Hepatitis B virus, hepatitis C virus and human immunodeficiency virus infection in undocumented migrants and refugees in southern Italy, January 2012 to June 2013

Screening of undocumented migrants or refugees for hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections has been offered free of charge and free from bureaucratic procedures since 2012 at four primary-level clinical centres in Naples and Caserta, Italy. Of 926 undocumented migrants and refugees visiting one of the primary-level clinical centres from January 2012 to June 2013, 882 (95%) were screened for hepatitis B surface antigen (HBsAg), total hepatitis B core antibody (anti-HBc) and antibodies against HCV and HIV. Of the 882 individuals enrolled, 78 (9%) were HBsAg positive, 35 (4%) anti-HCV positive and 11 (1%) anti-HIV positive (single infections); seven (1%) had more than one infection (three were HBsAg positive). Of the 801 HBsAg-negative patients, 373 (47%) were anti-HBc positive. The HBsAg-positivity rate was high (14%; 62/444) in individuals from sub-Saharan Africa and intermediate in those from eastern Europe (6%; 12/198), northern Africa (2%; 2/80) and Bangladesh, India, Pakistan and Sri Lanka (the 'India-Pakistan area') (3%; 4/126). Anti-HCV was detected in 9/126 (7%) individuals originating from the India-Pakistan area, in 12/198 (6%) from eastern Europe, in 17/444 (4%) from sub-Saharan and in 2/80 (2%) from northern Africa. The HBV, HCV and HIV infections in the undocumented migrants and refugees screened serve as a reminder to the Italian healthcare authorities to carry out extensive screening and educational programmes for these populations.

As a consequence of socio-economic and political crises in certain parts of the world, mostly due to war and civil war in recent decades, countries of western Europe have received migrants and refugees from areas that have a level of endemicity higher than 2% for HBV, HCV and HIV infections [10].Surveillance data from 2014 showed that nearly 4.9 million legal migrants lived in Italy, making up 8% of the resident population [11].The Italian immigration authorities estimate that nearly 500,000 migrants without a residence permit ('undocumented migrants') or refugees were living in Italy in 2014 [11], originating mainly from northern and sub-Saharan Africa, eastern Europe and central and eastern Asia [11,12].Once in Italy, these individuals -with a low income most frequently from casual day-to-day work, broken family ties and of no fixed abode [11] have limited access to healthcare services [11].Despite the intermediate or high level of endemicity of HBV, HCV and HIV infections in their geographical area of origin, the majority of them are unaware of their HBV, HCV and HIV status [11].
According to Italian policy, refugees should be regularly admitted to all healthcare facilities of the national healthcare system, whereas access of undocumented migrants is limited to minors, pregnant women, patients with serious pathological conditions and to individuals with transmissible diseases including HBV, HCV and HIV infections [11].In our experience, shared by numerous other investigators in Italy, undocumented migrants and refugees prefer primary-level clinical centres with proven experience in the clinical, psychological and legal management of vulnerable groups, operating with the help of cultural mediators (Asli Ahmed Abdulle, personal communication, September 2013).
Prospective screening of migrants and refugees for HBV, HCV and HIV infection started in January 2012 in Naples and Caserta and is still ongoing.It involves a large number of people (more than 1,200 participants up to December 2014) and is monitored by individuals outside the programme.Our study involved six participating centres: four primary-care clinical centres (two in Naples and two in Caserta) and two tertiary units in infectious diseases (one in Naples and one in Caserta).These two cities are located in the Campania region in southern Italy, 30 kilometres from each other, have a large population of refugees and undocumented migrants from Africa, central and eastern Asia, eastern Europe and Latin America [11].
In this article, we present the results of screening carried out from January to June 2013 for HBV, HCV and HIV infections in undocumented migrants and refugees living in Naples and Caserta, Italy.The study was carried out to help undocumented migrants and refugees to attend to the screening and to assess the extent to which these vulnerable groups take part in the screening.

Study protocol and setting
Senior investigators from the six participating centres prepared the study protocol and an anonymous questionnaire; they also established all screening and postscreening procedures.
The questionnaire was used to record age, sex, geographical origin, date (month and year) of migration, level of education, religion, family history, cohabitation details, sexual orientation and practices including condom use, history of HBV vaccination, surgery, dental care, tattooing, body piercing, use of drugs, blood transfusion, tribal rituals, abortion and information on previously documented personal and family HBV, HCV and HIV infections.
During clinical consultations, asked for by the patient, a physician from the clinical centre and a cultural mediator explain to the undocumented migrants and refugees the importance of being tested for HBV, HCV and HIV infection and offer testing free of charge, in anonymity (recording only the centre number and patient's number), in full accordance with Italian privacy law regarding observational studies [13].In each case, the clinical history was obtained with the help of a physician and a cultural mediator during a prolonged, in-depth clinical consultation and counselling.All questionnaires were checked by the senior investigators of the primary-care clinical centres and found suitable for the study, as they had been correctly completed.
The study population consisted of all the undocumented migrants (including the citizens from other European countries who might not qualify for the right of residence for more than three months in Italy) and refugees seen consecutively for clinical consultation at one of the four primary-care clinical centres from January 2012 to June 2013, who agreed to participate in the investigation.The most frequent clinical conditions leading the patients to seek care at one of these centres were lumbago, headache, pruritus, cough, high blood pressure and allergy symptoms.
Written informed consent to participate in the study, in the person's native language, was obtained on a voluntary basis.As explained in the informed consent form, a serum sample would be taken from study participants.All participants received the results of their serological screening and information on preventing infection and transmission of HBV, HCV and HIV.
The primary-level clinical centres involved in our study were hospitals in the national healthcare system or clinical centres (general outpatient clinics) of international charity organisations such as Caritas and the Sisters of Mother Teresa of Calcutta.
Participants who were positive for total hepatitis B core antibody (anti-HBc), positive or negative for hepatitis B surface antigen (HBsAg), anti-HCV positive or anti-HIV positive were referred to one of the two tertiary units of infectious diseases for further investigation, monitoring and possible treatment.These tertiary units offer refugees and undocumented migrants with serum markers of HBV, HCV or HIV infection the same clinical management given to legal migrants and to Italian citizens.These two tertiary units of infectious diseases are both affiliated with the Second University of Naples and have cooperated for nearly 15 years in several clinical investigations on HIV, HBV and HCV infections using the same clinical approach and the same laboratory methods [14][15][16][17][18].
The study was approved by the Ethics Committee of the Azienda Ospedaliera Universitaria of the Second University of Naples.

Testing procedure
Serum samples were tested for HBsAg, total anti-HCV, anti-HIV, anti-HBc and hepatitis B surface antibodies (anti-HBs) by commercial immunoenzymatic assays (Abbott Laboratories, North Chicago, IL, United States: AxSYM HBsAg (V.2) M/S for HBsAg, AXSYM HCV 3.0 for anti-HCV, AXSYM HIV 0.5 COMBO for anti-HIV, AXSYM core for anti-HBc and AXSYM AUSAB for anti-HBs).Anti-HIV reactivity was always confirmed by a western blot assay (Genelabs Diagnostics, Science Park Drive, Singapore) that identifies both HIV-1 and HIV-2 strains.Circulating HCV RNA was quantified by real-time polymerase chain reaction (PCR) in a Light cycler 1.5 (Roche Diagnostics, Branchburg, NJ, United States) as previously described [15].

Statistical analysis
Continuous variables were summarised as mean and standard deviation, and categorical variables as absolute and relative frequencies.Differences in mean values were evaluated by Student's t-test and chi-squared test was applied to categorical variables.A p value < 0.05 was considered to be statistically significant.

Results
Of the 926 undocumented migrants and refugees who attended one of the four primary-care clinical units during the study period, 882 agreed to participate in the study: 625 undocumented migrants and 257 refugees.The group of 625 undocumented migrants includes 105 citizens from Romania (n = 63), Bulgaria (n = 35) and Poland (n = 7) with no right of residence in Italy for more than three months, but living there for more than two years.The demographic and serological data of the patients obtained at the time of enrolment in the study are shown in Table 1.These participants were relatively young (median age: 34.5 years; range: 14-74), mostly male (72%) and had been living in Italy for a mean period of 58 months (standard deviation (SD): 55).A total of 444 (50%) came from sub-Saharan Africa, 198 (22%) from eastern Europe, 80 (9%) from northern Africa, 126 (14%) from Bangladesh, India, Pakistan and Sri Lanka (the 'India-Pakistan area'), 25 (3%) from other areas of Asia and 9 (1%) from South America (Table 2).Details of the country of origin of these 882study participants are shown in Table 2. for more than three months, but living there for at least two years.
All participants with a detectable serum marker of HBV, HCV or HIV infection were unaware of their serological status.The differences between the 625 undocumented migrants and the 257 refugees were small (Table 1), and were of no or limited clinical value.
The characteristics of participants grouped according to the four major geographical groups (northern Africa,  Anti-HBc: total hepatitis B core antibody; HBsAg; hepatitis B surface antigen; HCV: hepatitis C virus; HIV: human immunodeficiency virus; NC: not calculated; SD: standard deviation.
a Unless otherwise indicated.b Defined as consumption of alcohol exceeding 30g per day for women and 40g per day for men over the last six months.c The group of 625 undocumented migrants included 105 individuals from Romania, Bulgaria 35 and Poland with no right of residence for more than three months, but living in Italy from at least two years.d Bangladesh, India, Pakistan, Sri Lanka.e Both in the country of origin and in Italy.f No use of condoms with more than two sexual partners.g Unsafe intravenous or intramuscular injections, acupuncture, tattoo, body piercing or tribal practices.
In individuals from sub-Saharan Africa, the cumulative prevalence of markers of ongoing or past HBV infection was very high (more than 50%) in the age groups 16-30, 31-45 and 46-60 years, with a tendency to increase with age, from 98/183 in the youngest age group to 23/32 in those aged 46-60 years (Figure 1); this differed from that seen in individuals from the Anti-HBc: total hepatitis B core antibody; HBsAg; hepatitis B surface antigen; HCV: hepatitis C virus; HIV: human immunodeficiency virus; SD: standard deviation.a Unless otherwise indicated.b Bangladesh, India, Pakistan, Sri Lanka.c Defined as consumption of alcohol exceeding 30g per day for women and 40g per day for men over the last six months.d The group of the undocumented migrants included 105 individuals from Romania, Bulgaria and Poland with no right of residence for more than three months, but living in Italy from more than two years.e Both in the country of origin and in Italy.f No use of condoms with more than two sexual partners.g Unsafe intravenous or intramuscular injections, acupuncture, tattoo, body piercing or tribal practices.

Serological markers of infection and demographic and epidemiological characteristics
Correlation between the presence of serological markers of HBV, HCV and HIV infection and the demographic and epidemiological characteristics of the 882 migrants enrolled in the study is shown in Table 5.The individuals who were either HBsAg positive, HBsAg negative/ anti-HBc positive, anti-HCV positive or anti-HIV positive (aetiological subgroups), compared with those with no serum marker, had fewer years of schooling, were more frequently male and more frequently came from sub-Saharan Africa (Table 5).The percentages of migrants reporting the risk factors stated in None of the 882 undocumented migrants or refugees had been vaccinated against HBV.This is supported in part by the observation that 68 (17%) of the 408 anti-HBc negative individuals were tested for anti-HBs and found negative.

Discussion
The socio-economic and cultural backgrounds of the undocumented migrants and refugees in our study made their access to the Italian healthcare services difficult.Nevertheless, the presence of a skilled physician and a cultural mediator in the four centres overcame any language or cultural barrier and allowed successful screening, with an over-95% acceptance rate.Indeed, the rate of the interviewed undocumented migrants and refugees who agreed to be screened seems a useful parameter to evaluate the effectiveness of the screening.The success of screening to identify serum markers of HBV, HCV or HIV infection in undocumented migrants or refugees varies from one study to another, mainly due to the different composition of the populations studied according to their place of origin, legal status, length of stay in the foreign country and the quality of the strategies used to improve the access of these vulnerable populations to a screening programme [22][23][24][25][26][27][28][29][30][31].In our study, individuals who were HBsAg positive, HBsAg negative/anti-HBc positive, anti-HCV positive and anti-HIV positive were referred to a tertiary-level clinical centre to complete the diagnostic course and if necessary to receive therapeutic follow-up.In addition, all those participating in the screening had the opportunity to improve their knowledge of the transmission and prevention of HBV, HCV and HIV infections.Consequently, our screening strategy may be an example of how to overcome language and cultural barriers in a population of undocumented migrants and refugees.
In agreement with recommendations of the United States Centers for Disease Control and Prevention, the data from our study underscore the need for universal screening for HBV infection in people from countries with HBsAg prevalence higher than 2% [23].Our data also suggest the need for universal screening for HCV and HIV infections in people from countries with anti-HCV or anti-HIV prevalences higher than 2%.
An ongoing or past/occult HBV infection was frequently detected in the population in our study: nearly 9% of the undocumented migrants and refugees were HBsAg positive and nearly 40% HBsAg negative/anti-HBc positive.The HBsAg positivity prevalence ranged from 2.2% to 13.6% in other studies carried out in Italy investigating undocumented migrants in Verona (northern Italy) in 2004-05 [24] and in the Campania region (southern Italy) from 1999 to 2008 [22] and refugees in Bari in 2001-10 [25][26][27][28] and Foggia [29] in 2005 in southern Italy.The highest prevalence of HBsAg positivity was observed in refugees from Albania (13.6%), and the lowest (2.9%) in refugees from Kosovo under UN Security Council Resolution 1244 and in a Kurdish refugee population from Iraq (2.2%) in Apulia in 2000-03 [26][27][28].These high prevalences most probably reflect widespread HBV infection in the countries of origin, as the rate of HBsAg positivity is under 1% in Italy, a country with an ongoing mass vaccination programme currently covering Italian citizens aged 0-34 years [32].HCV infection was identified in 4.8% of the population in our study: it was 2.7% in undocumented migrants enrolled in 2004-05 in Verona [23], 4.3% in refugees in 2008 in Bari [25] and 3.6% in migrants, frequently undocumented, from 1999 to 2009 the Campania region [22].HIV infection was identified in 2% of the individuals in our study, whereas it was 5% of those investigated in the Campania region [22] and 1.5% of those investigated in Bari [25].Thus the prevalence of HBV, HCV or HIV infection in the populations investigated in the studies performed in Italy to date is high, but this provides an incomplete picture on a national scale due to the limited number of published papers and the lack of a systematic survey.Individuals from sub-Saharan Africa accounted for just over half of the people enrolled in our study, one eighth of whom had an ongoing infection and more than a half had a past or occult HBV infection.These very high rates, with wide variation in the country subgroups (data not shown), indicate acquisition of HBV at birth from infected mothers or in early youth from parents or siblings [30].The HBsAg positivity rates were intermediate in individuals from eastern Europe and the India-Pakistan area, again with wide variation in the country subgroups (data not shown).In agreement with the information available on the epidemiology of HIV infection [6,31,33,34], the highest prevalence of anti-HIV-positivity was seen in the individuals from sub-Saharan Africa, with some variations between single countries (data not shown).
The data in our study suggest that healthcare strategies for screening should differ by sex, as the prevalence of individuals with an ongoing or past HBV infection was higher among those who were male.Compared with individuals in the subgroup with no serum markers, those in the four aetiological subgroups were less frequently female, had fewer years of schooling and more frequently came from sub-Saharan Africa.In these aetiological subgroups, the percentages of individuals reporting risk factors for acquiring HBV, HCV or HIV were very high but similar to those found in the subgroup with no serum marker and, therefore, infectionassociated risk factors may be difficult to determine.It is noteworthy that many of the infections were detected in individuals who had experienced unsafe healthcare practices and that only a few individuals reported drug use or having had a blood transfusion in all the aetiological subgroups.
Only individuals aged 16-30, 31-45 and 46-60 years were numerous enough to be analysed by age.Widespread HBV infection (ongoing or past) in individuals from sub-Saharan Africa (65% aged 16-30 years and a progressive increase in older age groups up to 80% in individuals aged 45-60 years) should alert the Italian healthcare authorities to implement measures to control parenteral infections in undocumented migrants and refugees.Universal HBV vaccination has been mandatory in Italy since 1991 [32,35] and nearly all Italian citizens aged 0-34 years have been vaccinated [36].In contrast, none of the 882 undocumented migrants or refugees in our study had been vaccinated against HBV or tested for HBV markers after a mean stay in Italy of 58 months.Thus extending HBV  HBc: total hepatitis B core; HBsAg; hepatitis B surface antigen; HCV: hepatitis C virus; HIV: human immunodeficiency virus; SD: standard deviation.a Unless otherwise specified.b P value for A vs C = 0.003; B vs C <0.0001.c P value for D vs G < 0.0001; E vs G < 0.0001; F vs G = 0.042.d Defined as consumption of alcohol exceeding 30 g per day for women and 40 g per day for men over the last six months.e The group of the undocumented migrants included 105 individuals from Romania, Bulgaria and Poland with no right of residence for more than three months, but living in Italy from more than two years.f P value for G vs J <0.0001; H vs J =0.0001;I vs J =0.003.g Bangladesh, India, Pakistan, Sri Lanka.h Both in the country of origin and in Italy.i No use of condoms with more than two sexual partners.j Unsafe intravenous or intramuscular injections, acupuncture, tattoo, piercing, or tribal practices.
vaccination to legal and undocumented migrants and refugees living in Italy should be considered, especially for those who stay in the country a long time.Of course, some undocumented migrants and refugees will chose another country as their final destination, but some may remain in Italy for years or forever, as shown by the length of stay in our study (mean: 58 months; SD: 55).Indeed, taking care of this vulnerable group of individuals should be a moral duty of the governments of all countries [37][38][39].
The prevalence of anti-HCV positive individuals reached 10% in those aged 16-30 and 31-45 years from eastern Europe, whereas no positive individual was seen in the older age group.This finding possibly indicates spread of HCV infection in young adults in the countries of origin in recent years.Also of epidemiological importance was the percentage of individuals who were anti-HIV positive among those from sub-Saharan Africa: the prevalence increased with increasing age, with prevalence doubling (from 1.6% to 3%) in the 16-30 year-olds to the 31-45 year-olds and from the 31-45 year-olds to the 45-60 year-olds (from 3% to 6.2%).
It is difficult to assess the representativeness of the refugees and undocumented migrants in our study, but the percentages of cases with HBV, HCV or HIV infection in the different geographical groups in our study do not differ substantially from those reported in the literature for the corresponding areas of origin [1,2,5,6].Although the individuals investigated in this study may not be representative of the whole population in their geographical area of origin, the observed rates of HBV, HCV and HIV infection may be useful to help to devise appropriate healthcare strategies for undocumented migrant and refugee populations from different geographical areas.

Figure 1
Figure 1Serological status of study participants originating from sub-Saharan Africa, by age, Caserta and Naples, Italy, January 2012-June 2013 (n = 444)

Figure 2
Figure 2Serological status of study participants originating from northern Africa, by age, Caserta and Naples, Italy, January 2012-June 2013 (n = 80)

Figure 3
Figure 3Serological status of study participants originating from eastern Europe, by age, Caserta and Naples, Italy, January 2012-June 2013(n = 194)

Figure 4
Figure 4Serological status of study participants originating from eastern Europe, by age, Caserta and Naples, Italy, January 2012-June 2013(n = 194)

Table 1
Characteristics of study participants, Caserta and Naples, Italy, January 2012-June 2013 (n=882) Anti-HBc: total hepatitis B core antibody; HBsAg; hepatitis B surface antigen; HCV: hepatitis C virus; HIV: human immunodeficiency virus; NC: not calculated, SD: standard deviation.aUnlessotherwise stated.bThe group of 625 undocumented migrants included 105 individuals from Romania, Bulgaria 35 and Poland with no right of residence in Italy

Table 3
Characteristics of study participants by sex, Caserta and Naples, Italy, January 2012-June 2013 (n = 882)

Table 4
Characteristics of study participants by geographical area of origin, Caserta and Naples, Italy, January 2012-June 2013 (n = 848)

Table 5
no individual reported use of drugs.In individuals from eastern Europe, only a few (n = 7) reported having had a blood transfusion, 166/198 (84%) reported invasive medical procedures and 154 (78%) other parenteral exposure.Unsafe sex was reported by 10% (n = 19) and use of drugs by only a few (n=2) individuals from this area.

Table 5
Demographic and other characteristics at enrolment of study participants according to serum markers of HBV, HCV and HIV infection (n = 882)