Use of dried blood samples for monitoring hepatitis B virus infection.
BACKGROUND
Hepatitis B virus (HBV) infection is a problem in several regions of the world with limited resources. Blood samples dried on filter paper (DBS) have been successfully used to diagnose and monitor several infectious diseases. In Mexico there is an urgent need for an affordable and easy sampling method for viral load (VL) testing and monitoring of chronic HBV infection. The purpose of this work was to validate the utility of DBS samples for monitoring HBV infection in patients from Mexico City.


METHODS
Matched samples of plasma and DBS on filter paper from 47 HBV infected patients from the Instituto Mexicano del Seguro Social (IMSS), were included. To evaluate the DNA stability and purity from DBS stored at different temperature conditions, samples from ten patients were stored at 4 degree, 25 degree, and 37 degree C for 7 days. After DBS elution and DNA extraction, the purity of these samples was determined measuring the O.D. rate 260/280. The DBS utility for molecular studies was assessed with PCR assays to amplify a 322 bp fragment from the "a" determinant region of the HBV "S" gene. The VL from all samples was determined to evaluate the correlation between plasma and DBS matched samples.


RESULTS
The quality of the DNA from DBS specimen is not adversely affected by storage at 4 degree, 25 degree and 37 degree C for up 7 days. Statistical ANOVA analyses did not show any significant difference. The same amplification efficiency was observed between DNA templates from samples stored at different temperatures. The Pearson correlation between the VL from DBS and plasma matched samples was 0.93 (p = 0.01). The SD was 1.48 for DBS vs.1.32 for Plasma, and an average of log10 copies/mL of 5.32 vs. 5.53. ANOVA analysis did not show any statistically significant difference between the analyzed groups (p = 0.92).


CONCLUSIONS
The results provide strong evidence that the isolation and quantification of DNA-HBV from DBS is a viable alternative for patient monitoring, and molecular characterization of the virus variants circulating in Mexico.
Unidad de Investigacion Medica en Enfermedades Infecciosas y Parasitarias, Hospital de Pediatria, Centro Medico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Cuauhtemoc 330 Col, Doctores, Delegacion Cuauhtemoc, Mexico City, Mexico. rolica36@yahoo.com
Rosalia
R
Lira
Angelica
A
Maldonado-Rodriguez
Othon
O
Rojas-Montes
Martha
M
Ruiz-Tachiquin
Rocio
R
Torres-Ibarra
Carlos
C
Cano-Dominguez
Hilda
H
Valdez-Salazar
Alejandro
A
Gomez-Delgado
Onofre
O
Muñoz
Ma-Teresa
MT
Alvarez-Muñoz
D000328:Adult; D001769:Blood / Q000821:virology*; D003890:Desiccation; D005260:Female; D006509:Hepatitis B / Q000175:diagnosis*; D006515:Hepatitis B virus / Q000302:isolation & purification*; D006801:Humans; D008297:Male; D008800:Mexico; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*; D013696:Temperature; D013997:Time Factors; D055815:Young Adult