Hepatitis C virus genotype 4 in Ugandan children and their mothers.In Kampala, Uganda, in 2001, hepatitis C virus
The prevalence of hepatitis C virus (HCV HCV
hepatitis C virus
HCV 1 Hepatitis C virus, see there 2. Human coronavirus. See Coronavirus. ) infection in sub-Saharan African populations is an estimated 3%, with modest regional variation (1). The true prevalence may be lower because some positive HCV ELISA ELISA (e-li´sah) Enzyme-Linked Immuno-Sorbent Assay; any enzyme immunoassay using an enzyme-labeled immunoreactant and an immunosorbent.
n. results may be due to cross-reactivity, and the reliability of recombinant immunoblot assay (RIBA RIBA Royal Institute of British Architects ) as a confirmatory assay is unclear (2,3). Few studies have confirmed antibody-positive results by molecular methods, and data on HCV genotypes in Africans are limited.
In Uganda, 2 studies showed a low HCV seroprevalence seroprevalence Immunology The proportion of a population that is seropositive–ie, has been exposed to a particular pathogen or immunogen; the seropositivity of a population is calculated as the number of individuals who produce a particular antibody divided (2,4), but neither examined samples for viral RNA RNA: see nucleic acid.
in full ribonucleic acid
One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic . In 2001, we conducted a survey of Ugandan children with sickle cell disease sickle cell disease or sickle cell anemia, inherited disorder of the blood in which the oxygen-carrying hemoglobin pigment in erythrocytes (red blood cells) is abnormal. and their mothers and found that human herpesvirus herpesvirus, any of the family (Herpesviridae) of common DNA-containing viruses, many of which are associated with human disease. See cytomegalovirus; Epstein-Barr virus; herpes simplex; herpes zoster. 8 infection was associated with transfusion in these children (5). Children with sickle cell anemia sickle cell anemia
A chronic, usually fatal inherited form of anemia marked by crescent-shaped red blood cells, occurring almost exclusively in Blacks, and characterized by fever, leg ulcers, jaundice, and episodic pain in the joints. receive frequent injections during their care, and transfusions are frequently required for anemia. We therefore assumed that our population would be at increased risk for HCV infection. We tested for HCV antibodies and, on antibody-positive samples, sought HCV RNA to confirm antibody reactivity. For RNA-positive samples, we identified genotypes by sequencing and phylogenetic analysis.
In 2001, we enrolled 603 children (1-16 years of age) attending the Sickle Cell Clinic at Mulago Hospital, Kampala, in our study. By design, approximately half of the children had a history of blood transfusion. When available, the mothers of these children were also enrolled. Participants provided standardized information and blood samples. Plasma and buffy coats were immediately prepared and frozen at -80[degrees]C until testing.
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 [micro]L plasma by using the Qiagen Viral RNA Mini kit (Qiagen, Valencia, CA, USA). RT-PCR RT-PCR
reverse transcriptase-polymerase chain reaction. See PCR1. was performed in a Thermo Hybaid MBS See Mb/sec.
MBS - mobile broadband services 0.2S (Fisher Scientific International, Hampton, NH, USA) in triplicate reactions that used 10 [micro]L RNA per reaction. After cDNA synthesis, quantitative PCR PCR polymerase chain reaction.
polymerase chain reaction
Polymerase chain reaction (PCR) was performed by using an ABI Abi (ā`bī) [short for Abijah], in the Bible, King Hezekiah's mother.
(Application Binary Interface) A specification for a specific hardware platform combined with the operating system. 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. Briefly, purified RNA was used to generate cDNA by reverse transcription. Nested PCR was performed with sets of published PCR primers to amplify DNA DNA: see nucleic acid.
or deoxyribonucleic acid
One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. 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) was used to prepare products for sequencing in an Applied BioSystems 310 automated sequencer See MIDI sequencer.
(music) sequencer - Any system for recording and/or playback of music via a programmable memory which stores music not as audio data, but as some representation of notes. .
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). Additional genotype 4 reference sequences used in the analysis are listed in the Figure. 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 (www.psc.edu/biomed/genedoc). In Mega version 2.1 (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 F distribution (K80+[GAMMA]) distance model. Free parameters were reduced to the K80 model, and [alpha] values were determined by using a maximum likelihood approach in PAUP PAUP Phylogenetic Analysis Using Parsimony *4.0 (Sinauer Associates, Inc. Publishers, Sunderland, MA, USA).
Of 603 children (median age 6.8 years, interquartile range 3.9-10.9 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 seropositive /se·ro·pos·i·tive/ (-poz´i-tiv) showing positive results on serological examination; showing a high level of antibody.
adj. , compared with 16 (3%) of 456 children born to seronegative seronegative /se·ro·neg·a·tive/ (-neg´ah-tiv) showing negative results on serological examination; showing a lack of antibody.
adj. mothers (p = 0.58). Eleven (3%) of 322 children with a history of transfusion (mean 1.5 transfusions, range 1-10) 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 scarification /scar·i·fi·ca·tion/ (skar?i-fi-ka´shun) production in the skin of many small superficial scratches or punctures, as for introduction of vaccine.
n. 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 2.3x[10.sup.3], 2.8x[10.sup.4], and 3.8x[10.sup.6] 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 6.6x[10.sup.3] to 5.7x[10.sup.5] HCV IU/mL (median 8.1x[10.sup.4]). 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 (Figures 1 and 2). In the Core/E1 phylogeny (Figure 2), 2 clusters of HCV strains and 1 outlier outlier /out·li·er/ (out´li-er) an observation so distant from the central mass of the data that it noticeably influences results.
an extremely high or low value lying beyond the range of the bulk of the data. (UG1) appeared. Each of the NS5B sequences obtained (Figure 1) was from a different Core/E1 group and they did not cluster. Sequences from this study have been deposited in GenBank (accession nos. AY577578-AY577586).
[FIGURES 1-2 OMITTED]
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 Seropositivity is the presence of a certain antibody in a blood sample. A patient with seropositivity for a particular antigen or agent is termed seropositive. 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.
These results might indicate that false-positive ELISA results for HCV are frequent. False-positive results could be due to nonspecific nonspecific /non·spe·cif·ic/ (non?spi-sif´ik)
1. not due to any single known cause.
2. not directed against a particular agent, but rather having a general effect.
1. 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 viremia /vi·re·mia/ (vi-re´me-ah) the presence of viruses in the blood.
The presence of viruses in the bloodstream. ; however, in the industrialized in·dus·tri·al·ize
v. in·dus·tri·al·ized, in·dus·tri·al·iz·ing, in·dus·tri·al·iz·es
1. To develop industry in (a country or society, for example).
2. 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. How HCV is transmitted in sub-Saharan Africa is not known. We did not find correlations between infection in children and either seropositivity or viremia in their mothers. We expected to see iatrogenic iatrogenic /iat·ro·gen·ic/ (i-a´tro-jen´ik) resulting from the activity of physicians; said of any adverse condition in a patient resulting from treatment by a physician or surgeon. 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 El 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. Our HCV Core/El tree showed 3 clusters. Neither the Core/E1 nor NS5B sequences in Uganda clustered closely with genotype 4 sequences reported from other areas (Figures 1 and 2). Because the number of sequences was limited, we have not assigned subtypes to our newly described variants.
In sub-Saharan Africa, genotype frequencies vary by location (10). We found only genotype 4 in Uganda. Genotype 4 has been found in the west-central nations of Africa (Nigeria, Cameroon, Gabon, and Central African Republic Central African Republic, republic (2005 est. pop. 3,800,000), 240,534 sq mi (622,983 sq km), central Africa. The landlocked nation is bordered by Chad (N), Sudan (E), Congo (Kinshasa) and Congo (Brazzaville) (S), and Cameroon (W). ), but it also occurs in Tanzania (11). Phylogenetic analysis shows that types 1 and 4 share a common ancestor (12). Ndjomou et al. (13) noted that genotypes 1 and 4 in Cameroon have considerable genetic diversity and suggested that these types may have arisen there. They postulate that genotype 4 spread east into central Africa. 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 schistosomiasis (shĭs`təsōmī`əsĭs), bilharziasis, or snail fever, parasitic disease caused by blood flukes, trematode worms of the genus Schistosoma. eradication campaigns from 1960 to 1980 (in which needles for injecting potassium antimony antimony (ăn`tĭmō'nē) [Lat. antimoneum], semimetallic chemical element; symbol Sb [Lat. stibium,=a mark]; at. no. 51; at. wt. 121.75; m.p. 630.74°C;; b.p. 1,750°C;; sp. gr. (metallic form) 6. tartrate tartrate /tar·trate/ (tahr´trat) a salt of tartaric acid.
A salt or ester of tartaric acid.
a salt of tartaric acid. 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.
This project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract NO1-CO-12400. We thank Christine Gamache for technical support with the HCV ELISA, Wendell Miley for the HCV TaqMan data, and Denise Whitby for critical reading of the manuscript.
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Robert J. Biggar, * Betty A. Ortiz-Conde, ([dagger]) Rachel K. Bagni, ([dagger]) Paul M. Bakaki, ([double dagger]) Cheng-Dian Wang, ([dagger]) Eric A. Engels, * Sam M. Mbulaiteye, * and Christopher M. Ndugwa ([double dagger])
* National Cancer Institute, Bethesda, Maryland, USA; ([dagger]) National Cancer Institute-Frederick, Frederick, Maryland, USA; and ([double dagger]) Makerere University Medical School and Mulago Hospital, Kampala, Uganda
Dr Biggar is a senior investigator in the Viral Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute. He leads studies in Africa and elsewhere on the epidemiology and molecular aspects of viral infections, especially those caused by retroviruses and herpesviruses Herpesviruses
A family of viruses responsible for cold sores, chicken pox, and genital herpes.
Mentioned in: Skin Resurfacing .
Address for correspondence: Robert J. Biggar, National Cancer Institute, 6120 Executive Blvd, EPS (Encapsulated PostScript) A PostScript file format used to transfer a graphic image between applications and platforms. EPS files contain PostScript code as well as an optional preview image in TIFF, WMF, PICT or EPSI, the latter being an ASCII-only format. 8014, Bethesda, MD 20852, USA; email: email@example.com