in 2001 , we enrolled 603 children ( 116 years of age ) attending the sickle cell clinic at mulago hospital , kampala , in our study . by design ,
plasma specimens were tested by using an elisa for hcv antibodies to recombinant antigens c22 , c200 , and ns5b ( elisa-3.0 , ortho - clinical diagnostics , raritan , nj , usa ) according to the manufacturer 's instructions . to conserve samples
we did not confirm positive results by riba but instead tested for virus in plasma by using a real - time reverse transcription ( rt)pcr assay for hcv rna developed in our laboratory .
this quantitative assay amplified a conserved 155-nucleotide target sequence within the hcv 5  untranslated region and had an absolute sensitivity of 9 iu / ml of viral load ( 43 copies / ml ) .
total rna was extracted from 140 l plasma by using the qiagen viral rna mini kit ( qiagen , valencia , ca , usa ) .
rt - pcr was performed in a thermo hybaid mbs 0.2s ( fisher scientific international , hampton , nh , usa ) in triplicate reactions that used 10 l rna per reaction .
after cdna synthesis , quantitative pcr was performed by using an abi prism 7700 or 7900 sequence detection system ( applied biosystems , foster city , ca , usa ) . hcv rna 
positive specimens were further characterized by sequencing parts of the core / e1 and ns5b regions .
nested pcr was performed with sets of published pcr primers to amplify dna from core / e1 or ns5b regions ( 6,7 ) .
the amplification products were separated in an agarose gel and purified by using the promega dna purification kit ( promega , madison , wi , usa ) . the bigdye terminator kit ( applied biosystems )
separate alignments were generated for the ns5b and core / e1 regions , each including sequences from our samples and genotype 1 to 6 reference sequences obtained from genbank ( 7 ) .
these sequences were aligned in clustalx ( version 1.81 , plate - forme de bio - informatique , illkirch , france ) by using the clustalw ( version 1.6 , plate - forme de bio - informatique ) matrix and edited in genedoc version 2.6 ( http://www.psc.edu/biomed/genedoc ) . in mega version 2.1 ( http://www.megasoftware.net ) , the clustal alignments for core / e1 and ns5b
were used to generate neighbor - joining trees by using the kimura 2-parameter plus g distribution ( k80+g ) distance model .
free parameters were reduced to the k80 model , and  values were determined by using a maximum likelihood approach in paup*4.0 ( sinauer associates , inc .
estimated phylogenies of hepatitis c virus genotype 4 ; ns5b phylogenetic analysis based on 350 bp of ns5b nucleotide sequence .
numerical values ( presented when > 60% ) represent the statistical support for the tree topology as determined by 1,000 bootstrap replicates .
reference sequences for genotypes 13 , 5 , and 6 ( 7 ) were included in both analyses and retained as the outgroup .
of 603 children ( median age 6.8 years , interquartile range 3.910.9 years ) , 27 ( 4% ) were hcv antibody positive .
no significant trends were found between hcv status and demographic ( age , sex , tribe ) or household ( urban / rural location , water supply , room density ) variables .
three ( 5% ) of 61 children born to hcv - seropositive mothers were hcv seropositive , compared with 16 ( 3% ) of 456 children born to seronegative mothers ( p = 0.58 ) .
eleven ( 3% ) of 322 children with a history of transfusion ( mean 1.5 transfusions , range 110 ) were hcv seropositive , compared with 12 ( 5% ) of 245 children with no history of transfusion ( p = 0.38 ) .
prevalences in children with ( 2% ) and without ( 4% ) history of scarification were also similar ( p = 0.38 ) .
using rt - pcr to test for hcv rna , we examined 58 samples from children : all 27 samples with antibody - positive results , 11 with high - negative ( near the positive cutoff ) results , 10 with negative results from children with a history of frequent transfusions , and 10 with low - negative results from children with no history of transfusion . only 3 samples , all from seropositive children ( ages 6 , 7 , and 13 years ) , had positive results by rt - pcr , and viral rna levels in these samples were 2.310 , 2.810 , and 3.810 hcv iu / ml plasma .
of 525 serum samples available from mothers , 62 ( 12% ) were antibody positive .
we sought hcv rna in all 62 mothers with positive elisa results and in 20 mothers with high - negative results .
five ( 8% ) antibody - positive mothers ( ages 28 , 30 , 38 , 39 , and 42 years ) had rna - positive samples .
viral levels varied from 6.610 to 5.710 hcv iu / ml ( median 8.110 ) .
all hcv - seronegative mothers ( median age 32.3 years , mean 33.2 years , standard deviation 7.6 ) were rna negative .
we amplified samples for sequencing from 5 mothers and 1 child and obtained core / e1 sequences for all 6 samples and ns5b sequences for 3 samples .
none of the sequences was identical to any other or to any sequence in genbank .
phylogenetic analysis of both the ns5b and core / e1 regions showed that all viruses amplified clustered within genotype 4 ( figure 1 and figure 2 ) . in the core / e1 phylogeny ( figure 2 ) , 2 clusters of hcv strains and 1 outlier ( ug1 ) appeared .
each of the ns5b sequences obtained ( figure 1 ) was from a different core / e1 group , and they did not cluster .
estimated phylogenies of hepatitis c virus genotype 4 ; core / e1 phylogenetic analysis based on 340 bp spanning the junction between the core and e1 regions .
numerical values ( presented when > 60% ) represent the statistical support for the tree topology as determined by 1,000 bootstrap replicates .
reference sequences for genotypes 13 , 5 , and 6 ( 7 ) were included in both analyses and retained as the outgroup .
we found hcv infection to be uncommon in uganda , consistent with reports from other ugandan studies .
this cohort of patients with sickle cell disease had frequent blood transfusions , but only 4% of the children were hcv positive by elisa .
of the seropositive children , only 11% had hcv rna detectable by rt - pcr .
seropositivity in their mothers was higher , 12% , but only 8% of the seropositive women had positive rt - pcr results . if we assume that untested seronegative patients were not viremic , the overall prevalence of hcv rna was 0.5% in children and 1% in mothers .
false - positive results could be due to nonspecific antibody binding or to cross - reactivity with other tropical pathogens , e.g. , flaviviruses found in africa ( 2,3 ) .
alternatively , perhaps true antibody - positive participants did not have positive rt - pcr results because they had cleared hcv viremia ; however , in the industrialized world , most antibody - positive persons have persistent viremia .
we did not confirm elisa positivity with riba because , in africa , few hcv elisa - positive samples are confirmed by riba ( 3,8 ) .
our rt - pcr results confirm that hcv is present in uganda , albeit at a low prevalence .
we did not find correlations between infection in children and either seropositivity or viremia in their mothers .
we expected to see iatrogenic transmission ( 1,8 ) because the children in this study all had sickle cell disease and more than half had had blood transfusions , and many had had multiple transfusions .
most children would have also had potential exposure to hcv from contaminated needles used during the course of their medical care or blades used in scarification by traditional healers ( 9 ) . however , despite these potential exposures , documented hcv infections were uncommon .
most hcv isolates can be classified into 6 major genotypes on the basis of sequence data from the ns5b , core , and e1 genomic regions . in this study , as in others ( 8) , multiple sets of primers were needed to amplify these regions because different primer sets worked for different samples , which reflects the diversity of hcv .
neither the core / e1 nor ns5b sequences in uganda clustered closely with genotype 4 sequences reported from other areas ( figure 1 and figure 2 ) .
because the number of sequences was limited , we have not assigned subtypes to our newly described variants . in sub - saharan africa ,
genotype 4 has been found in the west - central nations of africa ( nigeria , cameroon , gabon , and central african republic ) , but it also occurs in tanzania ( 11 ) .
phylogenetic analysis shows that types 1 and 4 share a common ancestor ( 12 ) .
( 13 ) noted that genotypes 1 and 4 in cameroon have considerable genetic diversity and suggested that these types may have arisen there .
our detection of hcv genotype 4 in uganda is consistent with their hypothesis , but we do not know the direction of spread .
type 4 is also found in egypt , where its epidemic distribution is attributed to widespread iatrogenic transmission during schistosomiasis eradication campaigns from 1960 to 1980 ( in which needles for injecting potassium antimony tartrate were reused after rudimentary cleaning ) ( 7,14 ) , and in the arabian peninsula ( 15 ) . for many centuries ,
arab traders have had extensive contact with african populations of the nile watershed region , including uganda , which provides routes for genotype 4 to spread between east africa and the middle east .
 in kampala , uganda , in 2001 , hepatitis c virus antibodies were found in 27 ( 4% ) of 603 children and in 62 ( 12% ) of 525 of their mothers . 
 however , only 10% of positive results were confirmed by reverse transcription  pcr , which suggests frequent false - positive results or viral clearance . 
 all sequenced types were genotype 4 . 