Background Lamivudine inhibits replication of both human immunodeficiency virus (HIV) and hepatitis B virus (HBV) and is commonly used as part of antiretroviral therapy. The main limitation in the use of lamivudine is resistant mutation selection. Most of these mutations affect the YMDD motif of the HBV DNA polymerase. The resistance occurs through M550V or M550I aminoacid replacements. The M550V variation may be accompanied by L526M mutation, notably in HIV-HBV co-infected patients. The aim of this study was to investigate mutations associated with lamivudine resistance in a hemodialysis patient chronically co-infected with HIV-1 and HBV, who was submitted to several antiretroviral treatments.
Background
Lamivudine inhibits replication of both human immunodeficiency virus (HIV) and hepatitis B virus (HBV) and is commonly used as part of antiretroviral therapy. The main limitation in the use of lamivudine is resistant mutation selection. Most of these mutations affect the YMDD motif of the HBV DNA polymerase. The resistance occurs through M550V or M550I aminoacid replacements. The M550V variation may be accompanied by L526M mutation, notably in HIV-HBV co-infected patients. The aim of this study was to investigate mutations associated with lamivudine resistance in a hemodialysis patient chronically co-infected with HIV-1 and HBV, who was submitted to several antiretroviral treatments.Methods HBV isolates derived from three blood samples collected at different times of antiretroviral therapies with and without lamivudine, were titred and submitted to nucleotide sequencing.
Methods
HBV isolates derived from three blood samples collected at different times of antiretroviral therapies with and without lamivudine, were titred and submitted to nucleotide sequencing.Results HBV isolate derived from a sample collected in 1999 during an antiretroviral treatment with lamivudine showed the lamivudine resistant double mutation (L526M, M550V). However, no mutation associated with lamivudine resistance was observed in the HBV genome derived from the sample collected during a period of treatment without lamivudine (2001). After reinstitution of lamivudine (2002), the predominant HBV population exhibited a rare triple mutation (V519L, L526M, M550V), which has previously been associated with an  in vitro  reduction of virus antigenicity (escape mutant). HBV DNA was detected at high levels (10 8 –10 9  copies/ml) in the three blood samples.
Results
HBV isolate derived from a sample collected in 1999 during an antiretroviral treatment with lamivudine showed the lamivudine resistant double mutation (L526M, M550V). However, no mutation associated with lamivudine resistance was observed in the HBV genome derived from the sample collected during a period of treatment without lamivudine (2001). After reinstitution of lamivudine (2002), the predominant HBV population exhibited a rare triple mutation (V519L, L526M, M550V), which has previously been associated with an  in vitro  reduction of virus antigenicity (escape mutant). HBV DNA was detected at high levels (10 8 –10 9  copies/ml) in the three blood samples.in vitroConclusions Reintroduction of lamivudine as part of antiretroviral treatment in a patient who had developed lamivudine resistant HBV strains favored the predominance of an HBV isolate with reduced antigenicity. The absence of hepatitis acute exacerbation in this patient may be correlated to the absence of significant variations of the viral load, which was independent of the presence of mutations in the HBV DNA polymerase.
Conclusions
Reintroduction of lamivudine as part of antiretroviral treatment in a patient who had developed lamivudine resistant HBV strains favored the predominance of an HBV isolate with reduced antigenicity. The absence of hepatitis acute exacerbation in this patient may be correlated to the absence of significant variations of the viral load, which was independent of the presence of mutations in the HBV DNA polymerase.

Background Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) share common routes of transmission, mainly sexual, parenteral and vertical. Therefore, the prevalence of HBV serological markers is higher among HIV infected patients than in non-HIV infected individuals [ 1 , 2 ]. Considerable variations in the prevalence of HBV markers have been observed in HIV patients according to the geographical region and risk of exposure [ 3 ]. Recently, a prevalence of 68% of antibodies against hepatitis B core antigen (anti-HBc) was observed in HIV infected patients living in Rio de Janeiro, Brazil [ 4 ]. Co-infection with HIV interferes with the natural history of HBV infection and is associated with higher HBV DNA levels [ 5 , 6 ]. A more common progression to cirrhosis, despite a milder histological necro-inflammatory activity, has also been observed in cases of HIV-HBV co-infection [ 5 ]. Lamivudine is a nucleoside analogue that inhibits the reverse transcriptase activity of both HIV and HBV [ 7 ] and is commonly used in the treatment of both viral infections [ 8 , 9 ]. The HBV DNA loss due to lamivudine treatment is usually accompanied by significant histological and biochemical improvement [ 10 ]. The major limitation in the use of lamivudine is the selection of resistant mutations that may arise and accumulate during therapy. Most of these mutations usually affect the YMDD motif of the HBV DNA polymerase, by replacement of a methionine residue at position 550 with either valine (M550V) or isoleucine (M550I). Such mutations have notably been reported in HIV-HBV co-infected patients [ 11 , 12 ] who develop lamivudine resistance at an annual rate of 20%, with a projected rate of 90% after four years of therapy [ 13 ]. The consequences of drug resistance mutations for the evolution of HBV induced liver disease are currently under study. Hepatitis B acute exacerbation has been described after withdrawal of lamivudine therapy [ 14 ]. Such an exacerbation has also been associated with the appearance of YMDD mutants [ 14 , 15 ] and with a rapid increase of viral load [ 16 ]. The present study was performed to characterize HBV variants and genetic patterns of lamivudine resistant HBV strains in a patient co-infected with HIV-1 at different periods of an antiretroviral treatment with and without lamivudine.
Background
Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) share common routes of transmission, mainly sexual, parenteral and vertical. Therefore, the prevalence of HBV serological markers is higher among HIV infected patients than in non-HIV infected individuals [ 1 , 2 ]. Considerable variations in the prevalence of HBV markers have been observed in HIV patients according to the geographical region and risk of exposure [ 3 ]. Recently, a prevalence of 68% of antibodies against hepatitis B core antigen (anti-HBc) was observed in HIV infected patients living in Rio de Janeiro, Brazil [ 4 ]. Co-infection with HIV interferes with the natural history of HBV infection and is associated with higher HBV DNA levels [ 5 , 6 ]. A more common progression to cirrhosis, despite a milder histological necro-inflammatory activity, has also been observed in cases of HIV-HBV co-infection [ 5 ].Lamivudine is a nucleoside analogue that inhibits the reverse transcriptase activity of both HIV and HBV [ 7 ] and is commonly used in the treatment of both viral infections [ 8 , 9 ]. The HBV DNA loss due to lamivudine treatment is usually accompanied by significant histological and biochemical improvement [ 10 ]. The major limitation in the use of lamivudine is the selection of resistant mutations that may arise and accumulate during therapy. Most of these mutations usually affect the YMDD motif of the HBV DNA polymerase, by replacement of a methionine residue at position 550 with either valine (M550V) or isoleucine (M550I). Such mutations have notably been reported in HIV-HBV co-infected patients [ 11 , 12 ] who develop lamivudine resistance at an annual rate of 20%, with a projected rate of 90% after four years of therapy [ 13 ].The consequences of drug resistance mutations for the evolution of HBV induced liver disease are currently under study. Hepatitis B acute exacerbation has been described after withdrawal of lamivudine therapy [ 14 ]. Such an exacerbation has also been associated with the appearance of YMDD mutants [ 14 , 15 ] and with a rapid increase of viral load [ 16 ].The present study was performed to characterize HBV variants and genetic patterns of lamivudine resistant HBV strains in a patient co-infected with HIV-1 at different periods of an antiretroviral treatment with and without lamivudine.Methods Patient characteristics and serological markers A 30-year-old male patient initiated a hemodialysis treatment in August 1995. At that time, he was negative for hepatitis B surface antigen (HBsAg) as well as for anti-HBc and anti-HIV antibodies. The patient received three blood transfusions during the first trimester of 1996 and developed a posttransfusional hepatitis. He became HBsAg and anti-HIV positive and remained positive for both markers in all routine tests performed between 1996 and 2002. Data of other tests, such as serum transaminases, CD4 levels, and HIV load, performed in external laboratories between 1995 and 2002, were available. Resistance to different HIV therapies was evaluated by the medical staff based on HIV load and CD4 levels. HIV treatment started in April 1996, with a combination of zidovudine (AZT) and didanosine (ddI). In November 1996, this treatment was modified to a combination of AZT and lamivudine which was maintained until March 1997, when treatment was once again changed to triple therapy with AZT, lamivudine and invirase. In May 1999, high HIV load (3.1 × 10 5  copies/ml) was observed, indicating HIV resistance. At that time the patient started an anti-tuberculosis treatment with rifampicin. However, due to interaction of antiretroviral drugs with rifampicin, the administration of antiretroviral drugs was discontinued. This resulted in a decrease of CD4 counts to 156 cells/ml, while elevated levels of HIV persisted at the end of 1999. Antiretroviral treatment resumed in January 2000, composed of four drugs, namely ddI, stavudine, nevirapine (NVP) and nelfinavir (NFV). As a consequence, CD4 cell counts increased noticeably, and HIV RNA became undetectable. Unfortunately, resistance to therapy was observed during the second semester of 2001, when HIV load reached 1.2 × 10 5  copies/ml and CD4 counts decreased to 120 cells/ml. The treatment was then replaced by another including lamivudine associated to AZT, NVP and NFV. No HIV resistance was observed until the end of the follow-up (November 2002). The patient died of renal failure at the end of 2003. DNA extraction and PCR assays for DNA sequencing Three serum samples collected in March 1999, August 2000 and November 2002, were available for HBV DNA analysis. Sera were submitted to DNA extraction by the phenol-chloroform method after treatment with proteinase K, as described previously [ 17 ]. HBV pre-S/S genomic region was amplified by PCR using sense primer PS1 (5'-CCATATTCTTGGGAACAAGA-3', nt 2826-2845) and a mix of antisense primers S2 (5'-GGGTTTAAATGTATACCCAAAGA-3', nt 841-819) and S22 (5'-GTATTTAAATGGATACCCACAGA-3', nt 841-819), able to amplify all HBV genotypes. PCR assays were performed under the following conditions: 94°C, 30 s; 52°C, 1 min; 72°C, 2 min; 35 cycles, followed by a final elongation of 7 min at 72°C. Amplification products (10 μL) were loaded on a 2% agarose gel, electrophoresed, stained with ethidium bromide, and visualized under UV light. Quantification of HBV DNA Quantification of HBV DNA was performed by endpoint dilution. DNA samples were diluted to a tenfold series up to 10 -8  dilution. Each dilution was submitted to PCR with oligonucleotide pairs PS1–PS2, designed in the pre-S region and C1–C2 (core region), as described previously [ 4 ]. Serial dilutions and PCR assays for HBV DNA quantification were performed in triplicate. The sensitivity of the method has been estimated to about 100 copies per PCR reaction [ 18 ], equivalent to 10 4  HBV genome copies per milliliter of serum. Quantitative results were estimated by dividing 10 4  copies/ml by the last positive dilution. Molecular cloning and nucleotide sequencing Pre-S/S PCR products were cloned into pCRII plasmid vector using TA cloning kit (Invitrogen, San Diego, CA). For nucleotide sequencing, recombinant plasmid DNAs were purified by a commercially available kit (Plasmid midi kit, Qiagen, Hilden, Germany). Nucleotide sequences were determined using the Cy5 auto read sequencing kit (Amersham Biosciences, Little Chalfont, UK) with M13 universal and reverse primers, as well as internal, HBV specific primers. For direct sequencing (S region), PCR products were extracted from low melting agarose gels (Qiaquick gel extraction kit, Qiagen). Sequencing reactions with HBV specific primers were done using the thermo sequenase Cy5 dye terminator sequencing kit and analyzed on an ALFexpress automated sequencer (Amersham Biosciences). Independent plus and minus strand sequencing was completed. Phylogenetic analysis Nucleotide sequences were aligned using PILEUP (Wisconsin Sequences Analysis Package GCG, Madison, WI). A phylogenetic tree was generated by neighbour-joining analysis of genetic distances, using the TREECON software package for Windows [ 19 ]. HBV sequences available from the GenBank database (accession numbers AY090458, M57663, U55220-U55222, J02201 and X51970) were used for the construction of a phylogenetic tree.
Methods
Patient characteristics and serological markers A 30-year-old male patient initiated a hemodialysis treatment in August 1995. At that time, he was negative for hepatitis B surface antigen (HBsAg) as well as for anti-HBc and anti-HIV antibodies. The patient received three blood transfusions during the first trimester of 1996 and developed a posttransfusional hepatitis. He became HBsAg and anti-HIV positive and remained positive for both markers in all routine tests performed between 1996 and 2002. Data of other tests, such as serum transaminases, CD4 levels, and HIV load, performed in external laboratories between 1995 and 2002, were available. Resistance to different HIV therapies was evaluated by the medical staff based on HIV load and CD4 levels. HIV treatment started in April 1996, with a combination of zidovudine (AZT) and didanosine (ddI). In November 1996, this treatment was modified to a combination of AZT and lamivudine which was maintained until March 1997, when treatment was once again changed to triple therapy with AZT, lamivudine and invirase. In May 1999, high HIV load (3.1 × 10 5  copies/ml) was observed, indicating HIV resistance. At that time the patient started an anti-tuberculosis treatment with rifampicin. However, due to interaction of antiretroviral drugs with rifampicin, the administration of antiretroviral drugs was discontinued. This resulted in a decrease of CD4 counts to 156 cells/ml, while elevated levels of HIV persisted at the end of 1999. Antiretroviral treatment resumed in January 2000, composed of four drugs, namely ddI, stavudine, nevirapine (NVP) and nelfinavir (NFV). As a consequence, CD4 cell counts increased noticeably, and HIV RNA became undetectable. Unfortunately, resistance to therapy was observed during the second semester of 2001, when HIV load reached 1.2 × 10 5  copies/ml and CD4 counts decreased to 120 cells/ml. The treatment was then replaced by another including lamivudine associated to AZT, NVP and NFV. No HIV resistance was observed until the end of the follow-up (November 2002). The patient died of renal failure at the end of 2003.
Patient characteristics and serological markers
A 30-year-old male patient initiated a hemodialysis treatment in August 1995. At that time, he was negative for hepatitis B surface antigen (HBsAg) as well as for anti-HBc and anti-HIV antibodies. The patient received three blood transfusions during the first trimester of 1996 and developed a posttransfusional hepatitis. He became HBsAg and anti-HIV positive and remained positive for both markers in all routine tests performed between 1996 and 2002. Data of other tests, such as serum transaminases, CD4 levels, and HIV load, performed in external laboratories between 1995 and 2002, were available. Resistance to different HIV therapies was evaluated by the medical staff based on HIV load and CD4 levels. HIV treatment started in April 1996, with a combination of zidovudine (AZT) and didanosine (ddI). In November 1996, this treatment was modified to a combination of AZT and lamivudine which was maintained until March 1997, when treatment was once again changed to triple therapy with AZT, lamivudine and invirase. In May 1999, high HIV load (3.1 × 10 5  copies/ml) was observed, indicating HIV resistance. At that time the patient started an anti-tuberculosis treatment with rifampicin. However, due to interaction of antiretroviral drugs with rifampicin, the administration of antiretroviral drugs was discontinued. This resulted in a decrease of CD4 counts to 156 cells/ml, while elevated levels of HIV persisted at the end of 1999. Antiretroviral treatment resumed in January 2000, composed of four drugs, namely ddI, stavudine, nevirapine (NVP) and nelfinavir (NFV). As a consequence, CD4 cell counts increased noticeably, and HIV RNA became undetectable. Unfortunately, resistance to therapy was observed during the second semester of 2001, when HIV load reached 1.2 × 10 5  copies/ml and CD4 counts decreased to 120 cells/ml. The treatment was then replaced by another including lamivudine associated to AZT, NVP and NFV. No HIV resistance was observed until the end of the follow-up (November 2002). The patient died of renal failure at the end of 2003.DNA extraction and PCR assays for DNA sequencing Three serum samples collected in March 1999, August 2000 and November 2002, were available for HBV DNA analysis. Sera were submitted to DNA extraction by the phenol-chloroform method after treatment with proteinase K, as described previously [ 17 ]. HBV pre-S/S genomic region was amplified by PCR using sense primer PS1 (5'-CCATATTCTTGGGAACAAGA-3', nt 2826-2845) and a mix of antisense primers S2 (5'-GGGTTTAAATGTATACCCAAAGA-3', nt 841-819) and S22 (5'-GTATTTAAATGGATACCCACAGA-3', nt 841-819), able to amplify all HBV genotypes. PCR assays were performed under the following conditions: 94°C, 30 s; 52°C, 1 min; 72°C, 2 min; 35 cycles, followed by a final elongation of 7 min at 72°C. Amplification products (10 μL) were loaded on a 2% agarose gel, electrophoresed, stained with ethidium bromide, and visualized under UV light.
DNA extraction and PCR assays for DNA sequencing
Three serum samples collected in March 1999, August 2000 and November 2002, were available for HBV DNA analysis. Sera were submitted to DNA extraction by the phenol-chloroform method after treatment with proteinase K, as described previously [ 17 ]. HBV pre-S/S genomic region was amplified by PCR using sense primer PS1 (5'-CCATATTCTTGGGAACAAGA-3', nt 2826-2845) and a mix of antisense primers S2 (5'-GGGTTTAAATGTATACCCAAAGA-3', nt 841-819) and S22 (5'-GTATTTAAATGGATACCCACAGA-3', nt 841-819), able to amplify all HBV genotypes. PCR assays were performed under the following conditions: 94°C, 30 s; 52°C, 1 min; 72°C, 2 min; 35 cycles, followed by a final elongation of 7 min at 72°C. Amplification products (10 μL) were loaded on a 2% agarose gel, electrophoresed, stained with ethidium bromide, and visualized under UV light.Quantification of HBV DNA Quantification of HBV DNA was performed by endpoint dilution. DNA samples were diluted to a tenfold series up to 10 -8  dilution. Each dilution was submitted to PCR with oligonucleotide pairs PS1–PS2, designed in the pre-S region and C1–C2 (core region), as described previously [ 4 ]. Serial dilutions and PCR assays for HBV DNA quantification were performed in triplicate. The sensitivity of the method has been estimated to about 100 copies per PCR reaction [ 18 ], equivalent to 10 4  HBV genome copies per milliliter of serum. Quantitative results were estimated by dividing 10 4  copies/ml by the last positive dilution.
Quantification of HBV DNA
Quantification of HBV DNA was performed by endpoint dilution. DNA samples were diluted to a tenfold series up to 10 -8  dilution. Each dilution was submitted to PCR with oligonucleotide pairs PS1–PS2, designed in the pre-S region and C1–C2 (core region), as described previously [ 4 ]. Serial dilutions and PCR assays for HBV DNA quantification were performed in triplicate. The sensitivity of the method has been estimated to about 100 copies per PCR reaction [ 18 ], equivalent to 10 4  HBV genome copies per milliliter of serum. Quantitative results were estimated by dividing 10 4  copies/ml by the last positive dilution.Molecular cloning and nucleotide sequencing Pre-S/S PCR products were cloned into pCRII plasmid vector using TA cloning kit (Invitrogen, San Diego, CA). For nucleotide sequencing, recombinant plasmid DNAs were purified by a commercially available kit (Plasmid midi kit, Qiagen, Hilden, Germany). Nucleotide sequences were determined using the Cy5 auto read sequencing kit (Amersham Biosciences, Little Chalfont, UK) with M13 universal and reverse primers, as well as internal, HBV specific primers. For direct sequencing (S region), PCR products were extracted from low melting agarose gels (Qiaquick gel extraction kit, Qiagen). Sequencing reactions with HBV specific primers were done using the thermo sequenase Cy5 dye terminator sequencing kit and analyzed on an ALFexpress automated sequencer (Amersham Biosciences). Independent plus and minus strand sequencing was completed.
Molecular cloning and nucleotide sequencing
Pre-S/S PCR products were cloned into pCRII plasmid vector using TA cloning kit (Invitrogen, San Diego, CA). For nucleotide sequencing, recombinant plasmid DNAs were purified by a commercially available kit (Plasmid midi kit, Qiagen, Hilden, Germany). Nucleotide sequences were determined using the Cy5 auto read sequencing kit (Amersham Biosciences, Little Chalfont, UK) with M13 universal and reverse primers, as well as internal, HBV specific primers. For direct sequencing (S region), PCR products were extracted from low melting agarose gels (Qiaquick gel extraction kit, Qiagen). Sequencing reactions with HBV specific primers were done using the thermo sequenase Cy5 dye terminator sequencing kit and analyzed on an ALFexpress automated sequencer (Amersham Biosciences). Independent plus and minus strand sequencing was completed.Phylogenetic analysis Nucleotide sequences were aligned using PILEUP (Wisconsin Sequences Analysis Package GCG, Madison, WI). A phylogenetic tree was generated by neighbour-joining analysis of genetic distances, using the TREECON software package for Windows [ 19 ]. HBV sequences available from the GenBank database (accession numbers AY090458, M57663, U55220-U55222, J02201 and X51970) were used for the construction of a phylogenetic tree.
Phylogenetic analysis
Nucleotide sequences were aligned using PILEUP (Wisconsin Sequences Analysis Package GCG, Madison, WI). A phylogenetic tree was generated by neighbour-joining analysis of genetic distances, using the TREECON software package for Windows [ 19 ]. HBV sequences available from the GenBank database (accession numbers AY090458, M57663, U55220-U55222, J02201 and X51970) were used for the construction of a phylogenetic tree.Results Markers of HBV infection The patient under study developed an acute hepatitis and became anti-HIV positive in 1996, soon after receiving blood transfusions during the initial period of hemodialysis treatment. He became an HBV chronic carrier, with all blood samples collected between 1996 and 2002 being positive for HBsAg and HBeAg. During this period, the patient remained asymptomatic without clinical signs for HBV infection. Aspartate aminotransferase and alkaline phosphatase levels remained normal all during the follow-up. However, an increase of alanine aminotransferase (ALT) levels to 180 IU/L was observed in March 1999 (Figure  1 ). HIV treatment started in April 1996. Lamivudine was introduced as part of antiretroviral treatment in November 1996. HBV DNA was detected at high levels (10 8 copies/ml) in the blood sample collected in March 1999, during this first period of lamivudine treatment (Figure  1 ). In May 1999, lamivudine was discontinued, and the sample analyzed during the period of lamivudine interruption (August 2001) showed the highest (10 9  copies/ml) HBV DNA titre mesured in this study. HBV DNA was again detected at high levels (10 8 copies/ml) after reintroduction of lamivudine (November 2002, Figure  1 ). HBV variants Nucleotide sequences of HBVs (pre-S/S region) derived from HBsAg/HBeAg positive samples collected in March 1999, August 2001 and November 2002 were determined (three clones each). Phylogenetic analysis showed that all nine clones belonged to genotype A. This genotype has been subdivided into two subgenomic groups, designated A-A' (genotype A excluding A') and A' [ 20 ]. Recently, subgroups A-A' and A' were designated respectively as A1 and A2 [ 21 ] or Ae and Aa [ 22 ]. Isolates belonging to subgroup A' have been first identified in South Africa and circulate in a high proportion among HBV Brazilian isolates [ 23 ]. As can be observed on the phylogenetic tree represented in Figure  2 , all clones belonged to subgroup A' and were closely related to each other. The six clones obtained during the two periods of lamivudine treatment (1999 and 2002) clustered separately (with a bootstrap value of 86%) from those obtained in 2001, during the period of lamivudine interruption. Table  1  shows the main amino acid changes observed in the S region of the genome. Two substitutions, H359Y in the polymerase and Y100C in the surface antigen (small S), were observed in all HBV sequences. These two changes were natural variation, characteristics of the HBV strain infecting the patient under study. All three clones derived from the sample collected in 1999, during the first lamivudine treatment, showed two lamivudine resistant mutations (L526M and M550V), also detected in the major viral population by direct sequencing. One clone (1-B57) showed an additional mutation related to drug resistance (V519L) that was not detected by direct sequencing. None of these three mutations associated with lamivudine resistance was observed in sequences derived from the second sample (2001). In contrast, all three clones derived from the third sample (2002) showed the three lamivudine resistant mutations. However, by direct sequencing of PCR products of this last sample, the electropherogram could detect two nucleotides (A and G) at the same sequence position indicating a mixture of V519 and L519 residues. Curiously, all HBV sequences obtained during the period without lamivudine treatment (2001) displayed a unique G473E substitution in the polymerase gene. Stop mutations were also observed in the small S protein, which was truncated at position 182 in clone 2-B14 and at position 216 in clone 2-B62 (Table  1 ). Due to overlapping of polymerase and S genes on the HBV genome, mutations at positions 473, 519 and 550 of the polymerase were accompanied by mutations at positions 119, 164 and 195 of the small S protein (Table  1 ).
Results
Markers of HBV infection The patient under study developed an acute hepatitis and became anti-HIV positive in 1996, soon after receiving blood transfusions during the initial period of hemodialysis treatment. He became an HBV chronic carrier, with all blood samples collected between 1996 and 2002 being positive for HBsAg and HBeAg. During this period, the patient remained asymptomatic without clinical signs for HBV infection. Aspartate aminotransferase and alkaline phosphatase levels remained normal all during the follow-up. However, an increase of alanine aminotransferase (ALT) levels to 180 IU/L was observed in March 1999 (Figure  1 ). HIV treatment started in April 1996. Lamivudine was introduced as part of antiretroviral treatment in November 1996. HBV DNA was detected at high levels (10 8 copies/ml) in the blood sample collected in March 1999, during this first period of lamivudine treatment (Figure  1 ). In May 1999, lamivudine was discontinued, and the sample analyzed during the period of lamivudine interruption (August 2001) showed the highest (10 9  copies/ml) HBV DNA titre mesured in this study. HBV DNA was again detected at high levels (10 8 copies/ml) after reintroduction of lamivudine (November 2002, Figure  1 ).
Markers of HBV infection
The patient under study developed an acute hepatitis and became anti-HIV positive in 1996, soon after receiving blood transfusions during the initial period of hemodialysis treatment. He became an HBV chronic carrier, with all blood samples collected between 1996 and 2002 being positive for HBsAg and HBeAg. During this period, the patient remained asymptomatic without clinical signs for HBV infection. Aspartate aminotransferase and alkaline phosphatase levels remained normal all during the follow-up. However, an increase of alanine aminotransferase (ALT) levels to 180 IU/L was observed in March 1999 (Figure  1 ).HIV treatment started in April 1996. Lamivudine was introduced as part of antiretroviral treatment in November 1996. HBV DNA was detected at high levels (10 8 copies/ml) in the blood sample collected in March 1999, during this first period of lamivudine treatment (Figure  1 ). In May 1999, lamivudine was discontinued, and the sample analyzed during the period of lamivudine interruption (August 2001) showed the highest (10 9  copies/ml) HBV DNA titre mesured in this study. HBV DNA was again detected at high levels (10 8 copies/ml) after reintroduction of lamivudine (November 2002, Figure  1 ).HBV variants Nucleotide sequences of HBVs (pre-S/S region) derived from HBsAg/HBeAg positive samples collected in March 1999, August 2001 and November 2002 were determined (three clones each). Phylogenetic analysis showed that all nine clones belonged to genotype A. This genotype has been subdivided into two subgenomic groups, designated A-A' (genotype A excluding A') and A' [ 20 ]. Recently, subgroups A-A' and A' were designated respectively as A1 and A2 [ 21 ] or Ae and Aa [ 22 ]. Isolates belonging to subgroup A' have been first identified in South Africa and circulate in a high proportion among HBV Brazilian isolates [ 23 ]. As can be observed on the phylogenetic tree represented in Figure  2 , all clones belonged to subgroup A' and were closely related to each other. The six clones obtained during the two periods of lamivudine treatment (1999 and 2002) clustered separately (with a bootstrap value of 86%) from those obtained in 2001, during the period of lamivudine interruption. Table  1  shows the main amino acid changes observed in the S region of the genome. Two substitutions, H359Y in the polymerase and Y100C in the surface antigen (small S), were observed in all HBV sequences. These two changes were natural variation, characteristics of the HBV strain infecting the patient under study. All three clones derived from the sample collected in 1999, during the first lamivudine treatment, showed two lamivudine resistant mutations (L526M and M550V), also detected in the major viral population by direct sequencing. One clone (1-B57) showed an additional mutation related to drug resistance (V519L) that was not detected by direct sequencing. None of these three mutations associated with lamivudine resistance was observed in sequences derived from the second sample (2001). In contrast, all three clones derived from the third sample (2002) showed the three lamivudine resistant mutations. However, by direct sequencing of PCR products of this last sample, the electropherogram could detect two nucleotides (A and G) at the same sequence position indicating a mixture of V519 and L519 residues. Curiously, all HBV sequences obtained during the period without lamivudine treatment (2001) displayed a unique G473E substitution in the polymerase gene. Stop mutations were also observed in the small S protein, which was truncated at position 182 in clone 2-B14 and at position 216 in clone 2-B62 (Table  1 ). Due to overlapping of polymerase and S genes on the HBV genome, mutations at positions 473, 519 and 550 of the polymerase were accompanied by mutations at positions 119, 164 and 195 of the small S protein (Table  1 ).
HBV variants
Nucleotide sequences of HBVs (pre-S/S region) derived from HBsAg/HBeAg positive samples collected in March 1999, August 2001 and November 2002 were determined (three clones each). Phylogenetic analysis showed that all nine clones belonged to genotype A. This genotype has been subdivided into two subgenomic groups, designated A-A' (genotype A excluding A') and A' [ 20 ]. Recently, subgroups A-A' and A' were designated respectively as A1 and A2 [ 21 ] or Ae and Aa [ 22 ]. Isolates belonging to subgroup A' have been first identified in South Africa and circulate in a high proportion among HBV Brazilian isolates [ 23 ]. As can be observed on the phylogenetic tree represented in Figure  2 , all clones belonged to subgroup A' and were closely related to each other. The six clones obtained during the two periods of lamivudine treatment (1999 and 2002) clustered separately (with a bootstrap value of 86%) from those obtained in 2001, during the period of lamivudine interruption.Table  1  shows the main amino acid changes observed in the S region of the genome. Two substitutions, H359Y in the polymerase and Y100C in the surface antigen (small S), were observed in all HBV sequences. These two changes were natural variation, characteristics of the HBV strain infecting the patient under study. All three clones derived from the sample collected in 1999, during the first lamivudine treatment, showed two lamivudine resistant mutations (L526M and M550V), also detected in the major viral population by direct sequencing. One clone (1-B57) showed an additional mutation related to drug resistance (V519L) that was not detected by direct sequencing. None of these three mutations associated with lamivudine resistance was observed in sequences derived from the second sample (2001). In contrast, all three clones derived from the third sample (2002) showed the three lamivudine resistant mutations. However, by direct sequencing of PCR products of this last sample, the electropherogram could detect two nucleotides (A and G) at the same sequence position indicating a mixture of V519 and L519 residues. Curiously, all HBV sequences obtained during the period without lamivudine treatment (2001) displayed a unique G473E substitution in the polymerase gene. Stop mutations were also observed in the small S protein, which was truncated at position 182 in clone 2-B14 and at position 216 in clone 2-B62 (Table  1 ).Due to overlapping of polymerase and S genes on the HBV genome, mutations at positions 473, 519 and 550 of the polymerase were accompanied by mutations at positions 119, 164 and 195 of the small S protein (Table  1 ).Discussion Among lamivudine resistant mutations, those in the YMDD motif of the HBV DNA polymerase are the most common. The resistance occurs by replacement of a methionine residue at position 550 by either valine (M550V) or isoleucine (M550I). More rarely, HBV variant presenting M550S replacement may be selected during lamivudine treatment [ 24 ] The M550V variant may be accompanied by a mutation (leucine to methionine) at position 526 [ 25 ]. In the absence of HIV infection, the mutation at position 550 alone has been found in up to two-thirds of patients with lamivudine-resistant chronic hepatitis B. However, more than 90% of HIV-HBV co-infected patients display the double lamivudine resistant mutation at positions 526 and 550 [ 12 ]. Furthermore, the presence of L526M mutation in addition to mutation at position 550 seems to be associated with prolonged lamivudine treatment [ 26 ]. The double mutant has been shown to exhibit a 15-fold decrease of the  in vitro  susceptibility to lamivudine [ 27 ], since each mutation contributes to the loss of lamivudine sensitivity [ 28 ]. It is believed that the L526M mutation, when not accompanied with a mutation in the YMDD motif, does not confer lamivudine resistance [ 29 ]. Even so, the L526M mutation has been found alone in patients under lamivudine therapy [ 30 ]. Here, both L526M and M550V mutations were detected in all HBV sequences derived from the two blood samples collected during lamivudine treatment (1999 and 2002). Besides these common mutations, a third lamivudine resistant, rare mutation [ 11 , 31 ], namely V519L, was detected in one clone derived from the sample collected in 1999 as well as in all HBV sequences derived from the sample collected in 2002. This was in agreement with recent observations, showing that long lamivudine treatment may result in the predominance of this rare mutant [ 32 ]. The triple mutation V519L, L526M, M550V causes the concomitant amino acid substitutions E164D and I195M in the small S protein. It has been shown that this triple mutant has a reduced  in vitro  affinity to anti-HBs antibodies, similar to the hepatitis B vaccine escape mutant G145R [ 11 , 31 ]. The accumulation of mutations in the HBV genome should be monitored in order to guide patient management adequately. There is a general consensus that the lamivudine resistant single mutants in the YMDD motif (M550V/I) replicate substantially more slowly than the wild type. The addition of the mutation at position 526 may act as a compensatory change that partially restores the replication fitness of the virus [ 33 ]. Here, in agreement with this observation, HBV DNA was detected at high levels (about 10 8  copies/ml) in the blood sample collected in 1999, in which the double mutant represents the major viral population. Similar high HBV loads were observed in the sample containing the triple mutant (2002). This result is in agreement with a recent study, showing that V519L mutation also enhances viral replication [ 34 ]. The HBV population that emerged during the interruption of lamivudine treatment did not show mutations at polymerase positions 519, 526 and 550. However, another substitution, namely G473E, was observed that was associated to G119R substitution in the small S protein. Such a virus might be present as a minor population but not detected during the first lamivudine treatment. Although two out three clones of this population possessed stop codon mutations in S gene, HBV load (10 9  copies/ml) was moderately higher during the lamivudine interruption period than that found during lamivudine periods. HBV chronically infected patients submitted to lamivudine treatment may have acute exacerbation after withdrawal of drug therapy or when lamivudine resistance emerges [ 8 , 14 - 16 , 26 , 35 - 37 ]. In HIV-HBV co-infected patients, withdrawal of lamivudine may result in severe [ 8 , 35 , 37 ] or fulminant [ 37 ] hepatitis. Factors that trigger severe hepatitis in these cases are not well known. Both viral load and genome variations have been implicated in the pathogenesis of acute exacerbation. Here, the patient under study did not present clinical signs of HBV infection. The absence of acute exacerbation after the emergence of lamivudine resistant variants may be correlated to the absence of significant variations of the viral load. Indeed, HBV DNA was detected at high levels (10 8 –10 9  copies/ml) at different periods of antiretroviral treatment with and without lamivudine.
Discussion
Among lamivudine resistant mutations, those in the YMDD motif of the HBV DNA polymerase are the most common. The resistance occurs by replacement of a methionine residue at position 550 by either valine (M550V) or isoleucine (M550I). More rarely, HBV variant presenting M550S replacement may be selected during lamivudine treatment [ 24 ] The M550V variant may be accompanied by a mutation (leucine to methionine) at position 526 [ 25 ]. In the absence of HIV infection, the mutation at position 550 alone has been found in up to two-thirds of patients with lamivudine-resistant chronic hepatitis B. However, more than 90% of HIV-HBV co-infected patients display the double lamivudine resistant mutation at positions 526 and 550 [ 12 ]. Furthermore, the presence of L526M mutation in addition to mutation at position 550 seems to be associated with prolonged lamivudine treatment [ 26 ]. The double mutant has been shown to exhibit a 15-fold decrease of the  in vitro  susceptibility to lamivudine [ 27 ], since each mutation contributes to the loss of lamivudine sensitivity [ 28 ]. It is believed that the L526M mutation, when not accompanied with a mutation in the YMDD motif, does not confer lamivudine resistance [ 29 ]. Even so, the L526M mutation has been found alone in patients under lamivudine therapy [ 30 ]. Here, both L526M and M550V mutations were detected in all HBV sequences derived from the two blood samples collected during lamivudine treatment (1999 and 2002). Besides these common mutations, a third lamivudine resistant, rare mutation [ 11 , 31 ], namely V519L, was detected in one clone derived from the sample collected in 1999 as well as in all HBV sequences derived from the sample collected in 2002. This was in agreement with recent observations, showing that long lamivudine treatment may result in the predominance of this rare mutant [ 32 ]. The triple mutation V519L, L526M, M550V causes the concomitant amino acid substitutions E164D and I195M in the small S protein. It has been shown that this triple mutant has a reduced  in vitro  affinity to anti-HBs antibodies, similar to the hepatitis B vaccine escape mutant G145R [ 11 , 31 ]. The accumulation of mutations in the HBV genome should be monitored in order to guide patient management adequately.in vitroin vitroThere is a general consensus that the lamivudine resistant single mutants in the YMDD motif (M550V/I) replicate substantially more slowly than the wild type. The addition of the mutation at position 526 may act as a compensatory change that partially restores the replication fitness of the virus [ 33 ]. Here, in agreement with this observation, HBV DNA was detected at high levels (about 10 8  copies/ml) in the blood sample collected in 1999, in which the double mutant represents the major viral population. Similar high HBV loads were observed in the sample containing the triple mutant (2002). This result is in agreement with a recent study, showing that V519L mutation also enhances viral replication [ 34 ].The HBV population that emerged during the interruption of lamivudine treatment did not show mutations at polymerase positions 519, 526 and 550. However, another substitution, namely G473E, was observed that was associated to G119R substitution in the small S protein. Such a virus might be present as a minor population but not detected during the first lamivudine treatment. Although two out three clones of this population possessed stop codon mutations in S gene, HBV load (10 9  copies/ml) was moderately higher during the lamivudine interruption period than that found during lamivudine periods.HBV chronically infected patients submitted to lamivudine treatment may have acute exacerbation after withdrawal of drug therapy or when lamivudine resistance emerges [ 8 , 14 - 16 , 26 , 35 - 37 ]. In HIV-HBV co-infected patients, withdrawal of lamivudine may result in severe [ 8 , 35 , 37 ] or fulminant [ 37 ] hepatitis. Factors that trigger severe hepatitis in these cases are not well known. Both viral load and genome variations have been implicated in the pathogenesis of acute exacerbation. Here, the patient under study did not present clinical signs of HBV infection. The absence of acute exacerbation after the emergence of lamivudine resistant variants may be correlated to the absence of significant variations of the viral load. Indeed, HBV DNA was detected at high levels (10 8 –10 9  copies/ml) at different periods of antiretroviral treatment with and without lamivudine.Conclusions A rare HBV triple mutant, belonging to genotype A, subgroup A', appeared predominantly in a patient submitted to lamivudine as part of HIV treatment. This type of mutant, previously found in isolates belonging to genotypes A, D and G [ 15 , 36 ], may behave as a vaccine escape mutant. Understanding the circumstances leading to the appearance of such HBV strains may help to guide future therapies in HIV-HBV co-infected patients. A correlation may exist between acute exacerbation of hepatitis B and HBV load in lamivudine treated patients.
Conclusions
A rare HBV triple mutant, belonging to genotype A, subgroup A', appeared predominantly in a patient submitted to lamivudine as part of HIV treatment. This type of mutant, previously found in isolates belonging to genotypes A, D and G [ 15 , 36 ], may behave as a vaccine escape mutant. Understanding the circumstances leading to the appearance of such HBV strains may help to guide future therapies in HIV-HBV co-infected patients. A correlation may exist between acute exacerbation of hepatitis B and HBV load in lamivudine treated patients.Competing interests None declared.
Competing interests
None declared.Authors' contributions EAS and MVS carried out cloning and sequencing of HBV DNA. JA was involved in clinical evaluation of the patient and supervised the antiretroviral treatment. SAG conceived the study, participated in its design and coordination, and wrote the manuscript. All authors read and approved the final manuscript.
Authors' contributions
EAS and MVS carried out cloning and sequencing of HBV DNA. JA was involved in clinical evaluation of the patient and supervised the antiretroviral treatment. SAG conceived the study, participated in its design and coordination, and wrote the manuscript. All authors read and approved the final manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:


Did you ever wish you could peek inside someone's brain and see what was going on in there? In research reported in this issue of  PLoS Biology , Hajime Hirase and his colleagues at Rutgers University have done just that by focusing their microscope on the brains of living rats in order to examine how certain cells called astrocytes function in vivo.PLoS BiologyIn the longstanding quest to understand how the brain works, scientists have focused on neurons. Neurons conduct action potentials, electrical signals that transmit information in the nervous system. But the brain also contains several other types of cells called glia. ( Glia  is derived from the Latin for “glue”; these cells were thought to “hold it all together.”) One type of glial cell, the astrocyte (named for its starlike shape), is the most populous cell in the brain and forms an intimate association with neurons and their synapses. It was thought that these cells played a supporting role in the brain, ensuring the proper chemical environment for synapses.GliaRecent research, however, has suggested that astrocytes and other glial cells may play a more significant role. When examining astrocytes cultured in the lab, scientists have observed behavior suggesting that astrocytes can communicate with neurons. Though astrocytes cannot propagate electrical signals like neurons do, they can sense the transmission of such signals at the synapse between two neurons. Furthermore, astrocytes are able to propagate a different kind of signal, a chemical signal based on the release of calcium ions. Calcium signaling is a mechanism of chemical signaling that has been observed in many other cell types. The exact properties of neuron–astrocyte communication, however, are not clear because different preparations of these tissues have yielded different results. It has also not been established that this type of communication occurs in the living brain.To explore such questions, Hirase and colleagues have taken the next step by investigating the calcium signaling properties of astrocytes in the brains of living rats. To accomplish this feat, the researchers used a combination of two technologies. They monitored calcium signaling using a fluorescent dye called Fluo-4, which fluoresces in response to calcium ions. Then they used a special type of microscope called a two-photon laser scanning microscope to visualize the dye. Since this type of microscope uses a lower energy laser, it can image the dye in living tissue without causing harm.The researchers applied the dye to the brains of anesthetized rats, washed out the excess dye that had not penetrated into cells, and then imaged the tissue under the microscope. They first confirmed that they indeed were examining astrocytes and noticed that cells displayed a moderate level of baseline calcium signaling activity. They then used a drug called bicuculline to stimulate neurons and observed a significant increase in the calcium signaling activity of the astrocytes. Because bicuculline only affects neurons, this implies that the astrocytes are responding to the activity of the neurons. The researchers also found that neighboring astrocytes often also displayed coordinated calcium signaling activity, suggesting that the communication among astrocytes is facilitated by increased neuronal activity.This research confirms the complexity of astrocyte signaling functions in the living brain and demonstrates that astrocytes play far more than a supporting role in brain function. It also establishes an important experimental system for scientists seeking to understand how these distinct elements of the brain—neurons and astrocytes—work together. Though this research makes it clear that signaling exists both among astrocytes and between neurons and astrocytes, scientists have yet to understand the effect of this signaling. Some possibilities include regulation of synapse formation, modification of synaptic strength, or more complicated roles in information processing resulting from the coordination of neuronal activity. Future research using this and other systems will help reveal these functions.


Background Critically Appraised Topics (CATs) are a useful tool that helps physicians to make clinical decisions as the healthcare moves towards the practice of Evidence-Based Medicine (EBM). The fast growing World Wide Web has provided a place for physicians to share their appraised topics online, but an increasing amount of time is needed to find a particular topic within such a rich repository.
Background
Critically Appraised Topics (CATs) are a useful tool that helps physicians to make clinical decisions as the healthcare moves towards the practice of Evidence-Based Medicine (EBM). The fast growing World Wide Web has provided a place for physicians to share their appraised topics online, but an increasing amount of time is needed to find a particular topic within such a rich repository.Methods A web-based application, namely the CAT Crawler, was developed by Singapore's Bioinformatics Institute to allow physicians to adequately access available appraised topics on the Internet. A meta-search engine, as the core component of the application, finds relevant topics following keyword input. The primary objective of the work presented here is to evaluate the quantity and quality of search results obtained from the meta-search engine of the CAT Crawler by comparing them with those obtained from two individual CAT search engines. From the CAT libraries at these two sites, all possible keywords were extracted using a keyword extractor. Of those common to both libraries, ten were randomly chosen for evaluation. All ten were submitted to the two search engines individually, and through the meta-search engine of the CAT Crawler. Search results were evaluated for relevance both by medical amateurs and professionals, and the respective recall and precision were calculated.
Methods
A web-based application, namely the CAT Crawler, was developed by Singapore's Bioinformatics Institute to allow physicians to adequately access available appraised topics on the Internet. A meta-search engine, as the core component of the application, finds relevant topics following keyword input. The primary objective of the work presented here is to evaluate the quantity and quality of search results obtained from the meta-search engine of the CAT Crawler by comparing them with those obtained from two individual CAT search engines. From the CAT libraries at these two sites, all possible keywords were extracted using a keyword extractor. Of those common to both libraries, ten were randomly chosen for evaluation. All ten were submitted to the two search engines individually, and through the meta-search engine of the CAT Crawler. Search results were evaluated for relevance both by medical amateurs and professionals, and the respective recall and precision were calculated.Results While achieving an identical recall, the meta-search engine showed a precision of 77.26% (±14.45) compared to the individual search engines' 52.65% (±12.0) (p < 0.001).
Results
While achieving an identical recall, the meta-search engine showed a precision of 77.26% (±14.45) compared to the individual search engines' 52.65% (±12.0) (p < 0.001).Conclusion The results demonstrate the validity of the CAT Crawler meta-search engine approach. The improved precision due to inherent filters underlines the practical usefulness of this tool for clinicians.
Conclusion
The results demonstrate the validity of the CAT Crawler meta-search engine approach. The improved precision due to inherent filters underlines the practical usefulness of this tool for clinicians.

Background Healthcare has been steadily moving towards Evidence-Based Medicine (EBM) since the term was formally introduced in 1992 by a group led by Gordon Guyatt at McMaster University, Canada [ 1 - 3 ]. EBM promotes systematic literature review, critical appraisal skills and integrates scientific evidence with clinical expertise in the daily management of patients. The first three steps involved in the practice of EBM can comprehensively be summarized as a one-page written paper on a particular clinical topic, which is most commonly called a 'Critically Appraised Topic' (CAT) [ 4 ]. Different acronyms have emerged in various specialties, such as Best Evidence Topics (BET) [ 5 ] in emergency medicine and Evidence-Based Journal Club Reviews (EBJCR) [ 6 ] in pediatric critical care medicine. All these essentially provide physicians with a systematic method of formulating a clinical question and then critically evaluating the literature to answer the question posed. With the use of resources on the World Wide Web becoming common practice, several academic and healthcare organizations have built online CAT libraries for knowledge sharing with peer physicians. The repository of CATs has been growing steadily since the setup of the first accessible CATBank developed by the Centre for Evidence Based Medicine, Oxford in 1992 [ 7 ]. Among those, BestBETs developed by the Emergency Department, Manchester Royal Infirmary [ 8 ] and UMHS by the Department of Pediatric, University of Michigan Health System, Ann Arbor [ 9 ] hold hundreds of distinct topics. They are furnished with individual search engines for fast and direct access to a particular topic. Given the wealth of such medical information scattered in cyberspace, the effectiveness of locating the correct information has become an important issue [ 10 ]. The CAT Crawler application It is believed that more CATs will be added into the repositories as more people participate in EBM practice. However, the non-standardized electronic format of CATs has created much difficulty for physicians to access a particular topic. Accordingly, the CAT Crawler was developed at the Bioinformatics Institute, Singapore [ 11 , 12 ] to provide a one-stop search and download site for physicians by setting up a common platform to access eight popular online CAT libraries. CAT Crawler is freely accessible online [ 12 ]. The core component of the CAT Crawler is a meta-search engine. Its search is currently based on CAT resources from eight public online libraries [ 11 ]. Once the user chooses the libraries he intends to use in the search, information tailored to his needs can be produced. The matched results are sorted according to their origins. Following the user input of a query keyword, a partial search is done through information extracted during an off-line process from six websites that do not hold search engines. The remaining search is carried out by querying the two individual search engines at BestBETs and UMHS. Use of the CAT Crawler is expected to have a quantitative and qualitative improvement of the retrieved results by post-processing obtained raw results from both libraries. Motivation of the evaluation The work presented here aims to evaluate the quantity and quality of the obtained results from the CAT Crawler meta-search engine, and thus to evaluate the validity and the usefulness of the application. Recall and precision were estimated to measure the performance of this meta-search engine versus the two individual search engines at BestBETs and UMHS.
Background
Healthcare has been steadily moving towards Evidence-Based Medicine (EBM) since the term was formally introduced in 1992 by a group led by Gordon Guyatt at McMaster University, Canada [ 1 - 3 ]. EBM promotes systematic literature review, critical appraisal skills and integrates scientific evidence with clinical expertise in the daily management of patients. The first three steps involved in the practice of EBM can comprehensively be summarized as a one-page written paper on a particular clinical topic, which is most commonly called a 'Critically Appraised Topic' (CAT) [ 4 ]. Different acronyms have emerged in various specialties, such as Best Evidence Topics (BET) [ 5 ] in emergency medicine and Evidence-Based Journal Club Reviews (EBJCR) [ 6 ] in pediatric critical care medicine. All these essentially provide physicians with a systematic method of formulating a clinical question and then critically evaluating the literature to answer the question posed.With the use of resources on the World Wide Web becoming common practice, several academic and healthcare organizations have built online CAT libraries for knowledge sharing with peer physicians. The repository of CATs has been growing steadily since the setup of the first accessible CATBank developed by the Centre for Evidence Based Medicine, Oxford in 1992 [ 7 ]. Among those, BestBETs developed by the Emergency Department, Manchester Royal Infirmary [ 8 ] and UMHS by the Department of Pediatric, University of Michigan Health System, Ann Arbor [ 9 ] hold hundreds of distinct topics. They are furnished with individual search engines for fast and direct access to a particular topic. Given the wealth of such medical information scattered in cyberspace, the effectiveness of locating the correct information has become an important issue [ 10 ].The CAT Crawler application It is believed that more CATs will be added into the repositories as more people participate in EBM practice. However, the non-standardized electronic format of CATs has created much difficulty for physicians to access a particular topic. Accordingly, the CAT Crawler was developed at the Bioinformatics Institute, Singapore [ 11 , 12 ] to provide a one-stop search and download site for physicians by setting up a common platform to access eight popular online CAT libraries. CAT Crawler is freely accessible online [ 12 ]. The core component of the CAT Crawler is a meta-search engine. Its search is currently based on CAT resources from eight public online libraries [ 11 ]. Once the user chooses the libraries he intends to use in the search, information tailored to his needs can be produced. The matched results are sorted according to their origins. Following the user input of a query keyword, a partial search is done through information extracted during an off-line process from six websites that do not hold search engines. The remaining search is carried out by querying the two individual search engines at BestBETs and UMHS. Use of the CAT Crawler is expected to have a quantitative and qualitative improvement of the retrieved results by post-processing obtained raw results from both libraries.
The CAT Crawler application
It is believed that more CATs will be added into the repositories as more people participate in EBM practice. However, the non-standardized electronic format of CATs has created much difficulty for physicians to access a particular topic. Accordingly, the CAT Crawler was developed at the Bioinformatics Institute, Singapore [ 11 , 12 ] to provide a one-stop search and download site for physicians by setting up a common platform to access eight popular online CAT libraries. CAT Crawler is freely accessible online [ 12 ].The core component of the CAT Crawler is a meta-search engine. Its search is currently based on CAT resources from eight public online libraries [ 11 ]. Once the user chooses the libraries he intends to use in the search, information tailored to his needs can be produced. The matched results are sorted according to their origins.Following the user input of a query keyword, a partial search is done through information extracted during an off-line process from six websites that do not hold search engines.The remaining search is carried out by querying the two individual search engines at BestBETs and UMHS. Use of the CAT Crawler is expected to have a quantitative and qualitative improvement of the retrieved results by post-processing obtained raw results from both libraries.Motivation of the evaluation The work presented here aims to evaluate the quantity and quality of the obtained results from the CAT Crawler meta-search engine, and thus to evaluate the validity and the usefulness of the application. Recall and precision were estimated to measure the performance of this meta-search engine versus the two individual search engines at BestBETs and UMHS.
Motivation of the evaluation
The work presented here aims to evaluate the quantity and quality of the obtained results from the CAT Crawler meta-search engine, and thus to evaluate the validity and the usefulness of the application. Recall and precision were estimated to measure the performance of this meta-search engine versus the two individual search engines at BestBETs and UMHS.Methods The workflow of this study is demonstrated in Figure  1 . Selection of ten query keywords To find a viable sample of keywords for a test search, the titles of all CATs stored in the two CAT libraries, namely BestBETs and UMHS were submitted to  AnalogX Keyword Extractor , which is freely available online [ 13 ]. This led to a list of around 2000 keywords, of which approximately 500 were present in both libraries, of which ten were randomly chosen. In a second step, that list was curated so that only medically relevant keywords remained, excluding words such as  and  and  day . Search for technically relevant documents in the dataset In order to be able to calculate recall as detailed below, the  technical relevance  of all documents in the dataset must be assessed. In this study, a document is called technically relevant for a given search term if it contains this term in the full-text.  Perl  scripts were developed to examine all CATs in the two libraries BestBETs and UMHS and the total number of relevant documents as per the above definition in each library was collected for further calculation. This was done for each selected keyword and the process was independent from the search using the three search engines: the CAT Crawler, BestBETs and UMHS. Relevance evaluation of the retrieval results In the next step, those ten keywords were submitted to the search engines at BestBETs and UMHS, and to the CAT Crawler meta-search engine. The retrieved links were evaluated for their relevance by 13 volunteers, who are categorized into three groups. Among them, one physician in Group I represents medical professionals, six persons in Group II represent people who were trained in biology or medicine, and six persons in Group III represent people who do not have any medical background. Calculation of recall and precision Recall and precision are two accepted measurements to determine the utility of an information retrieval system or search strategy [ 14 ]. They are defined as: Despite the relevance evaluation from 13 volunteers, it is necessary to know the total number of the relevant documents in a database for each query keyword in order to estimate the recall. In the present study, a particular CAT in a database was defined as technically relevant if the keyword could be found in its full-text article. The CAT Crawler is designed not to hold permanently any full-text CATs [ 11 ]. When a query is done choosing the option to search only BestBETs and UMHS, the total number of relevant document in its acute database is equivalent to the sum of the number of relevant documents in the two libraries BestBETs and UMHS. Accordingly, the recall and precision of the CAT Crawler meta-search engine are revised as: Similarly, the recall and precision of the search engines at BestBETs and UMHS are estimated based on the combined repository of the two individual sites. The revised formula are shown below: Performance evaluation of the CAT Crawler versus BestBETs and UMHS The averaged precision and recall over all evaluators are used to evaluate the performance of the CAT Crawler meta-search engine. These values are compared to the estimate based on the search results from the two individual search engines at BestBETs and UMHS.
Methods
The workflow of this study is demonstrated in Figure  1 .Selection of ten query keywords To find a viable sample of keywords for a test search, the titles of all CATs stored in the two CAT libraries, namely BestBETs and UMHS were submitted to  AnalogX Keyword Extractor , which is freely available online [ 13 ]. This led to a list of around 2000 keywords, of which approximately 500 were present in both libraries, of which ten were randomly chosen. In a second step, that list was curated so that only medically relevant keywords remained, excluding words such as  and  and  day .
Selection of ten query keywords
To find a viable sample of keywords for a test search, the titles of all CATs stored in the two CAT libraries, namely BestBETs and UMHS were submitted to  AnalogX Keyword Extractor , which is freely available online [ 13 ]. This led to a list of around 2000 keywords, of which approximately 500 were present in both libraries, of which ten were randomly chosen. In a second step, that list was curated so that only medically relevant keywords remained, excluding words such as  and  and  day .AnalogX Keyword ExtractoranddaySearch for technically relevant documents in the dataset In order to be able to calculate recall as detailed below, the  technical relevance  of all documents in the dataset must be assessed. In this study, a document is called technically relevant for a given search term if it contains this term in the full-text.  Perl  scripts were developed to examine all CATs in the two libraries BestBETs and UMHS and the total number of relevant documents as per the above definition in each library was collected for further calculation. This was done for each selected keyword and the process was independent from the search using the three search engines: the CAT Crawler, BestBETs and UMHS.
Search for technically relevant documents in the dataset
In order to be able to calculate recall as detailed below, the  technical relevance  of all documents in the dataset must be assessed. In this study, a document is called technically relevant for a given search term if it contains this term in the full-text.  Perl  scripts were developed to examine all CATs in the two libraries BestBETs and UMHS and the total number of relevant documents as per the above definition in each library was collected for further calculation. This was done for each selected keyword and the process was independent from the search using the three search engines: the CAT Crawler, BestBETs and UMHS.technical relevancePerlRelevance evaluation of the retrieval results In the next step, those ten keywords were submitted to the search engines at BestBETs and UMHS, and to the CAT Crawler meta-search engine. The retrieved links were evaluated for their relevance by 13 volunteers, who are categorized into three groups. Among them, one physician in Group I represents medical professionals, six persons in Group II represent people who were trained in biology or medicine, and six persons in Group III represent people who do not have any medical background.
Relevance evaluation of the retrieval results
In the next step, those ten keywords were submitted to the search engines at BestBETs and UMHS, and to the CAT Crawler meta-search engine. The retrieved links were evaluated for their relevance by 13 volunteers, who are categorized into three groups. Among them, one physician in Group I represents medical professionals, six persons in Group II represent people who were trained in biology or medicine, and six persons in Group III represent people who do not have any medical background.Calculation of recall and precision Recall and precision are two accepted measurements to determine the utility of an information retrieval system or search strategy [ 14 ]. They are defined as: Despite the relevance evaluation from 13 volunteers, it is necessary to know the total number of the relevant documents in a database for each query keyword in order to estimate the recall. In the present study, a particular CAT in a database was defined as technically relevant if the keyword could be found in its full-text article. The CAT Crawler is designed not to hold permanently any full-text CATs [ 11 ]. When a query is done choosing the option to search only BestBETs and UMHS, the total number of relevant document in its acute database is equivalent to the sum of the number of relevant documents in the two libraries BestBETs and UMHS. Accordingly, the recall and precision of the CAT Crawler meta-search engine are revised as: Similarly, the recall and precision of the search engines at BestBETs and UMHS are estimated based on the combined repository of the two individual sites. The revised formula are shown below:
Calculation of recall and precision
Recall and precision are two accepted measurements to determine the utility of an information retrieval system or search strategy [ 14 ]. They are defined as:Despite the relevance evaluation from 13 volunteers, it is necessary to know the total number of the relevant documents in a database for each query keyword in order to estimate the recall. In the present study, a particular CAT in a database was defined as technically relevant if the keyword could be found in its full-text article.The CAT Crawler is designed not to hold permanently any full-text CATs [ 11 ]. When a query is done choosing the option to search only BestBETs and UMHS, the total number of relevant document in its acute database is equivalent to the sum of the number of relevant documents in the two libraries BestBETs and UMHS. Accordingly, the recall and precision of the CAT Crawler meta-search engine are revised as:Similarly, the recall and precision of the search engines at BestBETs and UMHS are estimated based on the combined repository of the two individual sites. The revised formula are shown below:Performance evaluation of the CAT Crawler versus BestBETs and UMHS The averaged precision and recall over all evaluators are used to evaluate the performance of the CAT Crawler meta-search engine. These values are compared to the estimate based on the search results from the two individual search engines at BestBETs and UMHS.
Performance evaluation of the CAT Crawler versus BestBETs and UMHS
The averaged precision and recall over all evaluators are used to evaluate the performance of the CAT Crawler meta-search engine. These values are compared to the estimate based on the search results from the two individual search engines at BestBETs and UMHS.Results Ten keywords for the search engine evaluation According to the predefined selection criteria, the ten keywords listed in Table  1  were selected as the seed for a test search. The number of retrieved results from each search engine was gathered with respect to each keyword query. For the selected ten medically relevant keywords, the total number of matched results are 116, 65 and132 corresponding to the three search engines at BestBETs, UMHS and CAT Crawler. The difference of 49 retrievals between the CAT Crawler and the sum of BestBETs and UMHS reflects the meta-search engine's inherent filter function which is described previously [ 11 ]. Performance evaluation of the CAT Crawler versus BestBETs and UMHS To compare the performance of the CAT Crawler meta-search engine to that of the two individual search engines, recall and precision were computed and averaged over the evaluation of all 13 participators. The data recorded are shown in Table  2 . As the CAT Crawler meta-search engine is built upon the two individual search engines, the document collection for evaluation is the combined repository of BestBETs and UMHS. The retrieved relevant documents from the CAT Crawler are the same as that from the individual search engines. This leads to the identical recall for both cases (Table  2 ). The average precision is increased from the individual search engines' 52.65% (±12.0) to the CAT Crawler's 77.26% (±14.45). Figure  2  provides a more intuitive comparison corresponding to each keyword.
Results
Ten keywords for the search engine evaluation According to the predefined selection criteria, the ten keywords listed in Table  1  were selected as the seed for a test search. The number of retrieved results from each search engine was gathered with respect to each keyword query. For the selected ten medically relevant keywords, the total number of matched results are 116, 65 and132 corresponding to the three search engines at BestBETs, UMHS and CAT Crawler. The difference of 49 retrievals between the CAT Crawler and the sum of BestBETs and UMHS reflects the meta-search engine's inherent filter function which is described previously [ 11 ].
Ten keywords for the search engine evaluation
According to the predefined selection criteria, the ten keywords listed in Table  1  were selected as the seed for a test search. The number of retrieved results from each search engine was gathered with respect to each keyword query. For the selected ten medically relevant keywords, the total number of matched results are 116, 65 and132 corresponding to the three search engines at BestBETs, UMHS and CAT Crawler. The difference of 49 retrievals between the CAT Crawler and the sum of BestBETs and UMHS reflects the meta-search engine's inherent filter function which is described previously [ 11 ].Performance evaluation of the CAT Crawler versus BestBETs and UMHS To compare the performance of the CAT Crawler meta-search engine to that of the two individual search engines, recall and precision were computed and averaged over the evaluation of all 13 participators. The data recorded are shown in Table  2 . As the CAT Crawler meta-search engine is built upon the two individual search engines, the document collection for evaluation is the combined repository of BestBETs and UMHS. The retrieved relevant documents from the CAT Crawler are the same as that from the individual search engines. This leads to the identical recall for both cases (Table  2 ). The average precision is increased from the individual search engines' 52.65% (±12.0) to the CAT Crawler's 77.26% (±14.45). Figure  2  provides a more intuitive comparison corresponding to each keyword.
Performance evaluation of the CAT Crawler versus BestBETs and UMHS
To compare the performance of the CAT Crawler meta-search engine to that of the two individual search engines, recall and precision were computed and averaged over the evaluation of all 13 participators. The data recorded are shown in Table  2 . As the CAT Crawler meta-search engine is built upon the two individual search engines, the document collection for evaluation is the combined repository of BestBETs and UMHS. The retrieved relevant documents from the CAT Crawler are the same as that from the individual search engines. This leads to the identical recall for both cases (Table  2 ). The average precision is increased from the individual search engines' 52.65% (±12.0) to the CAT Crawler's 77.26% (±14.45). Figure  2  provides a more intuitive comparison corresponding to each keyword.Discussion The performance evaluation clearly places the CAT Crawler meta-search engine on par with the individual search engines at BestBETs and UMHS as far as recall is concerned, and well above them for precision (see Table  2  and Figure  2 ). According to these results, the application can be called successful: by using the CAT Crawler to look for relevant information at specific sites, the medical professional will obtain as much information as by going to the sites directly, but the precision of the obtained results will be higher. Benoit [ 15 ] has analyzed various methods of information retrieval and their impact on user behavior. He finds that users wish for greater interactive opportunities to determine for themselves the potential relevance of documents, and that a parts-of-document approach is preferable for many information retrieval situations. At present, the CAT Crawler allows a number of interactive opportunities [ 11 ], but their implementation would have no impact on the calculation of recall and precision under the condition of the present study. Benoit's reasoning should be kept in mind, however, for improving the user friendliness in the sense that some further useful filter functions can be included in future versions of the application. While such advanced search functions will be profitable when large datasets are studied, the currently still manageable information in the online CAT libraries [ 11 ] will serve the user better if initially displayed in a broader way. For example, some of the information displayed here may be older than 18 months, which makes it undesirable according to the strict rules for CAT updating as defined by Sackett et al [ 3 ]. Formally outdated information, however, may in a given situation still be "best evidence" and positively influence the decision-making. Use of filters to block aged information will certainly influence this process. Despite the encouraging results, some fundamental questions regarding the evaluation of this meta-search engine in particular, and also meta-search engines in general remain unsolved. With regard to recall, there is the theoretical possibility that manually searching all documents at a given repository will yield a higher recall for a given search term. In view of hundreds of CAT documents per repository, however, it seems unlikely that a human evaluator's attention will not wander, leading to less than optimal scrutiny of the documents and introducing a non-quantifiable error to the evaluation. This is a general problem of knowledge databases, especially when indexing is done by humans, whose decisions are not consistent. In a study of 700 Medline references indexed in duplicate, the consistency of main subject-heading indexing was only 68% and that for heading-subheading combinations was significantly less [ 16 ]. Also, in two studies [ 17 , 18 ] on Medline searching, there was considerable disagreement by those judging relevance of the retrieved documents regarding which documents were relevant to a given query. In order to overcome this problem, the number of documents that contained a given keyword as found by the keyword extractor was used as the basis for calculating the technical recall. This may (or may not) lead to numerical results for recall that differ from the absolute true value as determined above. As the same numbers are used throughout, however, the comparison of search results obtained by the individual search engines and the CAT Crawler meta-search engine remains valid. Critics have pointed out the over-reliance of researchers on the use of recall and precision in evaluation studies [ 18 ] and the difficulty to design an experiment that allows both laboratory-style control and operational realism [ 19 ]. For instance, recall may be of only little consequence once the user has found a useful document. Rhodes and Maes [ 20 ] evaluated both with a traditional field user test and then asked for relevance feedback. In their experiment, users gave a score 1–5 to each document that was delivered to calculate an overall average value for perceived precision. While a document can get a high score for precision, it may at the same time get a low score for practical usefulness. This was often due to the fact that the documents were already known to the users, in some cases had even been written by them. Accordingly, Rhodes and Maes [ 20 ] added features to the system that weeded out relevant documents that by some predefined criteria would not be useful. As a result, the measurable precision could be worse, but the overall usefulness could be better. In the study presented here, a similar approach was chosen in the instructions to the evaluators in the sense that they could make the distinction between 'irrelevant' (e.g. the retrieved document was only a web hosted clinical question) and 'medically irrelevant' (e.g. the word  Appendicitis  appeared only in the reference section of a document dealing with questions of abdominal pain relief). Due to the relatively small number, no difference could be detected between the various grades of relevance, and results were pooled to relevant/irrelevant and used for calculating recall and precision as described above. If a larger number of volunteers could be recruited, repetition of this evaluation might yield interesting results. Other approaches have been spawned to evaluating system effectiveness in order to minimize these problems with recall and precision. One example are task-oriented methods that measure how well the user can perform certain tasks [ 21 - 24 ]. These different approaches were not chosen in this study for a reason: the primary aim was to compare the search engines. Under the present restrictions, recall and precision allow to answer this question.
Discussion
The performance evaluation clearly places the CAT Crawler meta-search engine on par with the individual search engines at BestBETs and UMHS as far as recall is concerned, and well above them for precision (see Table  2  and Figure  2 ). According to these results, the application can be called successful: by using the CAT Crawler to look for relevant information at specific sites, the medical professional will obtain as much information as by going to the sites directly, but the precision of the obtained results will be higher.Benoit [ 15 ] has analyzed various methods of information retrieval and their impact on user behavior. He finds that users wish for greater interactive opportunities to determine for themselves the potential relevance of documents, and that a parts-of-document approach is preferable for many information retrieval situations. At present, the CAT Crawler allows a number of interactive opportunities [ 11 ], but their implementation would have no impact on the calculation of recall and precision under the condition of the present study. Benoit's reasoning should be kept in mind, however, for improving the user friendliness in the sense that some further useful filter functions can be included in future versions of the application. While such advanced search functions will be profitable when large datasets are studied, the currently still manageable information in the online CAT libraries [ 11 ] will serve the user better if initially displayed in a broader way. For example, some of the information displayed here may be older than 18 months, which makes it undesirable according to the strict rules for CAT updating as defined by Sackett et al [ 3 ]. Formally outdated information, however, may in a given situation still be "best evidence" and positively influence the decision-making. Use of filters to block aged information will certainly influence this process.Despite the encouraging results, some fundamental questions regarding the evaluation of this meta-search engine in particular, and also meta-search engines in general remain unsolved.With regard to recall, there is the theoretical possibility that manually searching all documents at a given repository will yield a higher recall for a given search term. In view of hundreds of CAT documents per repository, however, it seems unlikely that a human evaluator's attention will not wander, leading to less than optimal scrutiny of the documents and introducing a non-quantifiable error to the evaluation. This is a general problem of knowledge databases, especially when indexing is done by humans, whose decisions are not consistent. In a study of 700 Medline references indexed in duplicate, the consistency of main subject-heading indexing was only 68% and that for heading-subheading combinations was significantly less [ 16 ]. Also, in two studies [ 17 , 18 ] on Medline searching, there was considerable disagreement by those judging relevance of the retrieved documents regarding which documents were relevant to a given query.In order to overcome this problem, the number of documents that contained a given keyword as found by the keyword extractor was used as the basis for calculating the technical recall. This may (or may not) lead to numerical results for recall that differ from the absolute true value as determined above. As the same numbers are used throughout, however, the comparison of search results obtained by the individual search engines and the CAT Crawler meta-search engine remains valid.Critics have pointed out the over-reliance of researchers on the use of recall and precision in evaluation studies [ 18 ] and the difficulty to design an experiment that allows both laboratory-style control and operational realism [ 19 ]. For instance, recall may be of only little consequence once the user has found a useful document. Rhodes and Maes [ 20 ] evaluated both with a traditional field user test and then asked for relevance feedback. In their experiment, users gave a score 1–5 to each document that was delivered to calculate an overall average value for perceived precision. While a document can get a high score for precision, it may at the same time get a low score for practical usefulness. This was often due to the fact that the documents were already known to the users, in some cases had even been written by them. Accordingly, Rhodes and Maes [ 20 ] added features to the system that weeded out relevant documents that by some predefined criteria would not be useful. As a result, the measurable precision could be worse, but the overall usefulness could be better. In the study presented here, a similar approach was chosen in the instructions to the evaluators in the sense that they could make the distinction between 'irrelevant' (e.g. the retrieved document was only a web hosted clinical question) and 'medically irrelevant' (e.g. the word  Appendicitis  appeared only in the reference section of a document dealing with questions of abdominal pain relief). Due to the relatively small number, no difference could be detected between the various grades of relevance, and results were pooled to relevant/irrelevant and used for calculating recall and precision as described above. If a larger number of volunteers could be recruited, repetition of this evaluation might yield interesting results.AppendicitisOther approaches have been spawned to evaluating system effectiveness in order to minimize these problems with recall and precision. One example are task-oriented methods that measure how well the user can perform certain tasks [ 21 - 24 ]. These different approaches were not chosen in this study for a reason: the primary aim was to compare the search engines. Under the present restrictions, recall and precision allow to answer this question.Conclusions In summary, the data obtained from the analysis of search results obtained from identical queries submitted to the two CAT libraries at BestBETs and UMHS, using either their respective search engines or the CAT Crawler meta-search engine, showed a competitive recall, and superior precision of the meta-search engine compared to the individual search engines.
Conclusions
In summary, the data obtained from the analysis of search results obtained from identical queries submitted to the two CAT libraries at BestBETs and UMHS, using either their respective search engines or the CAT Crawler meta-search engine, showed a competitive recall, and superior precision of the meta-search engine compared to the individual search engines.Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contributions PD participated in the design of the study, data analysis and drafting of the manuscript. LLW and SN generated raw data for the study. ML was involved in drafting the manuscript. AM designed the study and participated in the drafting of the manuscript.
Authors' contributions
PD participated in the design of the study, data analysis and drafting of the manuscript. LLW and SN generated raw data for the study. ML was involved in drafting the manuscript. AM designed the study and participated in the drafting of the manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here: