cervical cancer is one of the most critical health issues , affecting women in developing countries . it is regarded as the commonest malignancy of the female genital tract in nigeria ( 1 ) . there are 36.59 million nigerians , aged 15 years and older , who are at risk of cervical cancer ( 2 ) . cervical cancer is regarded as the second most common cancer amongst women worldwide , and it accounts for 250,000 deaths and 500,000 new patients worldwide , annually ( 3 ) . persistent infection by human papillomavirus ( hpv ) is considered to be the main causative agent of cervical cancer and other anogenital cancers . transmission of genital hpv infection occurs predominantly through sexual contact , although the infection is mostly self - limiting and clears within 6 months to 2 years , in most cases . persistent infection by high risk hpv is a critical factor in the development of cervical cancer . also , factors that contribute to consistent occurrence of hpv infection and development of cervical cancer in women include the number of sexual partners , the age of initiation of sexual activity and the sexual behaviour of the male partner ( 4 ) . cervical cytology screening , utilizing papanicolaou ( pap ) staining techniques , remains the most used method for screening for cervical cancer in nigeria , despite the high rates of false negatives ( 5 ) . the severe restriction on the availability of infrastructure , resources and funding , in developing countries , has made it difficult to adopt hpv dna detection , as a routine cervical cancer prevention strategy ( 5 ) . therefore , this research utilizes these molecular techniques in detecting the presence of hpv in abnormal cytology , rendering it as a confirmatory test to cytology . over 80% of cervical cancers in sub saharan africa are detected in late stages , which is largely due to the lack of information about cervical cancer and prevention services ( 3 ) . the control of cervical cancer is directly linked to prevention of hpv infection and identification of precancer cases , through screening and subsequent management of identified invasive diseases . these techniques are generally unavailable in nigeria . in cases , were cervical cancer screening is performed , the pap test is often the only screening test done . consequently , the use of hpv dna typing , which is a major tool used in developed countries , is hardly utilized in nigeria . this study aimed to use hpv dna typing , as a means of ascertaining the presence of high risk hpv in cytology samples , positive for cervical intraepithelial neoplasia ( cin ) , using the pap screening procedure . lokoja town has a heterogeneous population comprising the igala , ebira , yoruba , nupe , okun , oworo , bassa - nge , kakanda , egbura and hausa ethnic groups . the town is the most centrally located in nigeria , at the confluence of the rivers niger and benue . the study was carried out among women attending the obstetrics and gynecology clinic of the federal medical centre , lokoja , nigeria , aged 16 - 65 years old , who were willing to participate and met the inclusion criteria . a structured close ended questionnaire was administered to these patients , after an informed consent had been obtained , followed by clinical examination . approval for this study was obtained from the ethical review committee of the federal medical centre , lokoja . the study did not implicate any cost to the subjects and information from the patients and/or parents / tutors were confidential . a structured close ended questionnaire was administered to the participating population , using the convenience sampling technique , after due consent had been taken . ( 6 ) , each of the participants being placed in a dorsal position , with her legs flexed at the hip and knee and abducted . a cusco s bivalve speculum , which was not lubricated , was then passed and fixed to visualize the cervix , under a bright light source . the hook end of the ayres spatula was inserted into the cervix and swept through 360 movements , either in a clockwise or counterclockwise direction , to scrape the entire squamocolumnar junction of the transformation zone . the smears collected were immediately fixed to slides , before being transferred to the laboratory for processing . the fixed smears were stained by a cytotechnologist , using the pap staining procedure and read by a histopathologist , under a light microscope . the slides were reported as normal , inflammatory , abnormal ( epithelial lesion ) or unsatisfactory . the slides with abnormal smear were then tested for the presence of hpv dna , with several of the slides with normal cytology , which had been previously confirmed for the absence of hpv , utilized for control . to start the molecular analysis , the cover slips on the slides were removed , carefully . after the cover slips were removed , the smears were carefully taken from the slides , using sterile scalpel blades , as described by sharifah et al . a dna extraction kit ( integrated dna technologies , coralville , ia , usa ) was utilized for dna extraction . buffy coats , as well as proteinase k , were used to lyse the cells of the organism , with the dna extraction done using chloroform . equal volumes of 100% ethanol were added to 20 l of 3 m sodium acetate . , the tubes were centrifuged at maximum speeds , for 10 - 30 minutes , in a cold centrifuge , in the same orientation . a volume of 400 l 70% ethanol was added to the tubes containing the dna pellets and spun at maximum speed , for 5 minute , at 4c . the dna were dried out by leaving the tube open for 3 - 5 minutes . volumes of 1 l , 5 l , and 10 l were run on an agarose gel , to estimate quantity and integrity . quantities of 100 - 800 ng were used for polymerase chain reaction ( pcr ) . due to a 1.5% thickness required , 3 g of agarose were used . the agarose was heated in solution in a microwave , until it was completely dissolved . this was then allowed to cool in a water bath , set at 50 - 55c . a volume of 5 l of ethidium bromide was added to the cooled gel and poured into a gel tray . this mixture was allowed to cool for 15 - 30 minutes , at room temperature . the combs were removed and placed in electrophoresis chamber and covered with tris - acetate - edta ( tae ) buffer . the dna and standard ( ladder ) were loaded onto gel and this was electrophoresed for at least 1 hour . the dna bands were visualized , using a gel imaging system ( gel doc 2000 , bio - rad laboratories inc . , hercules , ca , usa ) . the pcr components were produced by integrated dna technologies ( coralville , ia , usa ) , and the reaction contained primers gp5 : 5tttgttactgtggtagatactac-3and gp6 : 5gaaaaataaactgtaaatcatattc-3. the reaction was programmed as follow : initial denaturing step , at 95c , for 15 minute , 10 cycles of 30 second at 94c , 90 second at 72c , followed by 30 cycles of 30 second at 94c , 90 second at 63c and 90 second at 72c , with final extension at 72c , for 10 minute . a fresh stop solution / glycogen mixture was prepared , as follows , per sequencing reaction : 2 l of 3 m sodium acetate , 2 l of 100 nmm na2-edta and 1 l of 20 mg / ml of glycogen ( provided in the kit ) . to each of the labeled tubes , the sequencing reactions were transferred to each of the appropriately labeled tube and mixed thoroughly . a volume of 60 l cold 95% ( v / v ) ethanol from a -20c freezer was added and mixed thoroughly . the pellet was rinsed with 200 l 70% ( v / v ) ethanol , from a -20c freezer , and centrifuged at 14,000 rpm at 4c , for a minimum of 2 minute . the sample was resuspended in 40 l of the sample loading solution ( provided in the kit ) . lokoja town has a heterogeneous population comprising the igala , ebira , yoruba , nupe , okun , oworo , bassa - nge , kakanda , egbura and hausa ethnic groups . the town is the most centrally located in nigeria , at the confluence of the rivers niger and benue . the study was carried out among women attending the obstetrics and gynecology clinic of the federal medical centre , lokoja , nigeria , aged 16 - 65 years old , who were willing to participate and met the inclusion criteria . a structured close ended questionnaire was administered to these patients , after an informed consent had been obtained , followed by clinical examination . approval for this study was obtained from the ethical review committee of the federal medical centre , lokoja . the study did not implicate any cost to the subjects and information from the patients and/or parents / tutors were confidential . a structured close ended questionnaire was administered to the participating population , using the convenience sampling technique , after due consent had been taken . ( 6 ) , each of the participants being placed in a dorsal position , with her legs flexed at the hip and knee and abducted . a cusco s bivalve speculum , which was not lubricated , was then passed and fixed to visualize the cervix , under a bright light source . the hook end of the ayres spatula was inserted into the cervix and swept through 360 movements , either in a clockwise or counterclockwise direction , to scrape the entire squamocolumnar junction of the transformation zone . the smears collected were immediately fixed to slides , before being transferred to the laboratory for processing . the fixed smears were stained by a cytotechnologist , using the pap staining procedure and read by a histopathologist , under a light microscope . the slides were reported as normal , inflammatory , abnormal ( epithelial lesion ) or unsatisfactory . the slides with abnormal smear were then tested for the presence of hpv dna , with several of the slides with normal cytology , which had been previously confirmed for the absence of hpv , utilized for control . to start the molecular analysis , the cover slips on the slides were removed , carefully . after the cover slips were removed , the smears were carefully taken from the slides , using sterile scalpel blades , as described by sharifah et al . ( integrated dna technologies , coralville , ia , usa ) was utilized for dna extraction . buffy coats , as well as proteinase k , were used to lyse the cells of the organism , with the dna extraction done using chloroform . equal volumes of 100% ethanol were added to 20 l of 3 m sodium acetate . , the tubes were centrifuged at maximum speeds , for 10 - 30 minutes , in a cold centrifuge , in the same orientation . a volume of 400 l 70% ethanol was added to the tubes containing the dna pellets and spun at maximum speed , for 5 minute , at 4c . the dna were dried out by leaving the tube open for 3 - 5 minutes . volumes of 1 l , 5 l , and 10 l were run on an agarose gel , to estimate quantity and integrity . quantities of 100 - 800 ng were used for polymerase chain reaction ( pcr ) . the agarose was heated in solution in a microwave , until it was completely dissolved . this was then allowed to cool in a water bath , set at 50 - 55c . a volume of 5 l of ethidium bromide was added to the cooled gel and poured into a gel tray . this mixture was allowed to cool for 15 - 30 minutes , at room temperature . the combs were removed and placed in electrophoresis chamber and covered with tris - acetate - edta ( tae ) buffer . the dna and standard ( ladder ) were loaded onto gel and this was electrophoresed for at least 1 hour . the dna bands were visualized , using a gel imaging system ( gel doc 2000 , bio - rad laboratories inc . the pcr components were produced by integrated dna technologies ( coralville , ia , usa ) , and the reaction contained primers gp5 : 5tttgttactgtggtagatactac-3and gp6 : 5gaaaaataaactgtaaatcatattc-3. the reaction was programmed as follow : initial denaturing step , at 95c , for 15 minute , 10 cycles of 30 second at 94c , 90 second at 72c , followed by 30 cycles of 30 second at 94c , 90 second at 63c and 90 second at 72c , with final extension at 72c , for 10 minute . a fresh stop solution / glycogen mixture was prepared , as follows , per sequencing reaction : 2 l of 3 m sodium acetate , 2 l of 100 nmm na2-edta and 1 l of 20 mg / ml of glycogen ( provided in the kit ) . to each of the labeled tubes the sequencing reactions were transferred to each of the appropriately labeled tube and mixed thoroughly . a volume of 60 l cold 95% ( v / v ) ethanol from a -20c freezer was added and mixed thoroughly . the pellet was rinsed with 200 l 70% ( v / v ) ethanol , from a -20c freezer , and centrifuged at 14,000 rpm at 4c , for a minimum of 2 minute . the sample was resuspended in 40 l of the sample loading solution ( provided in the kit ) . the study population for this research comprised of 203 women who met the inclusion criteria . those between the ages of 16 - 25 years accounted for 4.93% of the study population , while those between the ages of 26 - 35 years , while 36 - 45 years , 46 - 55 years and 56 - 65 years accounted for 29.56% , 34.48% , 23.65% and 7.59% of the study population , respectively . out of the 203 women , who participated in the study , 193 ( 95.07% ) had normal cytology ( they were negative for intraepithelial lesion or malignancy ) , while 10 ( 4.93% ) women had abnormal cytology . out of the 10 women with abnormal cytology , four of them were reported as having atypical squamous cells of uncertain significance ( ascus ) ( figure 1 ) , one had squamous cell carcinoma ( figure 2 ) , while the remaining five were reported as having low - grade squamous intraepithelial lesion ( figure 3 ) . arrow ( b ) depicts the intermediate cells , with the circled regions labeled c , depicting the affected cells with enlarged nucleus ( hematoxylin and eosin , 20 ) . arrow indicated cell , which has completely changed to a carcinoma ( hematoxylin and eosin , 500 ) . entire smear showing hyperchromasia ( nuclei darker than those of intermediate cells , cells are enlarged with slight increase in nucleus / cell ratio , with no prominent nucleoli ( hematoxylin and eosin , 40 ) . abbreviations : ascus , atypical squamous cells of uncertain significance ; df , degree of freedom ; lsil , low grade squamous intraepithelial lesion ; sc , squamous cell carcinoma ; x , chi square . p < 0.05 is statistically significant . results also showed that several of the slides had inflammatory smear 16 ( 7.88% ) . two ( 20% ) out of 10 women with abnormal cytology were in the age range of 36 - 45 years , six ( 60% ) of those with abnormal cytology were in the age range of 46 - 55 years , while two ( 20% ) were between 56 - 65 years ( table 1 ) . two of these slides were those with normal cytology , even though they presented inflammatory cells and the other four had hemorrhage , and these were to serve as experimental controls . after carrying out gene blasting , using the ncbi basic local alignment search tool ( blast ) system , results showed that hpv 35 , 31 , 18 and 16 strains were present in the sample sequenced , while the samples used as experimental control did not show the presence of hpv dna ( figure 4 ) . m , marker ; n , negative control ; hpv genotypic variants 16 , 18 , 31 , and 35 . out of the 203 women who participated in the study , 193 ( 95.07% ) had normal cytology ( they were negative for intraepithelial lesion or malignancy ) , while 10 ( 4.93% ) women had abnormal cytology . the dna typing was successfully used to confirm the presence of hpv in the abnormal cytology . genotypic variants of high risk hpv present in this study included hpv genotypes 16 , 18 , 31 , and 35 . to a certain extent , this finding is consistent with the type of variants present in west african region , with genotypes 16 and 18 , regarded as the most common variants of hpv available worldwide ( 8) . hamidi - fard et al . ( 9 ) also reported that the most prevalent hpv in their study , in iran , was hpv 16 . results showed a 100% presence of high risk hpv in all the samples with abnormal cytology , which would have not been known via the conventional pap test . the hpv genotype 35 had the highest percentage of the high risk hpvs present , with 40% of samples affected . the hpv genotype 31 accounted for 30% , while hpv genotype 16 and 18 accounted for 20% and 10% of samples , respectively . ( 7 ) , where 95% of all the abnormal smears , subjected to hpv dna typing , had high risk hpv present in them . the cervical smear that had squamous cell carcinoma had hpv genotype 16 present in it . two of the smears that were presented as atypical squamous cells of undetermined significance ( ascus ) had hpv genotype 35 , while the other two smears that had ascus were hpv genotypes 31 and 18 , respectively . the smears that were presented as low grade squamous intraepithelial lesion ( lsil ) had two hpv genotypes 35 , two hpv genotypes 31 and one hpv genotype 16 , respectively . ( 7 ) obtained high risk hpv genotypic variants of 16 , 18 , 31 , 51 , 52 , 56 , 58 , and 66 , from abnormal cervical smears , with the highest frequency of genotypes occurring in lsil smears . ( 10 ) obtained high risk hpv genotypic variants of 16 , 18 , 31 , 33 , 45 and 51 , from similar abnormal cervical smears . thomas et al . ( 11 ) obtained similar results in ibadan , nigeria , where the common high risk hpv genotypic variants found among the subjects were 16 , 31 , 35 , and 58 . the high prevalence of hpv genotypes 31 and 35 , in this study , supports suggestions that various genotypic variants are peculiar to particular regions and areas ( 11 ) . to the best of our knowledge , the only study , concerning genotypic variants of high risk hpv in nigeria , was conducted in ibadan , by thomas et al . they reported that the frequency of hpv types , both in single and multiple infections were combined , and hpv 16 and 35 were the most common high risk types , followed by hpv 31 , 58 , and 56 . however , in our study , which is the first in the north central nigeria , the hpv genotype 16 was the variant found in the cervical smear , with squamous cell carcinoma . this has certain significance , as various studies have indicated extensively the role of hpv genotypes 16 and 18 in the development of cervical cancer . the hpv 16 is regarded as a much stronger viral carcinogen , and persists longer than any other type of high risk hpv ( 8 , 12 ) . the use of dna typing , as a means of confirming the presence of high risk hpv , is essential to the early detection of cervical cancer , since continuous infection by high risk hpv is a prerequisite for the development of cervical cancer . early detection is crucial in the fight against this cancer and , therefore , hpv dna typing provides a fast and effective confirmatory test , compared to the 3 month repetitive test , which is recommended in most cases , and also provides solid information regarding the prevalent hpv genotypes , in a region that is instrumental to the development of vaccines . adopting molecular techniques , in the routine diagnoses of hpv infection and cervical cancer in nigeria ,
background : cervical cancer is regarded as the second highest cause of cancer deaths in nigeria , with an overall prevalence similar to most developing countries . screening for cervical cancer 
 is primarily performed using papanicolaou ( pap ) staining procedure , in nigeria.objectives:this study aimed to use human papillomavirus ( hpv ) dna typing , as a means of ascertaining the presence of high risk hpv in cytology samples , which are positive for the presence of cervical intraepithelial neoplasia ( cin ) , using the pap screening procedure.patients and methods : amplification of dna was done using polymerase chain reaction . 
 gene sequencing was carried out to determine the presence of high risk hpv from cervical smears that were positive for abnormal cytology , from a cross - sectional study involving women between the ages of 16 - 65 years , screened for cin and cervical cancer , in lokoja , nigeria.results:result showed a 100% presence of high risk hpv in all the samples with abnormal cytology . 
 the hpv genotype 35 accounted for the highest percentage of the hpvs cases , with a 40% incidence . 
 the hpv genotype 31 accounted for 30% of samples , while hpv genotype 16 and 18 accounted for 20% and 10% of samples , respectively.conclusions:the high prevalence of hpv in abnormal cytology underlines to the fact that the presence of hpv is a critical factor in the development of cervical cancer . 
 the use of hpv dna techniques could actually become an effective and fast means of ascertaining the presence of hpv in abnormal cytology .