Diagnostic Microbiology and Infectious Disease 78 (2014) 229–231
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Virology
Detection of hepatitis B virus infection markers in dried plasma spots among patients in Congo-Brazzaville Enagnon Kazali Alidjinou a,1, Donatien Moukassa b, 1, Famara Sané a, Séraphin Twagirimana Nyenyeli b, Estina Chandrelle Akoko b, Michèle Valy Mountou b, Laurence Bocket a, Jean-Rosaire Ibara c, Didier Hober a,⁎ a b c
Université Lille 2, Faculté de Médecine, CHRU Lille, Laboratoire de virologie EA3610, Lille, France Hôpital Général de Loandjili, Pointe Noire, Congo Centre Hospitalier et Universitaire de Brazzaville (CHUB), Brazzaville, Congo
a r t i c l e
i n f o
Article history: Received 1 June 2013 Received in revised form 31 August 2013 Accepted 2 September 2013 Available online 26 November 2013 Keywords: Dried plasma spots HBV antigens HBV antibodies HBV DNA Congo-Brazzaville
a b s t r a c t The detection of hepatitis B virus (HBV) infection markers by using dried plasma spots from 32 patients living in Congo has been assessed. Considering frozen plasma samples as gold standard, the sensitivity and specificity of HBV serologic markers detection in dried plasma eluted from filter paper were 100%. The sensitivity and the specificity of HBV DNA detection reached 96% and 100%, respectively, with plasma samples dried on filter paper compared to standard samples. Dried plasma samples can represent an alternative to conventional sampling for HBV detection and management of the infection in developing countries. © 2014 Elsevier Inc. All rights reserved.
The diagnosis and monitoring of hepatitis B virus (HBV) infection are based on serologic and molecular markers, the detection of which needs technologies and storage conditions rarely available in developing countries such as sub-Saharan African countries where the infection is highly endemic (Franco et al., 2012). In Republic of Congo-Brazzaville, the epidemiology of HBV infection has not been extensively studied. The prevalence reported by previous studies ranges from 6.5 to 15% (Ibara et al., 1991; Itoua-Ngaporo et al., 1995; Taty-Taty and Yala, 1990). Usually in this country, the panel of HBV markers available in routine analysis is limited to HBs antigen (Ag). The use of dried blood spots (DBS) on filter paper could greatly facilitate sample storage and transport from routine laboratories to reference centres. A few studies have reported the use of DBS for the characterization of some markers of HBV infection (Forbi et al., 2010; Gupta et al., 1992; Jardi et al., 2004; Komas et al., 2010; Lira et al., 2009; Mendy et al., 2005; Mohamed et al., 2013; Villa et al., 1981; Villar et al., 2011; Zoulek et al., 1985). This study aimed to evaluate the relevance of dried plasma samples to investigate the whole panel of HBV markers in patients living in Congo-Brazzaville. Samples were collected from 32 patients over 18 years old attending the hepatology department of Loandjili general hospital
⁎ Corresponding author. Tel.: +33-(0)3-20-44-66-88; fax: +33-(0)3-20-44-48-95. E-mail address: [email protected] (D. Hober). 1 Alidjinou EK and Moukassa D contributed equally to this work. 0732-8893/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2013.09.019
(Congo-Brazzaville), after informed and written consent in accordance with local ethics recommendations. One tube of EDTA-anticoagulated blood was collected by venipuncture. After centrifugation, the plasma was conditioned in 2 ways: as frozen plasma in cryotubes and as dried plasma spots (DPS). DPS were prepared by spotting 30 μL of plasma onto filter paper (PerkinElmer 226 Sample Collection Device For the Collection of Human Blood Spots, PerkinElmer Health Sciences, Greenville, USA) in 9-mm diameter-preprinted circles. Filter papers were air dried for 1 h, packed individually in a paper envelope, and then stored at room temperature. Cryotubes were stored at −80 °C. Samples were shipped to the Laboratory of Virology in Lille (France) for analysis. For serologic markers, DPS was eluted as previously described (Mendy et al., 2005) with some modifications. Briefly, each circle was punched from the filter paper. It was then incubated at room temperature in 150 μL of elution buffer (PBS+ 0.05% of Tween 20) for 30 min on a rotating device and then overnight without agitation. After a 14,000 rpm centrifugation for 5 min, the supernatant was collected and used for testing. Considering that 30 μL of dried plasma was recovered in 150 μL of buffer, the dilution factor of plasma in eluted samples was 5. The elution buffer has been tested before use successfully. For each patient, plasma, 5-fold diluted plasma, and DPS eluate were tested for serologic markers (100 μL for each). HBs Ag, anti-HBs, and Anti-HBc antibodies (Ab) were performed using Enzygnost® ELISA tests (Siemens Healthcare Diagnostics, Saint-Denis Cedex, France). HBe Ag and Anti-HBe Ab were tested with Architect® assays (Abbbott Diagnostics, Rungis Cedex, France). HBV DNA from the DPS was eluted using TECK (Tris EDTA–
E.K. Alidjinou et al. / Diagnostic Microbiology and Infectious Disease 78 (2014) 229–231
Table 1 Detection of HBV serological markers in plasma and dried plasma samples. Serologic markers
Plasma (n = 32)
1/5 Diluted plasma (n = 32)
DPS eluate (n = 32)
Sensitivity (%)
Specificity (%)
HBs Ag
26 6 5 27 32 0 7 25 9 23
26 6 5 27 32 0 7 25 9 23
26 6 5 27 32 0 7 25 9 23
100
100
100
100
100
NA
100
100
100
100
HBe Ag Anti-HBc Ab Anti-HBs Ab Anti-HBe Ab
Pos Neg Pos Neg Pos Neg Pos Neg Pos Neg
Serologic markers were detected in 3 types of samples. Sensitivity and specificity of DPS were calculated. NA = non-applicable.
sodium chloride–proteinase K, pH 8) buffer, as previously described (Vauloup-Fellous et al., 2006). One spot was cut and placed in a 2-mL tube; 400 μL of TECK buffer was added, and the tube was incubated at 53 °C for 1 hour. Then, tubes were centrifuged twice at 14,000 rpm, 20 °C for 2 min to obtain 350 μL of supernatant and to eliminate paper residues. 650 μL of phosphate-buffered saline (PBS) solution was added to the eluate to reach 1000 μL that is needed for the molecular detection sampling. Considering that 30 μL of dried plasma was recovered in 400 μL of buffer and that 350 μL of this buffer was diluted in 650 μL of PBS, the dilution factor was 38. DNA extraction and quantitative detection of HBV DNA were performed using the COBAS® AmpliPrep/COBAS® Taqman® HBV test v1.0 (Roche Diagnostics, Meylan Cedex, France) in both plasma and DPS eluate. Plasma was used as reference to calculate the sensitivity and specificity of markers detection in DPS, and the correlation between quantitative values was determined. The same pattern of serologic results was observed for the 3 types of samples. HBs Ag was detected in 26 patients, and among them, 5 were positive for HBe Ag. In the 3 types of samples, anti-HBs Ab was positive for 7 patients including 1 patient positive for HBs Ag, whereas anti-HBc and anti-HBe Ab were found positive, respectively, in 32 and 9 patients (see Table 1). There was a correlation of absorbance values for HBs Ag or relative light unit ratios for HBe Ag in plasma and DPS eluate with a Spearman correlation coefficient r of 0.89 and 0.8, respectively, for HBs and HBe antigens. HBV DNA was detected in plasma from the 26 patients who were HBs Ag positive. HBV DNA was found positive in dried plasma eluate of 25 out of these 26 subjects, giving a sensitivity of 96% and a specificity of 100% for DPS. The only 1 patient negative in DPS had in plasma a low rate of HBV DNA, below the quantification limit. In 2 patients, HBV DNA was detected but unquantifiable in any sample (plasma or DPS). In 3 other patients, the viral load in plasma was 152, 250, and 1727 IU/mL, respectively, whereas HBV DNA could not be quantified in DPS. HBV DNA was
Table 2 Detection and quantification of HBV DNA in plasma and dried samples. Samples
Frozen plasma (n = 32)
Dried plasma eluate (n =32)
Dried plasma eluate, normalized data (n =32)
HBV DNA detecteda HBV DNA quantifiedb Median viral load Interquartiles (25%, 75%) Mean viral load SD
26 23 12606 433.8, 2.4.105 1.7.107 3.107
25 20 1489 2820, 2.1.107 5.1.105 1.8.106
25 20 56563 16483; 9.2.106 1.9.107 6.8.107
The detection and quantification of HBV DNA were performed in plasma and DPS. The viral load is expressed as international units per milliliter. a The detection limit was 12 IU/mL. b The quantification limit was 54 IU/mL.
7
HBV DNA in DPS (log10 IU/mL)
230
6 5 4 3 2 1 2
3
4
5
6
7
8
9
HBV DNA in plasma (log10 IU/mL) Fig. 1. Individual representation of viral load in plasma and dried samples from 20 patients. For each patient, the viral load value in frozen sample was plotted on the X-axis, and the one in dried sample was plotted on the Y-axis. Viral loads were expressed in log10 IU/mL. The Spearman correlation coefficient r was 0.84.
successfully quantified in both plasma and DPS for 20 patients. The mean viral load in plasma was 33-fold higher than in DPS (see Table 2); however, the difference was not statistically significant when the values obtained in DPS were corrected by the dilution factor 38. Thereafter, for the 20 patients with quantified HBV DNA in the 2 types of samples, a log-log plot between HBV-DNA concentrations in DPS versus plasma was drawn (see Fig. 1) and found a linear correlation (R 2 = 0.61) between the 2 groups. The Spearman correlation coefficient r was 0.84. This is the first report regarding the detection of both serologic and molecular markers in dried plasma samples (DPS). Our results demonstrate an excellent specificity (100%) and sensitivity (100%) of DPS in HBV antigens and antibodies detection by using automated and standardized commercial assays. Moreover, we assessed in this study the use of dried samples for the whole panel of HBV serologic markers, whereas other reports focused mainly on HBs antigen or 3 serologic markers (Mohamed et al., 2013; Villar et al., 2011). Previous studies have evaluated dried blood samples for the detection of HBV serologic markers using various methods such as radioimmunoassay (Villa et al., 1981; Zoulek et al., 1985), immunochromatographic assay (Mendy et al., 2005), or enzyme-linked immunosorbent assay (Forbi et al., 2010; Mohamed et al., 2013; Villar et al., 2011) with reported sensitivities ranging from 78% to 96% and specificities from 88.6% to 100%. The higher performance in this study compared to others can be due to the spotting of plasma instead of whole blood on filter paper. The elution buffer used could also explain these results. Interestingly, in the present study, the spots were made in field conditions in tropical Africa and were not deteriorated by storage at ambient temperature (N25 °C) for more than 2 months. DPS showed very good sensitivity and specificity in HBV DNA detection. The discrepancies in HBV DNA quantification between plasma and DPS were probably due to the dilution of recovered DNA in elution buffer, since there was no statistic difference between the mean viral loads after correction with the dilution factor. In conclusion, filter paper is an efficient support of dried plasma for the detection of HBV infection markers even if this method needs to be improved for optimal quantification of HBV DNA and assessed by a large scale study.
Acknowledgments This study was supported by University Lille 2 and CHRU Lille, France, and Loandjili General Hospital, Pointe Noire, Congo.
E.K. Alidjinou et al. / Diagnostic Microbiology and Infectious Disease 78 (2014) 229–231
References Forbi JC, Obagu JO, Gyar SD, Pam CR, Pennap GR, Agwale SM. Application of dried blood spot in the sero-diagnosis of hepatitis B infection (HBV) in an HBV hyper-endemic nation. Ann Afr Med 2010;9:44–5. Franco E, Bagnato B, Marino MG, Meleleo C, Serino L, Zaratti L. Hepatitis B: epidemiology and prevention in developing countries. World J Hepatol 2012;4(3):74–80. Gupta BP, Jayasuryan N, Jameel S. Direct detection of hepatitis B virus from dried blood spots by polymerase chain reaction amplification. J Clin Microbiol 1992;30: 1913–6. Ibara JR, Itoua-Ngaporo A, Maniez-Montreuil M, Dupressoir MV, Huart JJ, Paris JC. Serological markers of hepatitis B virus in hospitalized patients (Gastroenterology and Internal Medicine Service of the University Hospital Center of BrazzavilleCongo). Med Trop 1991;51(1):9–13. Itoua-Ngaporo A, Sapoulou MV, Ibara JR, Iloki LH, Denis F. Prevalence of hepatitis B viral markers in a population of pregnant women in Brazzaville (Congo). J Gynecol Obstet Biol Reprod 1995;24:534–6. Jardi R, Rodriguez-Frias F, Buti M, Schaper M, Valdes A, Martinez M, et al. Usefulness of dried blood samples for quantification and molecular characterization of HBV-DNA. Hepatology 2004;40:133–9. Komas NP, Baï-Sepou S, Manirakiza A, Léal J, Béré A, Le Faou A. The prevalence of hepatitis B virus markers in a cohort of students in Bangui, Central African Republic. BMC Infect Dis 2010;10:226.
231
Lira R, Maldonado-Rodriguez A, Rojas-Montes O, Ruiz-Tachiquin M, Torres-Ibarra R, Cano-Dominguez C. Use of dried blood samples for monitoring hepatitis B virus infection. Virol J 2009;6:153. Mendy M, Kirk GD, van der Sande M, Jeng-Barry A, Lesi OA, Hainaut P, et al. Hepatitis B surface antigenaemia and alpha-foetoprotein detection from dried blood spots: applications to field-based studies and to clinical care in hepatitis B virus endemic areas. J Viral Hepat 2005;12(6):642–7. Mohamed S, Raimondo A, Pénaranda G, Camus C, Ouzan D, Ravet S, et al. Dried blood spot sampling for hepatitis B virus serology and molecular testing. PLoS One 2013;8(4):e61077. Taty-Taty R, Yala F. Carrier state for HBs antigen and HBc antibody in Brazzaville (Congo): sero-epidemiological study in the hospital and non hospital environment. Bull Soc Pathol Exot 1990;83:149–54. Vauloup-Fellous C, Dubreuil P, Grangeot-Keros L. Optimisation of retrospective diagnosis of cytomegalovirus congenital infection from dried blood spots. Pathol Biol 2006;54:551–5. Villa E, Cartolari R, Bellentani S, Rivasi P, Casolo G, Manenti F. Hepatitis B virus markers on dried blood spots. A new tool for epidemiological research. J Clin Pathol 1981;34(7):809–12. Villar LM, de Oliveira JC, Cruz HM, Yoshida CF, Lampe E, Lewis-Ximenez LL. Assessment of dried blood spot samples as a simple method for detection of hepatitis B virus markers. J Med Virol 2011;83(9):1522–9. Zoulek G, Burger P, Deinhardt F. Markers of hepatitis viruses A and B: direct comparison between whole serum and blood spotted on filter-paper. Bull World Health Organ 1985;63:935–9.
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Virology
Detection of hepatitis B virus infection markers in dried plasma spots among patients in Congo-Brazzaville Enagnon Kazali Alidjinou a,1, Donatien Moukassa b, 1, Famara Sané a, Séraphin Twagirimana Nyenyeli b, Estina Chandrelle Akoko b, Michèle Valy Mountou b, Laurence Bocket a, Jean-Rosaire Ibara c, Didier Hober a,⁎ a b c
Université Lille 2, Faculté de Médecine, CHRU Lille, Laboratoire de virologie EA3610, Lille, France Hôpital Général de Loandjili, Pointe Noire, Congo Centre Hospitalier et Universitaire de Brazzaville (CHUB), Brazzaville, Congo
a r t i c l e
i n f o
Article history: Received 1 June 2013 Received in revised form 31 August 2013 Accepted 2 September 2013 Available online 26 November 2013 Keywords: Dried plasma spots HBV antigens HBV antibodies HBV DNA Congo-Brazzaville
a b s t r a c t The detection of hepatitis B virus (HBV) infection markers by using dried plasma spots from 32 patients living in Congo has been assessed. Considering frozen plasma samples as gold standard, the sensitivity and specificity of HBV serologic markers detection in dried plasma eluted from filter paper were 100%. The sensitivity and the specificity of HBV DNA detection reached 96% and 100%, respectively, with plasma samples dried on filter paper compared to standard samples. Dried plasma samples can represent an alternative to conventional sampling for HBV detection and management of the infection in developing countries. © 2014 Elsevier Inc. All rights reserved.
The diagnosis and monitoring of hepatitis B virus (HBV) infection are based on serologic and molecular markers, the detection of which needs technologies and storage conditions rarely available in developing countries such as sub-Saharan African countries where the infection is highly endemic (Franco et al., 2012). In Republic of Congo-Brazzaville, the epidemiology of HBV infection has not been extensively studied. The prevalence reported by previous studies ranges from 6.5 to 15% (Ibara et al., 1991; Itoua-Ngaporo et al., 1995; Taty-Taty and Yala, 1990). Usually in this country, the panel of HBV markers available in routine analysis is limited to HBs antigen (Ag). The use of dried blood spots (DBS) on filter paper could greatly facilitate sample storage and transport from routine laboratories to reference centres. A few studies have reported the use of DBS for the characterization of some markers of HBV infection (Forbi et al., 2010; Gupta et al., 1992; Jardi et al., 2004; Komas et al., 2010; Lira et al., 2009; Mendy et al., 2005; Mohamed et al., 2013; Villa et al., 1981; Villar et al., 2011; Zoulek et al., 1985). This study aimed to evaluate the relevance of dried plasma samples to investigate the whole panel of HBV markers in patients living in Congo-Brazzaville. Samples were collected from 32 patients over 18 years old attending the hepatology department of Loandjili general hospital
⁎ Corresponding author. Tel.: +33-(0)3-20-44-66-88; fax: +33-(0)3-20-44-48-95. E-mail address: [email protected] (D. Hober). 1 Alidjinou EK and Moukassa D contributed equally to this work. 0732-8893/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2013.09.019
(Congo-Brazzaville), after informed and written consent in accordance with local ethics recommendations. One tube of EDTA-anticoagulated blood was collected by venipuncture. After centrifugation, the plasma was conditioned in 2 ways: as frozen plasma in cryotubes and as dried plasma spots (DPS). DPS were prepared by spotting 30 μL of plasma onto filter paper (PerkinElmer 226 Sample Collection Device For the Collection of Human Blood Spots, PerkinElmer Health Sciences, Greenville, USA) in 9-mm diameter-preprinted circles. Filter papers were air dried for 1 h, packed individually in a paper envelope, and then stored at room temperature. Cryotubes were stored at −80 °C. Samples were shipped to the Laboratory of Virology in Lille (France) for analysis. For serologic markers, DPS was eluted as previously described (Mendy et al., 2005) with some modifications. Briefly, each circle was punched from the filter paper. It was then incubated at room temperature in 150 μL of elution buffer (PBS+ 0.05% of Tween 20) for 30 min on a rotating device and then overnight without agitation. After a 14,000 rpm centrifugation for 5 min, the supernatant was collected and used for testing. Considering that 30 μL of dried plasma was recovered in 150 μL of buffer, the dilution factor of plasma in eluted samples was 5. The elution buffer has been tested before use successfully. For each patient, plasma, 5-fold diluted plasma, and DPS eluate were tested for serologic markers (100 μL for each). HBs Ag, anti-HBs, and Anti-HBc antibodies (Ab) were performed using Enzygnost® ELISA tests (Siemens Healthcare Diagnostics, Saint-Denis Cedex, France). HBe Ag and Anti-HBe Ab were tested with Architect® assays (Abbbott Diagnostics, Rungis Cedex, France). HBV DNA from the DPS was eluted using TECK (Tris EDTA–
E.K. Alidjinou et al. / Diagnostic Microbiology and Infectious Disease 78 (2014) 229–231
Table 1 Detection of HBV serological markers in plasma and dried plasma samples. Serologic markers
Plasma (n = 32)
1/5 Diluted plasma (n = 32)
DPS eluate (n = 32)
Sensitivity (%)
Specificity (%)
HBs Ag
26 6 5 27 32 0 7 25 9 23
26 6 5 27 32 0 7 25 9 23
26 6 5 27 32 0 7 25 9 23
100
100
100
100
100
NA
100
100
100
100
HBe Ag Anti-HBc Ab Anti-HBs Ab Anti-HBe Ab
Pos Neg Pos Neg Pos Neg Pos Neg Pos Neg
Serologic markers were detected in 3 types of samples. Sensitivity and specificity of DPS were calculated. NA = non-applicable.
sodium chloride–proteinase K, pH 8) buffer, as previously described (Vauloup-Fellous et al., 2006). One spot was cut and placed in a 2-mL tube; 400 μL of TECK buffer was added, and the tube was incubated at 53 °C for 1 hour. Then, tubes were centrifuged twice at 14,000 rpm, 20 °C for 2 min to obtain 350 μL of supernatant and to eliminate paper residues. 650 μL of phosphate-buffered saline (PBS) solution was added to the eluate to reach 1000 μL that is needed for the molecular detection sampling. Considering that 30 μL of dried plasma was recovered in 400 μL of buffer and that 350 μL of this buffer was diluted in 650 μL of PBS, the dilution factor was 38. DNA extraction and quantitative detection of HBV DNA were performed using the COBAS® AmpliPrep/COBAS® Taqman® HBV test v1.0 (Roche Diagnostics, Meylan Cedex, France) in both plasma and DPS eluate. Plasma was used as reference to calculate the sensitivity and specificity of markers detection in DPS, and the correlation between quantitative values was determined. The same pattern of serologic results was observed for the 3 types of samples. HBs Ag was detected in 26 patients, and among them, 5 were positive for HBe Ag. In the 3 types of samples, anti-HBs Ab was positive for 7 patients including 1 patient positive for HBs Ag, whereas anti-HBc and anti-HBe Ab were found positive, respectively, in 32 and 9 patients (see Table 1). There was a correlation of absorbance values for HBs Ag or relative light unit ratios for HBe Ag in plasma and DPS eluate with a Spearman correlation coefficient r of 0.89 and 0.8, respectively, for HBs and HBe antigens. HBV DNA was detected in plasma from the 26 patients who were HBs Ag positive. HBV DNA was found positive in dried plasma eluate of 25 out of these 26 subjects, giving a sensitivity of 96% and a specificity of 100% for DPS. The only 1 patient negative in DPS had in plasma a low rate of HBV DNA, below the quantification limit. In 2 patients, HBV DNA was detected but unquantifiable in any sample (plasma or DPS). In 3 other patients, the viral load in plasma was 152, 250, and 1727 IU/mL, respectively, whereas HBV DNA could not be quantified in DPS. HBV DNA was
Table 2 Detection and quantification of HBV DNA in plasma and dried samples. Samples
Frozen plasma (n = 32)
Dried plasma eluate (n =32)
Dried plasma eluate, normalized data (n =32)
HBV DNA detecteda HBV DNA quantifiedb Median viral load Interquartiles (25%, 75%) Mean viral load SD
26 23 12606 433.8, 2.4.105 1.7.107 3.107
25 20 1489 2820, 2.1.107 5.1.105 1.8.106
25 20 56563 16483; 9.2.106 1.9.107 6.8.107
The detection and quantification of HBV DNA were performed in plasma and DPS. The viral load is expressed as international units per milliliter. a The detection limit was 12 IU/mL. b The quantification limit was 54 IU/mL.
7
HBV DNA in DPS (log10 IU/mL)
230
6 5 4 3 2 1 2
3
4
5
6
7
8
9
HBV DNA in plasma (log10 IU/mL) Fig. 1. Individual representation of viral load in plasma and dried samples from 20 patients. For each patient, the viral load value in frozen sample was plotted on the X-axis, and the one in dried sample was plotted on the Y-axis. Viral loads were expressed in log10 IU/mL. The Spearman correlation coefficient r was 0.84.
successfully quantified in both plasma and DPS for 20 patients. The mean viral load in plasma was 33-fold higher than in DPS (see Table 2); however, the difference was not statistically significant when the values obtained in DPS were corrected by the dilution factor 38. Thereafter, for the 20 patients with quantified HBV DNA in the 2 types of samples, a log-log plot between HBV-DNA concentrations in DPS versus plasma was drawn (see Fig. 1) and found a linear correlation (R 2 = 0.61) between the 2 groups. The Spearman correlation coefficient r was 0.84. This is the first report regarding the detection of both serologic and molecular markers in dried plasma samples (DPS). Our results demonstrate an excellent specificity (100%) and sensitivity (100%) of DPS in HBV antigens and antibodies detection by using automated and standardized commercial assays. Moreover, we assessed in this study the use of dried samples for the whole panel of HBV serologic markers, whereas other reports focused mainly on HBs antigen or 3 serologic markers (Mohamed et al., 2013; Villar et al., 2011). Previous studies have evaluated dried blood samples for the detection of HBV serologic markers using various methods such as radioimmunoassay (Villa et al., 1981; Zoulek et al., 1985), immunochromatographic assay (Mendy et al., 2005), or enzyme-linked immunosorbent assay (Forbi et al., 2010; Mohamed et al., 2013; Villar et al., 2011) with reported sensitivities ranging from 78% to 96% and specificities from 88.6% to 100%. The higher performance in this study compared to others can be due to the spotting of plasma instead of whole blood on filter paper. The elution buffer used could also explain these results. Interestingly, in the present study, the spots were made in field conditions in tropical Africa and were not deteriorated by storage at ambient temperature (N25 °C) for more than 2 months. DPS showed very good sensitivity and specificity in HBV DNA detection. The discrepancies in HBV DNA quantification between plasma and DPS were probably due to the dilution of recovered DNA in elution buffer, since there was no statistic difference between the mean viral loads after correction with the dilution factor. In conclusion, filter paper is an efficient support of dried plasma for the detection of HBV infection markers even if this method needs to be improved for optimal quantification of HBV DNA and assessed by a large scale study.
Acknowledgments This study was supported by University Lille 2 and CHRU Lille, France, and Loandjili General Hospital, Pointe Noire, Congo.
E.K. Alidjinou et al. / Diagnostic Microbiology and Infectious Disease 78 (2014) 229–231
References Forbi JC, Obagu JO, Gyar SD, Pam CR, Pennap GR, Agwale SM. Application of dried blood spot in the sero-diagnosis of hepatitis B infection (HBV) in an HBV hyper-endemic nation. Ann Afr Med 2010;9:44–5. Franco E, Bagnato B, Marino MG, Meleleo C, Serino L, Zaratti L. Hepatitis B: epidemiology and prevention in developing countries. World J Hepatol 2012;4(3):74–80. Gupta BP, Jayasuryan N, Jameel S. Direct detection of hepatitis B virus from dried blood spots by polymerase chain reaction amplification. J Clin Microbiol 1992;30: 1913–6. Ibara JR, Itoua-Ngaporo A, Maniez-Montreuil M, Dupressoir MV, Huart JJ, Paris JC. Serological markers of hepatitis B virus in hospitalized patients (Gastroenterology and Internal Medicine Service of the University Hospital Center of BrazzavilleCongo). Med Trop 1991;51(1):9–13. Itoua-Ngaporo A, Sapoulou MV, Ibara JR, Iloki LH, Denis F. Prevalence of hepatitis B viral markers in a population of pregnant women in Brazzaville (Congo). J Gynecol Obstet Biol Reprod 1995;24:534–6. Jardi R, Rodriguez-Frias F, Buti M, Schaper M, Valdes A, Martinez M, et al. Usefulness of dried blood samples for quantification and molecular characterization of HBV-DNA. Hepatology 2004;40:133–9. Komas NP, Baï-Sepou S, Manirakiza A, Léal J, Béré A, Le Faou A. The prevalence of hepatitis B virus markers in a cohort of students in Bangui, Central African Republic. BMC Infect Dis 2010;10:226.
231
Lira R, Maldonado-Rodriguez A, Rojas-Montes O, Ruiz-Tachiquin M, Torres-Ibarra R, Cano-Dominguez C. Use of dried blood samples for monitoring hepatitis B virus infection. Virol J 2009;6:153. Mendy M, Kirk GD, van der Sande M, Jeng-Barry A, Lesi OA, Hainaut P, et al. Hepatitis B surface antigenaemia and alpha-foetoprotein detection from dried blood spots: applications to field-based studies and to clinical care in hepatitis B virus endemic areas. J Viral Hepat 2005;12(6):642–7. Mohamed S, Raimondo A, Pénaranda G, Camus C, Ouzan D, Ravet S, et al. Dried blood spot sampling for hepatitis B virus serology and molecular testing. PLoS One 2013;8(4):e61077. Taty-Taty R, Yala F. Carrier state for HBs antigen and HBc antibody in Brazzaville (Congo): sero-epidemiological study in the hospital and non hospital environment. Bull Soc Pathol Exot 1990;83:149–54. Vauloup-Fellous C, Dubreuil P, Grangeot-Keros L. Optimisation of retrospective diagnosis of cytomegalovirus congenital infection from dried blood spots. Pathol Biol 2006;54:551–5. Villa E, Cartolari R, Bellentani S, Rivasi P, Casolo G, Manenti F. Hepatitis B virus markers on dried blood spots. A new tool for epidemiological research. J Clin Pathol 1981;34(7):809–12. Villar LM, de Oliveira JC, Cruz HM, Yoshida CF, Lampe E, Lewis-Ximenez LL. Assessment of dried blood spot samples as a simple method for detection of hepatitis B virus markers. J Med Virol 2011;83(9):1522–9. Zoulek G, Burger P, Deinhardt F. Markers of hepatitis viruses A and B: direct comparison between whole serum and blood spotted on filter-paper. Bull World Health Organ 1985;63:935–9.
