Arch Virol (2016) 161:95–101 DOI 10.1007/s00705-015-2623-8
ORIGINAL ARTICLE
Hepatitis C virus genotypes in Kenya Joseph Mwangi1,4 • Zipporah Nganga4 • Solomon Mpoke2 • Raphael Lihana1 Joyceline Kinyua1 • Nancy Lagat1 • Joseph Muriuki1 • Rency Lel1 • Sheila Kageha1 • Saida Osman1 • Hiroshi Ichimura3
•
Received: 14 November 2013 / Accepted: 21 September 2015 / Published online: 23 October 2015 Ó Springer-Verlag Wien 2015
Abstract Hepatitis C virus is a great public-health concern worldwide. Phylogenetic analysis of the HCV genome has identified six different genotypes that have generally been divided into several subtypes. There is very little information on HCV seroprevalence and genotypes in Kenya. To determine the genotypes of HCV circulating in Kenya, blood donor samples were serologically tested and confirmed by polymerase chain reaction (PCR). Positive samples were cloned and sequenced, and phylogenetic analysis conducted to determine the HCV genotypes. One hundred Murexseropositive samples were re-tested using a passive hemagglutination test, and 16 of these were identified as seropositive. Further testing of all of the samples by
This work was supported financially in part by Kenya Medical Research Institute, Japan International Cooperation Agency and Kanazawa University. The results of this study have not been submitted to any other journal for publication. This research work has received approval from national research and ethical review committees in Kenya. & Joseph Mwangi [email protected]; [email protected] 1
Center for Virus Research, Kenya Medical Research Institute, P.O. Box 54628, Nairobi, Kenya
2
Kenya Medical Research Institute, Nairobi, Kenya
3
Department of Viral Infection and International Health, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan
4
Institute of Tropical Medicine and Infectious Disease, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya
PCR identified only 10 of the 16 samples as positive. Thus, only 10 % (10/100) of the samples were viremic. Six were from females (60 %), and four were from males (40 %). The mean age of the positive donors was considerably low, at 25 ?/- 9 years. Genotypic testing indicated the presence of genotype 1a (10 %) and genotype 2b (90 %). This study reports on HCV genotypes in a blood donor population in Kenya where little had been done to provide information on HCV genotypes.
Introduction Hepatitis C virus (HCV), hepatitis B virus (HBV) and human immunodeficiency virus (HIV) are viruses that cause great public-health concern worldwide. These three viruses cause over 562 million infections globally, affecting 9.4 % of the world population. Approximately 3 % of the world’s population (170 million people) is infected with HCV, which is a serious cause of chronic liver disease that may progress to liver cirrhosis and hepatocarcinoma. Thus, HCV continues to be a major disease burden on the world. The prevalence of HCV varies in different regions of the world. In Europe, the general prevalence of HCV is about 1 % but varies among the different countries [1], ranging from 0.87 % in Belgium [2], 3.2 % and 8.4 %-22.4 % in Italy [3] and 1.3 % in France [4]. In Central and South America, the estimated prevalence of HCV is 6.3 % [5]. In Japan, it is 0.49 %-0.98 % [6, 7], and in Thailand it is 3.25.6 % [8, 9]. In Africa, there have been few studies on genotypes of HCV when compared to other parts of the world, but
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seroprevalence studies have been done in a number of countries. In sub-Saharan Africa, the HCV seroprevalence has been reported to range from 0.1 to 13.8 [13], with some countries reporting a much higher prevalence: 20.7 % in Gabon [10], 17.1 % in Cameroon [11], and 13.9 % in the Republic of Congo [12]. In Egypt, higher seroprevalence rates have been reported, ranging from 15 %-28 % [13, 14]. In the eastern part of Africa, the reported prevalence in Tanzania is 17.3 % among patients, 8.8 %–11.0 % among blood donors [15], and 1.2 % in the general population [16]. In Kenya, results from selected population groups have shown that HCV prevalence ranges from 0.7 % to 0.9 % [17–20], and, in a recent study, a prevalence of 22.2 % was found among injecting drug users in Kenya [20]. Thus, HCV is a public-health problem that needs to be addressed in this part of the world. Transmission of hepatitis C virus has been strongly associated with intravenous and percutaneous drug and needle use in the USA, Europe, Asia and Australia [21–23]. Transfusion of blood products has been a leading cause of transmission of HCV; however, due to improved screening, transmission through transfusions has decreased in most developed countries [24]. However, the situation is different in other parts of the world, where blood transfusion is the most common risk factor [25]. Despite better screening for selecting blood donors, there is still a great need for laboratory tests for HCV screening. Other modes of transmission include sexual transmission [8], hemodialysis [26], and kidney transplantation [27]. Hepatitis C virus shows substantial nucleotide sequence diversity distributed throughout the viral genome [28]. Six different genotypes identified have generally been divided into several subtypes [29]. Interestingly, the distribution and prevalence of HCV genotypes depend on geographical location [30]. So far, the HCV genotype distribution exhibits three broad patterns [31]: one characterized by high genetic diversity, distributed in West Africa, with types 1 and 2 [32, 33, 46], Central Africa, with type 4 [14, 34], and Asia, with types 3 and 6 [35, 36]. The second pattern involves areas with subtypes circulating in specific risk groups such as subtype 3a in drug addicts [37]. The last pattern has areas where a single subtype is present, such as in Egypt, with subtype 4a [38], and South Africa, with subtype 5a [39]. The only study reporting on HCV genotypes in Kenya recently found genotype 1a and 4 among injecting drug users (IDUs) [23]. In the present study, we analyzed the genotype distribution of HCV in Kenya and sequenced samples collected from blood donors. This is the first report on HCV genotypes in Kenya that includes subjects other than injecting drug users.
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Materials and methods Blood donor samples A total of 100 samples were collected from a blood donor centre in Nairobi, Kenya, following standard operation procedures for sample collection. Most of the blood samples were collected from volunteer donors based at institutions of learning or in workplaces. Only those samples that were positive for antibody to HCV by Murex anti– HCV version 4.0 (Abbot Laboratories, Abbott Park, Illinois) were used in the study. Samples were re-tested by PHA (passive hemagglutination test, Abbot Japan) and polymerase chain reaction (PCR) using specific primers. Previously, we had experienced high false positivity with Murex, and we therefore retested the samples with another kit (data not shown). Serological tests were carried out in accordance with the manufacturers’ instructions (Table 1). RNA extraction RNA was extracted from 100 ll of plasma using an extraction kit (SMITEST R&D RNA/DNA, Genome Science Co. Ltd, Tokyo, Japan) according to the manufacturer’s instructions. The extracted nucleic acid was resuspended in 20 ll of RNAse/DNAse-free water and stored at -30°C until use. Polymerase chain reaction and sequencing Extracted HCV RNA was detected by nested PCR using primers KY80 (sense; 5’-GCAGAAAGCGTCTAGCCAT GGCGT-3’) and KY78 (antisense; 5’-CTCGCAAGCACC CTATCAGGCAGT-3’). Second-round PCR was done with hep21b (sense; 5’-GAGTGTYGTRCAGCCTCCAGG-3’) and hep22 (antisense; 5’-GCRACCCAACRCTACTCTCG Table 1 Demographic characteristics of donors and number of isolates from each No.
Sample ID
Sex
Age
No. of isolates
1
663c
F
16
2
2
674c
F
17
1
3
865c
F
18
1
4
1411c
F
50
1
5
1430c
F
24
1
6
1441c
M
29
2
7
1498c
M
22
1
8
1517c
M
33
1
9
1519c
F
23
2
HCV genotypes in Kenya
GCT) primers [40]. This targeted the HCV 5’UTR region. Briefly, extracted viral RNA was subjected to cDNA synthesis and PCR in a single tube with gene-specific primers using a SuperscriptTM III one-step RT-PCR system with a Platinum Taq High Fidelity Kit (Invirtogen, Carlsbad, CA) according to the manufacturer’s instructions. The second PCR was performed with an AmpliTaq Gold PCR Kit (Applied Biosystems, Foster City, CA) as described previously [41, 42]. Detection of amplified products was done by electrophoretic analysis on a 2 % agarose gel containing 0.005 % ethidium bromide, followed by examination under UV light. PCR products were cloned using a TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA). The cloning steps, cloning reaction, chemical transformation, recovery, plating, and analysis of positive clones were done according to the manufacturer’s instructions and as described previously [43]. Plasmid DNA isolation from recombinant E. coli cultures was carried out using a GenElute Plasmid Miniprep Kit according to the manufacturer’s instructions (Sigma, Hilden, Germany) and as reported previously [42]. Sequencing PCR was carried out with the primers M13 F (5’-GTAAAACGACGGCCAG-3’) (forward) and M13 R (5’-CAGGAAACAGCTATGAC-3’) (reverse) according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). DNA purification was carried out by the sodium acetateEDTA method. Briefly, 2 ll of 1.25 M EDTA, 2 ll of 3 mM sodium acetate, and 50 ll of absolute ethanol were mixed in a tube. Twenty ll of the PCR product was added, and the mixture was vortexed and incubated for 15 min. The sample was then centrifuged at 1400 rpm for 20 min, and the supernatant was discarded. Following a washing step with 70 % ethanol, pellets were air-dried, and 20 ll of TSR (template separation reagent) was added. This mixture was heated at 95°C for 30 seconds before loading into a sequencer. DNA sequencing was carried out using DyeDeoxy terminator chemistry on an ABI 310 automatic sequencer (Applied Biosystems, Foster City, CA). At least four clones per sample were analyzed to obtain consensus sequences [43]. Sequences were aligned with subtype reference sequences from the Los Alamos database, using CLUSTAL W (version 1.81). A phylogenetic tree was constructed by the neighbor-joining method, and its reliability was estimated by 1000 bootstrap replications. The profile of the tree was visualized with Tree View PPC version 1.6.5 (Institute of Biomedical and Life Sciences, Scotland, UK).
Results Serology and PCR In this study, PCR testing was carried out on all 100 of the Murex anti-HCV-positive donations. In addition, all 100
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samples were also re-tested by PHA (passive hemagglutination test, Abbot Japan). On retesting with PHA, only 16 of the 100 samples were seropositive. A polymerase chain reaction test on all of the samples identified only 10 of the 16 samples as positive. Thus, only 10 % (10/100) of the serologically positive samples were viremic. Six were from females (60 %), and four were from males (40 %). The majority of the infections (60 %) were in the age group above 22 years, while 40 % were aged below 22 years. The mean age of the positive donors was 25 ?/- 9 years. Phylogenetic analysis of the 5’UTR of HCV virus Phylogenetic analysis of the 5’UTR sequences showed that the HCV strains that were detected belonged to genotypes 1a and 2b, with subtype 2b being more prevalent (Fig. 1). While 10 samples were amplified by PCR, only nine samples were successfully sequenced. Despite several attempts, one sample could not be successfully cloned and sequenced. The isolates formed three clusters: one with isolate 663 alone, clustering with genotype 1a; one with isolates 1441, 1498, 1430 and 159, clustering with genotype 2b; and one with isolates 865, 1519, and 674, also clustering with genotypes 2b.
Discussion This study was carried out to determine HCV genotypes in a blood donor population. Considering the rarity of HCV genotype information in Kenya, this study is among the first reporting on genotypes of HCV. There is limited information on HCV in Kenya, probably because testing for HCV is not routinely done except at blood donor centers for transfusion purposes. Few hospitals in Kenya conduct tests for HCV in clinical settings. However, a few serological studies have been carried out in selected population groups. The prevalence rates available are those from blood donors: 0.2 %-0.9 % [24, 27] and 3.7 %-4.7 % among inpatients [44] and 7.1 % among icteric patients [45]. The highest prevalence of HCV reported in Kenya was found in intravenous drug users, at 22.2 % [20]. The estimated prevalence of HCV for the 33 countries of sub-Saharan Africa is 3.0 % (range, 0.1-3.7 %) [13], indicating a relatively low prevalence. The highest HCV prevalence in Africa has been reported in Egypt, Cameroon and Burundi at 17.5 %, 13.8 %, and 11.3 % respectively [46]. Because information on HCV genotypes is scarce, the results of this study, though not representative, since only a few samples were sequenced, nevertheless provide crucial data on genotypes of HCV, not only in Kenya but in this region as well. Based on the findings of this study we report presence of genotypes 1a and 2b in Kenya (Fig. 1). The predominant
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98 Fig. 1 Genotypes of HCV detected in Kenya (underlined). The rest are reference strains obtained from GenBank. Sample numbers are 663c, 1519c, 1517c, 1441c, 1411c, 1498c, 1674c, 865c and 1430c. GenBank accession numbers for the sequences reported in this study are as follows: DQ460666, FJ710110. FJ710111, FJ710112, FJ710114, FJ710115, FJ710116, FJ710118, FJ710119
J. Mwangi et al.
1000
999
851
D17763 D63821 663c-12F HM641727.1a 654 JQ012250.1a 730 525 AF009606 751 663c-13F 557 D14853 M62321 2.1a 482 633 AY051292 633 M62321.Ia 512 M58335.1b 643 D90208 643 L02836.1b D50409 1498c-3F 159 1411C-9F 132 L34369 121 EF115606.2 70 AY766688 69 AY232746.2b 71 1441C-2Rc 197 1430c-14F 228 1517c-12F EF564605 521 1441c-15Rc 865c-11Rc 345 1519c-8F 635 345 674C-8F X78861.2b 202 AY766672 7 KC197226.2b 260 96 EF564608.2B 119 KC844048.2B 149 KC844048.2b 7 191 1519c-15Rc KF548005.2b 14 D10988 6 AM502643
Isolates from Kenya are bolded and underlined genotype (90 %) in circulation is 2b (Fig. 1). This is also the first study to report the presence of genotype 2b in Kenya. The only other study on HCV genotypes in Kenya reported the presence of genotypes 1a and 4, with genotype 1a being more prevalent, at 73 % [20]. Globally, six major genotypes and more than 83 subtypes of HCV have been identified, with genotypes 1, 2 and 3 being the most prevalent [47, 48]. In Africa, various genotypes have been reported, including 4a in Egypt, 4c and 5a in Central Africa [49], and 3a, 1, 2, 4c and subtype 4g [50] in South Africa. Genotype 4 subtypes 4e, 4c, 4p and 4r have been reported in Gabon [13]. Closer to Kenya, subtype 4a has been reported in Tanzania [51], and genotype 4 in Uganda [52]. It is likely that diverse genotypes of HCV are present in Kenya.
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The presence of genotype 2b in Kenya is of particular interest. HCV genotypes 1a, 1b, and 3a are highly prevalent ‘‘epidemic’’ strains that are found globally [53]. There is glowing evidence to support the origin of genotype 2 being West Africa [54, 55]. The presence of this genotype in Kenya could indicate transmission from western to eastern Africa. In the last few years, bilateral relations between countries in the west and Kenya have been growing significantly. Notably, there are now direct flights to Cameroon, Nigeria and Ghana. A population of West Africans and Congolese from Zaire constitute a significant business community in Kenya, especially in the small-scale market sector. A number of countries in West and Central Africa have reported diverse genotypes in their populations. This
HCV genotypes in Kenya
could explain the emergence of related genotypes in Kenya. Genotype 2, together with 1, 4 and 5, have also been reported recently in Ethiopia [56], probably indicating further movement of this genotype in Eastern African countries. Muasya et al. [20] reported the presence of genotype 4 in a population of injecting drug users in Kenya. In that study, the two genotypes identified, 1a and 4, formed two clusters of related isolates. In the current study, the isolates clustered into three different groups. Since the two study populations are different, the current one being blood donors, it is most likely that HCV genotypes among blood donors could be more diverse than those in the previous group. Sharing of needles and close association among individuals injecting drugs could contribute to similar genotypes among the group, whereas this may not be the case in the diverse population from which blood donations are normally obtained. Although the limitations of the two studies done in Kenya prevent them from providing information about the topography of HCV genotypes, they nevertheless indicate an unfolding situation where diverse genotypes could be present in the country. Notably, Kenya is host to over 600,000 refugees from different countries, including Somalia, Ethiopia, Sudan, and Democratic Republic of the Congo, as well as other countries who have experienced internal civil war and unrest. Moreover, bilateral relations with Far East countries have increased in the recent past, with citizens from countries such as China now playing a major role in the business and construction sector. These populations could constitute a hot spot for divergent genotypes of HCV, perhaps pointing to a scenario where additional genotypes could be identified in Kenya. HCV genotype variation is associated with therapeutic response. Patients infected with HCV genotype 1 respond less well to therapy, while patients infected with HCV type 2 or 3 have been shown to have the best response[57, 58]. Patients with genotype 4 have been shown to respond well to treatment. With increasing identification of divergent genotypes in Kenya and the possibility of further ‘alien ‘genotypes being introduced, there is a need to reassess the treatment options available and further direct re-evaluation of policy guideline on control and management of HCV in Kenya. The average age of the infected population in this study was quite low (25 ?/- 9 years). It is important to note that the donation with subtype 1a detected in this study was from a 16-year-old female, while the majority (60%) of the other HCV-positive donors (subtype 2b) were above 22 years of age (mean 33 years). Blood donations in Kenya are collected mainly from learning institution and workplaces. This represents young and middle age groups in our population, with those below 22 years being high school or
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college donors. Other studies have shown age-related differences in subtype distribution, with patients infected with genotypes 1b and 2 being older than those infected with genotypes 3a and 1a [59]. Genotypes 1a and 3a have been linked to intravenous drug use [59], and the main risk factor for infection by genotypes 1b and 2 is blood transfusion [59]. In this study, the samples analysed were from blood donors, thus confirming the findings of Elghouzzi et al. [60]. Currently, injection drug use is a problem in some settings in Kenya. The only study targeting this population group reported the presence of genotype 1a (73 %) and genotype 4 (27 %) [20]. In the current study, there was only one sample with genotype 1a, while the rest were 2b. This appears to be consistent with the association of different subtypes with different risk factors, as indicated earlier. It would be interesting to see the genotypes and subtypes that are detectable in drug users and their correlation with those in other population groups where the HCV infection rate could be higher.
Conclusion This study provides data on genotypes of HCV in Kenya, where genotypes 1a and 2b were detected in blood donors. More data are required from other population subgroups in order to provide baseline data on the prevalence and genotypes of HCV in Kenya. Acknowledgement We acknowledge the director of the Kenya Medical Research Institute and the director of the Centre for Virus Research, KEMRI, all HIV laboratory staff at the Centre for Virus Research, and the director and staff of the National Blood Transfusion Services. Special thanks to Japan International Corporation and Kanazawa University for funds supporting part of this work. Compliance with ethical standards Conflict of interest No author has a commercial or other association that might pose a conflict of interest.
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ORIGINAL ARTICLE
Hepatitis C virus genotypes in Kenya Joseph Mwangi1,4 • Zipporah Nganga4 • Solomon Mpoke2 • Raphael Lihana1 Joyceline Kinyua1 • Nancy Lagat1 • Joseph Muriuki1 • Rency Lel1 • Sheila Kageha1 • Saida Osman1 • Hiroshi Ichimura3
•
Received: 14 November 2013 / Accepted: 21 September 2015 / Published online: 23 October 2015 Ó Springer-Verlag Wien 2015
Abstract Hepatitis C virus is a great public-health concern worldwide. Phylogenetic analysis of the HCV genome has identified six different genotypes that have generally been divided into several subtypes. There is very little information on HCV seroprevalence and genotypes in Kenya. To determine the genotypes of HCV circulating in Kenya, blood donor samples were serologically tested and confirmed by polymerase chain reaction (PCR). Positive samples were cloned and sequenced, and phylogenetic analysis conducted to determine the HCV genotypes. One hundred Murexseropositive samples were re-tested using a passive hemagglutination test, and 16 of these were identified as seropositive. Further testing of all of the samples by
This work was supported financially in part by Kenya Medical Research Institute, Japan International Cooperation Agency and Kanazawa University. The results of this study have not been submitted to any other journal for publication. This research work has received approval from national research and ethical review committees in Kenya. & Joseph Mwangi [email protected]; [email protected] 1
Center for Virus Research, Kenya Medical Research Institute, P.O. Box 54628, Nairobi, Kenya
2
Kenya Medical Research Institute, Nairobi, Kenya
3
Department of Viral Infection and International Health, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan
4
Institute of Tropical Medicine and Infectious Disease, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya
PCR identified only 10 of the 16 samples as positive. Thus, only 10 % (10/100) of the samples were viremic. Six were from females (60 %), and four were from males (40 %). The mean age of the positive donors was considerably low, at 25 ?/- 9 years. Genotypic testing indicated the presence of genotype 1a (10 %) and genotype 2b (90 %). This study reports on HCV genotypes in a blood donor population in Kenya where little had been done to provide information on HCV genotypes.
Introduction Hepatitis C virus (HCV), hepatitis B virus (HBV) and human immunodeficiency virus (HIV) are viruses that cause great public-health concern worldwide. These three viruses cause over 562 million infections globally, affecting 9.4 % of the world population. Approximately 3 % of the world’s population (170 million people) is infected with HCV, which is a serious cause of chronic liver disease that may progress to liver cirrhosis and hepatocarcinoma. Thus, HCV continues to be a major disease burden on the world. The prevalence of HCV varies in different regions of the world. In Europe, the general prevalence of HCV is about 1 % but varies among the different countries [1], ranging from 0.87 % in Belgium [2], 3.2 % and 8.4 %-22.4 % in Italy [3] and 1.3 % in France [4]. In Central and South America, the estimated prevalence of HCV is 6.3 % [5]. In Japan, it is 0.49 %-0.98 % [6, 7], and in Thailand it is 3.25.6 % [8, 9]. In Africa, there have been few studies on genotypes of HCV when compared to other parts of the world, but
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seroprevalence studies have been done in a number of countries. In sub-Saharan Africa, the HCV seroprevalence has been reported to range from 0.1 to 13.8 [13], with some countries reporting a much higher prevalence: 20.7 % in Gabon [10], 17.1 % in Cameroon [11], and 13.9 % in the Republic of Congo [12]. In Egypt, higher seroprevalence rates have been reported, ranging from 15 %-28 % [13, 14]. In the eastern part of Africa, the reported prevalence in Tanzania is 17.3 % among patients, 8.8 %–11.0 % among blood donors [15], and 1.2 % in the general population [16]. In Kenya, results from selected population groups have shown that HCV prevalence ranges from 0.7 % to 0.9 % [17–20], and, in a recent study, a prevalence of 22.2 % was found among injecting drug users in Kenya [20]. Thus, HCV is a public-health problem that needs to be addressed in this part of the world. Transmission of hepatitis C virus has been strongly associated with intravenous and percutaneous drug and needle use in the USA, Europe, Asia and Australia [21–23]. Transfusion of blood products has been a leading cause of transmission of HCV; however, due to improved screening, transmission through transfusions has decreased in most developed countries [24]. However, the situation is different in other parts of the world, where blood transfusion is the most common risk factor [25]. Despite better screening for selecting blood donors, there is still a great need for laboratory tests for HCV screening. Other modes of transmission include sexual transmission [8], hemodialysis [26], and kidney transplantation [27]. Hepatitis C virus shows substantial nucleotide sequence diversity distributed throughout the viral genome [28]. Six different genotypes identified have generally been divided into several subtypes [29]. Interestingly, the distribution and prevalence of HCV genotypes depend on geographical location [30]. So far, the HCV genotype distribution exhibits three broad patterns [31]: one characterized by high genetic diversity, distributed in West Africa, with types 1 and 2 [32, 33, 46], Central Africa, with type 4 [14, 34], and Asia, with types 3 and 6 [35, 36]. The second pattern involves areas with subtypes circulating in specific risk groups such as subtype 3a in drug addicts [37]. The last pattern has areas where a single subtype is present, such as in Egypt, with subtype 4a [38], and South Africa, with subtype 5a [39]. The only study reporting on HCV genotypes in Kenya recently found genotype 1a and 4 among injecting drug users (IDUs) [23]. In the present study, we analyzed the genotype distribution of HCV in Kenya and sequenced samples collected from blood donors. This is the first report on HCV genotypes in Kenya that includes subjects other than injecting drug users.
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Materials and methods Blood donor samples A total of 100 samples were collected from a blood donor centre in Nairobi, Kenya, following standard operation procedures for sample collection. Most of the blood samples were collected from volunteer donors based at institutions of learning or in workplaces. Only those samples that were positive for antibody to HCV by Murex anti– HCV version 4.0 (Abbot Laboratories, Abbott Park, Illinois) were used in the study. Samples were re-tested by PHA (passive hemagglutination test, Abbot Japan) and polymerase chain reaction (PCR) using specific primers. Previously, we had experienced high false positivity with Murex, and we therefore retested the samples with another kit (data not shown). Serological tests were carried out in accordance with the manufacturers’ instructions (Table 1). RNA extraction RNA was extracted from 100 ll of plasma using an extraction kit (SMITEST R&D RNA/DNA, Genome Science Co. Ltd, Tokyo, Japan) according to the manufacturer’s instructions. The extracted nucleic acid was resuspended in 20 ll of RNAse/DNAse-free water and stored at -30°C until use. Polymerase chain reaction and sequencing Extracted HCV RNA was detected by nested PCR using primers KY80 (sense; 5’-GCAGAAAGCGTCTAGCCAT GGCGT-3’) and KY78 (antisense; 5’-CTCGCAAGCACC CTATCAGGCAGT-3’). Second-round PCR was done with hep21b (sense; 5’-GAGTGTYGTRCAGCCTCCAGG-3’) and hep22 (antisense; 5’-GCRACCCAACRCTACTCTCG Table 1 Demographic characteristics of donors and number of isolates from each No.
Sample ID
Sex
Age
No. of isolates
1
663c
F
16
2
2
674c
F
17
1
3
865c
F
18
1
4
1411c
F
50
1
5
1430c
F
24
1
6
1441c
M
29
2
7
1498c
M
22
1
8
1517c
M
33
1
9
1519c
F
23
2
HCV genotypes in Kenya
GCT) primers [40]. This targeted the HCV 5’UTR region. Briefly, extracted viral RNA was subjected to cDNA synthesis and PCR in a single tube with gene-specific primers using a SuperscriptTM III one-step RT-PCR system with a Platinum Taq High Fidelity Kit (Invirtogen, Carlsbad, CA) according to the manufacturer’s instructions. The second PCR was performed with an AmpliTaq Gold PCR Kit (Applied Biosystems, Foster City, CA) as described previously [41, 42]. Detection of amplified products was done by electrophoretic analysis on a 2 % agarose gel containing 0.005 % ethidium bromide, followed by examination under UV light. PCR products were cloned using a TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA). The cloning steps, cloning reaction, chemical transformation, recovery, plating, and analysis of positive clones were done according to the manufacturer’s instructions and as described previously [43]. Plasmid DNA isolation from recombinant E. coli cultures was carried out using a GenElute Plasmid Miniprep Kit according to the manufacturer’s instructions (Sigma, Hilden, Germany) and as reported previously [42]. Sequencing PCR was carried out with the primers M13 F (5’-GTAAAACGACGGCCAG-3’) (forward) and M13 R (5’-CAGGAAACAGCTATGAC-3’) (reverse) according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). DNA purification was carried out by the sodium acetateEDTA method. Briefly, 2 ll of 1.25 M EDTA, 2 ll of 3 mM sodium acetate, and 50 ll of absolute ethanol were mixed in a tube. Twenty ll of the PCR product was added, and the mixture was vortexed and incubated for 15 min. The sample was then centrifuged at 1400 rpm for 20 min, and the supernatant was discarded. Following a washing step with 70 % ethanol, pellets were air-dried, and 20 ll of TSR (template separation reagent) was added. This mixture was heated at 95°C for 30 seconds before loading into a sequencer. DNA sequencing was carried out using DyeDeoxy terminator chemistry on an ABI 310 automatic sequencer (Applied Biosystems, Foster City, CA). At least four clones per sample were analyzed to obtain consensus sequences [43]. Sequences were aligned with subtype reference sequences from the Los Alamos database, using CLUSTAL W (version 1.81). A phylogenetic tree was constructed by the neighbor-joining method, and its reliability was estimated by 1000 bootstrap replications. The profile of the tree was visualized with Tree View PPC version 1.6.5 (Institute of Biomedical and Life Sciences, Scotland, UK).
Results Serology and PCR In this study, PCR testing was carried out on all 100 of the Murex anti-HCV-positive donations. In addition, all 100
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samples were also re-tested by PHA (passive hemagglutination test, Abbot Japan). On retesting with PHA, only 16 of the 100 samples were seropositive. A polymerase chain reaction test on all of the samples identified only 10 of the 16 samples as positive. Thus, only 10 % (10/100) of the serologically positive samples were viremic. Six were from females (60 %), and four were from males (40 %). The majority of the infections (60 %) were in the age group above 22 years, while 40 % were aged below 22 years. The mean age of the positive donors was 25 ?/- 9 years. Phylogenetic analysis of the 5’UTR of HCV virus Phylogenetic analysis of the 5’UTR sequences showed that the HCV strains that were detected belonged to genotypes 1a and 2b, with subtype 2b being more prevalent (Fig. 1). While 10 samples were amplified by PCR, only nine samples were successfully sequenced. Despite several attempts, one sample could not be successfully cloned and sequenced. The isolates formed three clusters: one with isolate 663 alone, clustering with genotype 1a; one with isolates 1441, 1498, 1430 and 159, clustering with genotype 2b; and one with isolates 865, 1519, and 674, also clustering with genotypes 2b.
Discussion This study was carried out to determine HCV genotypes in a blood donor population. Considering the rarity of HCV genotype information in Kenya, this study is among the first reporting on genotypes of HCV. There is limited information on HCV in Kenya, probably because testing for HCV is not routinely done except at blood donor centers for transfusion purposes. Few hospitals in Kenya conduct tests for HCV in clinical settings. However, a few serological studies have been carried out in selected population groups. The prevalence rates available are those from blood donors: 0.2 %-0.9 % [24, 27] and 3.7 %-4.7 % among inpatients [44] and 7.1 % among icteric patients [45]. The highest prevalence of HCV reported in Kenya was found in intravenous drug users, at 22.2 % [20]. The estimated prevalence of HCV for the 33 countries of sub-Saharan Africa is 3.0 % (range, 0.1-3.7 %) [13], indicating a relatively low prevalence. The highest HCV prevalence in Africa has been reported in Egypt, Cameroon and Burundi at 17.5 %, 13.8 %, and 11.3 % respectively [46]. Because information on HCV genotypes is scarce, the results of this study, though not representative, since only a few samples were sequenced, nevertheless provide crucial data on genotypes of HCV, not only in Kenya but in this region as well. Based on the findings of this study we report presence of genotypes 1a and 2b in Kenya (Fig. 1). The predominant
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98 Fig. 1 Genotypes of HCV detected in Kenya (underlined). The rest are reference strains obtained from GenBank. Sample numbers are 663c, 1519c, 1517c, 1441c, 1411c, 1498c, 1674c, 865c and 1430c. GenBank accession numbers for the sequences reported in this study are as follows: DQ460666, FJ710110. FJ710111, FJ710112, FJ710114, FJ710115, FJ710116, FJ710118, FJ710119
J. Mwangi et al.
1000
999
851
D17763 D63821 663c-12F HM641727.1a 654 JQ012250.1a 730 525 AF009606 751 663c-13F 557 D14853 M62321 2.1a 482 633 AY051292 633 M62321.Ia 512 M58335.1b 643 D90208 643 L02836.1b D50409 1498c-3F 159 1411C-9F 132 L34369 121 EF115606.2 70 AY766688 69 AY232746.2b 71 1441C-2Rc 197 1430c-14F 228 1517c-12F EF564605 521 1441c-15Rc 865c-11Rc 345 1519c-8F 635 345 674C-8F X78861.2b 202 AY766672 7 KC197226.2b 260 96 EF564608.2B 119 KC844048.2B 149 KC844048.2b 7 191 1519c-15Rc KF548005.2b 14 D10988 6 AM502643
Isolates from Kenya are bolded and underlined genotype (90 %) in circulation is 2b (Fig. 1). This is also the first study to report the presence of genotype 2b in Kenya. The only other study on HCV genotypes in Kenya reported the presence of genotypes 1a and 4, with genotype 1a being more prevalent, at 73 % [20]. Globally, six major genotypes and more than 83 subtypes of HCV have been identified, with genotypes 1, 2 and 3 being the most prevalent [47, 48]. In Africa, various genotypes have been reported, including 4a in Egypt, 4c and 5a in Central Africa [49], and 3a, 1, 2, 4c and subtype 4g [50] in South Africa. Genotype 4 subtypes 4e, 4c, 4p and 4r have been reported in Gabon [13]. Closer to Kenya, subtype 4a has been reported in Tanzania [51], and genotype 4 in Uganda [52]. It is likely that diverse genotypes of HCV are present in Kenya.
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The presence of genotype 2b in Kenya is of particular interest. HCV genotypes 1a, 1b, and 3a are highly prevalent ‘‘epidemic’’ strains that are found globally [53]. There is glowing evidence to support the origin of genotype 2 being West Africa [54, 55]. The presence of this genotype in Kenya could indicate transmission from western to eastern Africa. In the last few years, bilateral relations between countries in the west and Kenya have been growing significantly. Notably, there are now direct flights to Cameroon, Nigeria and Ghana. A population of West Africans and Congolese from Zaire constitute a significant business community in Kenya, especially in the small-scale market sector. A number of countries in West and Central Africa have reported diverse genotypes in their populations. This
HCV genotypes in Kenya
could explain the emergence of related genotypes in Kenya. Genotype 2, together with 1, 4 and 5, have also been reported recently in Ethiopia [56], probably indicating further movement of this genotype in Eastern African countries. Muasya et al. [20] reported the presence of genotype 4 in a population of injecting drug users in Kenya. In that study, the two genotypes identified, 1a and 4, formed two clusters of related isolates. In the current study, the isolates clustered into three different groups. Since the two study populations are different, the current one being blood donors, it is most likely that HCV genotypes among blood donors could be more diverse than those in the previous group. Sharing of needles and close association among individuals injecting drugs could contribute to similar genotypes among the group, whereas this may not be the case in the diverse population from which blood donations are normally obtained. Although the limitations of the two studies done in Kenya prevent them from providing information about the topography of HCV genotypes, they nevertheless indicate an unfolding situation where diverse genotypes could be present in the country. Notably, Kenya is host to over 600,000 refugees from different countries, including Somalia, Ethiopia, Sudan, and Democratic Republic of the Congo, as well as other countries who have experienced internal civil war and unrest. Moreover, bilateral relations with Far East countries have increased in the recent past, with citizens from countries such as China now playing a major role in the business and construction sector. These populations could constitute a hot spot for divergent genotypes of HCV, perhaps pointing to a scenario where additional genotypes could be identified in Kenya. HCV genotype variation is associated with therapeutic response. Patients infected with HCV genotype 1 respond less well to therapy, while patients infected with HCV type 2 or 3 have been shown to have the best response[57, 58]. Patients with genotype 4 have been shown to respond well to treatment. With increasing identification of divergent genotypes in Kenya and the possibility of further ‘alien ‘genotypes being introduced, there is a need to reassess the treatment options available and further direct re-evaluation of policy guideline on control and management of HCV in Kenya. The average age of the infected population in this study was quite low (25 ?/- 9 years). It is important to note that the donation with subtype 1a detected in this study was from a 16-year-old female, while the majority (60%) of the other HCV-positive donors (subtype 2b) were above 22 years of age (mean 33 years). Blood donations in Kenya are collected mainly from learning institution and workplaces. This represents young and middle age groups in our population, with those below 22 years being high school or
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college donors. Other studies have shown age-related differences in subtype distribution, with patients infected with genotypes 1b and 2 being older than those infected with genotypes 3a and 1a [59]. Genotypes 1a and 3a have been linked to intravenous drug use [59], and the main risk factor for infection by genotypes 1b and 2 is blood transfusion [59]. In this study, the samples analysed were from blood donors, thus confirming the findings of Elghouzzi et al. [60]. Currently, injection drug use is a problem in some settings in Kenya. The only study targeting this population group reported the presence of genotype 1a (73 %) and genotype 4 (27 %) [20]. In the current study, there was only one sample with genotype 1a, while the rest were 2b. This appears to be consistent with the association of different subtypes with different risk factors, as indicated earlier. It would be interesting to see the genotypes and subtypes that are detectable in drug users and their correlation with those in other population groups where the HCV infection rate could be higher.
Conclusion This study provides data on genotypes of HCV in Kenya, where genotypes 1a and 2b were detected in blood donors. More data are required from other population subgroups in order to provide baseline data on the prevalence and genotypes of HCV in Kenya. Acknowledgement We acknowledge the director of the Kenya Medical Research Institute and the director of the Centre for Virus Research, KEMRI, all HIV laboratory staff at the Centre for Virus Research, and the director and staff of the National Blood Transfusion Services. Special thanks to Japan International Corporation and Kanazawa University for funds supporting part of this work. Compliance with ethical standards Conflict of interest No author has a commercial or other association that might pose a conflict of interest.
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