poland is a central european country with a population of more than 38 million inhabitants . from the beginning of the hiv epidemic in 1985 to 2004
, 8491 cases of hiv infection , 1421 aids cases , and 676 hiv / aids - associated deaths have been reported and confirmed . at the beginning of 2004 , more than 2000
hiv - positive individuals were receiving antiretroviral treatment . in silesia , which has 4.7 million citizens and is the second largest population among polish provinces , the number of hiv infections from the beginning of the epidemic to 2004 was 1123 , which constitutes 13.2% of the total number of hiv infections detected in poland . in that time ,
185 aids cases and 87 hiv / aids  associated deaths have been recognized in silesia .
the mean number of newly diagnosed hiv cases during this time was less than 60 per year in our region .
the epidemiologic and clinical situation regarding hiv infections in silesia seems to be similar to that observed in other parts of poland .
inability of the viral reverse transcriptase ( rt ) to proofread nucleotide sequences during replication results in a high degree of hiv-1 genome variability , which together with rapid viral turnover , contributes to drug - resistant mutant development . in the absence of antiretroviral treatment ,
antiretroviral drugs incompletely suppressing viral replication exert selective pressure that results in resistant - strain dominance .
drug selection is not the only possible way of the resistant variants development , because the transmission of drug - resistant mutants to treatment - nave subjects has been reported in many cases [ 612 ] . to date
, hiv isolates resistant to each class of antiretroviral drugs were identified , and drug resistance is considered a major contributor to treatment failure .
currently approved antiretrovirals are targeted against viral rt , protease , integrase , and envelope glycoprotein .
the nucleoside inhibitors of hiv-1 rt were introduced as the first antiretroviral drugs in 1987 , and they are still the most widely used drug class .
for this reason , screening for the occurrence of rt inhibitors resistance mutations in the hiv-1 pol gene seems to be a suitable tool for presenting retrospective drug resistance studies .
such retrospective investigations were undertaken to enable comparisons with the present situation and to follow the dynamics of possible future changes in the drug resistance patterns .
although knowledge of the global situation concerning drug resistance mutation frequencies and types is permanently growing , in many local populations , such information is still rather limited and unsatisfactory .
, we have undertaken retrospective studies on drug resistance mutations among the 101 hiv-1positive silesian individuals who acquired infection before 2004 .
our studies have focused on estimations of the drug resistance mutations types , frequencies , and the level of their influence on drug effectiveness , in the group with almost 35% treatment - nave subjects .
enrollment of patients not administered with antiretroviral drugs in the studied population sheds some light on a potential transmission of drug - resistant mutants in the history of hiv-1 epidemic in silesia .
presented results may serve as an indispensable starting point for the further analysis of hiv-1 drug resistance and possible changes in this field in our region .
we included a group of 101 hiv-1  seropositive individuals infected before 2004 ( table 1 ) .
all patients were silesian residents and were attending the department of diagnostics and therapy for aids in chorzw , poland .
antiretroviral therapy was introduced before samples collection in 66 patients ( 65.3% ) , 7 of them ( 10.6% ) were treated with the nucleoside reverse transcriptase inhibitors ( nrtis ) exclusively , 12 ( 18.2% ) received nrtis with nonnucleoside reverse transcriptase inhibitors ( nnrtis ) , 30 ( 45.5% ) were using nrtis and protease inhibitors ( pis ) , and 17 patients ( 25.7% ) were treated with the drugs from nrtis , nnrtis , and pis classes .
thirty - five subjects ( 34.7% ) had received no antiretroviral treatment by the time of specimen collection .
eighty individuals ( 79.2% ) were intravenous drug users ( idus ) and 21 ( 20.8% ) reported no drug addiction , with 11 being heterosexuals ( 10.9% ) , and 10 being homosexual men ( 9.9% ) .
blood samples were obtained after patients signed informed consent ; the study fell under the agreement of the medical university of silesia bioethics committee ( nn-6501 - 191/i/05/06 ) .
peripheral blood mononuclear cells ( pbmcs ) were separated from edta - treated blood samples by ficoll - histopaque density gradient centrifugation ( 1.077 g / cm ) and immediately stored at 80c .
genomic dna extraction was performed from uncultured pbmc by the routine proteinase k and phenol - chloroform method .
proviral dna was amplified in the part of the pol gene covering the first 256 codons of the reverse transcriptase by nested polymerase chain reaction ( pcr ) using previously described primer pairs .
briefly , the pol gene fragment of 2017 bp spanning nucleotides 23774393 according to the hiv hxb2 reference sequence ( genbank accession number k03455 ) was amplified with the use of rt1 and rt2 as outer primers . in the second step , rt3 and rt4 inner primers were used to amplify 775-bp pol region located between 2545 and 3319 nucleotides in the hiv hxb2 .
the cycling parameters of the first amplification round with the rt1 and rt2 primers were an initial denaturation step at 94c/5 minutes , followed by 35 cycles of 94c/1 minute , 55c/1 minute , 72c/2.5 minutes , with the final extension at 72c/10 minutes , in a final volume of 50 l .
rt3-rt4 amplification was conducted with the following conditions : an initial denaturation step at 94c/5 minutes , followed by 35 cycles of 94c/30 seconds , 54c/30 seconds , 72c/1 minute , with the final elongation at 72c/5 minutes , in a final volume of 50 l .
purified nested - pcr products were further subjected to direct sequencing using the abi prism big dye terminator version 3.1 cycle sequencing kit ( applied biosystems , foster city , ca , usa ) with the rt3-rt4 primers for the pol gene fragment recognition .
sequences of both strands were determined separately using an abi prism 377 automated dna sequencer ( applied biosystems ) .
the nucleotide sequences reported in the presented study were submitted to the genbank under accession numbers eu910734eu910834 .
reverse transcriptase inhibitors resistance mutations in the pol gene fragments were determined and interpreted with the hivdb : genotypic resistance interpretation algorithm available at the stanford university hiv drug resistance database . according to this algorithm ,
 determining the hiv-1 genetic variant was performed with the ncbi genotyping tool , a program designed to identify the subtype sequence homologies with the use of the basic local alignment search tool ( blast ) algorithm .
reference sequences representing all hiv-1 genetic groups , subtypes , subsubtypes , and circulating recombinant forms were applied during ncbi genotyping tool analysis .
hypermutated sequences were recognized with the hypermut version 2.0 program . to compare data between patients who received antiretroviral therapy with treatment - nave subjects ,
the mann - whitney u test was used for analyses of quantitative variables as age , and the 2-tailed fisher exact test was used to compare qualitative variables such as sex and transmission risk group . to estimate the statistical values ,
we included a group of 101 hiv-1  seropositive individuals infected before 2004 ( table 1 ) .
all patients were silesian residents and were attending the department of diagnostics and therapy for aids in chorzw , poland .
antiretroviral therapy was introduced before samples collection in 66 patients ( 65.3% ) , 7 of them ( 10.6% ) were treated with the nucleoside reverse transcriptase inhibitors ( nrtis ) exclusively , 12 ( 18.2% ) received nrtis with nonnucleoside reverse transcriptase inhibitors ( nnrtis ) , 30 ( 45.5% ) were using nrtis and protease inhibitors ( pis ) , and 17 patients ( 25.7% ) were treated with the drugs from nrtis , nnrtis , and pis classes .
thirty - five subjects ( 34.7% ) had received no antiretroviral treatment by the time of specimen collection .
eighty individuals ( 79.2% ) were intravenous drug users ( idus ) and 21 ( 20.8% ) reported no drug addiction , with 11 being heterosexuals ( 10.9% ) , and 10 being homosexual men ( 9.9% ) .
blood samples were obtained after patients signed informed consent ; the study fell under the agreement of the medical university of silesia bioethics committee ( nn-6501 - 191/i/05/06 ) .
peripheral blood mononuclear cells ( pbmcs ) were separated from edta - treated blood samples by ficoll - histopaque density gradient centrifugation ( 1.077 g / cm ) and immediately stored at 80c .
genomic dna extraction was performed from uncultured pbmc by the routine proteinase k and phenol - chloroform method .
proviral dna was amplified in the part of the pol gene covering the first 256 codons of the reverse transcriptase by nested polymerase chain reaction ( pcr ) using previously described primer pairs .
this gene region is known for harboring most of the rt inhibitors resistance mutations . briefly
, the pol gene fragment of 2017 bp spanning nucleotides 23774393 according to the hiv hxb2 reference sequence ( genbank accession number k03455 ) was amplified with the use of rt1 and rt2 as outer primers . in the second step , rt3 and rt4 inner primers were used to amplify 775-bp pol region located between 2545 and 3319 nucleotides in the hiv hxb2 .
the cycling parameters of the first amplification round with the rt1 and rt2 primers were an initial denaturation step at 94c/5 minutes , followed by 35 cycles of 94c/1 minute , 55c/1 minute , 72c/2.5 minutes , with the final extension at 72c/10 minutes , in a final volume of 50 l .
rt3-rt4 amplification was conducted with the following conditions : an initial denaturation step at 94c/5 minutes , followed by 35 cycles of 94c/30 seconds , 54c/30 seconds , 72c/1 minute , with the final elongation at 72c/5 minutes , in a final volume of 50 l .
purified nested - pcr products were further subjected to direct sequencing using the abi prism big dye terminator version 3.1 cycle sequencing kit ( applied biosystems , foster city , ca , usa ) with the rt3-rt4 primers for the pol gene fragment recognition .
sequences of both strands were determined separately using an abi prism 377 automated dna sequencer ( applied biosystems ) .
the nucleotide sequences reported in the presented study were submitted to the genbank under accession numbers eu910734eu910834 .
reverse transcriptase inhibitors resistance mutations in the pol gene fragments were determined and interpreted with the hivdb : genotypic resistance interpretation algorithm available at the stanford university hiv drug resistance database . according to this algorithm ,
the resistance levels were specified as  potential low ,   low ,   intermediate ,  and  high .  determining the hiv-1 genetic variant was performed with the ncbi genotyping tool , a program designed to identify the subtype sequence homologies with the use of the basic local alignment search tool ( blast ) algorithm .
reference sequences representing all hiv-1 genetic groups , subtypes , subsubtypes , and circulating recombinant forms were applied during ncbi genotyping tool analysis .
to compare data between patients who received antiretroviral therapy with treatment - nave subjects , statistical analyses were performed .
the mann - whitney u test was used for analyses of quantitative variables as age , and the 2-tailed fisher exact test was used to compare qualitative variables such as sex and transmission risk group . to estimate the statistical values ,
all investigated samples collected from 101 hiv-1positive patients were successfully amplified and sequenced in the part of the pol gene spanning the first 256 codons for the rt . according to the ncbi genotyping tool
, all viral nucleotide sequences were classified as group m , subtype b. one viral sequence was recognized as hyper - mutated according to the hypermut program and was excluded from further analyses .
hypermutated sequence was derived from an idu who had experienced no anti - retroviral therapy . in the analyzed sequences
all of them were identified exclusively in the hiv-1 proviral dna obtained from patients receiving antiretroviral therapy . among all detected mutations corresponding to the drug resistance ,
7 ( m41l , a62v , d67n , k70r , m184v , t215y , and t215s ) were related to nrtis / ntrtis resistance .
the percentage of hiv-1 strains carrying any of the following substitutions : m41l , k70r , m184v , and t215y was 2% .
mutations : a62v , d67n , and t215s were present in 1% of hiv-1 strains .
the frequency of all nrtis / ntrtis resistance - associated mutations in the analyzed sequences was 10.9% .
further , 3 substitutions ( k103n , v179d , and y181c ) were related to the nnrtis resistance . in the studied population ,
the v179d change occurred in 1% of the investigated hiv-1 pol sequences . in the analyzed group of sequences ,
all rt inhibitors resistance - related mutations mentioned above were detected in 10 ( 9.9% ) of 101 pol gene sequences examined . in 7 analyzed pol gene fragments ,
only single resistance - associated mutations were identified , in 2 sequences , 2 drug resistance mutations were present , and in 1 fragment , 5 different substitutions were found . to evaluate the potential influence of recognized resistance - related substitutions on the level of drug resistance
sequence examinations revealed different extents of resistance to nrtis / ntrtis for 5 hiv-1 strains ( table 3 ) .
these viral strains were found among 5 patients who were receiving antiretroviral drugs for 116 months on average .
next , 5 strains exhibited different levels of nnrtis resistance , and they were present among 5 individuals with mean of 49 months of therapy ( table 3 ) .
we observed no hiv-1 strains with dual resistance to nrtis / ntrtis and nnrtis . in the nrtis / ntrtis drug classes , a high level of resistance
hiv-1 strains with mutations decreasing nnrtis susceptibility were highly resistant to delavirdine , nevirapine , and efavirenz .
the hiv-1 drug resistance development is currently one of the main factors limiting the efficacy of antiretroviral therapy . selection and transmission of drug - resistant mutants restricts the number of possible antiretroviral drug combinations and reduces the chance for life - long therapy responsiveness of individual patients .
identification of the mutations related to the drug resistance in the viral genome is widely used method for the drug resistance estimation . in europe
, drug resistance testing is recommended especially among patients with therapy failure and among pregnant women , as well as their hiv-1  infected children .
it is also strongly suggested to perform a test on hiv-1 drug susceptibility among subjects with the acute hiv-1 infection or recent seroconverters before introducing the first anti - retroviral therapy regimen to facilitate the optimal choice of the drug combination .
despite the fact that the most drug resistance investigation is focused on hiv-1 subtype b genotype , it has been shown that particular hiv-1 genetic variants may exhibit differences in natural susceptibility to the antiretroviral drugs .
for instance , group o is naturally resistant to nnrtis because of harboring the y181c and a98 g mutations in the rt coding region of the pol gene . innate resistance to nnrtis is also carried by some group m isolates , for example subtype c or d .
there also are indications that subtype - specific pathways for the development of resistance against certain drugs may exist .
although drug resistance mutations that were described for subtype b also have been observed among other viral genetic forms , it has been shown that some resistance - related substitutions may appear among various genetic variants with different frequency .
this is true for the nnrti resistance mutation , v106 m , which is much more frequent in the pol gene of subtype c than b , as development of this substitution may be dependent on the pre - existing genomic background . for these reasons
, it was important to determine genetic variants present among the investigated population . by 2004 ,
the only hiv-1 genetic form that was recognized among the silesian patients was subtype b. this hiv-1 genetic variant also remains the most - prevalent form in the majority of the european countries .
we analyzed 101 pol gene fragments covering the first 256 codons of a total of 440 for rt protein .
seven of 10 recognized different drug resistance mutations were involved in the reduced nrtis / ntrtis susceptibility .
they were m41l , a62v , d67n , k70r , m184v , t215y , and t215s .
presence of the m41l , d67n , k70r , and t215y mutations in hiv-1 pol gene results in the emergence of the phosphorolytic activity of the rt , allowing the enzyme to excise nucleotide analogues already incorporated to the dna chain .
the t215s substitution has been reported as a partial reversion change at codon 215 , conferring increased risk of virologic failure of azt and d4 t in antiretroviral - nave patients . in the presence of these drugs ,
strains harboring the t215s mutation may exhibit rapid development of the t215y mutation . the a62v substitution , together with unobserved in the analyzed pol gene fragments the q151 m mutation , is responsible for the cross - resistance between nrtis drugs . in the presence of a62v and q151
m rt can discern between the cellular dntps and activated nrtis molecules , which prevent nrtis incorporation to the growing proviral dna polymer .
strains with m184v substitution possess rt with decreased affinity to the nrtis molecules , which leads to resistance to lamivudine , emtricitabine , and to lesser extent , to abacavir . on the other hand , this mutation may increase viral susceptibility to zidovudine , stavudine , and tenofovir . another 3 identified pol gene mutations ( k103n , v179d , and y181c ) are described to be involved in nnrtis resistance .
all these substitutions generate changes in the hydrophobic site of rt where nnrtis are bound .
this results in the decreased affinity of rt to nnrtis and consequently , in the lack of the nnrtis antiretroviral activity . based on the identified and described resistance mutations
, we could establish that 10 viral strains in the investigated silesian population were , to a different extent , resistant to the rt inhibitors ( table 3 ) . because in the presented study hiv-1 strains with the drug resistance mutations were found only among persons receiving antiretroviral drugs
on the other hand , as the time between hiv-1 infection diagnosis and collecting samples was long ( on average 47 months ) , we can not exclude that the treatment - nave patients could have been infected with drug - resistant hiv-1 , but reversion to the sensitive wild - type virus could have occurred over time . however , there is some evidence that drug - resistant viruses can persist for years in the plasma of treatment - nave hiv-1-positive individuals .
the presence of such viruses among individuals without therapy experience may act as an indicator of the drug resistance transmission in a given population . in this view
, the lack of resistant viruses among the treatment - nave silesian patients hiv-1-infected before 2004 can indicate that there was no transmission of the antiretroviral drug - resistant viruses in the studied population to that time .
this finding may be meaningful for hiv-1 drug resistance testing strategies in our region , outlining the usefulness of storing the earliest sample available , to test the drug resistance before planned treatment introduction .
the possibility of superinfection , however , should be taken into account during interpretation of test results in such cases .
the frequency of the resistant viral strains in the studied population of hiv-1-positive individuals who acquired infection before the year 2004 was 9.9% , and as we noted earlier , drug - resistant viruses were not observed among the treatment - nave subjects . compared with our results , in 19 european countries ,
a 10.4% frequency of drug resistance was observed among persons with the hiv-1 infection recognized between 1996 and 2002 who had never received antiretroviral drugs .
this rate remained stable in the next study , which included a european population of treatment - nave persons , with the hiv-1 infection diagnosed in 20022003 .
surprisingly , in poland , the frequency of drug - resistant strains among untreated individuals with the infection detected between 2004 and 2006 has reached 14.7% .
mutations of this kind , such as k70r / e , t69s / n , and t215d / e were present in 2.6% samples . all recognized nnrtis resistance mutations ( a98 g , k101e , k103n , v108i , and m230l ) were identified in 1.7% hiv-1 strains . in our studies , we have observed only 2 mutations from those described above .
they were the k70r and k103n substitutions , each present in 2% of viral strains ; however , we found them exclusively in the samples from subjects with therapy experience .
the authors of the earlier quoted investigation described the group of patients that could have been hiv-1-infected later than subjects included in our retrospective studies .
this fact may be responsible for the different frequencies of the drug resistance among treatment - nave patients .
both studies taken together may represent a shift in the drug resistance frequencies that has occurred in a relatively short time .
we think that the wider access to the antiretroviral drugs results in the increased selective pressure on the virus .
this selective pressure seems to be the main factor responsible for the wider spread of the drug resistant variants causing the observed shift .
the second possibility is that relevant differences in drug - resistant mutants frequencies among drug - nave patients may exist in different parts of poland .
because we found no drug - resistant viruses among treatment - nave silesian patients hiv-1 infected before 2004 , it is highly possible that there was no drug - resistant viruses transmission in the studied population to that time .
forthcoming studies including recent hiv-1 infections will address the question if this unusual situation among the european regions is stable over time or if it changes as more antiretroviral drugs are applied in the population .
 summarybackgroundthis study was a retrospective analysis of drug resistance mutations among hiv-1 strains prevalent in silesia , poland , from the origin of the epidemic to 2004 . 
 the investigations included both type and frequency of the reverse transcriptase inhibitors resistance mutations and estimation of the drugs resistance levels.material/methodsproviral dna , obtained from peripheral blood mononuclear cells of the 101 hiv-1infected patients , was amplified and sequenced in the pol gene fragment covering the first 256 codons of the reverse transcriptase ( rt ) . 
 reverse transcriptase inhibitors resistance mutations were determined and interpreted with the hivdb : genotypic resistance interpretation algorithm available from the stanford university hiv drug resistance database . in the examined population , 35 subjects ( 34.7% ) received no antiretroviral treatment by the time of specimen collection.resultsthe overall frequency of the rt inhibitors resistance mutations in the studied population was 15.8% . 
 substitutions related to the reverse transcriptase inhibitors resistance were identified in 10 pol gene sequences ( 9.9% ) , all of them were present in the hiv-1 sequences obtained from persons receiving antiretroviral therapy.conclusionslack of drug - resistant viruses among treatment - nave silesian patients hiv-1-infected before the year 2004 may indicate that there was no transmission of the drug - resistant viruses in the studied population to that time . 
Background
Material and Methods
Study population
Samples preparation and DNA extraction
Amplification and sequencing of HIV-1 proviral DNA
Analysis of sequence data
Statistical analyses
Results
Discussion
Conclusions