key: cord-0324348-qmj9n3b3 authors: PISONI, A.; REYNAUD, E.; Douine, M.; Hureau, L.; ALCOCER CORDELLAT, C.; Schaub, R.; Poland, D.; Monkel, R.; LOMMEN, J.; YENKOYAN, K.; Nacher, M.; TUAILLON, E. title: Automatized and combined HIV, HBV, HCV, and syphilis testing among illegal gold miners in French Guiana using a standardized dried blood device date: 2022-05-02 journal: nan DOI: 10.1101/2022.05.02.22274368 sha: 20fb6545d7d55245fd1b1f7e1e3eb8574a5b4619 doc_id: 324348 cord_uid: qmj9n3b3 Abstract: Background: Blood spotted onto filter paper can be easy collected outside healthcare facilities and shipped to central laboratory for serological testing. However, dried blood testing generally request manual processing for elution. In this study, we used a standardized blood collection device combined with an automatized elution system to test illegal gold miners living in French Guiana for HIV, HBV, HCV and syphilis. Material and Methods: We included 378 participants in five resting sites of illegal gold mining. Serological status was determined by testing serum samples. Blood collected on the Ser-Col device (Labonovum), was eluted using an automated system (SCAUT Ser-Col automation, Blok System Supply) and an automatized analyzer (Alinity i, Abbott). Ser-Col results were compared to serum considered as the reference, and dried blood spot samples processed manually. Results: Two participants tested positive for HIV (0.5%), eight tested positive for hepatitis B surface antigen (HBsAg), four tested low positive for anti-HCV antibodies (1%) but negative for HCV RNA, 47 tested positive for syphilis (12.4%). We observed a full concordance of Ser-Col and DBS results for HIV diagnosis compared to serum results. Ser-Col and DBS samples tested positive in seven HBsAg carriers and negative for one participants having a low HBsAg level in serum (0.5 IU/mL). Three hundred and sixty-eight participants uninfected by HBV tested negative for HBsAg (99.5%). Two Ser-Col samples and two other DBS samples positive tested positive in HBV uninfected participants (false positive results with low S/CO indexes). All participants tested negative for HCV in Ser-Col and DBS samples, including in dried blood samples from the four participants tested low positive for HCV antibodies and HCV RNA negative in serum. The threshold of the treponemal assay was optimized for dried blood sample testing. Among syphilis seropositive participants, 35 (74.61%) tested positive for treponemal antibodies in Ser-Col and DBS samples. Among participants seronegative for syphilis, 326 (98.4%) and 325 (98.1%) tested negative in Ser-Col and DBS samples, respectively. Conclusion: The Ser-Col method allows to automatized dried blood testing of HIV, HBV, HCV and syphilis with performance comparable to DBS. Automated approaches to test capillary blood transported on dried blood devices may facilitate large scale surveys and improve testing of populations living in remote areas for infectious diseases. Reducing the burden of infectious diseases requires a global approach, without leaving underprivileged people aside. To date, access to reliable diagnosis of infectious diseases remains insufficient for a large part of the world's population. Migration, forced displacement of populations, weak infrastructures and distance from the health system are common unfavorable health factors. The lack of in vitro diagnostic tools contributes largely to the mortality and morbidity induced by infectious diseases in resource-limited countries [1] . Low number of laboratories and poor logistical infrastructure are a major problem in many of these countries. Laboratory equipment defined by WHO as essential for basic service readiness is also insufficient [2] . The weakness of human resources -health professionals and biologists -should also be underlined [3] . Capillary blood sampling performed outside healthcare facilities is one of the strategies to improve screening of hard-to-reach and key populations. DBS testing programs using dried blood spot (DBS) have been implemented for several years, targeting intravenous drug users [4] , sex workers [5] , homeless populations [6] , migrants and men who have sex with men [7] . Today, DBS sampling is considered by the WHO as a possible alternative to serum/plasma to improve access to HIV, HBV and HCV screening and therapeutic monitoring. However, DBS has several limitations such as a lack of standardization of blood sampling and elution, and the manual processing of DBS cards. These constrains restrict DBS set up in clinical laboratories. In French Guiana, about 10,000 illegal gold miners, mainly Brazilians (95%), work on more than 700 different mining sites located in the primary forest [8] . This neglected population presents a poor state of health [8] . People working on gold panning sites are particularly exposed to vector-borne diseases, including malaria [9] and leishmaniasis [8] . Data also suggest a higher prevalence of sexually transmitted infections HIV, hepatitis B (HBV) and syphilis than in other populations of French Guiana [10] . Having sex for presents or money is frequent in gold mining sites [10, 11] . According to a survey carried out in the city of Cayenne in French Guiana, transactional sex may contribute for 45% of HIV cases in men and 10.7% in women [12] . In a study carried out on the gold sites of French Guiana about 10% of women declared having already had a sex work activity [8] . In this study, we used an innovative blood collection device combined with an automatized elution system to test illegal gold miners living in French Guiana for HIV, HBV, HCV and syphilis. This study is part of the "SCAUT" study a EU-funded research projects (from finger to laboratory: personalized and automated serum collection for laboratory diagnostics). Participants were adults working in an illegal gold mining sites in French Guiana, (NCT03695770). Participants were included after having provided informed written consent. Testing for HIV, hepatitis B virus (HBV), hepatitis C virus (HCV) and syphilis were offered at the resting site less than seven days after leaving the mining sites. Samples were send by canoe or car from the resting site to the hospital center of Cayenne and then to the Montpellier University Hospital to be tested. Results were communicated within two weeks on each site during the post-test counselling. The processing of blood collected on the Ser-Col device (Labonovum, Netherlands) was performed using an automated system (SCAUT Ser-Col automation, Blok System Supply, Netherlands). The Ser-Col device contains a microfluidic paper separating plasma from whole blood. The automated system performed the elution from dried plasma contained in one centimeter of paper in 100µL of Ser-Col buffer. Blood spotted onto Whatman 903™ Protein Saver Card (Whatman GmbH, Dassel, Germany) was dried at ambient temperature for at least 24h. DBS processing and elution was manual. One punch 6 mm of diameter was eluted in 100µL of phosphate-buffered saline. HIV p24/antibodies, HBsAg, anti-HCV antibodies, and anti-treponemal antibodies were assessed using fully automated chemiluminescence immunoassays (Alinity i, Abbott, IL, USA). Sample tested positives for HIV were confirmed using a rapid confirmatory assay based on immune-chromatography and automated reading (Geenius™ HIV1/2 Confirmatory Assay, Bio-Rad, France). Serum samples positive for anti-treponemal antibodies were tested by a Rapid Plasma Reagin Assays (ASI RPR, Arlington Scientific Inc.). All assays were done in accordance with the manufacturer's instructions The HIV, HBV, HCV and syphilis status of participants was established by testing serum samples. Ser-Col results was compared to serum results considered as the reference. Ser-Col results were also compared to results obtained using the DBS considered as the reference sampling method using dried blood. Receiver operating characteristic curves (ROCs) were used to identify the positive threshold of All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2022. ; https://doi.org/10.1101/2022.05.02.22274368 doi: medRxiv preprint anti-treponemal antibodies in Ser-Col and DBS. Spearman's non-parametric test was used to assess correlation between results of anti-treponemal antibodies in Ser-Col and DBS, versus serum. Data results were analyzed using GraphPad Prism 9.3.1 (GraphPad Prism Software Inc.). We included 378 participants in five resting sites of illegal gold mining, 276 men and 102 women. The median (IQR) age was 39 years (31-48). We first tested participants for HIV, HBV, HCV and syphilis infections using serum to determine their infection status for these infections. Two participants tested positive for HIV. HIV infection was confirmed with the Geenius test. The pattern of antibodies showed IgG directed against gp160, gp41, p24 and p31 (low signal) of HIV-1 for the first subject and gp160, gp41, p24 with no p31 antibodies for the second one. We did not observe any reactivity for HIV-2 antigens (gp140, gp36). Two Ser-Col samples from HBsAg negative participants had signal over the cut-of and were considered as false positive. The S/CO were close to the positive threshold. All HCV seronegative participants were also found negative for anti-HCV antibodies using the Ser-Col device. The four participants with HCV Abs +/RNA-pattern were found negative using the Ser-Col device. All Ser-Col samples from participants seronegative for anti-treponeme antibodies in serum tested negative. 20 out of 47 (42.5%) participants tested positive for anti-treponemal antibodies in serum had S/CO results below the threshold of the All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2022. Our study shows the feasibility of serological screening for sexually transmitted infections in a vulnerable population living in remote areas using standardized dried blood device and a full automatized processing. The clinical performance of the assays using the Ser-Col device appeared as good as using DBS but overcomes some of the limitations related to DBS processing. This approach allows combined screening for HIV, HCV, HBV and syphilis infections on blood collected outside health care facilities in resting sites of gold mining. French Guiana has the highest prevalence of HIV and HBV among French regions [13, 14] . The HIV, HBV, HCV and syphilis prevalence observed in our study are consistent with those previously reported in this population [10] . With the exception of syphilis, the number of positive cases was relatively low, which leads to imprecise estimates of prevalence. These figures are close to HIV, HBV and HCV prevalence estimations in French Guiana. The prevalence of HIV has been estimated at 1.5% [13] , HBV 2.96% and HCV infection 0.67% [14] . A low proportion of persons living with HIV and viral hepatitis are unaware of their infection status in remote areas and vulnerable population [15] . It is of primary importance to increase the frequency of screening for HIV, HBV and HCV infections. Capillary blood All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2022. ; sampling and dried blood transportation could facilitate large scale health surveys. This type of population testing has been carried out on DBS, for example for viral hepatitis [16] or SARS-CoV-2 [17]. Guiana consist of migrants living far from transport networks and health infrastructures in a harsh tropical environment. In this study, sampling was done close to the field and in vitro diagnosis were performed centrally laboratory certified ISO 15189 for HIV, HBV, HCV and syphilis testing using a highthroughput serological platform. The sample transportation from the field to a central laboratory is facilitated when using dried blood because there is no risk of the biological fluid spilling and no risk of virus transmission. Serological and molecular tests on DBS have shown satisfactory analytical performance for HIV and viral hepatitis [18, 19] . The DBS is recommended by WHO for diagnosis of HIV and viral hepatitis B and C to improve access to in vitro diagnosis of populations with poor access to serum tests [20, 21] . Nevertheless, the DBS suffers from significant limitations that hamper its use, including the lack of standardization of the sample and the absence of automation of the pre-analytical steps: punching, transfer and elution. The manual processing of DBS is a source of error and limits its integration in medical laboratories. The Ser-Col device and its automated processing overcome these limitations. In our study, the screening on Ser-Col samples presents a high specificity, identifying HIV and HBV infections with exception of a weakly HBsAg positive sample. Two Ser-Col samples were false positive for HBsAg but with low S/CO values. The specificity for HCV appeared also high but the sensitivity could not be calculated since all participants tested seronegative for HCV RNA. The performance of the syphilis assay using the Ser-Col device is more uncertain since about a quarter of low serum positive subjects were not detected. These subjects had relatively weak positive results in serum suggesting old cured syphilis. However, a very recent infection would also be possible. Performance for syphilis screening was similar using the Ser-Col device and DBS. Even if the performance of screening for antitreponemal antibodies on Ser-Col or DBS is lower than serum, this method of blood sampling could be useful for syphilis testing in the field. A trade-off between the analytical performances of the test and its accessibility in the field should be considered to evaluate the benefit of in-vitro diagnosis methods in the real world, as underlined in the guidelines of WHO [20, 21] . Our study has several limitations. First, we did not evaluate loss of follow up before participants obtained the results. This is an important aspect of screening strategies. Compared to POC tests, laboratory analyzes whether carried out on serum or dried blood samples entail a longer delay before the result is returned and therefore a greater risk of patient loss to follow-up before the test results are given to the patient [22] . Another limitation is the low number of positive subjects for HIV, HBV All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2022. ; https://doi.org/10.1101/2022.05.02.22274368 doi: medRxiv preprint and HCV infections even though a large number of participants were tested. Finally, we only performed qualitative tests, whereas quantitative results may be important for diagnosis of infectious diseases. In conclusion, the Ser-Col device combined with an automated elution allows standardization and safety of the pre-analytical steps of analysis performed on dried blood samples. The clinical performances of the assays using the automated Ser-Col method appeared as good to those obtained with the DBS samples processed manually. Automated approaches to test capillary blood transported on dried blood devices are needed to enable testing of vulnerable population living outside healthcare facilities and facilitate large scale surveys assessing the burden of different infectious pathogens. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2022. ; (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2022. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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