- Email: [email protected]
Use of whole blood dried on filter paper for detection and genotyping of measles virus
Journal of Virological Methods 117 (2004) 97–99
Short communication
Use of whole blood dried on filter paper for detection and genotyping of measles virus Mar´ıa del Mar Mosquera a,∗ , Juan E. Echevarr´ıa a , Sabino Puente c , Félix Lahulla b , Fernando de Ory a a
Centro Nacional de Microbiolog´ıa, Instituto de Salud Carlos III, Carretera de Majadahonda-Pozuelo s/n, 28220 Majadahonda, Madrid, Spain b Hospital Regional de Bata, Bata, Equatorial Guinea c Hospital Carlos III, Madrid, Spain Received 19 August 2003; received in revised form 8 December 2003; accepted 9 December 2003
Abstract 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 chain capture ELISA. Genotype B3 was found in two positive samples by PCR. © 2003 Elsevier B.V. All rights reserved. Keywords: Measles; Polymerase chain reaction; Genotyping; Blood dried on filter paper
Laboratory diagnosis of measles virus infection is usually done by detection of specific IgM in serum samples. Recently, the use of blood samples dried in filter paper have been described as a potential good alternative, useful both for specific IgM (De Swart et al., 2001; Helfand et al., 2001; Riddell et al., 2002), and PCR (De Swart et al., 2001; Katz et al., 2002) for situations when serum cannot be easily obtained and stored. However, these studies were made under well controlled laboratory conditions. The present study shows results on samples obtained in a tropical area without adequate conditions for serum extraction and storing. Two commercial kits were used for the detection of measles virus specific IgM based either on indirect (Dade Behring, Germany) or chain capture (Chemicon USA) ELISA, and a multiplex nested reverse transcription PCR (RTMNPCR), designed for simultaneous detection of measles virus, rubella virus and parvovirus B19 (Mosquera Mdel et al., 2002), with the aim of diagnosing measles virus on dried blood samples. Genotyping measles virus was attempted on positive samples by sequencing a fragment of 456 bp corresponding to the COOH terminus on the nucleoprotein coding region amplified previously by
∗
Corresponding author. Tel.: +34-1-5097901; fax: +34-1-5097966. E-mail address: [email protected] (M.d.M. Mosquera).
0166-0934/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2003.12.004
a different reverse transcription nested PCR, currently under evaluation. Sequences were obtained with an automatic sequencer and compared with reference strains of measles virus genotypes. For this purpose, phylogenetic distance analysis of the aligned sequences was undertaken by the neighbor-joining algorithm as unrooted tree tested with 1000 bootstraps using the MEGA (Molecular Evolutionary Genetics Analysis, Tempe, Arizona, USA) software. Twenty samples were taken from 19 patients (age: 3 months–7.5 years) from a measles outbreak occurred in Equatorial Guinea from February to May of 2001. The specimens were taken during the first 3 days after the exanthema. The samples were collected at the Hospital Regional de Bata in a period of 4 weeks on May and maintained at room temperature (22–30 ◦ C with a relative humidity of 90%) until they were sent to the laboratory in Spain. Finally, they were stored at 4 ◦ C until tested, no more than 2 months later. All samples were tested by RTMNPCR, 14 by indirect ELISA, and 15 by chain capture ELISA. One circular filter paper piece of 3 mm in diameter corresponded to 1 l of serum (Evengard et al., 1988). In each serological assay, the adequate volume of dilution buffer was used to reach the corresponding dilution (1:40 in indirect ELISA and 1:200 in the capture assay). Serological tests were carried out according to manufacturer’s instructions. Both tests included antigen and control antigen wells each
98
M.d.M. Mosquera et al. / Journal of Virological Methods 117 (2004) 97–99
Table 1 Results of IgM and PCR on blood dried on filter paper No. of specimen
Age
PCR SAR
EIA SAR IND
EIA SAR CAP
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16a 17b 18b 19 20
21 months 3 months UN 3 years UN 5 years 6 years UN 8 months UN UN 7.5 years 14 months 5 years 4 months 1 years 7 years 7 years 9 months 6 months
Positive Positive Negative Negative Positive Negative Positive Negative Negative Negative Negative Positive Negative Positive Negative Negative Positive Negative Positive Negative
Equivocal Positive Negative Positive Equivocal NIR NT NT NT Equivocal NT Negative Positive NT Positive NIR Negative Negative Equivocal NT
Positive Positive Negative Positive Positive Equivocal NT NT NT Equivocal NT Positive Positive NT Positive Positive Positive Positive Positive Negative
8/20 (40%)
8/14 (57.1%)
13/14 (86.7%)
Positives/ total (%)
NIR: non-interpretable result; UN: unknown; NT: not tested; PCR SAR: RTMNPCR; EIA SAR IND: indirect IgM measles ELISA; EIA SAR CAP: chain capture IgM measles ELISA. a Patient B19V positive. b Samples from the same patient.
sample. In the indirect ELISA sera showing absorbance values higher than 0.20 were considered positive, equivocal if the absorbance ranged between 0.10 and 0.20, and negative if the absorbance was lower than 0.10. In the capture assay, the cut-off was established as the “absorbance of negative control × 3”. Samples with absorbance values higher than the cut-off were positive, equivocal if the absorbance was between the cut-off and cut-off × 0.8, and negative if the absorbance was lower than cut-off × 0.8. RTMNPCR were carried out with one dried spot, first allowing the filter paper to soak in 500 l of RNAse-free water for 15 min, then extracting 100 l of the blood soaked water as previously described (Casas et al., 1995). The results are shown in Table 1. By the indirect ELISA, four samples were positive, four equivocal, and four negative; the remaining two samples were non-interpretable, since high absorbance values were obtained in the control antigen wells. Both positive and equivocal results indicates recent infection with measles virus (de Ory, 2001; Riddell et al., 2002), thus the sensitivity of indirect ELISA was 57.1%. In relation to the capture assay, 11 samples were positive, 2 equivocal, and 2 negative; the sensitivity was 86.7%. Finally, measles virus RNA was detected in 40% of the samples, what is in agreement with the sensitivity obtained in a previous report (De Swart et al., 2001). B19 viral DNA was detected in one sample (case 16, Table 1), positive for measles virus IgM in the capture assay.
However, this sample was negative when tested for B19 specific IgM by a chain capture ELISA (Biotrin, Ireland). The patient suffered from Plasmodium falciparum infection, and died some days later. Sequencing of the 456 bp fragment containing the variable region of the N gene showed genotype B3 on samples no. 7 and 19, which is one of the genotypes found in this geographical area (Hanses et al., 1999; Kouomou et al., 2002; Mulders et al., 2001; Truong et al., 1999). The capture assay used in this study has been reported as highly sensitive, being able to detect positive results in early samples (de Ory, 2001; Ratnam et al., 2000). However, some problems of specificity have been detected due to false positive results on samples positive for other exanthematic diseases, as rubella, B19 or dengue (de Ory, 2001). This could be the cause of the result obtained by the capture assay on that sample positive for B19 by RTMNPCR. Despite the indirect ELISA being reported previously as sensitive and specific both for sera (de Ory, 2001; Ratnam et al., 2000), and dried blood samples (Helfand et al., 2001; Riddell et al., 2002) it seems to lack of sensitivity. Since high optical densitiy (OD) values were obtained in the control antigen well, causing a non-interpretable or negative results in positive RTMNPCR cases, blood residues in the eluate could act as interfering factors in the indirect ELISA. The use of alternative elution procedures could have avoided this problem (Riddell et al., 2002), but this study was not still available by the time of sample processing. Other factors such as early sampling or bad storing conditions could also account for this problem. RTMNPCR and indirect ELISA seemed to be complementary for diagnosis, since two RTMNPCR positive results were obtained in negative ELISA samples, while four RTMNPCR negative samples were positive or equivocal by indirect ELISA. In summary, dried blood samples are useful for the diagnosis of measles virus infection by serological and PCR approaches, allowing to get the viral genotype by direct sequencing. As sampling of blood on filter paper does not need venipunction and further refrigeration, this simple technique could be useful for the surveillance of measles activity as an alternative of venipunction in remote areas in developing countries or in patients refusing venipunction.
Acknowledgements This study was funded by the Dirección General de Salud Pública and the Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo) under the project “Development of the measles elimination program in Spain” (project number: SBVI 1284-3/02). The authors thank the collaboration of the technicians Teodora Minguito and Pilar Balfagón and the Biopolimers Unit of the National Centre of Microbiology (Instituto de Salud Carlos III).
M.d.M. Mosquera et al. / Journal of Virological Methods 117 (2004) 97–99
References Casas, I., Powell, L., Klapper, P.E., Cleator, G.M., 1995. New method for the extraction of viral RNA and DNA from cerebrospinal fluid for use in the polymerase chain reaction assay. J. Virol. Methods 53 (1), 25–36. de Ory, F., 2001. Evaluation of a capture enzyme-immunoassay (ELISA) for the diagnosis of measles virus infection. Enferm. Infect. Microbiol. Clin. 19 (8), 409–411. De Swart, R.L., Nur, Y., Abdallah, A., Kruining, H., El Mubarak, H.S., Ibrahim, S.A., Van Den Hoogen, B., Groen, J., Osterhaus, A.D., 2001. Combination of reverse transcriptase PCR analysis and immunoglobulin M detection on filter paper blood samples allows diagnostic and epidemiological studies of measles. J. Clin. Microbiol. 39 (1), 270– 273. Evengard, B., Linder, E., Lundbergh, P., 1988. Standardization of a filter-paper technique for blood sampling. Ann. Trop. Med. Parasitol. 82 (3), 295–303. Hanses, F., Truong, A.T., Ammerlaan, W., Ikusika, O., Adu, F., Oyefolu, A.O., Omilabu, S.A., Muller, C.P., 1999. Molecular epidemiology of Nigerian and Ghanaian measles virus isolates reveals a genotype circulating widely in western and central Africa. J. Gen. Virol. 80 (Pt 4), 871–877. Helfand, R.F., Keyserling, H.L., Williams, I., Murray, A., Mei, J., Moscatiello, C., Icenogle, J., Bellini, W.J., 2001. Comparative detection of measles and rubella IgM and IgG derived from fil-
99
ter paper blood and serum samples. J. Med. Virol. 65 (4), 751– 757. Katz, R.S., Premenko-Lanier, M., McChesney, M.B., Rota, P.A., Bellini, W.J., 2002. Detection of measles virus RNA in whole blood stored on filter paper. J. Med. Virol. 67 (4), 596–602. Kouomou, D.W., Nerrienet, E., Mfoupouendoun, J., Tene, G., Whittle, H., Wild, T.F., 2002. Measles virus strains circulating in Central and West Africa: geographical distribution of two B3 genotypes. J. Med. Virol. 68 (3), 433–440. Mosquera Mdel, M., de Ory, F., Moreno, M., Echevarria, J.E., 2002. Simultaneous detection of measles virus, rubella virus, and parvovirus B19 by using multiplex PCR. J. Clin. Microbiol. 40 (1), 111–116. Mulders, M.N., Truong, A.T., Muller, C.P., 2001. Monitoring of measles elimination using molecular epidemiology. Vaccine 19 (17–19), 2245– 2249. Ratnam, S., Tipples, G., Head, C., Fauvel, M., Fearon, M., Ward, B.J., 2000. Performance of indirect immunoglobulin M (IgM) serology tests and IgM capture assays for laboratory diagnosis of measles. J. Clin. Microbiol. 38 (1), 99–104. Riddell, M.A., Leydon, J.A., Catton, M.G., Kelly, H.A., 2002. Detection of measles virus-specific immunoglobulin M in dried venous blood samples by using a commercial enzyme immunoassay. J. Clin. Microbiol. 40 (1), 5–9. Truong, A.T., Kreis, S., Ammerlaan, W., Hartter, H.K., Adu, F., Omilabu, S.A., Oyefolu, A.O., Berbers, G.A., Muller, C.P., 1999. Genotypic and antigenic characterization of hemagglutinin proteins of African measles virus isolates. Virus Res. 62 (1), 89–95.
Short communication
Use of whole blood dried on filter paper for detection and genotyping of measles virus Mar´ıa del Mar Mosquera a,∗ , Juan E. Echevarr´ıa a , Sabino Puente c , Félix Lahulla b , Fernando de Ory a a
Centro Nacional de Microbiolog´ıa, Instituto de Salud Carlos III, Carretera de Majadahonda-Pozuelo s/n, 28220 Majadahonda, Madrid, Spain b Hospital Regional de Bata, Bata, Equatorial Guinea c Hospital Carlos III, Madrid, Spain Received 19 August 2003; received in revised form 8 December 2003; accepted 9 December 2003
Abstract 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 chain capture ELISA. Genotype B3 was found in two positive samples by PCR. © 2003 Elsevier B.V. All rights reserved. Keywords: Measles; Polymerase chain reaction; Genotyping; Blood dried on filter paper
Laboratory diagnosis of measles virus infection is usually done by detection of specific IgM in serum samples. Recently, the use of blood samples dried in filter paper have been described as a potential good alternative, useful both for specific IgM (De Swart et al., 2001; Helfand et al., 2001; Riddell et al., 2002), and PCR (De Swart et al., 2001; Katz et al., 2002) for situations when serum cannot be easily obtained and stored. However, these studies were made under well controlled laboratory conditions. The present study shows results on samples obtained in a tropical area without adequate conditions for serum extraction and storing. Two commercial kits were used for the detection of measles virus specific IgM based either on indirect (Dade Behring, Germany) or chain capture (Chemicon USA) ELISA, and a multiplex nested reverse transcription PCR (RTMNPCR), designed for simultaneous detection of measles virus, rubella virus and parvovirus B19 (Mosquera Mdel et al., 2002), with the aim of diagnosing measles virus on dried blood samples. Genotyping measles virus was attempted on positive samples by sequencing a fragment of 456 bp corresponding to the COOH terminus on the nucleoprotein coding region amplified previously by
∗
Corresponding author. Tel.: +34-1-5097901; fax: +34-1-5097966. E-mail address: [email protected] (M.d.M. Mosquera).
0166-0934/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2003.12.004
a different reverse transcription nested PCR, currently under evaluation. Sequences were obtained with an automatic sequencer and compared with reference strains of measles virus genotypes. For this purpose, phylogenetic distance analysis of the aligned sequences was undertaken by the neighbor-joining algorithm as unrooted tree tested with 1000 bootstraps using the MEGA (Molecular Evolutionary Genetics Analysis, Tempe, Arizona, USA) software. Twenty samples were taken from 19 patients (age: 3 months–7.5 years) from a measles outbreak occurred in Equatorial Guinea from February to May of 2001. The specimens were taken during the first 3 days after the exanthema. The samples were collected at the Hospital Regional de Bata in a period of 4 weeks on May and maintained at room temperature (22–30 ◦ C with a relative humidity of 90%) until they were sent to the laboratory in Spain. Finally, they were stored at 4 ◦ C until tested, no more than 2 months later. All samples were tested by RTMNPCR, 14 by indirect ELISA, and 15 by chain capture ELISA. One circular filter paper piece of 3 mm in diameter corresponded to 1 l of serum (Evengard et al., 1988). In each serological assay, the adequate volume of dilution buffer was used to reach the corresponding dilution (1:40 in indirect ELISA and 1:200 in the capture assay). Serological tests were carried out according to manufacturer’s instructions. Both tests included antigen and control antigen wells each
98
M.d.M. Mosquera et al. / Journal of Virological Methods 117 (2004) 97–99
Table 1 Results of IgM and PCR on blood dried on filter paper No. of specimen
Age
PCR SAR
EIA SAR IND
EIA SAR CAP
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16a 17b 18b 19 20
21 months 3 months UN 3 years UN 5 years 6 years UN 8 months UN UN 7.5 years 14 months 5 years 4 months 1 years 7 years 7 years 9 months 6 months
Positive Positive Negative Negative Positive Negative Positive Negative Negative Negative Negative Positive Negative Positive Negative Negative Positive Negative Positive Negative
Equivocal Positive Negative Positive Equivocal NIR NT NT NT Equivocal NT Negative Positive NT Positive NIR Negative Negative Equivocal NT
Positive Positive Negative Positive Positive Equivocal NT NT NT Equivocal NT Positive Positive NT Positive Positive Positive Positive Positive Negative
8/20 (40%)
8/14 (57.1%)
13/14 (86.7%)
Positives/ total (%)
NIR: non-interpretable result; UN: unknown; NT: not tested; PCR SAR: RTMNPCR; EIA SAR IND: indirect IgM measles ELISA; EIA SAR CAP: chain capture IgM measles ELISA. a Patient B19V positive. b Samples from the same patient.
sample. In the indirect ELISA sera showing absorbance values higher than 0.20 were considered positive, equivocal if the absorbance ranged between 0.10 and 0.20, and negative if the absorbance was lower than 0.10. In the capture assay, the cut-off was established as the “absorbance of negative control × 3”. Samples with absorbance values higher than the cut-off were positive, equivocal if the absorbance was between the cut-off and cut-off × 0.8, and negative if the absorbance was lower than cut-off × 0.8. RTMNPCR were carried out with one dried spot, first allowing the filter paper to soak in 500 l of RNAse-free water for 15 min, then extracting 100 l of the blood soaked water as previously described (Casas et al., 1995). The results are shown in Table 1. By the indirect ELISA, four samples were positive, four equivocal, and four negative; the remaining two samples were non-interpretable, since high absorbance values were obtained in the control antigen wells. Both positive and equivocal results indicates recent infection with measles virus (de Ory, 2001; Riddell et al., 2002), thus the sensitivity of indirect ELISA was 57.1%. In relation to the capture assay, 11 samples were positive, 2 equivocal, and 2 negative; the sensitivity was 86.7%. Finally, measles virus RNA was detected in 40% of the samples, what is in agreement with the sensitivity obtained in a previous report (De Swart et al., 2001). B19 viral DNA was detected in one sample (case 16, Table 1), positive for measles virus IgM in the capture assay.
However, this sample was negative when tested for B19 specific IgM by a chain capture ELISA (Biotrin, Ireland). The patient suffered from Plasmodium falciparum infection, and died some days later. Sequencing of the 456 bp fragment containing the variable region of the N gene showed genotype B3 on samples no. 7 and 19, which is one of the genotypes found in this geographical area (Hanses et al., 1999; Kouomou et al., 2002; Mulders et al., 2001; Truong et al., 1999). The capture assay used in this study has been reported as highly sensitive, being able to detect positive results in early samples (de Ory, 2001; Ratnam et al., 2000). However, some problems of specificity have been detected due to false positive results on samples positive for other exanthematic diseases, as rubella, B19 or dengue (de Ory, 2001). This could be the cause of the result obtained by the capture assay on that sample positive for B19 by RTMNPCR. Despite the indirect ELISA being reported previously as sensitive and specific both for sera (de Ory, 2001; Ratnam et al., 2000), and dried blood samples (Helfand et al., 2001; Riddell et al., 2002) it seems to lack of sensitivity. Since high optical densitiy (OD) values were obtained in the control antigen well, causing a non-interpretable or negative results in positive RTMNPCR cases, blood residues in the eluate could act as interfering factors in the indirect ELISA. The use of alternative elution procedures could have avoided this problem (Riddell et al., 2002), but this study was not still available by the time of sample processing. Other factors such as early sampling or bad storing conditions could also account for this problem. RTMNPCR and indirect ELISA seemed to be complementary for diagnosis, since two RTMNPCR positive results were obtained in negative ELISA samples, while four RTMNPCR negative samples were positive or equivocal by indirect ELISA. In summary, dried blood samples are useful for the diagnosis of measles virus infection by serological and PCR approaches, allowing to get the viral genotype by direct sequencing. As sampling of blood on filter paper does not need venipunction and further refrigeration, this simple technique could be useful for the surveillance of measles activity as an alternative of venipunction in remote areas in developing countries or in patients refusing venipunction.
Acknowledgements This study was funded by the Dirección General de Salud Pública and the Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo) under the project “Development of the measles elimination program in Spain” (project number: SBVI 1284-3/02). The authors thank the collaboration of the technicians Teodora Minguito and Pilar Balfagón and the Biopolimers Unit of the National Centre of Microbiology (Instituto de Salud Carlos III).
M.d.M. Mosquera et al. / Journal of Virological Methods 117 (2004) 97–99
References Casas, I., Powell, L., Klapper, P.E., Cleator, G.M., 1995. New method for the extraction of viral RNA and DNA from cerebrospinal fluid for use in the polymerase chain reaction assay. J. Virol. Methods 53 (1), 25–36. de Ory, F., 2001. Evaluation of a capture enzyme-immunoassay (ELISA) for the diagnosis of measles virus infection. Enferm. Infect. Microbiol. Clin. 19 (8), 409–411. De Swart, R.L., Nur, Y., Abdallah, A., Kruining, H., El Mubarak, H.S., Ibrahim, S.A., Van Den Hoogen, B., Groen, J., Osterhaus, A.D., 2001. Combination of reverse transcriptase PCR analysis and immunoglobulin M detection on filter paper blood samples allows diagnostic and epidemiological studies of measles. J. Clin. Microbiol. 39 (1), 270– 273. Evengard, B., Linder, E., Lundbergh, P., 1988. Standardization of a filter-paper technique for blood sampling. Ann. Trop. Med. Parasitol. 82 (3), 295–303. Hanses, F., Truong, A.T., Ammerlaan, W., Ikusika, O., Adu, F., Oyefolu, A.O., Omilabu, S.A., Muller, C.P., 1999. Molecular epidemiology of Nigerian and Ghanaian measles virus isolates reveals a genotype circulating widely in western and central Africa. J. Gen. Virol. 80 (Pt 4), 871–877. Helfand, R.F., Keyserling, H.L., Williams, I., Murray, A., Mei, J., Moscatiello, C., Icenogle, J., Bellini, W.J., 2001. Comparative detection of measles and rubella IgM and IgG derived from fil-
99
ter paper blood and serum samples. J. Med. Virol. 65 (4), 751– 757. Katz, R.S., Premenko-Lanier, M., McChesney, M.B., Rota, P.A., Bellini, W.J., 2002. Detection of measles virus RNA in whole blood stored on filter paper. J. Med. Virol. 67 (4), 596–602. Kouomou, D.W., Nerrienet, E., Mfoupouendoun, J., Tene, G., Whittle, H., Wild, T.F., 2002. Measles virus strains circulating in Central and West Africa: geographical distribution of two B3 genotypes. J. Med. Virol. 68 (3), 433–440. Mosquera Mdel, M., de Ory, F., Moreno, M., Echevarria, J.E., 2002. Simultaneous detection of measles virus, rubella virus, and parvovirus B19 by using multiplex PCR. J. Clin. Microbiol. 40 (1), 111–116. Mulders, M.N., Truong, A.T., Muller, C.P., 2001. Monitoring of measles elimination using molecular epidemiology. Vaccine 19 (17–19), 2245– 2249. Ratnam, S., Tipples, G., Head, C., Fauvel, M., Fearon, M., Ward, B.J., 2000. Performance of indirect immunoglobulin M (IgM) serology tests and IgM capture assays for laboratory diagnosis of measles. J. Clin. Microbiol. 38 (1), 99–104. Riddell, M.A., Leydon, J.A., Catton, M.G., Kelly, H.A., 2002. Detection of measles virus-specific immunoglobulin M in dried venous blood samples by using a commercial enzyme immunoassay. J. Clin. Microbiol. 40 (1), 5–9. Truong, A.T., Kreis, S., Ammerlaan, W., Hartter, H.K., Adu, F., Omilabu, S.A., Oyefolu, A.O., Berbers, G.A., Muller, C.P., 1999. Genotypic and antigenic characterization of hemagglutinin proteins of African measles virus isolates. Virus Res. 62 (1), 89–95.