Detection Of Measles Virus Research Articles

Use of whole blood dried on filter paper for detection and genotyping of measles virus

PCR, and two different ELISAs were used to detect measles virus on blood dried on filter paper samples from Equatorial Guinea. Sensitivity was 40% by PCR, 57.1% by indirect ELISA and 86.7% by μ chain capture ELISA. Genotype B3 was found in two positive samples by PCR.

Molecular epidemiology of measles virus in Kenya

Measles causes significant morbidity and mortality globally. Many countries have embarked on immunization programs to control and prevent measles outbreaks and eventually to eliminate endemic measles. Kenya is currently in the outbreak control and prevention stage for measles. Measles virus genotyping is important for molecular epidemiological purposes, including the documentation of the elimination of endemic measles virus strains from a country, and mapping of transmission pathways. In this study, we collected clinical specimens from measles outbreak cases in 2002 in Kenya for measles virus genotyping. We were able to isolate and/or detect measles virus in 10 cases from 5 of the 8 provinces in Kenya. All these Kenyan measles strains were determined to be genotype D4 strains when compared to the standard World Health Organization-designated measles virus reference strains. Interestingly, the Kenyan D4 strains clustered into two distinct D4 subgroups. In addition, the inclusion of other published D4 measles strains in this analysis indicated that there are four distinct D4 clusterings, or subgroups: Montreal-like, India-like, Johannesburg-like, and Ethiopia-like. This is the first measles molecular epidemiology study in Kenya and establishes the current endemic measles strain as genotype D4. Importantly, this study shows that the Kenyan D4 strains are distinct from the B3 measles strain found in West Africa and the D4 strains reported in Ethiopia.

Detection of Measles Virus Genomic RNA in Tear Samples from a Patient With Measles Keratitis

To report a case of measles keratitis, in which measles virus genomic RNA was detected in tear samples. Samples of tears and pharyngeal mucus were obtained from a 19-year-old man with active systemic measles on day 7 after illness onset. These specimens were then subjected to reverse-transcriptase polymerase chain reaction (RT-PCR), using measles-specific primers. One week after systemic measles outbreak, the patient presented with a chief complaint of irritation in both eyes. Slit-lamp examination revealed bilateral superficial punctate keratopathy in the central cornea with small diffuse subepithelial infiltrations, and RT-PCR detected measles virus genomic RNA in tears and pharyngeal mucus. The patient was treated with topical 0.1% fluorometholone and 0.5% levofloxacin three times a day for 1 week, and the keratopathy resolved without sequelae. This report demonstrates for the first time the presence of measles virus genomic RNA in the tears of a patient with measles keratitis. It also highlights the utility of RT-PCR for rapid detection of measles RNA in tear or mucus samples. Furthermore, the presence of virus in bodily fluids and secretions signifies the potential contagiousness of this type of keratopathy.

Detection of measles virus in children with ileo-colonic lymphoid nodular hyperplasia, enterocolitis and developmental disorder.

Detection of measles virus in children with ileo-colonic lymphoid nodular hyperplasia, enterocolitis and developmental disorder

Prolonged measles virus shedding in human immunodeficiency virus-infected children, detected by reverse transcriptase-polymerase chain reaction.

A reverse transcriptase-polymerase chain reaction assay was used to detect measles virus RNA in peripheral blood mononuclear cells, urine, and nasopharyngeal specimens from Zambian children during hospitalization and approximately 1-2 months after discharge. Of 47 children, 29 (61.7%) had prolonged measles virus shedding, as defined by detection of measles virus RNA in > or =1 specimen obtained 30-61 days after rash onset. Ten (90.9%) of 11 human immunodeficiency virus (HIV)-infected children had prolonged measles virus shedding, compared with 19 (52.8%) of 36 HIV-uninfected children (P=.02). Prolonged measles virus shedding did not correlate with levels of measles virus-specific antibody. HIV-infected children with measles may have a prolonged infectious period that potentially enhances measles virus transmission and hinders measles control.

No evidence of measles virus in stapes samples from patients with otosclerosis.

Otosclerosis is a localized bone dystrophy of unknown etiology mainly involving the stapes. The hypothesis of a persistent infection by the measles virus was based on the inconstant detection of the virus by various methods, including reverse transcription-PCR (RT-PCR) of patients' stapes samples. The aim of this work was to investigate the presence of the measles virus in stapedial otosclerosis foci by different sensitive methods. Pathologic stapes samples were obtained from 35 patients suffering from otosclerosis. Measles virus detection was performed by (i) cocultures of Vero cells and primary cell cultures of bone samples (n = 7), (ii) immunofluorescence study of these cocultures (n = 3), and (iii) RT-PCR on RNA directly obtained from fresh frozen samples (n = 28) and on RNA extracted from the primary cell cultures (n = 2). Viral genomic regions coding for N (nucleoprotein) and M (matrix) proteins were separately amplified. PCR sensitivity was optimized on the measles virus Edmonston strain. Glyceraldehyde-3-phosphate dehydrogenase mRNA was used as a marker of total RNA recovery. PCR products were tested by Southern blot hybridization technique to improve sensitivity and specificity. PCRs amplifying the M and the N protein genes were able to detect the control measles virus RNA at titers as low as 0.1 and 0.01 50% tissue culture infective dose, respectively. With these highly sensitive methods, we could not evidence the presence of the measles virus in any of our bone samples or primary bone cell cultures. Our results do not confirm the hypothesis of persistent measles virus infection in otosclerosis.

A simplified and standardized neutralization enzyme immunoassay for the quantification of measles neutralizing antibody

A simplified and standardized neutralization enzyme immunoassay (Nt-EIA) was developed to detect measles virus growth in Vero cells and to quantify measles neutralizing antibody. Heat-inactivated sera were diluted serially 4-fold and tested in duplicate. The 50% reduction point (50%RP) of virus growth was calculated using the Reed–Muench formula and the neutralizing antibody titre of test sera was converted into mIU/ml by comparing their 50%RP with that of the international standard serum. The optimal virus input and incubation time were found to be 50–100 plaque forming unit (PFU)/well and 64–72 h, respectively. The simplified Nt-EIA had a good reproducibility with only 3.7–4.2% of duplicate tests having a ratio >4 in an evaluation of intra assay variation and the coefficients of variance were 2–9% in an evaluation of inter assay variation. In addition, the simplified Nt-EIA had a high sensitivity(98.6%), specificity (100%) and agreement (98.8%) in qualitative comparison with plaque reduction neutralization test (PRNT). In quantitative comparison, the correlation coefficient between Nt-EIA and PRNT was 0.83 without log transformation or 0.77 after log transformation and 90% of 61 positive sera had a ratio <4 between antibody titre tested by the two methods. The simplified Nt-EIA is thus a suitable alternative to the PRNT for the quantification of measles neutralizing antibody.

Clinicopathological study on liver dysfunction in measles.

We analyzed the clinical course of eight patients with liver dysfunction in measles. All of the patients showed an elevation of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH), but no jaundice. These levels returned to normal about 3 weeks after the onset of the rash. A percutaneous liver biopsy was done in two cases. Histological examination showed slight necrosis of liver cells but no significant changes in portal area. On electron microscopy, virus particles were not detected. We detected measles virus RNA in the liver specimen by RT-PCR, which suggests that the measles virus affects liver cells directly in measles.

Direct in situ reverse transcriptase polymerase chain reaction for detection of measles virus.

New methods are described for combined intracellular reverse transcription (RT) and polymerase chain reaction (PCR) using single primer pairs, with direct incorporation of digoxigenin-11-dUTP into amplificants (direct in situ RT/PCR). Routinely used fixatives and minimal pre-treatments were employed. Target sequences of measles virus nucleocapsid (N) and phosphoprotein genes were detected within measles virus infected Vero cells, both in suspension and in formalin-fixed sections, that had been treated by in situ reverse transcription and 30 cycles of direct in situ PCR. Uninfected cells, omission of Taq polymerase, and irrelevant primers were used as controls. Distribution of measles virus within infected cells was determined by in situ hybridisation and immunocytochemistry for measles virus N gene and protein, respectively. Confirmation of amplification within sections was by gel electrophoresis, Southern blotting and sequencing of extracted amplicons. In the majority of cases, measles-infected cells exhibited intense cytoplasmic signal after direct in situ PCR; this was not seen in uninfected cells or infected cells reacted either with irrelevant primers or without Taq polymerase. Unfixed cells in suspension required nested reaction. Measles-specific in situ hybridisation and immunocytochemistry gave an identical signal distribution in sections. Nuclear artifact occurred in some sections and was unpredictable, although it was greatest either in areas of cellular damage, following DNase predigestion, or with vigorous protease pre-treatment. In situ RT-PCR is feasible for measles virus in acutely infected cells both in sections and in suspension. Further work is required to improve the procedure and to eliminate artefactual nuclear signal.

The use of microwave irradiation as a pretreatment to in situ hybridization for the detection of measles virus and chicken anaemia virus in formalin-fixed paraffin-embedded tissue.

Microwave irradiation was investigated as a pretreatment to in situ hybridization on formalin-fixed, paraffin-embedded tissue. Two probe/tissue systems were used: a single-stranded RNA probe for the detection of measles virus nucleocapsid genome in subacute sclerosing panencephalitis brain tissue, and a double stranded DNA probe for chicken anaemia virus in thymus of chicken infected with the virus. Microwaving, when used as sole pretreatment, was not as effective as the more traditional enzyme pretreatments for in situ hybridization. However, when used in combination with existing pretreatments, a significant increase was found in hybridization signal in both brain and thymus tissue. This was emphasized when combination enzyme/microwave pretreatments were used prior to detection of measles virus by in situ hybridization in a series of five archival subacute sclerosing panencephalitis cases. The use of microwave irradiation would be recommended as a means of supplementing in situ hybridization methods, especially when using long-term formalin fixed paraffin-embedded tissue.

Failure to Detect Measles Virus Rna, by Reverse Transcription-Polymerase Chain Reaction, in Peripheral Blood Leucocytes of Patients with Multiple Sclerosis

We have examined peripheral blood leucocytes (PBLs) from 17 multiple sclerosis patients, two patients with rheumatoid arthritis, one case of acute childhood measles and one case of subacute sclerosing panencephalitis, as well as 19 healthy adult controls for measles virus (MV) RNA, by the technique of reverse transcription-polymerase chain reaction. MV nucleocapsid gene specific primers were used to amplify all PBL-derived cDNA samples. These proved to be negative with the exception of the sample derived from the acute measles case. Selected cases were examined further, using fusion gene and matrix gene specific primers. MV RNA could not be detected.

Widespread, restricted low-level measles virus infection of brain in a case of subacute sclerosing panencephalitis.

In situ reverse transcriptase-polymerase chain reaction amplification with labeled-probe hybridization (in situ RT-PCR/LPH) was used to detect measles virus RNA within formalin-fixed, paraffin-embedded brain tissue sections from a patient who died with subacute sclerosing panencephalitis (SSPE). Many more infected neurons and oligodendrocytes were detected by in situ RT-PCR/LPH than by immunohistochemistry or by in situ hybridization alone. In addition, infection of vascular endothelial cells was demonstrated only by in situ RT-PCR/LPH. The observation that many cells contained only a few copies of viral RNA without detectable antigen is consistent with a persistent viral infection of the central nervous system. In situ RT-PCR/LPH, combining the sensitivity of PCR with the tissue localization of in situ hybridization, should prove useful in further studies to detect nucleic acids in situ in the central nervous system.

Otosclerosis: a measles virus associated inflammatory disease.

The etiology of otosclerosis is still unknown. Immunohistochemical studies have revealed the characteristics of chronic inflammation in the otospongiotic area. Paramyxoviral structures have been identified by electron microscopy and the expression of measles virus antigen has been observed by immunohistochemistry in active otosclerotic tissue. By use of the polymerase chain reaction, measles virus related sequences have been detected in otosclerotic bone tissue but not in control specimens. The aim of our study was i) to detect measles virus genome in affected patients using a more sensitive PCR system and ii) to search for anti-measles virus IgG in the perilymph. In 13 out of 14 specimens of bone fragments from surgically removed stapedes of patients suffering from otosclerosis, measles virus RNA sequences could be detected while other tissues from the same patients and from a negative control group lacked such sequences. Furthermore, IgG anti-measles virus antibodies were detected in the perilymph of 6 patients. Our results support our previously stated hypothesis that otosclerosis is a measles virus associated disease which provokes a local immune response within the inner ear.

Polymerase chain reaction for detection of measles virus in clinical samples

A rapid and sensitive one-step reverse transcription polymerase chain reaction assay was developed to detect measles virus (MV) in nasal aspirates from patients with suspected MV infection. Oligonucleotide primers and probe were targeted to highly conserved regions of the matrix gene. Assay conditions were optimized to allow detection of as little as 1 PFU of an MV stock whose titer was known. Extraction of RNA from 38 nasal aspirates and then reverse transcription and MV matrix gene amplification yielded a polymerase chain reaction product of the predicted size in 14 of 14 MV culture-positive patients. Matrix gene amplification provides a rapid, sensitive, and specific supplementary assay to the currently available modalities for MV detection.

Failure to detect measles virus sequences in lymphocytes of patients with multiple sclerosis

A role for measles virus (MV) infection in multiple sclerosis (MS) has been suggested by the finding that antibodies to MV are present in the cerebrospinal fluid of patients with MS. Our results do not support a role for MV infection of lymphocytes in MS.

Measles virus infection of cells in perivascular infiltrates in the brain in subacute sclerosing panencephalitis: confirmation by non-radioactive in situ hybridization, immunocytochemistry and electron microscopy.

As part of continuing multidisciplinary studies on the neuropathogenesis of subacute sclerosing panencephalitis (SSPE), in situ hybridisation, immunocytochemistry and electron microscopy were used to detect measles virus nucleic acid, protein and nucleocapsids in brain perivascular infiltrates of three cases. Perivascular cuffing cells which contained measles virus nucleic acid and antigens were found in all cases. Infected cuffs occurred predominantly in areas of general parenchymal cell infection and in many of these a high proportion of the infiltrating cells were infected. Other cuffs in these areas were either uninfected or contained only a few infected cells. Occasional infected cells were also seen in cuffs in non-infected areas. In contrast, no specific immunocytochemical reactions or in situ hybridisation for measles virus was observed in brain tissue from a patient with herpes encephalitis. By electron microscopy viral nucleocapsid, consistent with measles virus, was found within the cytoplasm of plasma cells in the inflammatory cuffs in SSPE brain tissue. Possible explanations for our results are that infiltrates become infected on arrival in the CNS or alternatively, that the infected infiltrates reflect a generalised infection of the reticuloendothelial system. The frequent presence of uninfected cuffs favours the former explanation.

Diagnosis of measles by fluorescent antibody and culture of nasopharyngeal secretions

An indirect fluorescent antibody test (IFA) was evaluated using commercial mouse anti-measles monoclonal antibody and FITC-labeled goat anti-mouse immunoglobulin. For measles isolation, specimens were inoculated into Rhesus monkey kidney (RMK) cells and, when available, CV-1 cells. 381 specimens were tested by IFA and 408 specimens were cultured from patients suspected of having measles. For the 381 specimens tested by both methods, IFA and culture were positive for 31%, culture alone for 14%, IFA alone for 15%, and both negative for 40%, This study indicates that both IFA and culture are required for maximum measles virus detection. Of the positive specimens, 48% were detected either by IFA only (24%) or culture only (24%). IFA was positive in 69% of the culture-positive specimens and therefore, provided rapid diagnosis for many patients.

Comparative study of peroxidase-labelled IgG and anti-IgG for the detection of measles virus antigens

The immunoperoxidase and immunoblotting techniques were used to detect measles virus antigens in nasopharyngeal samples taken from children visiting the Yaounde Health Centre (Cameroon). In order to make these diagnostic tests simpler, faster and less costly, peroxidase-labelled anti-measles IgG were prepared and compared with commercially available peroxidase-labelled mouse anti-IgG. Measles antigens were detected by immunoperoxidase and immunoblotting with the same sensitivity, no matter which type of conjugate was used. Moreover, the anti-measles IgG permitted the reduction of the duration of the test by half.

Postmortem detection of measles virus in non-neural tissues in subacute sclerosing panencephalitis.

Subacute sclerosing panencephalitis, a rare, progressive, fatal central nervous system disease of children, is caused by measles virus. Clinical signs occur months to several years after recovery from acute measles infection. It is not known where the virus persists while the disease is inapparent. Involvement of organs outside the central nervous system has rarely been documented. To search for possible peripheral reservoirs of measles virus we used in situ hybridization to probe for measles virus RNA and immunocytochemical studies to localize measles virus antigens ina variety of organs taken at autopsy from confirmed cases of subacute sclerosing panencephalitis. Seven of 9 cadavers were found to contain measles virus RNA or antigens, or both, in at least one location outside the central nervous system. These sites included lymphoid organs such as thymus, spleen, lymph nodes, and tonsil, suggesting a role for lymphocytes in disease pathogenesis. Virus was also detected in kidney, lung, and glandular tissues such as pancreas, adrenal, and pituitary. These reservoirs may provide the antigenic stimulus leading to the elevated response characteristic for subacute sclerosing panencephalitis.

Detection of viral infection by immunofluorescence in formalin-fixed tissues, pretreated with trypsin.

The presence of viral antigen in sections from formalin-fixed and paraffin-embedded human tissues was demonstrated by trypsin digestion followed by direct or indirect immunofluorescence. The specimens may be used for retrospective diagnosis. The immunofluorescence technique has to be adapted to the suspected virus infection on the basis of previous histopathologic study. Variations of trypsin concentration time and temperature of incubation, expose different viral antigens and have to be previously tested for each unknown system. For measles virus detection in lung a stronger digestion has to be applied as compared to adenovirus or respiratory disease viruses in the same tissue. Flavivirus in liver tissue needs a weaker digestion. The reproducibility of the method makes it useful as a routine technique in diagnosis of virus infection.