We read with great interest the article by Attaran and colleagues in APMIS (1). In their study, in a large group of HBsAg-positive Iranian blood donors, 18% were hepatitis B virus (HBV) DNA positive by PCR, 2% were anti-hepatitis D virus (HDV) Ab positive, and 0.6% were HDV RNA positive by RT-PCR and real-time PCR. Also, authors stated that 16.6% and 83.4% of patients with anti-HDV Ab were coinfected and superinfected with HBV/HDV, respectively (1). The large study population and collection of samples from different provinces of Iran and a complete panel of clinical, molecular, and virological laboratory studies have strengthened the study. However, we believe that there are few ambiguous points regarding the obtained results. First, the result of HBV DNA detection by PCR, which was detected in 18% of HBsAg-positive patients, was not compatible with previous similar studies, which detected HBV DNA in more than 85% of HBsAg-positive blood donors (2, 3). The low rate of positivity for HBV DNA PCR in this study could be as a consequence of unreliable PCR amplification method for detection of HBV DNA. Second, authors found the rate of HDV RNA positivity by RT-PCR to be 27.7% in the anti-HDV Ab-positive patients, which seems to be underestimated in this study. The frequency of HDV RNA positivity in similar group of patients was reported to be more than 60% in other studies (4, 5). This aberrant result can be as a consequence of unreliable pre-analytical procedures including sample processing, transportation, and storage and analytical procedures such as viral RNA extraction, cDNA synthesis, and PCR amplification methods. We should remember this point that HDV is a RNA virus, which is sensitive to the environmental condition especially multiple freeze and thaw of plasma sample. Third, authors used HBV DNA PCR and HDV RNA RT-PCR for differentiation of coinfection from superinfection in their patients, while there is no evidence for using HBV DNA and HDV RNA detection simultaneously for differentiation of coinfection from superinfection in asymptomatic and chronic hepatitis B-infected patients in literature.However, in patients with acute viral hepatitis, presence of anti-HBcAb IgM indicates HBV/HDV coinfection, whereas absence of anti-HBcAb IgM confirms HBV/HDV superinfection (6). We suggest that similar studies should be performed by valid and reliable molecular methods and beside analytical sensitivity (lower limit of detection), clinical sensitivity and specificity of the methods should be considered. Also, differentiation of HBV/HDV coinfection and superinfection should be performed just in acute phase of viral hepatitis by detection of anti-HBcAb IgM.