Ababa, Ethiopia, were investigated. Paired serum and oral fluid samples were collected 2-8 days after the onset of symptoms. A total of 53 of 55 outbreak cases and both sporadic cases were positive for serum measles virus-specific IgM. Oral fluid measles-specific IgM was positive in 71% of cases collected up to 5 days after onset and in 90% collected at 6-8 days. By contrast, 100% of oral fluid samples were positive for measles virus RNA by RT-PCR, suggesting that early collection of samples favoured the detection of measles virus RNA by RT-PCR. The measles virus strain in the outbreak was identified as genotype D4. One strain from a sporadic case was also genotype D4; the strain from the other sporadic case was assigned to clade D but was distinct. The degree of divergence from recognised clade D strains suggested that, together with three strains from the United Kingdom, it represents an additional genotype of clade D (GenBank accession numbers AF280800-280807).     

Investigation of measles and rubella outbreaks in Tamil Nadu, India-2003

The aims of the present study were to confirm measles outbreaks by detection of measles-specific IgM antibodies, isolation of measles virus, and genetic characterization to document the circulating genotypes in Tamil Nadu. Eight outbreaks were reported from six districts of Tamil Nadu, India during the period Jan-Dec 2003. Blood samples were collected for serology, urine, and throat swabs for virus isolation. Genotypic characterization of measles isolates was based on the sequence of the N gene. All the clinically suspected outbreaks (n = 8) were confirmed by serology; six out of the eight as measles and two as combination of measles and rubella highlighting the need to carry out rubella serology on measles-negative samples. Genetic characterization of three isolates obtained revealed one as genotype D4 and two as D8. Measles genotypes D4 and D8 were found to circulate in three districts of Tamil Nadu. It is necessary to be aware of the circulating genotypes within the geographical area. The information would be valuable to evaluate control measures and identify viral transmission and importation.     

Serological and molecular epidemiology of measles virus outbreaks reported in Ethiopia during 2000-2004

Twenty-eight outbreaks in six regions and two major cities in Ethiopia from 2000 to 2004 were investigated, with the collection of 207 venous blood and/or oral fluid samples. Measles diagnosis was confirmed by detection of measles-specific IgM and/or detection of measles virus by polymerase chain reaction (PCR). Of 176 suspected cases tested for specific measles IgM, 142 (81%) were IgM positive. Suspected cases in vaccinated children were much less likely to be laboratory confirmed than in unvaccinated children (42% vs. 83%, P < 0.0001). Of 197 samples analyzed by RT-PCR measles virus genome was detected in 84 (43%). A total of 58 wild-type measles viruses were characterized by nucleic acid sequence analysis of the nucleoprotein (N) and hemagglutinin (H) genes. Two recognized genotypes (D4 and B3) were identified. Each outbreak comprised only a single genotype and outbreaks of each genotype tended to occur in distinct geographical locations. B3 was first observed in 2002, and has now been the cause of three documented outbreaks near to the border of Sudan. D4 genotype was previously observed in an outbreak in 1999 and occurs in more diverse locations throughout the country. These data yield insights into geographical and age-related sources of continued transmission. Refinement of measles control measures might include targeting older age groups (5-14 years) and strengthening routine immunization particularly where importation of cases is a concern.     

Cocirculation of measles virus genotype B2 and B3.1 in Central African Republic during the 2000 measles epidemic

Many African countries have begun implementation of national programs to eliminate measles by the year 2015. However, measles continues to be endemic in Africa. This study describes the first molecular epidemiological study of measles virus circulating in Central African Republic. Two hundred and ten blood samples were tested for measles IgM. Sixty-seven urine samples were collected during measles outbreak in Bangui in 2000 and 2004 and used for genotyping studies. Two different methods were used to determine measles virus genotypes; the recently described real-time PCR-based method and the nucleotide sequencing and phylogenetic analysis methods. These tests revealed the cocirculation of two distinct viruses in Bangui. The proposed subgroup of the B3 genotype, B3.1 was found in 14 samples. This virus has been found in other neighboring countries. More surprising, genotype B2 was found in samples from four patients. The first measles genotype B2 viruses were isolated in Gabon in 1984, but have not been detected until recently when they were identified during a measles outbreak in 2003 in South Africa. This suggests that the circulation of measles genotype B2 has continued in Central Africa during the last 20 years. This study provides the baseline for genetic surveillance of measles virus in Central African Republic. Knowledge of currently circulating measles virus genotype in Central African Republic will help in monitoring the success of measles elimination program.     

Real-time PCR for mumps diagnosis on clinical specimens--comparison with results of conventional methods of virus detection and nested PCR.

BACKGROUND: Since November 2003, the UK has seen a dramatic rise in the number of mumps cases, resulting in increasing demands on virology laboratories to confirm mumps infection in a timely and efficient manner. Traditional mumps virus detection methods are often insensitive, lengthy, and cumbersome. Some laboratories in the UK now use molecular methods that are based on nested polymerase chain reaction (PCR). Early serological diagnosis often relies on detection of anti-mumps IgM, which may be absent in the first 10 days of illness. OBJECTIVES: We compared a one-step real-time RT-PCR with an established nested PCR (SH-PCR) and virus detection by culture and antigen detection, and assessed the clinical usefulness of mumps real-time PCR for diagnosis from CSF. STUDY DESIGN: In total, 280 clinical samples were investigated by real-time PCR, nested PCR and a combination of traditional virus detection methods (antigen detection on oral samples, cell culture on all samples). Furthermore, 88 CSF samples submitted for diagnosis of possible viral meningitis were analysed by real-time PCR. RESULTS: The real-time PCR detected the highest number of positive oral samples (119/180) compared to SH-PCR (92/180) and combined virus culture and antigen detection procedures (90/180). Sensitivity of mumps virus detection in urine was poor for all three methods: 34.0% (traditional detection), 29.8% (real-time PCR) and 2.1% (SH-PCR), respectively. Real-time PCR on 88 CSF samples identified five patients with mumps meningitis, significantly increasing viral diagnosis in this cohort. CONCLUSION: Real-time PCR on oral samples is the investigation of choice for mumps infection. Mumps virus detection in urine by any of the PCRs used was clearly less successful. Real-time PCR on CSF samples seems a promising adjunct for diagnosis of mumps meningitis, especially in an age group with high incidence of mumps.     

Detection of measles,mumps, and rubella antibodies in saliva using antibody capture radioimmunoassay

Antibody capture radioimmunoassays were developed for detecting virus specific IgM (MAC-RIA) and IgG (GACRIA) to measles, mumps, and rubella and used to investigate saliva as an alternative specimen to serum for diagnosis. Saliva was collected from 63 patients with measles, 19 with mumps, and 150 with rubella, which were all clinically diagnosed and serologically confirmed. Virus specific IgM was detected in 92% of measles, 75% of mumps, and 100% of rubella saliva samples collected during the first week of illness. Between 1 and 5 weeks after onset virus specific IgM was detected in 100% of saliva specimens. After the 5th week the proportion of reactive specimens declined. The specificity of the MACRIA tests was established by testing saliva samples collected from blood donors for measles (88), mumps (88), and rubella IgM (91). All of the saliva specimens tested for measles and rubella specific IgM were unreactive, 1/88 specimens tested for mumps specific IgM contained significant reactivity. Saliva specimens collected from acute cases of MMR were tested in all 3 MACRIAs. A small proportion of saliva samples contained detectable IgM of more than one virus infection. Rubella and measles specific IgG was detected in the saliva of all cases from the 4th or 5th day of illness, respectively. Detection of mumps specific IgG was less successful. We have demonstrated that virus specific IgM can be reliably detected in saliva samples collected from acute cases of measles, mumps, and rubella and identified 1–5 weeks after onset of illness as the optimum time for collection of samples. © 1993 Wiley-Liss, Inc.     

Optimized SYBR green real-time PCR assay to quantify the absolute copy number of measles virus RNAs using gene specific primers

A sensitive and specific RT-QPCR based on real-time analysis of PCR products stained with SYBR green, was designed and carefully optimised to quantify individual measles virus RNA species. Pairs of specific primers were designed to detect N, P, M, F, H, or L sequences. To detect the genome and/or antigenome, two primers were chosen so as to amplify a 221 nt fragment (L-Tr) encompassing L gene end and trailer. Every gene-specific PCR assay was able to detect = 10 copies/sample, with a dynamic range of 4-5 log10 copies. No significant fluorescent signal was detected from non-infected cell cDNA template. When measles virus microccocal nuclease resistant genomic RNA was reverse transcribed, a 1:1 ratio was observed between single gene amplicons except for L-Tr which displayed a 2.6-fold excess over the other genes. This likely reflects the presence of some shorter abortive genome since the use of a plasmid encoding the entire virus genome resulted in 1:1 ratio for L-Tr segment when compared to others amplicons. Thus, this RT-QPCR assay appears suitable for follow-up studies of viral RNA populations during infection and may also be useful for reliable detection of measles virus in clinical samples.     

Absence of detectable measles virus genome sequence in inflammatory bowel disease tissues and peripheral blood lymphocytes

A highly sensitive measles-specific RT-PCR-nested PCR system was established, which consistently amplified measles virus genome sequence from control samples containing as little as 5.5 × 10⁻³ pfu per reaction. This method failed to detect the presence of measles virus in 93 colonoscopic biopsies and 31 peripheral blood lymphocyte preparations, examined and obtained from patients with inflammatory bowel disease (IBD) and noninflammatory controls. All patients had detectable levels of serum neutralization antibody against measles virus. Each biopsy was estimated to have about one million cells, based on the amplification of the beta actin gene. The assay was calibrated by use of a known number of lymphocytes. The method applied was able to amplify measles virus RNA from a nucleic acid mixture equivalent to 18 cells derived from subacute sclerosing panencephalitis (SSPE) brain material. The level of measles RNA present, if any, in the biopsies is therefore at least 50,000-fold less than in SSPE. J. Med. Virol. 55:243–249, 1998. © 1998 Wiley-Liss, Inc.     

Comparison of laboratory diagnostic methods for measles infection and identification of measles virus genotypes in Hong Kong

The sensitivities of IgM detection, virus isolation, and RT‐PCR for the diagnosis of measles infection were assessed using samples collected from confirmed measles cases from 2006 to 2009. The optimal timing of specimen collection and the preferred specimen type(s) for these tests were also determined. IgM detection showed highest sensitivity when serum samples were collected ≥5 days after rash onset. Virus isolation gave the highest sensitivity when samples were collected ≤3 days after rash onset, with nasopharyngeal aspirate being the best specimen type, followed by urine and throat/combined throat and nasal swab. The highest RT‐PCR positive rate (81.0%) was obtained with serum samples collected ≤3 days after rash onset. RT‐PCR positive rate of 100% was observed with throat/combined throat and nasal swab, urine and nasopharyngeal aspirate collected ≤16, 4–16, and 4–7 days after rash onset, respectively. The genotype of each measles case was confirmed by sequencing. It was shown that the predominant measles viruses detected in Hong Kong during 2006–2009 belonged to genotype H1 (subtype a) and these strains were related closely to those detected in China. J. Med. Virol. 82:1773–1781, 2010. © 2010 Wiley‐Liss, Inc.     

Molecular characterization of measles virus circulating in the Indian Ocean Islands during 2005-2006 and in France in 2006

Despite the availability of safe and immunogenic vaccines, measles still causes significant morbidity and mortality especially in Africa. In this study, two measles outbreaks in the Indian Ocean Islands; Mayotte in 2005-2006 and Seychelles in 2006 were studied. Nasopharyngeal swabs, urine and/or blood samples were collected from patients with clinically diagnosed measles. Measles viruses were isolated in four cases from patients in Mayotte. Measles strains circulating in both outbreaks were determined to be genotype D4 when compared to the WHO reference strains. During this time, measles virus was isolated from patients in France and they were also found to belong to the same genotype. The viruses clustered into two distinct D4 subgroups; The Indian Ocean strains were similar to the Montreal-subgroup, whereas the French strains associated with the Johannesburg-subgroup. The Indian Ocean strains formed a homogeneous group. They shared four specific amino acids in the 3' region of the N gene and two amino acids in the H gene, which differed from other genotype D4 viruses. This suggests that the same measles lineage circulated in Mayotte and Seychelles. Sequence comparison of the French isolates with other measles strains showed that they were more closely related to strains circulating in Germany in 2005, which had their origin in Romania. This study provides the baseline for molecular epidemiology of measles virus in Mayotte and Seychelles. The knowledge of circulating measles virus will help in documenting measles elimination program. This report also highlights the fact that progress of measles elimination is blighted continually by the phenomenon of measles importation.