Queries

LUIZ CARLOS GARCEZ NOVAES, MD,MSc*, MARIA RITA CARVALHO GARBI NOVAES, PHD† AND AUGUSTO SIMÕES-BARBOSA, PHD*.

From *Program of Genomic Sciences and Biotechnology, Universidade Católica de Brasília, and

†

Escola Superior de Ciências da Saúde, Brasília DF, Brazil

The authors thank colleagues Doctors Evandro Silva, Maria Quitéria Santos, Jackson Pontes and Valquíria Primo for sample collection and Prof. Dr. Eduardo Leonardecz for statistical assistance. This work was supported by the Program of Genomic Science and Biotechnology at the Catholic University of Brasilia.

Correspondence: Augusto Simões-Barbosa, PhD, Professor and Research Leader, Universidade Católica de Brasília, Programa de Pós-graduação em Ciências Genômicas e Biotecnologia, SGAN 916, módulo B, Asa Norte, Brasília DF, CEP 70790-160, Brasil. Phone (61) 340 5550. Fax: (61) 347 4797. E-mail: [email protected].

Summary page

Cases of divergence between results of Hybrid Capture and Papanicolaou cytology for diagnosis of Human Papillomatosis were evaluated by PCR.

Abstract

Background: Human Papillomavirus has been related to cancer of the uterine cervix. Precocious

diagnosis of morphological and cytological alterations has been used as a tracking method. Diagnosis by molecular biology presents itself as a possible improvement.

Goal: Evaluate the divergence of results between Papanicolaou Cytology and Hybrid Capture through

the detection of the HPV DNA using PCR.

Study Design: A transversal study was conducted with 70 women attending private gynecological

clinics for the prevention of cervical cancer in Brasilia, Brazil. The samples came from patients who presented previous divergent and contemporary results between Papanicolaou and Hybrid Capture. PCR with specific primers for HPV and high risk HPV DNA was conducted, products were visualized by electrophoresis on agarose gels. The statistic analysis was done by the test of Chi-square - χ2, with a p-value ≤ 0.05. Comparative results of Papanicolaou Cytology, Hybrid Capture and PCR were evaluated.

Results: Accordance of PCR results was higher with Hybrid Capture. However, in the comparison

between Papanicolaou and any of the molecular biology methods, the χ2 test presented extremely high negative dependence (p-value = 0.002844), indicating that a positive Papanicolaou result will not necessarily indicate the presence of HPV. When comparing the molecular biology methods, the χ2 test presented very high dependence, thus indicating that the result of a given test must correspond to the same result on the other test.

Conclusion: Elucidating causes of divergence between Hybrid Capture and Papanicolaou results

were possible by PCR. The accordance between PCR and Hybrid Capture can be attributed to the fact that both methods detect latent infection, while Papanicolaou detects only microscopic cellular alterations. Due to the technical complexity, the need of specific material and sophisticated equipment inherent to the PCR and Hybrid Capture methods, costs are still considerably high.

Introduction

It is widely accepted that genital human papillomavirus (HPV) is the major contributing factor for cervical intraepithelial neoplasia and carcinoma [1,2]. In Brasilia, capital of Brazil, the regular methods for the diagnosis of this infection and its lesions are the oncologic cytology, by traditional Papanicolaou cytology (PC), considered election’s exam [3,4], and the Hybrid Capture® (HC) and its variant HC II. The last is able to detect deoxyribonucleic acid of the virus and, indirectly, infers the risk of evolution of cervical lesions to cancer, based on distinguished viral types, high-risk (HR) and low- risk (LR) HPV [5].

The classic clinical recommendation for the investigation of HPV by HC is the result of atypical squamous cells of undetermined significance (ASC-US) in PC, which means no possible evolution to cancer by cytological aspects. HC is quickly becoming popular and solicitation of the HC exam by gynecologists is now very common. The presence of a HR HPV requires a prompt decision by the physician, while the presence of a LR HPV implicates in more frequency of attendance by the patient throughout exams and a long observation period [6]. Therefore, the detection of HR HPV by a molecular biology diagnostic test would help physician’s decision, avoiding the risk of a pathological evolution and sequential PC exams [7].

The magnitude of preventive exams for the detection of uterine cervix cancer may be assessed considering that every woman, since beginning sexual practice, has indication to take PC or an equivalent exam. At first, the routine recommends one exam per year, and after 3 consecutive negative results, one exam every 3 years for the rest of a woman’s life, if exams are still negative, according to World Health Organization – WHO recommendation [8]. In Brazil, it has been estimated that 35 millions of PC exams are done every year, and about 800 thousand only in Brasília, according to the Health Department of the Capital of Brazil (1999). The HC exam is not a routine procedure on the public health centers in Brasilia, being generally solicited by physicians from private gynecology clinics. The fact is that such exam is about 50 to 100 times more expensive than conventional PC.

It has been observed that divergence between PC and HC may occur due to the fact that the sensibility and specificity of neither method is 100% accurate. Contemporary results of PC and HC may be contradictory. The specificity for PC is about 60 to 75% by comparative study considering gold standard histopathology results [9]. On the other hand, HC presents sensitivity and specificity up to 93% and 61%, respectively [10].Since the adequate examination, prognostic evaluation and treatment

are directly dependent on the definition of the infection status, discordance on HPV diagnostic results always causes embarrassment to physicians. Whenever PC and HC present no accordant results, the diagnosis of HPV by Polymerase Chain Reaction (PCR) may represent a third option [11,12]. Although more consuming, PCR can provide consistent information for a clinical decision. The objective of this experimental work is to evaluate the presence of HPV DNA using PCR in samples from patients with previous divergent results reported by PC and HC, and further analyze possible causes and consequences of the divergent results by investigating the inherent limitations of the current techniques.

Methods

Study population and clinical definitions

The transversal study population has included 70 women attending gynecology clinics of private health centers in Brasilia, Brazil, from November 2003 to July 2004. The age of the patients varied from 22 to 73 years old (average, 43 years old). This population was randomized in terms of behavior and clinical signs and symptoms. Following the advice of the national committee of ethics, all patients volunteered to participate. The criterion for inclusion of the patients in the study group was to present contemporary but divergent results between PC and HC, in a maximum period of 30 days. The HC and PC were processed in different specialized laboratories in Brasilia in order to counteract individual subjective aspects as well as specific variations of results. The divergent exam results were grouped as shown in Table 1.

Laboratory procedures and data analysis

Cervical samples were collected from the patients by gynecologists using a sterile cervix brush. The samples were immediately disposed into sterile microtubes with 600 microlitres (µL) containing preserving solution. The material was kept at 4ºC up to two days before DNA extraction. The DNA was obtained using the Easy-DNA TM Kit Version E, as recommended (Invitrogen, USA). Qualitative and quantitative analysis of the DNA samples were performed by electrophoresis in Tris- borate-EDTA buffer and ethidium bromide stained agarose gels, as described elsewhere [14]. For a quality control, DNA extraction and PCR were performed in separate rooms with different sets of micropipettes. A total of 50 to 100 ng DNA was used for each reaction of DNA amplification. Routinely, a negative control was performed by omitting DNA from the reaction. All cervicovaginal swab samples

were firstly tested by PCR with a set of primers for amplification of a human housekeeping gene, glyceraldheyde-3-phosphate-dehydrogenase (GAPDH), as previously described by Lobo [15]. The amplification with these primes indicated the absence of inhibitors and adequate quality of the DNA samples for PCR assays.

For the detection of HPV DNA by PCR and the determination of HR and LR virus type, the BIOPAP – Detection Kit® was employed as recommended (Biotools B&M Labs, Madrid, Spain). Briefly, the standardized procedure for PCR amplification for HPV included 5 minutes of predenaturation at 940C, followed by 35 cycles each consisting of 30 seconds of denaturation at 940C, 60 seconds of annealing at 550C, and 60 seconds of extension at 720C. A final step of 10 minutes of post-extension was added at the end. The enzymatic conditions were adjusted to a final volume of 25 µL. The 450 bp PCR product represents the universal HPV DNA amplicon, and the 250 bp PCR product indicates the presence of HR-type HPV. In other words, LR-type HPV is suggested when the first PCR product is detected but not the second one. In addition, each sample was tested twice by independent PCR assays. No conflicting or doubtful results were seen throughout the screening of the samples. Besides the positive control in the Biotools kit, DNA from HeLa and SeHa cells were included as positive controls routinely. Statistical agreement among diagnostic results and their correlations were calculated by the parametric test of Fisher, after translation of qualitative to quantitative dates. The translation was based on the conversion table of PC results of CIN system to Bethesda Cytology System for cervicovaginal smears [14]. In addition to that; the probability of significance (p ≤ 0.05) was determined by the Chi-square test.

Results

Figure 1 illustrates a set of PCR results with cervicovaginal DNA samples from patients included in this study group. All samples were initially tested with primers for amplification of a human DNA segment corresponding to GAPDH. All samples showed a single amplification product of 783 bp, indicating that DNA samples exhibited adequate quality for PCR assays (Figure 1 B). On the other hand, using specific primers to HPV, three possibilities are envisaged: (1) no products were amplified; (2) two amplification products of 450bp and 250bp were seen; (3) only a single amplification product of 450 bp was observed. (Figure 1 A). The former means absence of HPV infection (samples 1, 2, 5, 7 and 9). The second case means presence of HPV infection of HR-type (samples 3 and 4). In the third

case, although the presence of HPV is confirmed by the presence of the universal fragment of 450 bp, the infection is due to LR-type, since the presence of the HR-type is excluded by the lack of the 250bp amplicon (samples 6, 8 and 10).

Table 2 summarizes the comparative diagnostic results and illustrates the distribution of positive and negative cases in terms of concordance between PCR/PC and PCR/HC. Although a total of 25 cases of agreement between PCR/PC and 36 cases between PCR/HC have been observed, a total of 45 cases of disagreement between PCR/PC and 34 between PCR/HC have also been reported. Concordance of PCR results was higher with HC. In the comparison of PC and PCR, the Chi-square test (χ2) presented extremely high negative dependence among different forms of examination (p-value = 0.002844). In the comparison of HC and PCR exams, the χ2 test presented very high dependence, varying according to different forms of examination (p-value = 0.014429), thus indicating that the result of a given test (positive or negative), must correspond to the same result on the other test. In addition, when only the positivity or negativity of results are compared between HR- HPV by PCR and HR-HPV by HC, and between LR-HPV by PCR and LR-HPV by HC, the χ2 test presented very high dependence among different kinds of exams (p-value = 1.15 x 10-18), indicating that the result of a given test (high or low risk), must strongly correspond to the same result on the other test.

Discussion

HR-HPV and LR-HPV chronic infection in uterine cervix that causes morphocytologic changes in cells [16]. The HR-HPV may cause high grade lesions and cancer, but LR-HPV lesions do not evolve into cancer as demonstrated in a statistically significant number of cases [17]. Therefore, HPV diagnosis at early stages of the infection is of fundamental importance. Although diagnosis is still a critical aspect of this infection, a correct diagnosis will help reduce the incidence of cervix cancer and its complications [18]. Previous studies have demonstrated that the Papanicolaou test is not equally efficient and adequate when used as a sole method for diagnosis of HPV infection based of cytological alterations [21]. In fact, if a molecular biology based method is not employed to specifically distinguish subtypes of HPV, it will be necessary to repeat Papanicolaou exams several times during the follow- up of the patient in order to decide for clinical or chirurgical treatment. However, this is not the current

situation of the public health primary attendance in most developing countries. Determining the HPV type of infection in a low-cost and labor fashion is still an unresolved issue. The development of a more sensible and specific method might be expected. Once this issue is not settled, the epidemiological investigation with those improved molecular detection methods would reveal the necessity of adequate diagnosis and treatment of the infected population, dispensing unnecessary aggressive treatment and providing information for surveillance, policy and management programs.

PCR has been extensively applied for diagnosis of infectious diseases by amplification of a target DNA sequence, and several methods for PCR detection of HR- and LR-HPV have also been developed [19]. Although it isn’t a suitable method for routine diagnosis in most laboratories in developing countries, the PCR technique is a better choice for the detection of HPV in cervicovaginal smear samples in terms of sensibility and specificity. Furthermore, the use of a molecular technique such as PCR determines the real status of the infection and enables the distinction among pathotype strains in order to deal with specific treatment strategies [20]. Hybrid Capture® (HC) technology detects nucleic acid targets directly by immunology reaction after hybridization with RNA probes. That is comparable to target amplification methods by molecular biology with PCR. However, the Papanicolaou represents the most used methodology for primary screening of cellular abnormalities and some genital infections in most developing countries since it is a simple and a low-cost cytological technique available elsewhere.

The goal of this study was evaluate the divergence of results of Papanicolaou Cytology (PC) and HC through the detection of the HPV DNA using PCR. The rate of misdiagnosis, false-positives and false-negatives, analyzed in both methods is enough to characterize it as a real problem, though with uncertain proportions in a large scale analysis. PCR was able to detect a larger number of positives than HC and a more number of divergent samples with PC, indicating higher sensibility and sufficient clinical specificity. Nevertheless, PCR results do not guarantee absence of discrepancy. Multiple explanations for divergence of PC, HC and PCR results are possible [22]. The variability of results on a Papanicolaou exam may be determined by individual factors of the patient, by the experience of the examiner, and by the subjectivity of the examination itself [7]. Both PCR and HC, molecular biology techniques, are standardized and may be compared among different laboratories and research centers [23].

Patients included in this study presented divergence on PC and HC diagnosis. We have shown that the divergence probability is higher between PCR and PC than PCR and HC. This was expected according to previous studies [25]. The p-value by χ2 indicated high negative dependence when comparing PCR or HC to PC. This fact shows that a positive PC result will not necessarily indicate the presence of HPV. The opposite is also true. In other words, the virus may be present even if morphologic modifications in cells of the uterine cervix were not identified in a PC exam. In fact, we found out more cases of false-negatives (26/45) than false-positives (19/45) by PC, which could represent latent infections of the HPV. On the other hand, when comparing only positivity or negativity between PCR and HC, the p-value indicates that a result from one exam (positive or negative) must correspond to the same result by other exam. These data contradict a previous study in Brazil [26]. In cases of HR- and LR-HPV genotyping, PCR and HC are very concordant (p-value = 1,15 x 10-18), as expected when both strategies are employed [27].

It has been previously shown that PCR has a sensibility of 50% (47/94) and 37.2% (35/94) in comparison to PC and HCII, respectively. The findings indicated more chances of false-negative than false-positive misdiagnosis by the HCII. Moreover, the accordance between PCR and HCII was strongly decreased in cases of ASC-US, on contrary to other grade lesions [24]. Although quantitative HC data was not available for our study, we hypothesized that the cut-off values from HC may be a cause of misdiagnosis. It is becoming more evident that a PCR-based technology can be a better choice methodology for the detection and genotyping of HPV infection. Our results suggest that the accordance between PCR and HC results and their high sensitivity can be attributed to the fact that both methods detect latent infection, while PC is dependent on the subjective appearance of cellular alterations visualized through microscopy. PCR is a high specific and sensitive diagnosis tool for the detection of the HPV DNA, and could be indicated for tracking HPV of cervicovaginal smears. However, due to the complexity of the techniques, the necessity of specific material and sophisticated equipment, costs of PC and HC exams are still considerably high.

References

1. Walboomers JMM, Jacobs MV, Manos MM, et alli. Human Papillomavirus is a necessary cause of invasive cervical cancer world-wide, Journal of Pathology 1999;189: 12-19.

3. Novaes LCG, Novaes MRCG, Barbosa AMS. Molecular biology of human Papillomavirus and carcinogenesis. RSDF. 2002 Jul/Dez;13(3/4):7-16.

4. papillomavirus infection and cervical neoplasia. J Natl Cancer Inst. 2003 Mar; 19;95(6):E2. 5. Papanicolaou GN, Traut HF. Diagnosis of uterine cancer by vaginal smear. New York, NY.

Commonwealth Foundation., 1943.

6. O'Meara AT. Present standards for cervical cancer screening. Curr Opin Oncol. 2002 Sep;14(5):505-11

7. Lorincz AT. Molecular methods for the detection of human Papillomavirus infection. Obstet Gynecol Clin North Am. 1996 Sep;23(3):707-30.

8. Sellors JW; Karwalajtys TL; Kaczorowski J; et alli. Incidence, clearance and predictors of human Papillomavirus infection in women. CMAJ; 168(4):421-5, 2003 Feb 18

9. Hubbard RA. Human papillomavirus testing methods. Arch Pathol Lab Med. 2003 Aug;127(8):940-5

10. Dillner J. Primary screening for human Papillomavirus infection. Best Pract Res Clin Obstet Gynaecol. 2001 Oct;15(5):743-57.

11. Renshaw AA. Measuring sensitivity in gynecologic cytology: a review. Cancer. 2002 Aug; 25;96(4):210-7.

12. Lorincz AT, Hildesheim A, Herrero R, et al. Comparison of hybrid capture II HPV DNA detection and conventional PAP smear for cervical cancer screening in Costa Rica. 17th International Papillomavirus Conference, 26,1999.

13. Manos MM, Waldman J, Zhang TY, et alli. Epidemiology and partial nucleotide sequence of four novel genital human papillomaviruses. J Infect Dis. 1994 Nov;170(5):1096-9.

14. Jacobs MV, de Roda Husman AM, van den Brule AJ, et alli. Group-specific differentiation between high- and low-risk Human Papillomavirus genotypes by general primer-mediated PCR and two cocktails of oligonucleotide probes. J Clin Microbiol. 1995 Apr; 33(4):901-5.

15. Sambrook J, Fritsch EF, Maniatis T. New York, NY. Molecular cloning. A laboratory manual. Cold – Spring Harbor. 2001.

16. Lobo T, Feijó G, Carvalho S, et alli. A Comparative Evaluation of the Papanicolaou Test for the Diagnosis of Trichomoniasis. Sex Transm Dis 2003Set;9(2):694-699

17. World Health Organization. An overview of selected curable sexually transmitted disease. Global Program on AIDS report. Geneva: World Health Organization, 1995.

18. J. Pfister, WSLH. Summary: Epidemiology & Public Health. Cervical Cancer Screening Program Public Health.2002.

19. Laimins LA. The biology of human papillomaviruses: from warts to cancer. Infect Agents Dis. 1993 Apr; 2(2):74-86.

20. Dôres GC, Taromaru KE, Gallo C. Aspectos atuais do rastreamento das lesões HPV