Molecular diagnosis of the human immunodeficiency, Hepatitis B and C viruses among blood donors in Lomé (Togo) by multiplex real time PCR

This study aimed to compare the sensitivity of multiplex PCR to ELISA technique in the instantaneous detection of HBV, HCV and HIVin blood samples from donors of the National blood Transfusion Centre in Togo. A total of 440 blood samplesfrom volunteer were collected and tested by ELISA and multiplex PCR for HBV, HCV and HIV detection. Among the 440 volunteer blood donors, 83% were female and 17% were male. Age range of 20-29 years was more represented (73%). Whereas, multiplex PCR detected more cases of HBV than ELISA (50% vs 33%, P=0.0155);ELISA more detected HCV than PCR (34% vs 3%, P<0.0001) and HIV (26% vs 7%, P<0.0001). Confirming these observations our data showed that multiplex PCR was more sensitive in the detection of HBV. The sensitivity of ELISA for the detection of HCV and HIV was elevated compared to multiplex PCR. Multiplex PCR was more specific that ELISA for the detection of HCV and HIV.Interestingly, our data showed that the gender do not influenced the sensitivity of either ELISA or multiplex PCR to detect these viruses. This study showed the limit of both ELISA and multiplex PCR in the detection of HBV, HCV and HIV.


Introduction
The persistence of the risk of viral infection transmission in blood transfusion is a serious public health problem. To improve the quality of blood products (PSL) and ensure blood safety, a systematic serological screening of Human Immuno deficiency Virus (HIV), Hepatitis B virus (HBV), and Hepatitis C Virus (HCV)were introduced in the selection of blood donors [1][2][3]. In 2009,theprevalenceof these viruses in Togo as reported by the National Blood Transfusion Center were 1.15% for HIV, 4.70% for HBV and 2.52% for HCV [4]. In the same year, in Burkina Faso, this prevalence was 2.21%; 14.96%and8.69% respectively for HIV, HBV and HCV, according to the report in blood donors in Koudougou [5].
In South Africa, prevalence of73.9%and89.4%were reported respectively for HBV and HIV among first blood donors [6]. Despite the measures taken to reduce or eliminate the transmission of these viruses by blood transfusion, the residual post-transfusion risk remains [7]. This persistence is related to the serological box during which the serological techniques are less effective. For this reason, a good transfusion policy based on rigorous screening strategies must be implemented [8]. The molecular methods such as viral genomic diagnosis (DGV) using molecular techniques such as the Polymerase Chain Reaction (PCR) in real time, applied to the detection and quantification of viral genomes, are more sensitive and do not expose to false positives associated with crosscontamination [9]. Indeed, a study on the characterization of the genotype of HCV among 2200 blood donors in the regional blood transfusion center of Ouagadougou in Burkina Faso in 2011 showed the prevalence of HCV antibodies and viral RNA respectively 4.4% and 1.5% by ELISA and PCR [10]. Using the PCR in the detection of the viral genome (DGV) has the advantage of reducing the silent phase of the most sought virus in blood donors. It can be fully automated, and considerably reduce the analysis duration [11]. In Togo, as in almost sub-Saharan Africa, due to the limited financial resources, the DGV is not yet introduced in blood transfusion. In this study we evaluated the effectiveness of multiplex real-time PCR, that can simultaneously detect the three major viruses (HBV, HCV and HIV) diagnosed among blood donors of the National Blood Transfusion Centre of Lomé.

Study population
Voluntary blood donors aged from 18 to 53 years were recruited according to the standard procedure of the National Blood test was performed using two-side Fisher's exact test. The statistical threshold was defined as P ≤ 0.05.

Results
Study population characteristic: in order to compare the sensitivity of ELISA and multiplex PCR in the detection of HBV, HCV and HIV we collected sera from 440 donors aged from 18-53years. Table 1 shows the characteristics of the study population.
Men were more represented than women (82.90% vs 17.10%). The study population could be divided into four age groups as shown in the table. Among them, the age range from 20-29 years was significantly represented than the other groups. With regards to the professional occupation of the donors, students were also more represented compared to people working in the formal sector and in informal sector. Overall, 326 donors were at their first donation while 114 regularly donated the blood. (440) sera were screened by ELISA for the detection of the co-infection of the three viruses. Figure 1 indicates the co-infection rates between HBV/HCV, HBV/HIV, HCV/VIH and HBV/HCV/HIV. According to the figure, the co-infection HBV/HIV was more encountered, in more than 45%. This was followed in the order of importance by the coinfections HBV/HCV and HCV/VIH, rates under 30% and no statistically difference was observed between these frequencies.

Frequency of HBV and HIV infection: four hundred forty
Furthermore, the triple co-infection HBV/HCV/HIV was detected in approximately 10% cases.  Figure 3A, 27% vs 7%, P<0.0001; Figure 4A respectively). Confirming the higher frequencies of positive ELISA in the detection of HCV and HIV, our data showed that ELISA detected more cases of both viruses in male (36% vs 4%, P<0.0001; Figure   3B, 24% vs 4%, P=0.0002; Figure 4B respectively) and female (28% vs 1%, P<0.0001; Figure 3C and 27% vs 12%, P=0.0042; Figure 4C respectively) than multiplex PCR.Unlike HBV and HCV, there was no difference in the frequencies of detection of HIV in male and female by ELISA ( Figure 4D) and by multiplex PCR ( Figure 4E). In contrast, the multiplex PCR was more sensitive than ELISA but this was not statistically different ( Figure   5). For HCV and HIV detection, our data showed that ELISA was sensitive than the multiplex PCR (P<0.0001; Figure 6, Figure 7). In term of specificity, the multiplex PCR was more specific than ELISA for the detection of HCV (Figure 7; P<0.0001) and for HIV ( Figure   5; P=0.0004) detection.

Discussion
This study aimed to evaluate the efficacy of a multiplex real time PCR to simultaneously diagnose the three major viruses HBV, HCV  [13]. The sensitivity and the frequencies of HBV detection by the multiplex PCR were higher than the fourth generation ELISA which was more specific than the multiplex PCR for the detection of HBV. In contrast, ELISA showed more sensitivity for the detection of HCV and HIV than multiplex PCR. Furthermore, multiplex PCR was more specific for the detection of HCV and HIV than ELISA.
In the seroconversion phase, viruses transmitted by blood are