THE BRAZILIAN JOURNAL OFINFECTIOUS DISEASES

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THE BRAZILIAN JOURNAL OF INFECTIOUS DISEASES

An Official Publication of the Brazilian Society of Infectious Diseases

EDITOR Anastácio Q. Sousa

PUBLISHED BY CONTEXTO

October 2008

Printed in Brazil

Volume 12 • Number 5 • October 2008

ISSN 1413-8670

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348 BJID 2008; 12 (October)

THE BRAZILIAN SOCIETY OF INFECTIOUS DISEASES

The Brazilian Society of Infectious Diseases is conducted for scientific purposes, for the advancement and promulgation of knowledge relevant to infectious diseases.

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EDITOR

Anastácio Q. Sousa

ASSOCIATE EDITORS

Adauto Castelo (BR) André Villela Lomar (BR) Antônio C. Pignatari (BR) Carlos Brites Alves (BR) Hélio Sader (BR)

João Silva de Mendonça (BR) Márcio Nucci (BR)

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THE BRAZILIAN JOURNAL OF INFECTIOUS DISEASES

An Official Publication of the Brazilian Society of Infectious Diseases

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The Brazilian Journal of Infectious Diseases is an official publication of the Brazilian Society of Infectious Diseases and is published bi-monthly by Contexto - Rua Alfredo Magalhães, 04/Barra, 40140-140, Salvador-Bahia- Brazil. The editorial offices are at Contexto.

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Brief Communications

Quinine Levels in Patients with Uncomplicated Falciparum Malaria in the Amazon Region of Brazil ... 353

José Luiz Fernandes Vieira, Larissa Maria Guimarães Borges, Margareth Tavares Silva Nascimento and Andreza L.S. Gomes

Lamivudine for Chronic Hepatitis B: A Brief Review ... 355

Emilio Palumbo

Hepatitis C Virus Detection in the Semen of Infected Patients ... 358

Norma de Paula Cavalheiro, Ana Carolina de Oliveira Santos, Carlos Eduardo Melo, Suzana Rie Morimitsu and Antonio Alci Barone

Original Papers

Histological Response Study of Chronic Viral Hepatitis C Patients Treated With Interferon Alone or Combined With Ribavirin ... 362

Kleber Prado, Rosely Patzina, Denise Bergamaschi and Roberto Focaccia

Cost-Effectiveness of Entecavir versus Lamivudine for the Suppression of Viral Replication in Chronic Hepatitis B Patients in Brazil ... 368

Anna Maria N. Costa, Gilbert L.’Italien, Marcelo Eidi Nita and Evaldo Stanislau A. Araujo

Genetic Characterization and Evolutionary Inference of TNF- α Through Computational Analysis ... 374

Gauri Awasthi, Suchita Singh, A.P. Dash and Aparup Das

Prevalence of Resistance-Associated Mutations in Human Immunodeficiency Virus Type 1-Positive Individuals Failing HAART in Rio de Janeiro, Brazil ... 380

Rafael Brandão Varella, Selma Baía Ferreira, Márcia Braga de Castro, Marisa Dias Tavares and Mariano Gustavo Zalis

Invasive Aspergillosis in Hematopoietic Stem Cell Transplant Recipients: A Retrospective Analysis ... 385

Viviane Maria Hessel Carvalho-Dias, Caroline Bonamin Santos Sola, Clóvis Arns da Cunha, Sílvia Emiko Shimakura^, Ricardo Pasquini and Flávio de Queiroz-Telles

An Outbreak of Candida spp. Bloodstream Infection in a Tertiary Care Center in Bogotá, Colombia ... 390

Carlos A. DiazGranados, Adriana Martinez, Ceneth Deaza and Sandra Valderrama

THE BR THE BR THE BR

THE BR THE BRAZILIAN AZILIAN AZILIAN AZILIAN JOURNAL AZILIAN JOURNAL JOURNAL JOURNAL JOURNAL OF OF OF OF OF INFECTIOUS DISEASES INFECTIOUS DISEASES INFECTIOUS DISEASES INFECTIOUS DISEASES INFECTIOUS DISEASES

Volume 12 • Number 5 October 2008

Molecular Investigation of a Fungemia Outbreak Due to Candida parapsilosis in an Intensive Care Unit ... 395

Murat Dizbay, Ayse Kalkanci,Busra Ergut Sezer,Firdevs Aktas,Sibel Aydogan,Isil Fidan,Semra Kustimur and Takashi Sugita

Treatment of Invasive Fungal Infections: Stability of Voriconazole Infusion Solutions in PVC Bags ... 400

Andréa I.H. Adams, Lucia N. Morimoto, Leonardo Z. Meneghiniand Ana M. Bergold

Rates of Antimicrobial Resistance in Latin America (2004-2007) and in vitro Activity of the Glycylcycline Tigecycline and of Other Antibiotics ... 405

Flávia Rossi, Patricia García, Bernardo Ronzon, Daniel Curcio and Michael J. Dowzicky

Inadequate Timing Between Corticosteroid and Antibiotics Applications Increases Mortality Due to Sepsis ... 416

Marcus Vinícius Telles Fadel, João Carlos Repka, Cláudio Leinig Pereira da Cunha and Maria Terezinha C. Leão

Comparison of PCR-Based Molecular Markers for the Characterization of Proteus mirabilis Clinical Isolates .... 423

Lessandra Michelim, Gabriela Muller, Jucimar Zacaria, Ana Paula Longaray Delamare, Sérgio Olavo Pinto da Costa and Sergio Echeverrigaray

Ribotyping, Biotyping and Capsular Typing of Haemophilus influenzae Strains Isolated from Patients in Campinas, Southeast Brazil ... 430

Marcelo Lancellotti, Fernanda de Pace, Eliana Guedes Stehling, Maria Cecília Barisson Villares, Marcelo Brocchi and Wanderley Dias da Silveira

Postnatal Acquired Toxoplasmosis Patients in an Infectious Diseases Reference Center ... 438

Cassius Schnell Palhano Silva, Elizabeth de Souza Neves, Eliezer Israel Benchimol and Danielle Ribeiro de Moraes

Case Reports

Tatumella ptyseos Causing Severe Human Infection: Report of the First Two Brazilian Cases ... 442

Paulo Sérgio Gonçalves da Costa, Juliana Monteiro de Castro Mendes and Geyza Machado Ribeiro

Ventilator-Associated Pneumonia with Col–S Strains: A Successful Comeback of Colistin! ... 444

Mukhyopadhyay C., Krishna S., Vandana K.E., Shenoy A. and Bairy I.

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Instructions for Authors

Statement of Editorial Policy

Checklist for Submitted Manuscripts Extensively Drug-Resistant Tuberculosis: A Case Report and

Literature Review ... 447

João Alves de Araújo-Filho, Arioldo Carvalho Vasconcelos-Jr, Eduardo Martins de Sousa, Colombina da Silveira, Elisangela Ribeiro, André Kipnis and Ana Paula Junqueira-Kipnis

Primary Gastric Fundus Tuberculosis in Immunocompetent Patient: A Case Report and Literature Review ... 453

Fahmi Yousef Khan, Ahmed AlAni, Ammar Al-Rikabi, A. Mizrakhshi and Mohamed El-Mudathir Osman

Acute Hepatitis Due to Dengue Virus in a Chronic Hepatitis Patient ... 456

Souza L.J., Coelho J.M.C.O., Silva E.J., Abukater M., Almeida F.C.R., Fonte A. S. and Souza L.A.

Images in Clinical Infectious Diseases

Leprosy with Necrosis in Granulomatous Reaction ... 460

Maria Ângela Bianconcini Trindade, Brandt H., Teixeira R., Sotto M.N. and Fleury R.N.

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Quinine Levels in Patients with Uncomplicated Falciparum Malaria in the Amazon Region of Brazil

José Luiz Fernandes Vieira¹, Larissa Maria Guimarães Borges², Margareth Tavares Silva Nascimento²and Andreza de L.S. Gomes³

1Professor of Toxicology of the Department of Legal Medicine of Federal University of Pará; ²Graduate Student (Master’s Degree Program in Tropical Disease) of Federal University of Pará; ³Undergraduated Student of Federal University of Pará; Belém, PA, Brazil We examined the plasmatic concentrations of quinine in patients with uncomplicated falciparum malaria in an endemic area of the Amazon region in Brazil in a prospective clinical trial, in which a standard three-day course of oral quinine plus doxycycline was used. We measured the quinine in the plasma samples on days 0 and 3by high performance liquid chromatography. The mean concentration of quinine was 6.04 ±2.21 μμμμμg/mL in male patients and 5.98 ±1.95 μμμμμg/mL in female patients. No significant differences in quinine concentration were observed between these two groups. All samples collected before starting treatment were negative for quinine. This information could help in the development of strategies for the rational use of antimalarial drugs in Brazil.

Key-Words: Falciparum malaria, quinine, treatment.

Received on 12 May 2008; revised 10 September 2008.

Address for correspondence: Dr. Jose Luiz Fernandes Vieira. Toxicology Laboratory- Federal University of Pará. Rua Augusto Corrêa 01. Phone:

559132017733. E-mail: jvieira@ufpa.br. Belém, Para, Brazil.

The Brazilian Journal of Infectious Diseases 2008;12(5):353-354.

The monitoring of quinine blood concentration is an importa n t a p p r o a c h f o r b e t t e r u n d e r s t a n d i n g t h e epidemiology of falciparum malaria, especially for a clear distinction between treatment failure, i.e. the absence of resolution of clinical signs after antimalarial treatment associated with inadequate drug concentration, and true resistance to an antimalarial drug [1,2]. Much work has been done to determine how blood quinine concentration profiles correlate with subsequent therapeutic responses in malaria patients; however, the precise therapeutic range of plasma quinine levels remains uncertain, because the pharmacokinetics and the therapeutic response to quinine varies with drug formulation, patient age and immunity, the sensibility of the malaria strain [3,4], the fraction bound to α1-acid-glycoprotein, parasite clearance in the course of the disease and interracial differences in metabolism [5]. However, some authors have stated that continuous concentrations ≥ 6 μg/mL are required to ensure cure [2,4].

There is a lack of knowledge about the plasmatic concentration of quinine in Brazilian falciparum-malaria patients that have been treated with the standard three- day course of oral quinine. We examined the plasmatic concentrations of quinine in patients with uncomplicated falciparum malaria in an endemic area of the Amazon region, in a prospective clinical treatment trial [6].

This study was conducted from January 2006 to January 2007, on 15 adult male and 15 adult female patients with uncomplicated P. falciparum malaria admitted to the public Hospital of Tucurui, Amazonas, Brazil. Patients with severe or mixed malaria and pregnancy were excluded.

Patients who had taken any antimalarial drugs within the previous 48 hours were also excluded. All patients were monitored for at least 28 days within the malaria

transmission area. This study was approved by the ethics committee of the Tropical Medicine Center of Para Federal University. Informed consent was obtained from each subject.

After clinical assessment and disease confirmation through microscopic examination of blood smears, all patients were treated with the first line standard three- day oral treatment regimen of quinine (750 mg), twice a day, followed by doxycycline hydrochloride (100 mg), twice a day, for five days and primaquine phosphate (45mg) on the last day [6]. Reappearance of infection was assessed in these patients for at least 28 days.

Venous blood samples were taken for quinine level determination before and on the third day after treatment began. All blood samples were taken before quinine intake.

The samples (3 mL) were collected in heparinized tubes and immediately centrifuged at 1500 x for 15 minutes. The plasma samples were stored at -20 ⁰C until analysis. The Student’s t test was used to compare the two groups; P<

0.05 was considered significant.

Quinine was analyzed by high performance liquid chromatography, as described previously [7]. The analytical procedure validated in our laboratory gave within-day and day-to-day coefficients of variation of 6.7 and 8.1%, respectively. Mean extraction recovery of the quinine was 95%. The stability of blank plasma spiked with quinine was 60 days under the conditions described above. Primaquine, doxycycline mefloquine, amodiaquine, acetaminophen and chloroquine do not interfere in the detection of quinine.

All patients recovered following treatment. The mean time for parasite clearance was 70.4 hours, varying from 28 to 108 hours. None of the patients had recrudescent infection, checked until 28 days after treatment.

The mean concentration of quinine in the male plasma samples in the steady state was 6.04 ± 2.21 μg/mL, ranging from 3.08 to 10.04 μg/mL; in the female samples it was 5.98

± 1.95 μg/mL, ranging from 2.78 to 9.17 μg/mL. There were no significant differences between males and females in terms of quinine concentration in the plasma. These levels were similar to what was found in other studies, with plasmatic

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quinine concentration during treatment of falciparum malaria varying from 5.0 to 10.0 μg/mL; they are also similar to steady state concentrations of quinine in A f r i c a n a n d A s i a n p a t i e n t s w i t h u n c o m p l i c a t e d falciparum malaria [2,8].

All samples collected before treatment were negative for quinine and confirmed drug absence. This fact is commonly observed in the Amazon region; it could favor the development of resistance of P. falciparum to drugs used in the treatment of this disease.

Quinine concentrations must remain above levels that inhibit parasite multiplication throughout the course of treatment in order to eradicate infection, because there is a significantly higher probability that a resistant strain will emerge if the drug is present at suboptimal concentrations. It is very important to provide an adequate treatment course and to maintain complete adherence to the prescribed drug regimen in order to optimize cure rates and reduce the risks of side effects [1,3,5]. This information could be useful for developing strategies for the rational use of antimalarial drugs in Brazil.

References

1 . WHO. World Health Organization- Guidelines for the treatment of malaria. Switzerland. 2006.

2 . Pukrittayakamee S., Wanwimolruk S., Stepniewska K., et al.

Quinine Pharmacokinetic- Pharmacodynamic Relationships in uncomplicated falciparum malaria. Antimicrob Agents Chemother 2003;47:3458-63.

3 . Pukrittayakamee S., Looareesuwan S., Keeratithakul D., et al. A study of the factors affecting the metabolic clearance of quinine in malaria. Eur J Clin Pharmacol 1997;52:487-93.

4 . Supanaranond W., Davis T.M., Pukritayakamee S., et al.

Disposition of oral quinine in acute falciparum malaria. Eur J Clin Pharmacol 1991;40:49-52.

5. White N.J. Assessment of the pharmacodynamic properties of antimalarial drugs in vivo. Antimicrob Agents Chemother 1997;41:1413-22.

6 . Brasil. Fundação Nacional de Saúde. Manual de Terapêutica da Malária. 2nd. Brasilia, 2001.

7. Dua K.V., Sarin R., Prakash A. Determination of quinine in serum, plasma, red blood cells and whole blood in healthy and Plasmodium falciparum malaria cases by high-performance liquid chromatography.

J Chromatogr B Biomed Sci Appl 1993;614:87-93.

8. Flanag K.L., Sharp M.B., Doherty T., Whitty C.J.M. Quinine levels revisited: the value of routine drug level monitoring for those on parenteral therapy. Acta Trop 2006;97:233-7.

Quinine Levels and Uncomplicated Falciparum Malaria

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Received on 5 May 2008; revised 15 September 2008.

Address for correspondence: Dr. Emilio Palumbo. Department of Pediatric, Hospital of Sondrio. Via Stelvio, 25. Zip code: 23100. Sondrio, Italy. E-mail: emipalu2003@yahoo.it.

Lamivudine for Chronic Hepatitis B: A Brief Review

Emilio Palumbo

Pediatrics Department of Sondrio Hospital; Italy

Until recently, the only generally approved treatment for chronic hepatitis B was alpha-interferon; however, it gives only moderate efficacy in terms of sustained response (biochemical, virological and histological). In fact, only 20%

to 40% of treated patients respond to therapy, with lower percentages (~ 10%) among patients infected with precore- mutant strains of HBV (HBeAb HBV-DNA positive). The FDA of the USA approved the use of lamivudine in adult patients affected by chronic hepatitis B in 1998. In this review, we focused on the pharmacokinetic and pharmacodynamic properties and efficacy and tolerability of lamivudine in the treatment of chronic hepatitis B cases that are both HBeAg and anti-HBe-positive.

Key-Words: Lamivudine, hepatitis, HBV.

Despite the use of HBV vaccine, chronic hepatitis B virus (HBV) infection occurs in approximately 5% of the global population. This infection, if persistent, may lead to chronic hepatitis, cirrhosis and hepatocellular carcinoma in 25 to 40%

of infected patients and is among the 10 main causes of death throughout the world [1,2].

Until recently, the only generally approved treatment for chronic hepatitis B was alpha-interferon; however, it has demonstrated only moderate efficacy in terms of sustained response (biochemical, virological and histological). In fact, only 20 to 40% of treated patients respond to therapy, with lower percentages (~10%) among patients infected with precore-mutant strains of HBV (HBeAb HBV-DNA positive) [3,4].This form, prevalent in the Mediterranean region (~90%

of all patients with HBV infection), is due to a mutation at nucleotide 1896 in the precore region of the HBV-DNA genome.

The result of this mutation is a stop codon that blocks HBeAg synthesis but still permits HBV replication and hepatitis B core antigen production, leading to persistent viremia and persistent or intermittent elevated serum alanine aminotransferase (ALT) levels, with frequent evolution of the disease into cirrhosis and hepatocellular carcinoma. The suboptimal response of this form to alpha-interferon, with a high rate of non-responders or relapsers, has led to research and development of new antiviral drugs that could be used as alternative therapies.

The use of nucleotide analogues is a milestone in the treatment of chronic hepatitis B (CHB). The FDA of the USA approved the use of lamivudine in adult patients in 1998. This drug is advantageous because of oral administration and good safety parameters; however, it induces a sustained response (after withdrawal of therapy) in only a minority of patients.

Thus, the treatment should be given to most patients for long periods. In addition, the long-term efficacy of lamivudine is

limited by the frequent emergence of drug-resistant HBV mutants [5-7]. Adefovir is a nucleotide analogue associated with a low frequency of resistance; however, its antiviral efficacy is not optimal [8,9].

In this review, we focused on the pharmacokinetics and pharmacodynamics and efficacy and tolerability of lamivudine treatment of chronic hepatitis B cases that are both HBeAg and anti-HBe-positive. Relevant literature was identified through searches of MEDLINE (2002-October 2006). Search terms included, but were not limited to, lamivudine, hepatitis B, pharmacology, pharmacokinetics, adverse events, and therapeutic use.

Pharmacokinetic and Pharmacodynamic Properties Lamivudine (3TC), the negative enantiomer of 2'-deoxy-3'- thiacytidine, is a dideoxynucleoside analogue used in combination with other agents in the treatment of human immunodeficiency virus type 1 (HIV-1) infection and as monotherapy in the treatment of hepatitis B virus (HBV) infection. It is a nucleoside analog that inhibits HBV-DNA synthesis. Lamivudine undergoes anabolic phosphorylation by intracellular kinases to form lamivudine 5'-triphosphate, the active anabolite that prevents HIV-1 and HBV replication by competitively inhibiting viral reverse transcriptase and terminating proviral DNA chain extension. The pharmacokinetics of lamivudine are similar in patients with HIV-1 or HBV infection, and in healthy volunteers. The drug is rapidly absorbed after oral administration, with maximum serum concentrations usually attained 0.5 to 1.5 hours after administration. The absolute bioavailability is approximately 82 and 68% in adults and children, respectively. Lamivudine systemic exposure, as measured by the area under the serum drug concentration-time curve (AUC), is not altered when it is administered with food. Lamivudine is widely distributed in total body fluid, the mean apparent volume of distribution (Vd) being approximately 1.3 L/Kg following intravenous administration. In pregnant women, lamivudine concentrations in maternal serum, amniotic fluid, umbilical cord and neonatal serum are comparable, indicating that the drug diffuses freely across the placenta. In postpartum women, lamivudine is secreted into breast milk. The concentration of lamivudine in cerebrospinal fluid (CSF) is low to modest, reaching 4% to 8%

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of serum concentrations in adults and 9% to 17% of serum concentrations in children measured at two to four hours after administration. In patients with normal renal function, about 5% of the original compound ismetabolized to the trans- sulphoxide metabolite, which is pharmacologically inactive.

In patients with renal impairment, the amount of trans- sulphoxide metabolite recovered in the urine increases, presumably as a function of decreased lamivudine elimination.

As approximately 70% of an oral dose is eliminated in the urine as unchanged drug, the dose needs to be reduced in patients with renal insufficiency. Hepatic impairment does not affect the pharmacokinetics of lamivudine. Systemic clearance following single intravenous doses averages 20 to 25 L/h (approximately 0.3 L/h/Kg). The elimination half-life of lamivudine is approximately five to seven hours, and the in vitro intracellular half-life of its active 5'-triphosphate anabolite is 10.5 to 15.5 hours and 17 to 19 hours in HIV-1 and HBV cell lines, respectively.

Efficacy and Tolerance

Randomized controlled trials have demonstrated the efficacy of lamivudine in the treatment of HBeAg-positive and HBeAg-negative CHB. One randomized, placebo- controlled trialshowed that almost all patients treated with lamivudine (98%) had a reduction of serum HBV-DNA levels.

Serum HBV-DNA levels were undetectable after lamivudine therapy in 44% of patients, compared with 16% of patients treated with a placebo. After one year of treatment, the HBeAg seroconversion rates were 17 and 6% in the lamivudine and placebo groups, respectively. ALT levels were normalized in 41% of the lamivudine-treated patients compared with 7% of the placebo group. Histological improvement was observed in 52% of the lamivudine group compared with 23% of the placebo group [5]. The rates of virologic, biochemical and histological response in another randomized controlled trial were similar, with an HBeAg anti-HBeAb seroconversion rate of 21% [6].The tolerability and safety of lamivudine were excellent, and the frequency of adverse events was similar to that of the placebo group. Lamivudine therapy seems to be well-tolerated for up to five years; however, data on long-term therapy with lamivudine are limited to a small number of patients. The main limitation of lamivudine treatment is the high rate of viral resistance due to mutations in the YMDD motif of the HBV polymerase gene. Indeed, even if the HBeAg seroconversion rate is increased by continuing treatment, the frequency of resistance to lamivudine increases with time:

24% at one year, 38% at two years, 50% at three years, and 67% at four years [7]. The most important mutation is substitution of valine or isoleucine for methionine. In many patients, this mutation isaccompanied by a second mutation, in which methionine is substituted for leucine in an upstream region (rtL180M). Lamivudine resistance is more likely to occur in patients with high baseline levels of serum HBV-DNA.

Emergence of lamivudine resistant mutants is usually associated with a breakthrough of hepatitis, resulting in

moderately increased levels of serum HBV-DNA and ALT, although these levels may remain lower than baseline (pretreatment) for several months.

In another investigation, 76 HBeAg-negative patients with CHB were given 100 mg LAM daily in a five-year follow-up study [10]. The incidence of YMDD was 39% at month 12, 54% at month 24 and 57% at month 36; at the same time, the response declined from 67% at six months to 51%, 34% and 29% after 12, 24 and 36 months, respectively. A complete analysis at month 24 of follow-up was available. Increases in HBV-DNA and ALT levels were apparent in patients with YMDD variants. Among the patients who did not develop YMDD variants, 72% had normal ALT and negative HBV- DNA, whereas only 5% of the patients who developed YMDD mutants maintained normal ALT levels after one year.

In a recent study the efficacy of LAM administered for three years in patients with chronic active anti-HBe-positive hepatitis was evaluated. Thirty-four patients with chronic active anti-HBe-positive hepatitis were treated with LAM (100 mg) once daily for three years. Before treatment, all patients had serum ALT levels >two times normal levels for

>six months and HBV DNA positivity >5 pg/mL, as determined by a sandwich hybridization test for nucleic acids. Both ALT and HBV DNA were monitored during therapy. After 12 months of therapy, 24 of 34 patients (70.6%) showed evidence of HBV DNA clearance and normal ALT levels; 22 of 34 (64.7%) and 19 of 34 (55.8%) patients maintained a complete response after two and three years of therapy, respectively.

Long-term LAM therapy (>one year) was not associated with an increase in the response of initially nonresponding patients. The YMDD variant emerged in 17.6% of patients in the first year, in 35.2% during the second year, and in 52.9%

during the third year of treatment. LAM was well tolerated during the three-year therapy in all patients. In patients with chronic active anti-HBe-positive hepatitis, the LAM response rate tended to decrease over time due to the emergence of YMDD variants [11].

Conclusions

The goal of therapy for patients with HBV infection is to prevent the progression of liver disease to cirrhosis and hepatic cell cancer. In recent years, progress has been made in the treatment of CHB. Nucleos(t)ide analogs such as lamivudine have been approved as initial therapy for CHB.

Currently, lamivudine is the first line of treatment of chronic hepatitis B; however, efficacy is limited by the high frequency of resistance. Recent preliminary results show that adefovir dipivoxil and entecavir could be potent analogs for the treatment of HBV. Further studies are being made to assess the long-term efficacy and safety of these drugs [12,13].

References

1. Chu C.M., Liau Y.F. Natural history of chronic B virus infection:

an immunopathological study. J Gastroenterol Hepatol 1997;12, S218-S22.

Lamivudine for Chronic Hepatitis B

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2. Ganem D., Prince A.M. Hepatitis B virus infection-natural history and clinical consequences. N Engl J Med 2004;350:1118-29.

3. Olivero F., Santantonio T., Bellati G., et al. Long-term response to therapy of chronic anti-Hbe-positive hepatitis B is poor independent of type and schedule of interferon. Am J Gastroenterol 1999;94:1366-72.

4. van Zonneveld M., Honkoop P., Hansen B.E., et al. Long-term follow-up of alpha-interferon treatment of patients with chronic hepatitis B. Hepatology 2004;39:804-10.

5. Lai C.L., Chien R.N., Leung N.W., et al. A one year trial of l a m i v u d i n e f o r c h r o n i c h e p a t i t i s B . N E n g l J M e d 1998;339:61-8.

6. Dienstag J.L., Schiff E.R., Wright T.L., et al. Lamivudine as initial treatment for chronic hepatitis B in the United States. N Engl J Med 1999;341:1256-63.

7 . Lau D.T., Khokhar F., Doo E., et al. Long-term therapy of chronic hepatitis B with lamivudine. Hepatology 2000;32:828-34.

8. Zeng M., Mao Y., Yao G., et al. A double-blind randomized trial of adefovir dipivoxil in Chinese subjects with HBeAg.

p o s i t i v e c h r o n i c h e p a t i t i s B . H e p a t o l o g y 2 0 0 6; 4 4 ( 1 ) : 1 0 8 - 1 6 .

9. Hadziyannis S.J., Tassopoulos N., Heathcote E.J., et al. Adefovir dipivoxil for the treatment of hepatitis B e antigen-negative chronic hepatitis B. N Engl J Med 2003;348:800-7.

10. Rizzetto M., Tassopoulos N.C., Goldin R.D., et al. Extended lamivudine treatment in patients with HBeAg-negative chronic hepatitis B. J Hepatol 2005;42(2):158-62.

11. Scotto G., Palumbo E., Fazio V., et al. Prolonged lamivudine treatment in patients with chronic active anti-HBe positive hepatitis. Am J Ther 2006;13(3):218-22.

12. Hadziyannis S.J. New developments in the treatment of chronic hepatitis B. Expert Opin Biol Ther 2006;6(9):913-21.

13. Zhou X.X., Littler E. Nucleoside analogs as anti-HBV agents. Curr Top Med Chem 2006;6(9):851-65.

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358

Received on 26 May 2008; revised 10 August 2008.

Address for correspondence: Dr. Norma de Paula Cavalheiro, Ph.D.

Av. Dr. Enéas de Carvalho Aguiar, 500, 1°andar, sala 12. Bairro Cerqueira César. Zip code: 05403-000. São Paulo – SP, Brazil. Phone/fax: (11) 3085-1601. E-mail: norma@usp.br.

Hepatitis C Virus Detection in the Semen of Infected Patients

Norma de Paula Cavalheiro, Ana Carolina de Oliveira Santos, Carlos Eduardo Melo, Suzana Rie Morimitsu and Antonio Alci Barone Laboratory of Hepatitis LIM-47 – Department of Infectious Diseases of Clinical Hospital of University of São Paulo; São Paulo, SP, Brazil

Though HCV infection is a serious public health problem, some aspects of its biology are still not well understood, such as its transmission through seminal fluid and sexual transmission. We looked for HCV in the semen of infected patients. Thirteen patients were included. Semen fractions (seminal plasma, leukocytes and spermatozoa) were separated with 45% and 90% Percoll gradients. The HCV-RNA in blood and semen fractions was extracted using the same protocol (AMPLICOR Roche) and was detected using the qualitative Roche Amplicor test and by agarose gel electrophoresis, with ethidium bromide staining. The mean age of the patients was 40.7 years. Risk factors for the acquisition of HCV included injectable and inhaled drug use in six (42.8%), blood transfusion in four (28.6%), and no risk factors in four (28.6%) patients. Genotype 1 was detected in 62% of the patients, followed by genotype 3 in 23% and genotype 2 in 15%. All blood samples were positive, regardless of the technique used for detection. All semen samples identified by Roche Amplicor and analyzed by agarose gel electrophoresis were negative. Among the 52 semen samples (total and fractions) identified by the Roche Amplicor method, 45 (87%) were inhibited. A negative result was recorded for one (1.9%) total semen sample, one (1.9%) leukocyte and four (7.7%) seminal plasma fractions. Only one (1.9%) sample of the spermatozoon fraction was positive. The results obtained suggested false-negative reactions for the semen samples.

Key-Words: HCV, PCR, sexual transmission, semen.

Various studies suggest that the risk of sexual transmission of hepatitis C virus (HCV) is minimal or even inexistent, with its incidence ranging from 0 to 3% [1,2]. The first report discussing the sexual transmission of HCV, in which multiple sex partners were considered to be a risk factor, was published by Alter et al. in 1989 [3].

As is the case for sexual transmission, the risk of HCV transmission through seminal fluid is also controversial. Risk factors that may increase the probability of transmission are the type of relationship, since monogamic couples tend to present lower transmission rates than individuals reporting sex with multiple partners, sexual relations that involve trauma, co-infection with acquired immunodeficiency virus (HIV), partners using drugs, associated sexually-transmitted diseases, paid sex, and a long-term relationship (>10 years) with an HCV-positive partner[4-6].

The direction of sexual transmission of HCV from men to women has been reported by Rooney and Gilson [7], who showed that the estimated risk of HCV infection is 3.7 times higher in women with HCV-positive partners. Cavalheiro et al.

[8] studied a series of 24 couples with a diagnosis of hepatitis C; there was an average viral similarity of 98.3% for 22/24 (91.7%) couples. In that study, the NS5b-HCV region was chosen for phylogenetic analysis. Nine couples attracted attention because the women did not report any risk factor for acquisition of the virus, whereas all nine men reported one or more risk factors. In this case, the average genomic similarity was 98%. That study supports the hypothesis of infection from men to women.

Studies analyzing semen by molecular biology techniques have reported difficulty in eliminating natural inhibitors present in the sample, which frequently leads to false-negative results [9]. Cassuto et al.[10], in a study of semen from 35 men, reported difficulty in removing inhibitors during HCV-PCR, with only five men testing positive, suggesting false-negative results.

Various investigators have been able to detect HCV in semen, while others could not. The difficulty in isolating this virus from body fluids, especially semen, is probably due to the presence of inhibitors and the lack of standardized techniques and protocols for RNA extraction and reverse transcription and polymerase amplification (RT-PCR); these factors may contribute to false-negative results[9,11].

Our objective was to investigate whether HCV is present in semen samples from infected patients.

Material and Methods Patient Selection

Between June and December 2004, male patients with a clinical and laboratory diagnosis of HCV infection were recruited from the Hepatitis Outpatient Clinic of the Infectious Diseases Department. All recruited patients signed an informed consent agreement.

Blood and Semen Collection and Preparation

Blood was collected from patients into 10mL dry vacuum tubes, after an 8 to 12-h fast. Semen samples were obtained by self-masturbation, after a period of sexual abstinence of at least three days. Blood and semen samples were stored at - 80°C until the time of use.

In addition to an aliquot of total semen, fractions were isolated on 90 and 45% Percoll gradients. The samples were centrifuged for 30 min. at 3,000 rpm, and the following three phases were obtained: seminal plasma, leukocytes and spermatozoa.

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Serum

All serum samples were analyzed with the qualitative Amplicor HCV test, version 2.0 (Roche Diagnostics Corp., Indianapolis, IN, USA). If positive, the blood samples were genotyped with INNO-LiPA HCV II kit (INNO-LIPA HCV, Versant Bayer, Tarrytown, NY, USA).

Semen

All semen samples were analyzed with the qualitative Amplicor HCV test, version 2.0 (Roche Diagnostics Corp., Indianapolis, IN, USA). After the end of the PCR-HCV reaction, the presence of HCV in semen samples was revealed by two methods: i) electrophoresis in agarose gel and ii) Roche Amplicor test by colorimetric determination.

Electrophoresis Agarose Gel Detection

The PCR-HCV products were detected by 2% Agarose Gel electrophoresis, stained with ethidium bromide and observed under ultraviolet light[13-15].

Amplicor HCV Test, v 2.0

The Amplicor HCV Test, v 2.0, is an RT-PCR in a manual, microwell format that amplifies a 244-nucleotide segmentof the 5’-UTR of the HCV genome. The test was performed at all sites according to manufacturer’s instructions, as previously described. HCV RNA optical density (OD) values were interpretedas follows: <0.3, negative; ≥0.3 and <1.0, equivocal;

and ≥1.0,positive. An OD value of 0.3 was used as the cutoff for the internalcontrol RNA. Any specimen with an OD value

<0.3 for bothHCV and internal control wells was considered to be inhibited[12].

HCV Genotyping by the Line Probe Assay (LiPA)

RNA was isolated from serum and 5'-UTRgenotyping was performed with the INNO-LiPA HCV II kit (INNO-LIPA HCV, Versant Bayer, Tarrytown, NY, USA), according to manufacturer’s instructions.Briefly, the 5'-UTR region was amplified with biotinylated primers. The biotin-labeled PCR products were then reverse hybridized to specificprobes attached to nitrocellulose strips, and the resultswere developed as a purple precipitate that formed a positive line on the strip.

The HCV type was deduced on the basis of the patterns of hybridizingbands using the LiPA interpretation chart[16].

Results

The mean age of the 13 patients was 40.8 years (range: 28- 50, median: 45 years). The mean time since the diagnosis of HCV infection was 7.15 years (range: 2-14, median: 7 years).

Serum

Analysis was made of the same HCV-PCR product by the Roche Amplicor method and agarose gel electrophoresis with ethidium bromide staining; all serum samples tested positive by these two techniques, i.e., all patients presented viremia.

Semen

The products analyzed with the Amplicor HCV test and revealed by agarose electrophoresis gel detection were negative for all samples of total semen and fractions (spermatozoa, leukocytes and seminal plasma).

The same semen and seminal fractions (52 samples) analyzed by optical density (Roche Amplicor method) revealed 45 (86.5%) of the results to be inhibited. Negative results were detected for one (1.9%) total semen sample, one (1.9%) leukocyte and 4 (7.7%) seminal plasma fraction. Only one (1.9%) sample of the spermatozoon fraction was positive.

Discussion

The inhibition of HCV-PCR in semen samples was clearly and unquestionably demonstrated, with 45 (87%) of the 52 reactions involving total semen and fractions, spermatozoa, leukocytes and seminal plasma presenting inhibited results (Table 3). Amplicor HCV qualitative tests have an internal control, permitting the evaluation of the efficiency of the tests.

In contrast, when the products of PCR (Amplicor HCV qualitative tests, v. 2.0 (Roche Diagnostics) were examined on the agarose gel with ethidium bromide staining, all samples tested negative. False-negative results were obtained with the in-house nested HCV-PCR method used in this preliminary study (data not shown). The presence of HCV-PCR inhibitors in semen masks the results and impairs their interpretation, a fact explaining the wide disparity in HCV detection rates reported in the international literature, which vary from 0 to 36% [10,11,17-25].

Our results support the hypothesis of the presence of HCV in semen samples from infected patients and also indicate the need for more sensitive tests and/or laboratory measures that remove HCV-PCR inhibitors so that reliable results can be obtained. Analysis of the HCV-PCR products by electrophoresis of an agarose gel and ethidium bromide staining apparently yielded false-negative results for all the semen samples. The commercial Roche Amplicor test demonstrated interference from inhibitors present in the semen samples, because it uses an internal control that guarantees the efficacy of the reaction; but it was unable to eliminate the inhibitors.

According to Abou-Setta[26] the contradictory results of HCV-PCR in semen can be explained by i) the different techniques used, ii) different HCV-PCR sensitivities, and iii) presence of inhibitors in semen that lead to false-negative results. Furthermore, this author reported that viral concentrations in semen samples go through rapid variations and indicated that inhibition might be due to the action of RNases or lipoperoxidase. The enzyme Taq polymerase may be inhibited by seminal fluid enzymes, lactoferrin, peroxidase or zinc residues[26].

The two main adaptations needed for testing semen are elimination of PCR inhibitors, especially frequent in seminal plasma, and the use of small sample volumes. The choice of the extraction protocol is critical[9].

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Information regarding the quantity of circulating virus is important; it helps increase the probability of HCV detection in semen and demonstrates the potential of sexual transmission.

Nyamaty et al. [27] showed that the quantity of virus in blood is correlated with the virus in semen. The distribution of HCV viral types was compatible with the prevalence that was previously reported for Brazil, with most of the (infected) population being infected with HCV genotype 1, followed by genotypes 3 and 2 (Table 2)[28]. On average, 40 to 50% of patients infected with HCV indicate no specific source of acquisition of the virus[26].

We had 28.6% unknown risk factors (Table 1).

This preliminary study of semen samples by HCV-PCR indicates the need for new research protocols in order to obtain more reliable results. Other laboratory resources or even new techniques for viral detection should be tested for the evaluation of semen samples so that the results better correspond to reality.

Acknowledgements

We thank the Hepatitis Outpatient Clinic of the Infectious Diseases Department, University Hospital, University of São Paulo.

References

1 . Flamm S.L. Chronic hepatitis C virus infection. JAMA 2003;289(18):2413-7.

2 . Brook M.G. Sexually acquired hepatitis. Sex Transm Infect 2002;78(4):235-40.

3 . Alter M.J., Coleman P.J., Alexander W.J., et al. Importance of heterosexual activity in the transmission of hepatitis B and non-A, non-B hepatitis. JAMA 1989;262(9):1201-5.

4. Terrault N.A. Sexual activity as a risk factor for hepatitis C.

Hepatology 2002;36(5 Suppl 1):S99-105.

5. Clarke A., Kulasegaram R. Hepatitis C transmission - where are we now? Int J STD AIDS 2006;17(2):74-80.

6. Filippini P., Coppola N., Scolastico C., et al. Does HIV infection favor the sexual transmission of hepatitis C? Sex Transm Dis 2001;28(12):725-9.

7 . Rooney G., Gilson R.J. Sexual transmission of hepatitis C virus infection. Sex Transm Infect 1998;74(6):399-404.

8 . Cavalheiro N., Tengan F., Melo C., et al. P.224 Hepatitis C:

interfamilial or sexual transmission? J Clin Virol 2006;36(Suppl 2):S131.

9. Pasquier C., Souyris C., Moinard N., et al. Validation of an automated real-time PCR protocol for detection and quantitation of HIV and HCV genomes in semen. J Virol Methods 2006;137(1):156-9.

10. Cassuto N.G., Sifer C., Feldmann G., et al. A modified RT-PCR technique to screen for viral RNA in the semen of hepatitis C virus-positive men. Hum Reprod 2002;17(12):3153-6.

11. Pekler V.A., Robbins W.A., Nyamathi A., et al. Use of versant TMA and bDNA 3.0 assays to detect and quantify hepatitis C virus in semen. J Clin Lab Anal 2003;17(6):264-70.

12. Lee S.C., Antony A., Lee N., et al. Improved version 2.0 qualitative and quantitative AMPLICOR reverse transcription-PCR tests for hepatitis C virus RNA: calibration to international units, enhanced genotype reactivity, and performance characteristics.

J Clin Microbiol 2000;38(11):4171-9.

13. Smith D.B., Lawlor E., Power J., et al. A second outbreak of hepatitis C virus infection from anti-D immunoglobulin in Ireland. Vox Sang 1999;76(3):175-80.

14. Garson J.A., Ring C., Tuke P., Tedder R.S. Enhanced detection by PCR of hepatitis C virus RNA. Lancet 1990;336(8719):878-9.

HCV in Semen

Table 1. Distribution of the patients according to risk factors for the acquisition of HCV.

Epidemiology No. of %

patients

Use of injectable and/or inhaled drugs 6 42.8%

Blood transfusion 4 28.6%

Unknown 4 28.6%

Table 2. Distribution of the patients according to HCV subtype.

Genotype No. of patients (%)

1a 5 (38.5%)

1b 1 (7.7%)

1a/1b 2 (15.4%)

2b 2 (15.4%)

3a 3 (23.0%)

Total 13 (100%)

Table 3. Results of HCV-PCR with the Roche Amplicor test.

Patient Serum Total semen Spermatozoa Leukocytes Seminal plasma

1 Positive Inhibition Inhibition Inhibition Inhibition

3 Positive Inhibition Inhibition Inhibition Negative

4 Positive Negative Positive Negative Negative

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15. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.

Anal Biochem 1987;162(1):156-9.

16. Stuyver L., Wyseur A., van Arnhem W., et al. Second-generation line probe assay for hepatitis C virus genotyping. J Clin Microbiol.

1996;34(9):2259-66.

17. Hsu H.H., Wright T.L., Luba D., et al. Failure to detect hepatitis C virus genome in human secretions with the polymerase chain reaction. Hepatology 1991;14(5):763-7.

18. Fried M.W., Shindo M., Fong T.L., et al. Absence of hepatitis C viral RNA from saliva and semen of patients with chronic hepatitis C. Gastroenterology 1992;102(4 Pt 1):1306-8.

19. Terada S., Kawanishi K., Katayama K. Minimal hepatitis C infectivity in semen. Ann Intern Med 1992;117(2):171-2.

20. Caldwell S.H., Sue M., Bowden J.H., et al. Hepatitis C virus in body fluids after liver transplantation. Liver Transpl Surg 1996;2(2):124-9.

21. Semprini A.E., Persico T., Thiers V., et al. Absence of hepatitis C virus and detection of hepatitis G virus/GB virus C RNA sequences in the semen of infected men. J Infect Dis 1998;177(4):848-54.

22. Debono E., Halfon P., Bourliere M., et al. Absence of hepatitis C genome in semen of infected men by polymerase chain reaction, branched DNA and in situ hybridization. Liver 2000;20(3):257-61.

23. Levy R., Tardy J.C., Bourlet T., et al. Transmission risk of hepatitis C virus in assisted reproductive techniques. Hum Reprod.

2000;15(4):810-6.

24. Liou T.C., Chang T.T., Young K.C., et al. Detection of HCV RNA in saliva, urine, seminal fluid, and ascites. J Med Virol 1992;37(3):197-202.

25. Leruez-Ville M., Kunstmann J.M., De Almeida M., et al. Detection of hepatitis C virus in the semen of infected men. Lancet 2000;356(9223):42-3.

26. Abou-Setta A.M. Transmission risk of hepatitis C virus via semen during assisted reproduction: how real is it? Hum Reprod 2004;19(12):2711-7.

27. Nyamathi A., Robbins W.A., Fahey J.L., et al. Presence and predictors of hepatitis C virus RNA in the semen of homeless men. Biol Res Nurs 2002;4(1):22-30.

28. Cavalheiro N. de P., Barone A.A., Tengan F.M. HCV serotypes in Brazilian patients. Int J Infect Dis 2002;6(3):228-32.

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Received on 15 April 2008; revised 20 September 2008.

Address for correspondence: Dr. Kleber Dias do Prado. Emilio Ribas Infectious Diseases Institute, Scientific Department. Av. Dr. Arnaldo 165, São Paulo, SP, Brazil, Zip code: 01246-900. E-mail:

kdiasdoprado@gmail.com - Phone/Fax: (55 11) 3896-1241.

Histological Response Study of Chronic Viral Hepatitis C Patients Treated With Interferon Alone or Combined With Ribavirin

Kleber Prado¹, Rosely Patzina², Denise Bergamaschi³ and Roberto Focaccia¹

1Viral Hepatitis Clinic - Emilio Ribas Infectious Diseases Institute; ²Department of Pathology – Emilio Ribas Infectious Diseases Institute;

3Department of Statistics - Public Health College – University of São Paulo; São Paulo, SP, Brazil

Chronic hepatitis C is often a progressive, fibrotic disease that can lead to cirrhosis and other complications. The recommended therapy is a combination of interferon and ribavirin. Besides its antiviral action, interferon is considered to have antifibrotic activity. We examined the outcome of hepatic fibrosis and inflammation in chronic hepatitis C patients who were non-responders to interferon. We made a case series, retrospective study, based on revision of medical records and reassessment of liver biopsies. For inclusion, patients should have been treated with interferon alone or combined with ribavirin, with no virological response (non responders and relapsers) and had a liver biopsy before and after treatment. Histological evaluation included: i-outcome of fibrosis and necroinflammation; ii-annual fibrosis progression rate evaluation, before and after treatment. Seventy-five patients were included. Fifty-seven patients (76%) did not show progression of fibrosis after treatment, compared to six (8%) before treatment (p < 0.001). The mean annual fibrosis progression rate was significantly reduced after treatment (p = 0.036). Inflammatory activity improved in 19 patients (25.3%). The results support the hypothesis of an antifibrotic effect of interferon-based therapy, in non-responder patients. There was evidence of anti-inflammatory effects of treatment in some patients.

Key Words: Chronic hepatitis C, fibrosis, interferon-alfa, ribavirin, treatment result.

Hepatitis C virus (HCV) infection is a major worldwide public health issue. It is estimated that between 170 and 250 million people are chronic carriers [1]. There is no available information about the prevalence rate of chronic hepatitis C in Brazil, although Focaccia et al. found an estimated prevalence of 1.42% in São Paulo City, rising to over 3% within the 30 and older age group. We examined the medical records of patients from 2 to 80 years old in a serological survey of a stratified, randomized and residence-based population [2].

HCV infection becomes a chronic disease in a large proportion of patients, ranging from 55% to 90%; it has a progressive fibrotic nature. Up to 20% of the cases may culminate in cirrhosis; which may cause complications, such as hepatocellular carcinoma and end-stage liver disease [3-5].

The combination of pegylated interferon (PEG) and ribavirin, which is currently the best available treatment, given during 48 weeks, only reaches a sustained virological response (SVR) in 54%-56% of naive patients [6,7]. Non-responder patients to interferon alone or in combination with ribavirin have an even smaller frequency of virological response [8]. Currently, for non-responders to a combination of PEG and ribavirin, there is no consensus therapeutic option that considers the histological response among treatment efficacy end-points [3,9-11].

Several studies have demonstrated the inherent antifibrinogenic action of interferon, which is not related to

its antiviral and anti-inflammatory effects [12-16]. Furthermore, it has also been proven through several clinical trials that interferon-based treatments provoke histological improvements, even in virological non-responders [17-22]. We evaluated hepatic fibrosis and necro-inflammatory activity progression in chronic hepatitis C patients who were non- responders to interferon-based treatments or relapsers.

Material and Methods

A retrospective study was made of chronic hepatitis C patients who had been treated at the Viral Hepatitis Clinic of the Emilio Ribas Infectious Diseases Institute (ERIDI), in São Paulo, Brazil, from January 1997 to December 2002. We reviewed patient medical records to collect demographic, anthropometric, epidemiological and clinical information, as information on liver biopsies. The study protocol was approved by ERIDI’s Ethics Committee.

We identified 8,192 medical records of hepatitis patients within this period. A preliminary analysis ruled out acute hepatitis cases, HIV or HVB coinfections, HCV spontaneous clearance, patients who had never been under treatment or had not undergone liver biopsy before and after treatment. The remaining 615 medical charts were reviewed for inclusion. The inclusion criteria were: 1 – previous treatment for at least six months with interferon-alfa, with or without ribavirin, using standardized doses and routes of administration [6,7,23,24]; 2 – a liver biopsy performed or reviewed by an ERIDI pathologist, performed within six months prior to treatment and at least six months after treatment; 3 – non-response to treatment or relapse. The exclusion criteria were: 1 – chronic liver diseases not related to HCV; 2 – current alcohol or illegal drug abuse; 3 - use of hepatotoxic or immunosuppressive drugs;

4 - immunosuppressive illness and HIV coinfection.

Non response was defined as detectable HCV RNA by PCR technique (Amplicor 2.0 version kits, with a lower limit of

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sensitivity of 50 IU/mL) at the end of treatment and relapse based on undetectable serum HCV-RNA using the same technique at the end of treatment, which became detectable six months later. To determine the HCV genotype, the INNO- LIPA HCV II technique was used, with the Versant kit (Lipa)®.

Liver biopsies were read blind, always by the same pathologist from the Department of Pathology of ERIDI, applying the METAVIR score. Fibrosis qualitative evolution was evaluated by making a fibrosis stage comparison between the two biopsy tests (before and after treatment), with three possible outcomes: 1 – progression (after-treatment fibrosis stage more advanced than before-treatment stage); 2 – stabilization (after-treatment fibrosis stage not changed compared to before-treatment stage); and 3 – regression (after- treatment fibrosis stage lower than before-treatment stage).

Patients classified as stage four (cirrhosis) at the first liver biopsy were classified in the progression category whenever decompensated cirrhosis was identified. In such cases, a new biopsy was not required. Otherwise, patients were designated in the non-progression category if the new biopsy did not demonstrate regression.

The annual fibrosis progression rate, both pre and post- treatment, was calculated based on the following definitions:

1- pre-treatment annual fibrosis progression rate =the ratio between fibrosis stage measured in METAVIR units at the first biopsy (pre-treatment) and the estimated time of infection in years; 2 – post-treatment annual fibrosis progression rate

= the ratio between the difference in fibrosis stage measured in METAVIR units of the pre and post-treatment biopsies and the delay between the two biopsies in months multiplied by 12 [4,18].

The calculation of the probable infection date, and consequently, the time of infection, was based on: a – for transfusional infections, the date of the first blood and/or plasma derived products transfusion was considered, if the patient had gone through more than one transfusion session;

b – for infection caused by injectable drugs, the first year of use of such drugs [12,25,26]. For any other cause of HCV infection, only infection dates based on unequivocal evidence of a temporal relation between exposure and HCV infection were used.

The liver biopsy samples were at least 10mm long or had six portal tracts [27]. The liver tissue fragments were colored by hematoxilin-eosine, Masson’s trichrome, Perls and reticuline.

Statistical Analysis

For comparison of proportions (pre and post-treatment situations), we used the McNemar test for dependent samples and the Pearson’s chi-square for independent samples. For mean values comparison, we used the “Student” t test (on independent samples) and a paired student t (on dependent samples), when there were only two means involved in the comparison. When the variables did not conform to a normal distribution, we used the Wilcoxon signed-rank test [28], for

population comparisons. The statistical computer program Stata was used for this analysis [29]. Statistical decisions were made considering the descriptive value of the tests (p value).

Results

Seventy-five patients met the inclusion criteria. Table 1 shows the main characteristics of the 75 patients included in this study.

Seventy-nine percent of the patients had undergone a liver biopsy one year or more after the end of treatment (median time: 20 months; range: 1 - 67 months). Seventy-six percent of patients showed stabilization or regression of fibrosis (no progression) at the second liver biopsy (Table 2 and Figure 1). Before treatment, the vast majority of patients had evidence of fibrosis progression (fibrosis stage > 0). In contrast, after treatment, most patients displayed a tendency towards no progression (p< 0.001), at least over the period the patients were observed (Figure 2).

A significant association was found between fibrosis qualitative evolution and pre-treatment fibrosis stages (p=0.005), with higher rates of progression among patients at stages 0, 1 and 2, and regression among patients at stages 3 and 4 (Table 3).

The pre-treatment annual fibrosis progression rate was defined for 42 patients. There was a mean rate of 0.096 FMU/

year (fibrosis METAVIR units per year) (CI 95%: 0.075 – 0.117 FMU/year), a median value of 0.096 FMU/year, with minimum and maximum values of 0.000 and 0.272 FMU/year, respectively.

The post-treatment fibrosis annual progression rate was calculated for 75 patients. There was a mean rate of 0.007 FMU/year, (CI 95%: -0.066 – 0.081 FMU/year), a median value of 0.000 FMU/year, with minimum and maximum values of - 1.500 and 0.827 FMU/year, respectively. The paired pre and post –treatment rate analyses, restricted to the 42 patients with known pre-treatment rates, showed a significant decrease of the fibrosis annual progression rate. The post-treatment mean rate in these patients was -0.005 FMU/year (CI 95%: - 0.088 – 0.078 FMU/year); there was a difference of 0.101 FMU/

year (CI 95%: 0.007 – 0.194FMU/year, p=0.036) between the mean rates (paired Student-t test).

The post-treatment fibrosis annual progression rate in comparison with the fibrosis stage on the pre-treatment biopsy showed a significantly higher decrease of this progression among the patients that were in an advanced stage of fibrosis (F3/F4) at the time of the pre-treatment biopsy when compared to those who had had mild-to-moderate fibrosis (F0/F1/F2) (p=0.0143). Among the 31 patients that had showed mild-to- moderate fibrosis at pre-treatment, the mean decrease in fibrosis progression after treatment was 0.034 FMU/year, while in the 11 patients who had shown advanced fibrosis pre- treatment, the mean decrease was 0.287 FMU/year.

Table 4 summarizes the information about the progress in inflammatory activity. Table 5 shows the progression in inflammatory activity according to different ranks of fibrosis qualitative progress. There were significant differences among