Our study proposes to analyze the inmate population that represents a model of closed community subject to restrictive conditions and characterized by a multinational composition and a high incidence of disease. In prison, emerging problems such as drug use, HIV/AIDS and other infectious diseases are often present and they must be addressed in both Italian and non-Italian national inmates.
For an accurate surveillance, in prison the monitoring of viral epidemiological changes and circulation of virus strains are pivotal for both risk groups and the general population.
In our study, 30.8% of the enrolled inmates were of non-Italian origin. This data is in line with the 2018 official data (the average time period of sample collection) from the Ministry of Justice, which indicated a 33.9% of inmates of non-Italian nationality (https://www.giustizia.it/giustizia/it/mg_1_14_1.page?facetNode_1=1_5_40&contentId=SST165666&previsiousPage=mg_1_14).
It is well known that HIV-1 subtype B is predominant in Western Europe, including Italy, North America and North Africa27,28. However, in recent years, the frequency of non-B clade forms has been reported to increase in subtype B-restricted geographical areas and in Italy6,29.
The HIV strains phylogenetic analysis showed that the majority of inmates of Italian nationality were infected with the HIV-1 subtype B, except two, one infected with the recombinant form CRF72_BF1, and the other one with the HIV-1 sub-subtype A6, both previously not identified in inmates of Italian nationality30,31. CRF72_BF1 was isolated in Brazil32, and it has spread in Spain. HIV-1 sub-subtype A6 is present in the Former Soviet Union (FSU) countries likely originated from A1 strains of African origin, and is now spreading into non-FSU countries33. The presence of new strains within a closed community, such as a prison, can become a public health problem because this would allow the spread of new drug-resistant variants.
Conversely, a different pattern was found in the non-Italian inmate population. Ten out of 19 inmates of non-Italian nationality, were infected by subtype B strains. They came from countries where HIV-1 subtype B circulates with the exception of Liberia and Nigeria in which only very few cases of subtype B have been described in the recent past [Los Alamos https://www.hiv.lanl.gov/components/sequence/HIV/geo/geo.html accessed on march 7, 2023; 29,34]. Of the other non-Italian inmates, six were infected with subtypes and CRFs circulating in their countries of origin, and three were infected with CRF71_BF135, CRF72_BF132 and CRF108_BC36 respectively. Of note, these CRFs were isolated from inmates coming from Tunisia, Romania and Albany, where circulation of these three recombinant forms has not been previously described.
Despite considerable success in the treatment of HIV-1 infection, there continues to be a growing concern about the emergence of HIV-1 drug resistance mutations (DRMs) that can compromise the effectiveness of antiretroviral drugs. It has been described earlier that the prevalence of DRMs in cART naive individuals varies from 0 to 15% and may depend on several variables such as geographical area, HIV prevalence, transmission route and virus subtype37,38,39,40. However, it must be kept in mind that various factors, such as early treatment, use of appropriate drug regimens, and good adherence to therapy play a major role in blocking the emergence of DRMs41.
Since DRMs do not appear during ART in patients with suppressed viremia42, HIV-infected ART-naive individuals are the major source of drug-resistant viruses in both the developed and low- and middle-income countries43,44. Consequently, the DRMs, that can persist for a significant period of time even in the absence of drug treatment, can be transmitted (TDRMs)45. DRMs can be selected during antiviral treatment and persist in proviral DNA as integrated forms in viral reservoirs. Consequently, if treatment is interrupted or adherence compromised, there is a risk that the reactivated virus is drug resistant, potentially facilitating transmission to co-infection partners and hampering the effectiveness of antiviral treatments45.
In our study, we found DRMs in 7 HIV-1 subtyped inmates (6 with subtypes B isolated from inmates of Italian nationality, and 1 with CRF02_AG isolated from a inmate of Nigerian nationality). Six inmates had major DRMs in the PR and RT genes, and one inmate had major DRM in the IN gene. Of note, two mutations, K70R and I84V, were present in the same sequence from a single inmate. The simultaneous presence of multiple DRMs within the same viral genome can lead to a significant increase in resistance to antiviral drugs, as well as cross-resistance to inhibitors of the same class46. Three out of 6 inmates showed resistance mutations to drugs belonging to classes never administered to them. For one inmate the previous therapeutic regimen was unknown, therefore it cannot be excluded that the DRM could be related to the previous therapy. One inmate, treated with PIs and with InSTI showed a resistance mutation to NRTI drugs. It has been recently described that transmitted mutations to NRTI drugs in patients treated with InSTI can increase the risk of viral failure if treated with InSTI-based regimens47. We also found an inmate with the R263K mutation against InSTIs, a class of drugs never administered to him, which confers low-level resistance to InSTIs48,49. Other authors have also described a very low prevalence of transmitted drug resistance to InSTIs50,51,52. However, it has been observed that increased resistance rates is associated with incomplete adherence to therapy and low CD4 T-cells regardless of which INSTI was administered53. Thus, baseline resistance to INSTIs should be evaluated. In the case of emergence of DRMs to one class of drugs the retreatment with an antiviral of the same class could cause poor response, suggesting that switching to another combination type/class of drugs is preferable46. However, it must be appreciated that there are resistance mutations that may appear in patients receiving drugs that affect regions other than those in which the mutations were found. As in the case of DDA resistance for HCV infection, we can define these extra target mutations46. This suggests that DRM studies are important when deciding to treat HIV-infected inmates.
We found that 75.4% of HIV-infected inmates showed also markers of HBV and/or HCV infection. In particular, 56.9% of individual had infection markers for HBV, 60.0% for HCV and 41.5% + for both HBV and HCV co-infection. Of note, 14 inmates (13 Italian nationals and 1 Peruvian national, 21.5%) were found positive only for anti-HBs antibodies. Therefore, although we did not have information on the HBV vaccination status of the 65 inmates, we considered these inmates as vaccinated for HBV, as suggested by the CDC (https://www.cdc.gov/hepatitis/hbv/pdfs/serologicchartv8.pdf). Although the Ministerial Decree n. 251 of 25 October 1991 specifies that vaccination against hepatitis B must be administered to all prisoners, this is severely hampered by the high turnover of inmates in Italian prisons. For this reason, rapid vaccination schedules have been proposed and are being applied54,55. Stasi and colleagues found a similar prevalence of inmates (15.9%) with isolated anti-HBs marker in detention facilities in the Tuscany region55, and considered them as vaccinated.
We found that HBV infected inmates were mostly Italian nationals (81.3% vs 57.9% of non-Italian nationals). This highlights the importance of screening for HBV in accelerating vaccination in order to ensure short-term protection in the prison population55.
In our study, 11 (17%) inmates showed an isolated anti-HBc marker. This prevalence is in line with the frequency of anti-HBc marker in HIV-1-infected patients observed by Chang and colleagues. This frequency varied from 17 to 40%, representing, in most of cases, a waning host immune response to HBV infection that can result in HBV reactivation with varying consequences in morbidity56. Nine out of 11 inmates in our study showed an isolated anti-HBc pattern associated to HCV marker. The isolated anti-HBc pattern is particularly relevant in HIV/HCV co-infected patients and, most often, HCV is dominant, suppressing HBV replication57. When HCV is treated, its inhibitory effects on HBV replication is released possibly resulting in HBV reactivation. In fact, HBV reactivation has emerged in the era of DAA treatment of HCV infection. Due to this, screening for HBV should be performed also in prison prior to initiation of HCV therapy.
In our study, HBV sequencing was not attempted since all samples were found to be negative for HBV DNA. This was expected because HIV treatment includes inhibitors of reverse transcriptase that are also effective against the reverse transcriptase activity of the HBV polymerase.
Our data confirm the results of other studies that showed a high prevalence of HIV/HCV coinfection in inmates. HCV co-infection was significantly associated with the Italian nationality. In Italy, a high prevalence of HCV infection has been reported in drug users, both in the general and in the inmate populations58,59,60. In fact, we found a higher prevalence of this coinfection in inmates older than 45 years (74.2%), corresponding to inmates born in the years at major risk for drug abuse, one of the main transmission routes of HIV/HCV. The multivariate model showed that higher CD8 + T-cell counts, elderly age and drug abuse were associated with HIV infection alone, while nationality seemed not to be a risk factor for having co-infections. In agreement with our data, Schmidt et al.61 reported CD8 T-cell failure in HCV and HBV infections, due to T-cell exhaustion, deletion, and viral escape.
The prevalence of HBV and HCV infection markers in HIV-infected prisoners, although high, is lower than what we found in our previous study31. The greatest difference was observed considering the HCV infection that was present in 60.0% of inmates in this study, as compared to the 78.3% of the previous study. Similarly, 27.7% of inmates had HCV RNA, a much lower prevalence compared to the 65.2% found in our previous work. It must be specified that inmates enrolled in our previous work were not under anti-HCV DAA-based treatments31. Therefore, even if the detention centers involved in our two studies are different, we can speculate that the lower prevalence of HCV markers can be, at least partially, due to administration of DAAs against HCV, as observed in our present study and as already reported by others62,63. A recent study indicates that the prevalence of HCV markers among inmates is less than 20%, possibly due to the availability of DAAs64. However, other factors may also play a role, such as the improving strategies for the control of infectious diseases, and/or a different distribution of behaviors at-risk for HCV infection among the detention centers. More tailored studies are needed to evaluate the efficacy of DAAs drugs in reducing HCV infection prevalence in prison.
HCV genotype and subtype heterogeneity may be influenced by the route of infection and coinfection, regardless of the geographic variable65,66. In this work we found that the subtype 1a is the most frequent variant (66% prevalence), followed by subtypes 3a and 4d that are equally represented with a prevalence around 16.7%. In our previous study31, we found a similar HCV subtype distribution with the exception of the prevalence of HCV subtype 3a which was higher than in the present study. In addition, in both studies, we found a similar prevalence of the 4d subtype. This distribution of HCV subtypes is in line with the distribution observed in the population of drug addicts outside the prisons, both in Italy and in Europe58,67. However, in the general population, subtype 1b is predominant with a prevalence of more than 50%, followed by genotype 2 (35%), and by subtypes 1a and 3a that show a very low prevalence68.
Some limitations of our study should be addressed. Firstly, our study was based on voluntary enrollment and, therefore, we were unable to obtain an estimate of the percentage of HIV-positive people who were enrolled in the study, as compared to the total HIV-positive people in the 5 detention facilities. However, the prisons participating in the study were uniformly distributed in all the Italian territory.
Another limitation of our study is that HIV sequences were obtained from proviral DNA since the majority of the enrolled inmates had negative plasma viral load. In fact, the proviral DNA sequence can contain a variety of multiple DRMs, not present in plasma viral RNA69, which may thus reveal viral variants with mutations that are not actively expressed70. However, HIV DNA sequencing may provide a good method to gain unique preliminary insights into HIV‐1 subtype diversity or for an overall evaluation of HIV DRM in a population70. Data from previous studies have also shown that the proviral compartment can be reliably used for the investigation of DRMs in ART-naive patients71.
Finally in our study, we utilized Sanger sequencing as the standard HIV genotyping method. However, this methodology may not detect low–abundance HIV variants mutations, that are less represented within the viral pool. Although Next Generation Sequencing (NGS) is a more sensitive method capable of identifying minor resistant mutants within viral populations72,73, a high concordance exists between Sanger sequencing and NGS. Nevertheless, further investigation are needed for detection of low‐abundance HIV‐1 variants by NGS.
In conclusion, our data indicate the need for a systematic screening and monitoring both at the beginning of incarceration and during incarceration to limit the spread of HIV, HCV, and HBV.
Although public health interventions are underway, effective prevention programs should be implemented that can limit the spread and circulation of the different genetic forms of these viruses, in prisons and to the general population, as well as of variants with mutations that confer resistance to treatment.