Leishmaniainfantum are able to infect sandflies. Thus, individuals withcutaneous parasitic infections may act as reservoirs and allow interhumantransmission. Additionally, the skin may be responsible for reactivation ofthe disease after therapy. This study’s objective was to evaluate cutaneousparasitism in humans with VL in an American endemic area.  Visceral leishmaniasis (VL) represents a public health concern in severalareas of the world. In the American continent, VL transmission is typicallyzoonotic, but humans with active VL caused by  A cross-sectional hospital-based study was conducted in northeast Brazilfrom October 2016 to April 2017. Biopsies of healthy skin for histopathologyand immunohistochemistry were performed prior to treatment in all studypatients.  Twenty-two patients between the ages of five months to 78 years wereincluded in the study. Seven patients (31.8%) tested positive for HIV. Onlyone patient had cutaneous parasitism, as confirmed by immunohistochemistryprior to treatment. Parasitism was not detected after treatment.  Cutaneous parasitism in the healthy skin of humans with visceralleishmaniasis, although unusual, may be a source of infection forphlebotomine sandflies.  Leishmaniadonovani, while in the American continent and Southwestern Europe themain infectious agent is L. infantum. The transmission can beanthroponotic, zoonotic, or both, depending on the endemic areaL. infantum in the American continentis Lutzomyia longipalpis,,,Visceral Leishmaniasis (VL) is an endemic disease undergoing geographical expansionand represents a public health concern in many regions of the world,L.infantumPrograms focusing on disease control usually include vector control, culling sickdogs and early treatment of affected humanset al.Silva L. donovani carriers, foundthat the skin, more often than the blood, was the source of parasites. The reasonfor this was that although the volume of the micro-biopsies was ten times smallerthan finger prick blood samples, the rates of detection ofLeishmania DNA in micro-biopsies was significantly higher,,,A study with minimally invasive micro-biopsies, which were aimed at identifyingasymptomatic and potentially infectious L. infantum.Considering the possible epidemiological implications of cutaneous parasitism inhumans, which may facilitate its transmission and be responsible for post-therapyreactivation of the disease, we conducted a study in search of parasites living onthe healthy skin of people with American visceral leishmaniasis in an area endemicfor A cross-sectional study was performed with the selection of participants with adiagnosis of Visceral Leishmaniasis. Participants were of both genders and allages and were recruited in Teresina, Northeast Brazil.®) or polymerase chain reaction (PCR).Pregnant or breastfeeding women, patients with leishmaniasis-like skin wounds,patients with an absence of healthy skin regions on the forearms due to otherskin conditions such as ichthyoses and patients who had received anyanti-Leishmania drug in the last 6 months were excludedfrom the study.A patient was considered to have VL if they presented with the typical symptomsof fever, wasting, paleness and hepatosplenomegaly with confirmation by at leastone of the following tests: direct visualization of amastigotes in tissues,visualization of promastigote forms in culture, immunochromatographic test withrK39 antigen  for histological examination using light microscopy, andthe immunohistochemical slides were prepared.et alet alet alThe immunohistochemical reaction for quantifying parasitism was performed usingthe Novolink® kit (Novocastra® RE7260-K), according to the protocol described byMoreira Statistical analysis was performed using Stata/SE® 10.0 for Windows .This study was approved by the Ethics Committee in Research of the FederalUniversity of Piauí . All participants signed Free andInformed Consent Forms or the Free and Informed Assent Forms.Twenty-two patients were included in the study. From these, 18 (81.8%) patients werediagnosed using the parasitological method (microscopy or culture) and four (18.2%)patients were diagnosed using only serological reactivity. The ages of the patientsranged from 5 months to 78 years of age (median: 30 years). Four patients (18.2%)were less than 2 years old and 16 (72.7%) were older than 18 years. In the children(under 12 years old), the number of male and female patients was equal. However87.5% of the adults were male. The majority of the patients came from medium-sizedcities in Piauí and Maranhão states and had low education levels .3 . All of these patients were known to be HIV-infected priorto the diagnosis of VL, but two of them had not yet started anti-retroviraltherapy.Seven patients (31.8%) were co-infected with HIV. Their T-CD4+ lymphocyte countranged from 9 to 677 cells/mmLeishmania co-infected patient was classified as highcutaneous parasitism (more than 50 amastigotes / field) HIV// field) .3 (CD4+/CD8+=0.09), which was lowerthan his count at HIV diagnosis .This patient was a 78 year-old man who was diagnosed with VL in January 2017. Hereported pallor, apathy, increased abdominal volume, and coughing for 90 days, buthad no referred fever, weight loss, edema, or bleeding disorder. He was diagnosedwith immunodeficiency syndrome in September 2015 and had been on a highly activeantiretroviral therapy since. His viral load was below the minimum detection limitsince November 2016, but his T-CD4+ lymphocyte count performed before the diagnosisof VL, in August 2016, was 53 cells/mmHe was in good overall health, had a preserved nutritional state, and had lightpallor. His right retroauricular and cervical lymph nodes were approximately 0.5 cmin diameter, and splenomegaly and hepatomegaly were detected. There were no othersigns or symptoms such as edema, bleeding, or jaundice.3, neutrophil count was 1343/mm3, platelet countwas 147,000/mm3, serum albumin was 2.6 g/dL, globulin was 4.4 g/dL, serumcreatinine was 0.9 mg/dL and urea was 31 mg/dL.Laboratory tests revealed his serum hemoglobin was 9.24 g/dL, leukocyte count was4630/mmLeishmania staining (The patient presented a positive rapid immunochromatographic test (rK39 antigen), butboth direct amastigote screening and visualization of promastigotes in the bonemarrow NNN medium were positive. He received liposomal amphotericin B (4mg/kg/day)for 14 days with a satisfactory response. Skin biopsies performed 72 hours after theend of treatment in the same forearm, adjacent to the pre-treatment biopsy, werenegative for staining .3) and viral load .Secondary prophylaxis with liposomal amphotericin B every 14 days was prescribed forseven months. Laboratory post-treatment tests done in May 2017, three months afterVL treatment, revealed a slight and non-significant improvement in T-CD4+ . In Europe, the interhuman transmission of theparasite through shared contaminated syringes between individuals co-infected withHIV and Leishmania infantum has been described as one of the mainforms of transmission in the regionThe anthroponotic cycle of Leishmania-infected dogs as a diseasecontrol program and the demonstration by xenodiagnostic studies that people withactive VL, especially when co-infected with HIVL. infantum, strengthened thehypothesis that humans could be parasitic reservoirs,Leishmania infantum in endemic areas of VL,The ineffectiveness of culling Leishmania, representing one of the highest prevalence ofreported asymptomatic infectionsIn Teresina, Piauí, asymptomatic individuals living in households where a recent caseof active VL was diagnosed presented 71% more skin reactivity toOther studies have already evaluated the infectivity of animals by sandflies. Deane& DeaneL. infantum-infected dogs have demonstratedlasting parasitemia for over six months and the tendency of infections to remain onthe skin around the site of the infectious sandfly biteRecent experiments in Leishmania infantum inhumans with VL and apparently healthy skin have been previously described in Brazil,but there have not been any new publications on this topic in the last 20 years. InPará, a study found rare parasites on the smear biopsy of healthy skin in one of thefour individuals studied (25%)Previous cases of cutaneous parasitism by In Ceará, Deane & DeaneIn 1962, in a series of 43 patients with active VL and macroscopically normal skin,Deane & Deane found seven individuals with cutaneous parasitism (16.3%), one ofwhich was very prominent. They showed that all patients whose skin had beenparasitized during the visceral involvement were cured when re-examined aftertreatmentAlso in this state (CE), the researchers did not find any amastigote in tissuesamples from biopsies of the subscapular paraspinal region of healthy skin from 27individuals investigated, but identified promastigote forms in skin culture by meansof monoclonal antibodies and enzymatic electrophoresis in seven (38.9%) of 18patients studiedLeishmaniain the skin of one of them (14.3%). In Minas Gerais, investigators obtained anegative result for material obtained by scarification of the skin of all 23 studiedpatientsPrata & PivaLu. longipalpis is more attracted to humans than canine hosts,L. infantumshould not be neglected. Although asymptomatic carriers of L.infantum were less infectious for sandflies than individuals withactive VL, their vast numbers in an endemic city would constitute a surprisinglylarge reservoir of parasitesL. infantum is greater than was foundin this study.The population enrolled in this study differs from the general population of peoplewith VL due to the predominance of adults and HIV-infected men. The small sample ofpatients that could be included in the study restricts the power of generalizationof the data. It is also possible that the proportion of people with VL presentingcutaneous parasitism due to Lutzomyalongipalpis in Brazil can transmit infections to other individualssystematically and effectively. In conclusion, amastigotes were viewed in histopathological and immunohistochemicalexamination on the skin of a patient with VL who was co-infected with HIV. Furtherstudies are needed to prove whether humans infected with


Point-of-care (POC) technologies in resource-limited settings can circumvent challenges of centralized laboratory testing, improving clinical management. However, higher device costs and uncertain indications for use have inhibited scaling up POC modalities. To address this gap, we investigated the feasibility and cost of targeted near-POC viral load (VL) testing in 2 large HIV clinics in Lilongwe, Malawi.VL testing using GeneXpert was targeted for patients suspected of treatment failure or returning to care after a previously elevated VL (>1000 copies/mL). Descriptive analysis of retrospective clinical and cost data is presented.Two thousand eight hundred thirteen near-POC VL tests were conducted. One thousand five hundred eleven (54%) tests were for patients for whom results and reason for the test were documented: 57% (794/1389) of tests were to confirm a previously high VL, and 33% (462/1389) were due to clinical indications. Sixty-one percent (926/1511) of patients had a high VL, of whom 78% (719/926) had a recorded clinical action: 77% (557/719) switched to second line antiretroviral therapy, and 15% (194/719) were referred for intensive adherence counseling. Eighty-two percent (567/687) of patients received a clinical action on the same day as testing. The “all-in” cost was $33.71 for a valid POC VL test, compared with an international benchmark for a centralized VL test of $28.62.Targeted, near-POC VL testing was feasible and consistently enabled prompt clinical action. The difference between the “all-in” cost of near-POC VL and centralized testing of $5.09 could be further reduced in an optimized national program by combining targeted near-POC testing and centralized testing. Ninety-one percent of PLHIV on ART in South Africa had at least one VL performed in 2015,7 whereas only 53% of PLHIV on ART in Malawi met their monitoring milestone by the end of 2016.8 Enabling routine access to VL testing to ensure viral suppression is necessary to reduce the HIV burden and associated morbidity and mortality,10 but testing coverage is only impactful if results are used appropriately to differentiate patients to the model of care that meets their condition.11 In many countries, PLHIV on ART for at least 1 year with a suppressed VL (<1000 copies/mL) are considered stable and eligible for a less intense model of care, which could include decreased dispensing frequency or participation in a community-based adherence club. Conversely, PLHIV with an unsuppressed VL (>1000 copies/mL) require more intensified care that includes adherence counseling and subsequent regimen change if the VL does not resuppress with time.12 Despite these recommendations, many stable patients unnecessarily remain in more intense models of care,13 and the rates of ART regimen switch to second line do not correspond well to the estimated rates of unsuppressed VL, despite the known risk of poor outcomes.15The WHO guidelines recommend VL testing at 6 and 12 months after ART initiation, and then annually thereafter.16 These tests can be operated outside of conventional laboratory settings by laboratory or nonlaboratory personnel and closer to locations where patients are receiving care, either in the facility or the community. POC tests range from device-free rapid HIV or pregnancy tests to device-based near-POC technologies, such as the GeneXpert . By enabling testing on-site, near-POC technologies can remove the need for specimen transport to centralized laboratories that lead to long TAT between sample collection and result availability—nearly 7 weeks in some settings.7 However, decentralized testing brings additional challenges, including the need for decentralized quality control, equipment maintenance, supply chain, and waste management.17 In addition, there is a perception of increased expense because the cost for POC proprietary commodities and devices may be higher than those for centralized laboratory machines while the throughput may be substantially lower.18One proven approach to increase access to testing services and rates of clinical action is point-of-care (POC) technologies—tests that have rapid turnaround times (TAT) that enable result availability and clinical action in the same visit.19 Similarly, POC testing for early infant diagnosis (EID) has been shown to increase rates of ART initiation for HIV-positive infants across several countries in sub-Saharan Africa.22 Evidence is emerging on how near-POC testing could benefit VL testing programs. A recently completed study in South Africa replaced centralized testing with near-POC VL within a package of services for adult clients newly initiating efavirenz-based ART. The combination intervention increased the proportion of patients achieving viral suppression, retained in care, and referred into community-based care.23 In addition, a recent project in Malawi demonstrated that routine POC VL testing decreased overall TAT, time to intensified adherence counseling, and a shorter time to switch to second line for patients with a VL >1000 copies.24Despite these challenges, the evidence that near-POC testing improves the management of PLHIV continues to grow. Near-POC CD4 testing has been successfully rolled out in resource-limited settings for the past decade, resulting in increased ART initiations and retention in care.25 and nearly 70% are receiving 3 monthly refills13 so typically receive their VL result on their next visit or are called by phone if the test result is unsuppressed.In the resource-limited setting of Malawi, we hypothesized that an optimal testing program for nonpregnant or breastfeeding patients would combine (1) high-volume centralized testing for most patients requiring routine VL assessments with (2) targeted near-POC VL testing on low throughput machines for patients whose management requires the same day result, specifically patients suspected of treatment failure or returning to care after a previously elevated VL (>1000 copies/mL). The strategy was predicated on the evidence that most patients on ART are effectively managed through centralized VL testing, despite extended TAT for results. National data show that 91% of PLHIV on ART are virally suppressedTo improve the management of those who were at high risk for being unsuppressed, we provided targeted near-POC VL testing to eligible patients at 2 high-volume facilities in Lilongwe and assessed the process and cost implications in a programmatic setting.25 During the time of this project, the country guidelines recommended VL monitoring at 6 months and at 2 years after ART initiation and every 24 months thereafter, which differs from the WHO recommendations.12 The decision to reduce the frequency of treatment monitoring in Malawi was driven by a desire to maximize coverage while minimizing costs.26 This guidance was changed to annual VL monitoring in 2019. In addition, the national ART guidelines recommend regimens based on 2 nucleoside reverse transcriptase inhibitors (NRTIs), combined with a non-NRTI, mainly efavirenz as first-line ART. As second-line ART, a combination of 2 NRTIs with ritonavir-boosted protease inhibitors are recommended. Confirmed treatment failure occurs after a targeted or repeat VL is >1000 copies/mL, with good adherence in the previous 3 months. After a switch to second-line or third-line ART, patients reset their clock for routine VL monitoring.12The prevalence of HIV infection among adults aged 15–64 years in Malawi is estimated at 10.6%, and 67.6% of all PLHIV are virally suppressed. The Lighthouse (LH) Trust is a public trust and a WHO-recognized Center of Excellence for integrated HIV prevention, treatment, care, and support partly funded through the Malawi Ministry of Health but receives significant supplemental funding from the President’s Emergency Plan for AIDS Relief through the Centers for Disease Control and Prevention. LH operates 2 large urban patient-centered HIV clinics in Lilongwe: one at the Kamuzu Central Hospital (LH-KCH) with a current cohort of 10,729 patients, and the second called the Martin Preuss Center (LH-MPC) with 21,701 patients.From January to December 2017, the GeneXpert HIV-1 VL assay was available at the 2 LH facilities for nonpregnant adult and adolescent clients who met the following criteria: clinically suspected of ART failure or advanced HIV disease, such as those with WHO stage 3 and 4 diseases or those with low CD4 counts; in need of follow-up because of previous high VL; and switched to second-line ART 6 months previously, regardless of clinical status. Clinicians were trained on testing guidelines for VL monitoring and failure management in January 2017.One GeneXpert platform with 4 modules was placed in each clinic and was primarily operated by nurses. A laboratory technologist also operated the GeneXpert platform at LH-KCH and assisted with supply chain management and technical support for both sites. At LH-KCH, most tests were for VL , and at LH-MPC, the majority of tests were for VL .27 is approved by the European Union and WHO Prequalification Program. The GeneXpert technology enables the automated quantitation of HIV VL, requiring 1 mL of plasma in 10427 minutes. The assay has been validated in lower resourced clinical settings and detects virological failure (>1000 copies per mL) with 94% sensitivity and 99% specificity.29The Xpert HIV-1 VL test, which is run on the GeneXpert platform Near-POC VL testing and results data were extracted from the POC laboratory registers and POC VL request forms. LH clinics serve both patients within their primary cohort on-site and referral patients. VL results for referred patients are returned directly back to the patient without maintaining documentation at LH facilities; therefore, the data analysis was restricted to the primary cohort. Demographic and clinical data were abstracted from the electronic medical record system. Process outcomes from the targeted near-POC testing included accuracy of pretest risk assessment of treatment failure, time to clinical action, and costs to implement the targeted near-POC VL testing. Data were analyzed using Stata 14.2.As the targeted POC VL testing was implemented concurrently with an aggressive process to ensure all LH patients received at least one VL test, an excess number of routine centralized VL tests among LH patients occurred during the study period. Comparisons of the targeted approach with a routine testing approach within the same cohort were therefore not possible.P > 0.5). There were also no associations between categorical variables and missingness.Missing values were evaluated using Little's missing completely at random test and did not show any systematic patterns in missing data . Where exact figures were not available, estimates from similar contexts were used. The study reported costs in the United States Dollar.The following equipment was purchased for the purpose of this study: Cepheid GeneXpert IV device, service contract, centrifuge, and uninterrupted power supply. The total cost to set up a testing site for laboratory equipment was $22,316, with the majority $17,000 comprised the POC GeneXpert device. During the study, testing activities were conducted by 3 cadres of health workers who worked full time, senior nurses, junior nurses, and laboratory technicians. The model was populated with corresponding salary information, inclusive of benefits, for each cadre of health worker in a top-down costing approach. The materials required to run each test were GeneXpert HIV VL test cartridges; gloves, syringes, and EDTA tubes for specimen collection; and paper and toner for printing results. The prices for gloves, syringes, and EDTA tubes were reported from invoices from the study. The GeneXpert HIV VL cartridge was benchmarked to manufacturer's ex-works pricing. Toner and paper were assumed to be purchased at typical benchmark prices. There were additional costs incurred for freight, clearing and duty to import the test cartridges, consumables, and equipment; there was one initial training and one refresher training  conducted during the study period; waste management costs were incurred for the disposal of GeneXpert HIV VL, which contains guanidinium thiocyanate, a toxic substance. It was not possible to collect study-specific overhead data, so a fixed percentage of 15% was used across all other components of the all-in cost per test. There were assumed to be 20 working days in a month after accounting for weekends and holidays.Targeted POC VL testing was provided for as routine care and followed the national guideline protocols; therefore, no informed consent was required. The protocol was approved by the Human Research Ethics Committee of the National Health Science Research Committee of Malawi.During the study period, a total of 2813 VL tests were run on the GeneXperts at the 2 study sites, with an error rate of 4% (59/1364) at LH-KCH and 6% (87/1449) at LH-MPC.P > 0.5) (Table Of the 2813 POC VL tests conducted across both LH-KCH (n = 1364) and LH-MPC (n = 1449), 54% (1511) tests were for patients who received their primary HIV care at LH and thus had documented results, with the remainder testing volume for referred patients. Fifty-seven percent (n = 794/1389) of LH primary patients had a previous high VL, 33% (462/1389) patients had a clinical indication of advanced HIV disease, 3.6% (n = 50/1389) patients were being monitored after switching to second line, 6% (n = 83/1389) patients for other reasons, and 8% (n = 122/1511) patients were missing a reason for testing on their sample referral form and assumed to be missing completely at random  patients were women with a mean age of 32 years. Seventy-eight percent (719/926) patients had a documented clinical action captured in the register: 77% (557/719) patients were switched to second line, whereas 15% (104/719) of patients were referred to intensified adherence counseling  of the cost per test. The remaining 53% of the test cost comprised staff, equipment, other, and overhead. The other category includes freight, clearing and duty, initial training of device users, refresher trainings, and waste management, the largest of which is clearing and duty at $6.35 per test. The scale-up costs assumes 75% device utilization, whereas including estimates for costs not incurred in the study, such as external quality assurance and connectivity to enable remote monitoring of device performance. A sensitivity analysis shows that in the best-case scenario, the all-in cost per patient result could be as low as $21.03 and in a worst-case scenario as high as $60.15 per result. The primary driver of lower costs would be full utilization of the devices, running more tests than were observed during the study period. The main driver of increased cost in the worst-case scenario are primarily due to higher human resources cost from health workers not multitasking while assays are in the analyzer for 104 minutes and lower device utilization.The implementation of near-POC testing for targeted VL monitoring proved feasible at 2 high-volume urban clinics in Malawi. Near-POC VL testing enabled prompt and effective clinical management for nonpregnant adult and adolescent patients on ART who were suspected of treatment failure. Among patients deemed eligible for targeted testing, clinicians documented that 57% had a previously elevated VL and 33% had clinical signs or symptoms consistent with advanced disease or a serious coinfection such as tuberculosis. Sixty-one percent of patients tested using GeneXpert were found to have an elevated VL that required clinical attention, adherence counseling, or switch to second line. Across both clinics, 78% of patients with an elevated VL result had a documented clinical action, 82% of which happened on the same day as testing and 92% within 2 days of testing.30 in the same year 6% of VL tests performed nationally were >1000 copies/mL.31 Although not a one-to-one comparison and not all patients with an elevated VL should be switched to second line, the proportion of patients on second line is much lower than the proportion who have an elevated VL. In our study using GeneXpert for targeted POC VL testing, most patients with an elevated VL received a clinical action; 60% patients were switched to second line and 11% patients initiated adherence counseling, indicating the impact that rapid, immediate testing can have on effective clinical decision making in a nonresearch setting. Third, the same day clinical action not only benefits patients' health but also may reduce their out-of-pocket cost of returning to collect their test results at subsequent visits.These findings illustrate several potential advantages of near-POC VL testing, particularly prompt data-driven clinical action. First, had patients perceived to be failing been presumptively switched to second line without the use of VL testing up to 39% (585/1511) may have been unnecessarily switched, indicating that the access to on-site VL testing enables more appropriate clinical action. Second, although only 2% of ART patients were receiving a second-line (protease inhibitor based) regimen at the end of 2017 throughout Malawi,Assuming 240 working days per year, the average daily number of VL tests run was 5.7 at LH-KCH and 6.0 at LH-MPC; the average daily utilization during the study period was 6 tests, compared with a total theoretical throughput of 28 per day based on the 11-hour workday 5 days per week observed at the study sites. Comparing the cohort sizes, at LH-KCH, 1 near-POC VL was performed per 7.9 ART patients per year, and at LH-MPC, the ratio was 1 near-POC VL per 15.0 ART patients per year. With targeted testing in our cohort in high-volume centers of excellence, we reached only 32% utilization of the testing capacity of the GeneXpert devices, leaving plenty of capacity for other types of urgent near-POC testing that could be performed.The cost per test was $33.71 and cost per result, accounting for test errors, increased to $35.46. With 68% remaining capacity, economies of scale dictate the cost per test can continue to be reduced.32 or children and adolescents. Finally, near-POC VL testing would be useful in a remote or difficult-to-access location, which may not be part of routine sample transportation routes.In this study, the POC VL was mainly targeted to patients who had either a previous elevated VL, were suspected of advanced disease, or required a follow-up VL after switching to second line. In addition to these priority populations, the POC VL can be further expanded to other targeted populations, for example, pregnant and breastfeeding women33 Indeed, a large fleet of GeneXpert devices already exists in many resource-limited settings mostly for TB testing,35 which could be leveraged for targeted VL testing to benefit more patients living with HIV on ART, without requiring large capital investments in new devices.Several test types can be performed on the GeneXpert, such as EID, tuberculosis (TB), and human papillomavirus (HPV). Multidisease testing on GeneXpert across HIV VL, EID, and TB has been shown feasible in Zimbabwe.There are several limitations of our study, including that this is an observational study with no concurrent comparator in the project facilities. The implementation of the study in routine settings was intentional for purposes of assessing feasibility and process outcomes; however, record keeping was not optimal; we were only able to collect data for 54% of all POC VL test of interest. There is no reason to assume that the patients without test process documentation were systematically different from those with documentation. In addition, during the time of implementation, patient files without recent VL tests were actively flagged for testing, which likely led to overall increases in testing coverage during the study period, but may have not influenced testing among the targeted population substantially.The most encouraging findings of this intervention were the reduced time between VL sample collection and management actions for patients eligible for a targeted near-POC VL, with most clinical decisions occurring on the same or next day as sample collection as well as the very high proportion of at-risk clients who had a documented clinical action after their VL testing. Even at very high-volume centers of excellence, the less than 10 at-risk patients were identified each day—well within the testing capacity of a 4 module GeneXpert.This study shows the potential benefit of providing targeted near-POC VL technology for people in need of urgent clinical management, whereas most patients who require more routine clinical decisions receive centralized testing. Because of economies of scale, centralized VL testing is lower cost and may provide more consistent quality results and fewer operational challenges. It offers a valuable option for VL testing for most people on ART in lower resource settings who are stable and virally suppressed. Patients at risk of advanced disease, however, can feasibly be provided with and benefit from access to near-POC VL testing. The higher costs and facility resource required by near-POC testing are minimized by only using the intervention for the patients who will most benefit from prompt clinical action, including appropriate switch to a virally suppressing regimen. These costs can be further amortized by expanding the eligibility criteria for near-POC VL or using GeneXpert machines for additional purposes such as TB diagnoses. Therefore, targeted near-POC VL testing for the facilitation of high-quality patient care was both affordable and feasible. Further evaluation of how best to optimize use of near-POC VL with centralized VL testing, especially in the context of a multidisease testing program, is warranted.


Extreme weather events (EWE) are expected to increase as climate change intensifies, leaving coastal regions exposed to higher risks. South Florida has the highest HIV infection rate in the United States, and disruptions in clinic utilization due to extreme weather conditions could affect adherence to treatment and increase community transmission. The objective of this study was to identify the association between EWE and HIV-clinic attendance rates at a large academic medical system serving the Miami-Dade communities. The following methods were utilized: (1) Extreme heat index (EHI) and extreme precipitation (EP) were identified using daily observations from 1990–2019 that were collected at the Miami International Airport weather station located 3.6 miles from the studied HIV clinics. Data on hurricanes, coastal storms and flooding were collected from the National Oceanic and Atmospheric Administration Storms Database (NOAA) for Miami-Dade County. (2) An all-HIV clinic registry identified scheduled daily visits during the study period (hurricane seasons from 2017–2019). (3) Daily weather data were linked to the all-HIV clinic registry, where patients’ ‘no-show’ status was the variable of interest. (4) A time-stratified, case crossover model was used to estimate the relative risk of no-show on days with a high heat index, precipitation, and/or an extreme natural event. A total of 26,444 scheduled visits were analyzed during the 383-day study period. A steady increase in the relative risk of ‘no-show’ was observed in successive categories, with a 14% increase observed on days when the heat index was extreme compared to days with a relatively low EHI, 13% on days with EP compared to days with no EP, and 10% higher on days with a reported extreme weather event compared to days without such incident. This study represents a novel approach to improving local understanding of the impacts of EWE on the HIV-population’s utilization of healthcare, particularly when the frequency and intensity of EWE is expected to increase and disproportionately affect vulnerable populations. More studies are needed to understand the impact of EWE on routine outpatient settings. Science predicts an increase of 1.5 °C will lead to devastating extreme weather events (EWE), such as heavy rainfall or a high heat index , with coAll persons within a community are vulnerable to EWE, though the level of vulnerability is greater among those already vulnerable with regards to co-occurring health conditions. Another consideration is the disruption of access to treatment for those with infectious diseases. Metropolitan Miami (Miami) has the highest national average of HIV transmission in the United States (US), and adherence to both medical appointments and treatment is crucial to improve health outcomes in reducing the risk of transmission in the community .This study builds on previous research ,10,11, iIn order to derive locally relevant weather information, we first calculated the distribution of extreme precipitation (EP) and extreme heat index (EHI)  using daIn Miami-Dade County (MDC), the care of approximately 10% of the population of PLWH   is conceOur institution’s HIV services provide integral medical care to all adult PLWH through six clinics. An all-HIV clinic registry database was created from a large county hospital in South Florida, in collaboration with the department data manager. Clinics’ scheduled data were de-identified and organized into the following categories: attended, not attended, re-scheduled, lost, canceled, and no-show. ‘No-show’ was the variable of interest, as these patients failed to notify the HIV clinic about their appointment re-schedule or cancellation. This database runs during hurricane season (from May to October) from the years 2017–2019 during clinic operating hours from 8:00 am to 5:00 pm, excluding weekends, holidays, and any other non-operating clinic days .We used a time-stratified case crossover approach to assess how clinic visits were impacted on days with adverse weather . We controlled for the day of the week in a given month and compared the rate of no-show on days with adverse conditions to days with no adverse conditions. We used a Conditional Poisson regression model  to estimBased on 30 years of data, the EWE values were as follows: the EHI values corresponding to any value above the 90th percentile was 100.83 °F A,B; the There were 26,444 scheduled visits by the 383th day of the study period, equivalent to 26,444 scheduled visits and an average of 69 scheduled visits per day. From the 12,679 (100%) that had not attended their appointment, the no-show subset was 5328 (42%) .There were 111 (29%) days with an extreme heat index in the study period, equivalent to 7548 scheduled visits to an HIV clinic A, and 15The results from the regression model with ‘no-show’ as the dependent variable are presented in To the authors’ knowledge, this is the first study ever published demonstrating an association between extreme weather events  with lower outpatient healthcare visits. Little is known about what climate change may mean for access to healthcare in general. This study serves as a starting point to measure the expected subtle but progressive impact of EWE on healthcare utilization, particularly in outpatient clinic settings. The study focus was persons with HIV (a chronic and infectious condition requiring regular care and treatment) who were followed in Metropolitan Miami—the epicenter of new HIV infections in the US. Among the vulnerable groups of persons in the setting of climate change and its consequences are those with immunodeficiency, including those with HIV infection . In addiPLWH are a particularly vulnerable group in this context. The primary and secondary prevention of HIV represents one of the biggest challenges in this century worldwide . In the Patients not adherent to medical appointments are more likely to not adhere to their medications, thereby increasing the risk of transmission of HIV to sexual partners (men and women) and unborn babies (reproductive-aged women). From a clinic–policy perspective, for example, a systematic process evaluating weather and predicted weather or clinic operations such as extended hours or weekend dates could be modified to minimize clinic attendance disruption while mitigating potential exposure to an EWE. The use of technology could alleviate the burden of health systems; for example, through the implementation of tele-medicine, facilitating a home care discussion. These options, however, would need careful consideration to balance the availability of resources.The recent US National Climate Assessment highlights the increase in climate risk that communities in the Southeastern regions face from extreme weather, including extreme heat and extreme flooding. These climate risks pose new challenges to patients seeking routine healthcare, as well as for professionals providing these services . As a waHealth risk assessments like these will help identify a range of adverse weather conditions, which could inform the calibration of health-weather alerts. For example, we observed a statistically significant decline in clinic visits on days below the threshold of excessive heat warnings issued by the National Weather Service for the Miami region. We need to factor in the frequency and intensity of EWE, which could make the public health crisis worse. Analyses like ours can help start a dialogue among critical stakeholders to re-plan their HIV population’s adherence to treatment, reassess current efforts on extreme weather days, and train their providers about the impact of climate change on PLWH. This study strength resides in the link between climate and health with a methodology to derive weather extremes and test their associations with healthcare utilization patterns by geographic location. The implemented methods could be replicated in different geographic locations and could be extended to clinical service areas, other than HIV clinics at one institution.As an observational, descriptive, ecological study, this project has the strengths and weaknesses inherent in the design, including the ecological fallacy—the observed association between EWE and healthcare utilization by PLWH does not necessarily represent the association that exists at the individual level of someone living with HIV in Miami. There are several limitations that must be noted; first, because of limitations of the health data available, we were not able to control for important patient-level characteristics that can affect clinic visits. While the results of the impact of extreme weather on clinic visits are novel, they only partially explain the observed fluctuations in clinic visits. No causality can be established between the EWE and healthcare utilization. Precipitation data focuses on rainfall measurements during working hours, and does not include precipitation outside of working hours or daily precipitation data. The latter represents a limitation to this study, since flooding was not being considered.As the effects of climate change intensify, there is an increased need to understand how extreme weather patterns affect healthcare utilization by geographic location, given the potential for the disruption of medical treatment for individuals with the greatest need—patients reliant on treatment for their health conditions and persons with infectious diseases. This is the first study documenting a weather methodology in relation to healthcare utilization patterns overall, which was detailed within PLWH—a group dependent on treatment for their own health outcomes and their ability to avoid the further spread of HIV to others.From this study, we were able to derive a local definition for both an EHI and EDP, and a methodology to be able to replicate this approach at any location’s EWE with any patient clinic registry, given that weather data are publicly available. Our data have demonstrated an association between EWEs  with the healthcare utilization of PLWH. Even without considering weather events, these data highlight the challenges in terms of adherence to care in the study population. Large clinics with irregular attendance patterns may want to assess clinic attendance and explore the factors that may be driving increasing rates of patients not attending clinics, with the goal of improving healthcare utilization and patient health outcomes.Our findings demonstrate a significant inverse association between EHI and EP and clinic attendance. More studies are needed to identify the subtle but progressive influence of climate on health. Solutions are needed to provide care for PLWH and determine how to mitigate these potential effects through public health capacity building and collective resiliency.


A functional Human immunodeficiency Virus (HIV) cure has been proposed as an alternative to antiretroviral treatment for life, and therapeutic vaccines represent one of the most promising approaches. The goal of therapeutic vaccination is to augment virus-specific immune responses that have an impact on HIV viral load dynamics. To date, the agreed feature to evaluate the effects of these therapeutic interventions is analytical antiretroviral treatment interruption (ATI), at least until we find a reliable biomarker that can predict viral control. Different host, immunologic, and virologic markers have been proposed as predictors of viral control during ATI after therapeutic interventions. This review describes the relevance of ATI and the different surrogate markers of virological control assessed in HIV therapeutic vaccine clinical trials. Antiretroviral therapy (ART) is effective in maintaining viral suppression and reducing morbidity and mortality in patients with Human Immunodeficiency Virus (HIV) infection. Nevertheless, some patients have adherence problems with long-term ART, especially due to its adverse effects, leading to an increased risk of developing drug resistances. In addition, ART is a life-long therapy because the virus persists indefinitely in latent reservoirs, and there are currently no effective strategies to eliminate them . There aWhen ART combinations first came into use, there was uncertainty regarding how to balance adverse effects with the benefits of such treatment. This uncertainty created disagreement among experts and thus guidelines were constantly changing as to when HIV-infected individuals should start ART and whether ART should be taken continuously or intermittently. To address this issue, trials involving structured treatment interruptions (STI) were performed to outweigh the benefits and risks. STI were generally long interruptions and reintroducing ART was set to low levels of CD4+ T cells (mainly <250 cells/μL). It was first hypothesized that treatment interruptions could decrease ART toxicity and potentiate immunological response to HIV. However, these trials demonstrated a considerable increase in morbidity, mortality, and disease progression, and an increased risk of HIV transmission. Therefore, the initial hypothesis was rejected, and STI were no longer standard of care ,7. The oThere are two main strategies that can be used for ATI. The first one is extended interruption, which is a fixed period of treatment interruption, normally between 12 or 24 weeks, with the goal of measuring how effectively VL is controlled once it declines to a steady state, referred to as the set point. Viral set point is one of the most appropriate virological outcome measures to assess immunological interventions aiming to improve viral control after ART is interrupted. Results of successful cure and remission experiments in SIV/macaque models demonstrate that certain interventions induce VL containment for extended periods, but only after a significant increase in SIV replication after ART withdrawal . This saThere is no “gold standard”, so studies tend to use TtR or viral set point depending on the study’s objective. Consequently, the comparison of efficacy in interventions between ATI studies is highly difficult. Nevertheless, there are few available publications that directly address the correlations between different virological endpoints measured during ATI .In one study, Treasure G et al. assessed the relationship among VL outcomes in HIV ATI trials. A total of 235 participants undergoing ATI in six AIDS Clinical Trials Group (ACTG) studies were analyzed. There was no significant association between TtR and ATI VL set point in participants who had viral rebound at or before week 12. However, patients who rebounded after week 12 had significantly lower ATI VL set point, suggesting the presence of an immunologic and/or virologic factor that could have an impact on VL dynamics. Participants who were treated during early infection were also found to have a lower ATI VL set point; a finding consistent with other studies that favor treatment from early stages of infection. Moreover, VL set point tended to be lower than pre-ART VL .Feher et al.  conducteThe viral reservoir not only makes research on HIV remission or cure challenging, but also affects the ability to estimate risks from ATI themselves. This is due to the lack of evidence as to the size and composition of individual viral reservoirs, and thus the potential impact of ATI cannot be well evaluated. The absence of risk assessment is one of the main concerns of ATI, which are mainly based on theoretical and experienced risks  see Tab.Clinically significant adverse events have been reported in treatment interruption studies, probably related to the increase of VL. The described adverse events (AEs) found in the bibliography occurred during the ATI phase or during short-term follow-up. It is important to note that many of these studies used STI and thus interruptions had a longer duration, endpoints were more flexible, and inclusion criteria were less strict. Reported clinical adverse events include acute retroviral syndrome, cardiovascular, renal, or hepatic disease, among others.Symptoms resembling those of acute retroviral syndrome may occur during ATI due to viral rebound. Patients more frequently presented fever, lymphadenopathy, rash, myalgia, diarrhea, headache and pharyngitis. However, symptoms resolved quickly upon resuming treatment ,17,18,193 until an increase to over 350 was achieved. The results demonstrated that although the overall rate of events was relatively low, the risk of serious illness and mortality was more than double among those receiving intermittent ART. Higher incidence of opportunistic diseases and death from other causes were found in the interrupted ART groups and thus confirmed that immunosuppression increased the risk of death. They also showed that participants undergoing a STI had a higher rate of major cardiovascular, renal, or hepatic disease than those in the viral suppression group. Part of the difference in the rates of opportunistic diseases or death from any cause between participants in STI and the viral suppression group was explained by the differences in the CD4+ T cell count and HIV RNA level [The SMART group randomized over 5000 participants to continuous or interrupted ART to investigate whether ART could be used intermittently and maintain CD4+ T cell counts above a threshold. Participants in the interrupted ART arm were to reinitiate ART when CD4+ count decreased to <250 cells/mmNA level . The larNA level .Neurological events are poorly defined risks of ATI. However, previous experience with cerebrospinal fluid monitoring during prolonged ART interruption indicated that rebound HIV RNA accompanied by elevations in biomarkers of intrathecal inflammation and neuronal injury were found approximately 20 days after ATI .Many of the short-term clinical adverse events have been thoroughly studied in the past years. Due to this research, the use of STI are nowadays restricted to ATI in clinical trials to evaluate efficacy. Data from these large studies involve extensive treatment interruptions or delayed ART, and therefore, are difficult to extrapolate to time-limited, closely monitored ATI in current clinical trials. The main concerns nowadays are two potential effects of ATI, diminished responsiveness to future cure interventions, and increased chronic inflammatory processes, due to expanded viral reservoirs, even after reintroduction of effective ART . In a syThe latent HIV-1 reservoir is one of the primary targets for virus eradication and curative strategies. Upon discontinuation of ART, safety concerns related to the expansion of latent reservoir emerge. Therefore, it has been widely studied in these past years. Evidence to this date has concluded that short-term ATI does not lead to expansion of the persistent HIV reservoir . ClarridOne of the main adverse effects analyzed in studies is drug resistances. Previous studies with STI reported new cases of drug resistances. However, it has been revealed that the incidence of resistance mutations was higher when CD4+ T cell count was <500 cells/μL . NonetheAn increased risk of HIV transmission to sexual partners during treatment interruption can occur due to increased VL. Evidence of detection of HIV in semen has been documented . MoreoveATI mostly depend on the trial study, and therefore one single guideline for its design is not feasible. In order to expose patients to ATI, predefined goals must be achieved for the intervention being analyzed. This means that pre-existing data should support the intervention, and thus ATI should not be merely used to generate hypotheses. In a review by Julg et al. , a meeti3, fully suppressed HIV RNA while on stable ART, and otherwise healthy individuals without major comorbidities.The major inclusion criteria included stable CD4+ counts ≥500 cells/mm(a)Active or chronic hepatitis B virus infection and active hepatitis C infection;(b)Active Mycobacterium tuberculosis infection;(c)History of cancer ;(d)History of HIV-associated dementia or progressive multifocal leukoencephalopathy;(e)Resistance to two or more classes of antiretroviral drugs;(f)History of cardiovascular event or at high risk of an event;(g)History of AIDS-defining illness/CDC category C events;(h)3 during chronic stages of infection;History of CD4+ nadir <200 cells/mm(i)Women who are pregnant or breastfeeding;(j)Advanced non-alcoholic fatty liver and advanced non-alcoholic steatohepatitis if evidence for substantial fibrosis or evidence of cirrhosis; and(k)2).HIV-related kidney disease or moderate-to-severe decrease in estimated glomerular filtration rate  in patients recruited in a phase I therapeutic vaccine trial of a polyvalent MVA-B vaccine candidate. However, no influence of the presence of protective HLA class I alleles  or number of HLA footprints in Gag were associated with time to rebound. These data differ from some of the findings of other groups that suggest that vaccinated participants with neutral HLA alleles had lower median VL 16 weeks after ATI than vaccinated participants did with protective HLA alleles or placebo participants with neutral HLA alleles. In this study, factors independently associated with lower VL 16 weeks after ATI included greater Gag sequence divergence from the vaccine sequence and decreased proportion of HLA-associated polymorphisms in Gag [In addition, it seems that type of HLA of the host could influence VL rebound. Rosas-Umbert et al.  found ths in Gag .Lymphocyte CD4+ T cell proliferation after exposure to HIV antigens and IFN gamma secretion T lymphocytes are commonly used tests to measure immunogenicity in therapeutic HIV vaccine trials. Both parameters have been associated with VL control in chronic HIV infected patients ,39, so tOne of the first studies that described T cell responses in HIV vaccination used ALVAC vCP1452 in eleven chronic HIV infected patients. Absence of correlates between VL dynamics and T cell responses were described, but they were determined after ATI and not as predictors .Therapeutic vaccination with autologous monocyte derived-dendritic cells (MD-DCs) loaded with autologous heat-inactivated HIV-1 in patients with non-advanced chronic HIV-1 infection showed a decrease in the magnitude and the breadth of the CD8+ T cell responses after the immunization period. However, there was no difference in the CD8+ T cell dynamics between virologic responders (decrease of >0.5 log in relation to baseline VL after immunization and stopping treatment) and non-responders. In contrast, responders had a weak but statistically significant increase in HIV-1 specific CD4+ lymphoproliferative responses (LPR) during immunization . Eighty-Some studies with HIV therapeutic vaccines have demonstrated to be immunogenic, but with no beneficial effect on viral replication ,48 and sOnly two studies have studied the relation of vaccine induced antibodies and VL rebound during ATI after immunization and opposite results have been encountered . The ACTHigher soluble markers of inflammation have been related to disease progression in HIV. Data addressing whether soluble inflammation markers could influence viral control dynamics after ATI has been assessed in a peptide-based therapeutic HIV-1 p24 Gag vaccine candidate . Better viral control was observed in vaccinated patients with higher levels of TNF alpha and IL-6 between week 28 and week 1. In contrast, the same observations in IFN-γ and TNF alpha secretion in the placebo group were not associated with better control of VL after ATI, suggesting a possible qualitative difference in the responses induced by the vaccine . In relaOne of the major barriers to HIV functional cure is latency , and depQVOA is the “gold standard” test for measuring the size of the latent reservoir. This assay requires extracting resting CD4+ T cells to activate them in order to induce HIV replication by any latently infected cell. Although it has positive features, it has several important disadvantages such as time consuming, costs, and the need for many viable cells ,59 thereTotal DNA is the quantification of all forms of HIV-DNA, integrated and unintegrated, coding for both competent and defective viruses reflecting the global size of the reservoir giving an overview of its distribution in the body at all stages of the disease . SeveralDuring the viral replication, only a fraction of total DNA is integrated, and an even smaller fraction is capable of producing infectious virus . Memory A clinical trial using a poxvirus-based vaccine (MVA-B) alone or in combination with a drug to reactivate latent HIV-1 (disulfiram) assessed the impact on latent reservoir and pVL rebound during ATI and found that only participants receiving vaccinations alone had a marginal delay in TtR after treatment interruption, but failed to have an impact on the viral reservoir . When anAn autologous DC-based therapeutic vaccine (DCV2) was able to decrease the peak viral set point after ATI  and in aMore than 40 viral RNA are produced in HIV infected cells . CA-RNA Only very few therapeutic vaccine clinical trials have assessed CA-RNA as a reservoir marker and have studied if there are any relations with pVL dynamics during ATI ,50 Tabl. A doublSystems biology is a scientific approach to study complex biological systems as a whole, which has gained great popularity in our century due to the almost exponential increase in computational capacity in the last decades. The integrative study of a large set of biological data is referred to by the suffix—omics—such as genomics (genes), proteomics, (proteins), metabolomics (metabolites), microbiomics (microbiome) or transcriptomics (RNA), which is a fundamental part of systems biology. The application of such methodology in the development of vaccines gave birth to systems vaccinology, a young discipline that seems to be a useful tool in a number of stages of modern vaccine development strategies  includinAlthough the intersection of systems vaccinology and therapeutic HIV-1 vaccine research is very small thus far, there are some interesting data available. Interestingly, all available data comes from studies performed with DC-based vaccines.The pioneer study in this area was published five years ago by de Goede et al.  where thA recent substudy of the DALIA trial , is the Our group analyzed the relationship between post-vaccination transcriptomic patterns and virological response  in partiBoth of these studies underline the importance of the predominance of specific immune response in order to achieve better response to vaccination, similar to what was observed by others in the case of different preventive vaccines ,79 incluMany HIV therapeutic vaccines have been evaluated and some others are currently in clinical trials. In all of these studies, ATI is the main intervention to assess efficacy. Different surrogate markers of viral control after ATI have been studied with different outcomes. There have been classical assessments such as host factors, immunologic assays, and reservoir measurements and more recent approaches such as omics data, but still not one seemed to robustly predict post-intervention viral control, hence ATI seems currently irreplaceable to evaluate the effect of therapeutic vaccines alone or in combination, even though there are potential related risks. Further research is necessary for a reliable surrogate biomarker focused on improving classical strategies and pursuing new approaches.


CD4 cell and viral load count are highly correlated surrogate markers of human immunodeficiency virus (HIV) disease progression. In modelling the progression of HIV, previous studies mostly dealt with either CD4 cell counts or viral load alone. In this work, both biomarkers are in included one model, in order to study possible factors that affect the intensities of immune deterioration, immune recovery and state-specific duration of HIV-infected women.The data is from an ongoing prospective cohort study conducted among antiretroviral treatment (ART) naïve HIV-infected women in the province of KwaZulu-Natal, South Africa. Participants were enrolled in the acute HIV infection phase, then followed-up during chronic infection up to ART initiation. Full-parametric and semi-parametric Markov models were applied. Furthermore, the effect of the inclusion and exclusion viral load in the model was assessed.anemia, having a high liver abnormality score and patients who reported many sexual partners, had a significant increase in the intensities of immunological deterioration transitions. On the other hand, having high weight, higher education level, higher quality of life score, having high RBC parameters, high granulocyte component scores and high mononuclear component scores, significantly increased the intensities of immunological recovery transitions.Inclusion of a viral load component improves the efficiency of the model. The analysis results showed that patients who reported a stable sexual partner, having a higher educational level, higher physical health score and having a high mononuclear component score are more likely to spend more time in a good HIV state . Patients with TB co-infection, with Inclusion of both CD4 cell count based disease progression states and viral load, in the time-homogeneous Markov model, assisted in modeling the complete disease progression of HIV/AIDS. Higher quality of life (QoL) domain scores, good clinical characteristics, stable sexual partner and higher educational level were found to be predictive factors for transition and length of stay in sequential adversity of HIV/AIDS. This Markov chain process has an initial probability, denoted by P(S(0) = m), m ∈ E, which evolves over time and with a history (HE), which contains the states previously visited, durations and times of transitions [Let a Markov chain process {nsitions , 26. TheThe corresponding transition intensity is defined byWhere X.Besides modeling the ordinal transitional state of the CD4 progression, our object is to examine the effect of covariates or risk factors namely the education, marital status, age, sex under the influence of alcohol, TB-co infection status, anemia status, white blood cell parameters, RBC parameters, HR-QoL domain scores and weight on such transitions. We denote this set of covariates by m to state j and Xmj representing a set of covariates, while αmj is the effect of the covariates on the hazard intensity hmj. The transition m to j is defined as immunological deterioration if m < j, as immunological recovery if m > j; as the probability of staying in the same diseasing state if m = j. In other words, the state labeling is 1 for normal, 2 for mild, 3 for advanced and 4 for severe states  on CD4 cell counts (xi) to improve the efficiency of the model above. In order to create two new uncorrelated components, as explained by Chikobvu and Shoko [0) and slope (β1) parameters in the model: yi = β0 + β1xi+εi. We then define a viral load orthogonal variable (VLO)= εi= yi − (β0 + β1xi).Principal component analysis (PCA) is a statistical procedure that uses an orthogonal transformation to convert a set of observations, of possibly correlated variables, into a set of values of linearly uncorrelated variables. In this paper, a principal component variable is created by fitting a regression model of lognd Shoko , we carrVLO in the model explains the component of disease progression of HIV/AIDS that cannot be explained by the CD4 counts alone. In order to deal with multicollinearity of CD4 cell count and viral load count, the orthogonal viral load component was used. The residual from the fitted model was included with the original HIV/AIDS data to form the new viral load component.The estimates of full and semi-parametric multi-state models were compared with non-parametric estimates to assess model fit  flexsurv, survival and mstate packages in order to fit fully-parametric Markov, Semi-parametric Markov and to estimate the nonparametric Aalen-Johansen model, respectively. For visual graphical presentation of the estimates proc. SGPLOT in SAS 9.4 (https://www.sas.com/en_us/software/sas9.html) was used.All of the analyses were implemented using R-3.6.2 . Anemic patients had also significantly increased the intensity of immune deterioration from mild to advanced disease state  as compared to non-anemic patients. The result further showed that having TB co-infection significantly accelerates the transition time from normal to mild  and advance to severe  disease stages, compared to those without TB co-infection.  and mild to normal disease state . Similarly, an increase in level of independence score reduces the intensities of transition from normal to mild  and advanced to severe disease state . Furthermore, having high psychological well-being scores, is significantly associated with reduced intensities of transitions from normal to mild  and mild to advanced  disease state  had statistically significantly reduced intensities of transitions from normal to mild , mild to advanced  and advanced to severe  disease state, as compared to those with low level of education (grade < 8). Patients who reported stable sexual partners  and no sexual partner , had significantly increased immune recovery, from severe to advanced disease state, as compared to those who reported many sexual partners. Furthermore, as the weight of women in the study increased, the intensities of immune deterioration from normal to mild and mild to advanced disease state, decreased  and mild disease state ,  and no sexual partner  were more likely to stay longer in a normal disease state as compared to those who reported many sexual partners. Furthermore, patients with higher educational levels (grade > 11) were more likely to stay longer in the normal disease state , as compared to those with lower education (< 8 grade). (See Table We have presented both full-parametric and semi-parametric multi-state models, to model the transition intensity and length of stay of sequential adverse events of HIV/AIDS. We also improved the selected model, the Weibull multi-state model, by including an orthogonal viral load component, derived from PCA. The new orthogonal viral load component helped to explain the determinants of transitions and length of stay in sequential adverse events, which could not be explained by the CD4 count alone. This further improved the efficiency and predictive accuracy of the model. Results from the Akaike information criteria and likelihood ratio test showed that the model defined by CD4 cell counts states with the viral load orthogonal component, fitted significantly better than the model with the exclusion of orthogonal viral load components, as it was done by , 29.Among the different hematological parameters for HIV infected patients, as expected, a latent variable related to basophils counts and total lymphocytes was significantly associated with the intensities of immunological deterioration transitions, and this confirms that HIV infection targets T-cells. Many studies also suggested that total lymphocytes can adequately serve as a surrogate biomarker for predicting CD4 count progression –32. The We found that hematological abnormalities such as anemia had a significantly accelerated immune deterioration effect. This may be attributed to the fact that anemia is commonly due to the under-production of erythrocytes by the bone marrow cells . The bonWe observed that TB co-infection accelerated the deterioration of immunological functions, a finding that is in accordance with the literature –48, wherHIV patients who reported many sexual partners had significantly increased intensities of immune deterioration transitions. Moreover, patients who reported stable or no sexual partners were more likely to spend a long time in a normal disease state, as compared to those who reported many sexual partners. Indeed it has been reported  that patPatients with higher educational levels had significantly reduced rates of immune deterioration. Other researchers , 54 alsoHaving high body weight significantly reduces the intensities of immunological deterioration transitions. This finding is consistent with higher weight was significantly related to higher CD4 cell count levels , 58. RecA significant positive relationship between QoL domain scores and intensities of recovery of HIV infected patients is noted in our study. Previous studies –63 had rThis study has some limitations, including missing data, which are expected for a study conducted on data collected from patients’ files with many variables and a long term follow-up period. Moreover, some clinical covariates that influence the intensities of immune deterioration and immune recovery, may not have been included, for example, CD4/CD8 ratio and hepatitis status. Furthermore, the study findings were limited to adult females.From a clinical perspective, the study has revealed that having higher QoL domain scores, good clinical characteristics, stable sexual partners and higher educational levels, were found to be the significant predictive factors for intensities of immune deterioration, immune recovery and state-specific length of stay of HIV-infected women. Furthermore, we found that the identification of factors, such as QoL measurement items, clinical attributes, marital status, and educational status, associated with the current state of the patient, were important contributing factors to extend the survival of the patients and could potentially guide clinical interventions.From a methodological perspective, by treating the collinearity of the viral load and CD4 cell counts using orthogonal; transformation, the Weibull multi-state Markov model gives a better fit and insight than the semi-parametric methods. Moreover, our study will help researchers to uncover the critical areas of correcting and dealing with multicollinearity when including both CD4 cell and viral load count in parametric multi-state modelling of HIV/AIDS, that many researchers have not been able to explore. Other more robust parametric distributions other than the Weibull are also a possible area of further research.


Once-daily, single-tablet regimens (STRs) have been associated with improved patient outcomes compared to multi-tablet regimens (MTRs). This study evaluated real world adherence and persistence of HIV antiretroviral therapy (ART), comparing STRs and MTRs.Adult Medicaid beneficiaries (aged ≥ 18 years) initiating ART with ≥ 2 ART claims during the identification period  and continuous health plan enrollment for a 12-month baseline period were included. For STRs, the first ART claim date was defined as the index date; for MTRs, the prescription fill claim date for the last drug in the regimen was defined as the index date, and prescription fills were required to occur within a 5-day window. Adherence was assessed in 30-day intervals over a 6-month period, with adherence defined as having less than a 5-day gap between fills. Persistence was evaluated as median number of days on therapy and percent persistence at 12 months. Cox Proportional Hazard models were used to evaluate risk of discontinuation, controlling for baseline and clinical characteristics.A total of 1,744  and 2409  patients newly prescribed ART had available data concerning adherence and persistence, respectively. Average age ranged 40–42 years. The patient population was predominantly male. Adherence assessments showed 22.7% of STR initiators were adherent to their index regimens over a 6-month period compared to 11.7% of MTR initiators. Unadjusted persistence analysis showed 36.3% of STR initiators discontinued first-line therapy compared to 48.8% for MTR initiators over the 2-year study period. Controlling for baseline demographic and clinical characteristics, MTR initiators had a higher risk of treatment discontinuation . Among STRs, compared to the referent elvitegravir(EVG)/cobicistat(COBI)/emtricitabine(FTC)/tenofovir alafenamide(TAF), risk of discontinuation was higher for efavirenz(EFV)/FTC/tenofovir disoproxil fumarate(TDF) , EVG/COBI/FTC/TDF , and abacavir (ABC)/lamivudine (3TC)/dolutegravir (DTG) . Among backbones, FTC/TAF was associated with lower risk of discontinuation than FTC/TDF  and ABC/3TC .Among patients newly prescribed ART, STR initiators were significantly less likely to discontinue therapy and had greater adherence and persistence compared to MTR initiators. Regimens containing FTC/TAF as a backbone had higher persistence than those consisting of other backbones. At the end of 2015, ~ 1.1 million people aged ≥ 13 years were living with human immunodeficiency virus (HIV) in the United States [HIV treatment typically includes a combination of antiretroviral therapy (ART) regimens to prevent HIV disease progression and transmission . ART hasCombination ART was initially characterized by high pill burden and multiple daily doses –10. OverPrior research suggests that adherence and persistence may be higher among patients treated with STRs compared with those treated with MTRs , 19. HowThis retrospective real-world study utilized data from the Truven Health Medicaid database from January 1, 2014 through December 31, 2016 (study period), with an approximately 6 months lag time. The Truven Health Medicaid Database contains medical, surgical, and prescription drug claims for more than 44 million Medicaid enrollees from multiple states. The database includes records of inpatient services, inpatient admissions, outpatient services, and prescription drug claims as well as information about long-term and other medical care. Data on eligibility (by month), service, and provider type are also included. In addition to standard demographic variables including age, gender, and race, the database also included variables more specific for Medicaid populations including aid category .Adult patients aged ≥ 18 years) initiating ART after one pre-index year of not receiving ART (i.e. presumably beginning first-line ART for the majority of patients) with ≥ 2 claims for an ART of interest , number of unique medications on index date except ART, Deyo-modified Charlson comorbidity index (CCI) score, and baseline individual comorbidities  were assessed. All ICD-9-CM codes were mapped to ICD-10-CM codes based on the General Equivalence Mappings published by the Centers for Medicare & Medicaid Services (CMS) .Outcome measures, including adherence and persistence during the follow-up period were measured. In HIV, a high level of adherence to ART is required for viral suppression . HoweverPersistence for HIV treatments was compared between STRs versus MTRs, backbones, and third agents where persistence was measured from the index regimen start date until the start of the first 90-day gap between prescription fills or the end of the enrollment. Patients were considered to have discontinued first-line therapy if a gap of ≥ 90 days between fills was observed. Patients who restarted therapy after a ≥ 90-day gap remained classified as discontinued, as restarts were not captured in the analysis. MTR initiators were required to remain on all therapies in the initial regimen to be considered persistent. Among MTR initiators, a gap of ≥ 90 days for any drug in the regimen was considered discontinuation of that regimen. The proportion of patients who discontinued their first-line therapy, time to discontinuation, and the proportion of patients with 12 months of persistence were also measured. Additionally, persistence was assessed as the risk of treatment discontinuation among different treatment cohorts.All study variables, including baseline demographics and clinical characteristics, as well as outcome variables  were first examined descriptively. For continuous variables such as persistence measures, mean, median, and standard deviations were generated. For categorical variables, counts (frequencies) and percentages were reported.® statistical software .Overall time to discontinuation of first-line ART for STRs and MTRs was assessed via Kaplan–Meier curves. Log-rank tests were used to evaluate statistical differences between treatment discontinuation curves. To assess the factors associated with risk of treatment discontinuation for each regimen, a multivariable Cox proportional hazard model was constructed. Covariates included in the multivariable-adjusted model comprised age group, gender, race, insurance type, CCI score, baseline individual comorbidities (more specific than CCI comorbidities), pre-index medication use, and the number of unique medications on index date. All of the analyses were conducted using SASFor the adherence study, 1744 patients met inclusion criteria, of whom 1290 (74.0%) initiated STRs and 454 (26%) initiated MTRs. For the persistence study, 2409 patients met inclusion criteria, of whom 1782 (74.0%) initiated STRs and 627 (26.0%) initiated MTRs. Patients on STRs and MTRs were further stratified by specific ARTs to compare persistence within STRs and within MTRs  to 41 (adherence sample). The adherence study included 1744 patients, most (71.4%) of whom were aged 18–49 years, male (56.5%), black (61.0%), and were enrolled in a comprehensive insurance plan (74.4%). Among patients in the adherence sample, pre-index medication use was highest for respiratory drugs (54.9%) and antibiotics (43.5%); the most common comorbidities at baseline were mental health disorders (56.5%), substance abuse (41.5%), and central nervous system toxicity (40.9%). Mean CCI score during the baseline period was 4.2 ± 3.4. Patients taking STRs and MTRs had similar demographic and clinical characteristics  during the post-index period of 1-30 days were adherent, defined as a gap of ≤ 5 days between successive fills. However, adherence declined sharply by 38.4% during the post-index period of 31–60 days, corresponding to when patients would have had to refill a 30-day supply. Thereafter, a gradual decline was observed at each post-index period. Similarly, among patients who initiated MTRs, nearly all patients (98.9%) during the post-index period of 1–30 days were adherent, defined as having a gap of ≤ 5 days between successive fills for any drug in the regimen. Adherence declined by roughly 45.0% during the post-index period of 31–60 days, and decreased gradually at each post-index period afterwards. The proportion of adherent patients at each post-index period was higher among patients on STRs as compared to patients prescribed MTRs Fig. . LikewisAmong patients on STRs who were non-adherent, the mean refill gap at each post-index time interval ranged from 22–24 days with a cumulative mean refill gap of 71 days during the overall 6-month period. Likewise, among patients prescribed MTRs, the mean refill gap at each post-index time interval ranged from 18–25 days, with a cumulative mean refill gap of 81 days during the overall 6-month follow-up period. A greater proportion of patients prescribed MTRs had a cumulative refill gap of > 30 days as compared to STRs .When comparing those initiating STRs, the proportion of patients who were adherent during the post-index period of 1-180 days was highest among patients prescribed EVG/COBI/FTC/TAF (40.7%) followed by DTG/ABC/3TC (23.4%), EVG/COBI/FTC/TDF (22.4%), RPV/FTC/TDF (20.8%), and EFV/FTC/TDF (12.8%). When comparing patients who were non-adherent, the mean cumulative refill gap for those prescribed STRs was higher among those who initiated EFV/FTC/TDF (80 days) followed by RPV/FTC/TDF (76 days), EVG/COBI/FTC/TDF (70 days), ABC/3TC/DTG (66 days), and EVG/COBI/FTC/TAF (61 days)  followed by DRV/c + FTC/TDF (14.3%), ATV/c + FTC/TDF (12.8%), and DRV/r + FTC/TDF (5.4%). Furthermore, among patients taking DTG + FTC/TDF, adherence to the DTG component (25.8%) was more common than to the FTC/TDF component (18.5%). When comparing patients with MTR who were non-adherent, the mean cumulative refill gap was higher among those who initiated FTC/TDF + DRV/c (91 days), followed by FTC/TDF + ATV/c (86 days), FTC/TDF + DR/Vr (82 days), and FTC/TDF + DTG (76 days) . Likewise, findings based on sensitivity analyses using an adherence definition of ≤ 14 gap days between successive fills also yielded similar findings. Among the overall patients prescribed STRs, 39.8% were adherent during the post-index period of 1–180 days. By STR regimen type, patients prescribed EVG/COBI/FTC/TAF had the highest proportion of adherent patients during the post-index period of 1–180 days (58.5%).The median follow up time was 300 days (range: 4–730 days). Unadjusted analysis for persistence indicated that median time on treatment was significantly higher for patients prescribed STRs as compared to MTRs . Furthermore, 36.3% of STR patients discontinued the index regimen compared to 48.8% of MTR patients . Similarly, comparing STR patients, persistence was significantly higher for those treated with EVG/COBI/FTC/TAF as compared to EFV/FTC/TDF , RPV/FTC/TDF , EVG/COBI/FTC/TDF , and DTG/ABC/3TC .Comparing third agents, persistence was significantly higher for patients with EVG/COBI use as compared to DRV/r  and ATV/r  Fig. . FurtherThe discontinuation caused by poor tolerability, poor adherence, or complexity carries risks of the development of toxicity and resistance associated with compromised future treatment options. Improved rates of adherence and persistence are critically important for the long-term therapy of HIV patients  and imprART adherence has been shown to improve health outcomes and QoL, including HIV transmission, viral load suppression, drug resistance prevention, and survival rates . Most reAdherence and persistence are also considered cornerstones of effective ART therapy; hence, it is recommended that ART regimens should be efficacious, with no virologic failures, well-tolerated, and without toxicity, to improve adherence, and, in turn, improve long-term persistence , 40. TheThe current study examined regimen components in addition to specific STRs and MTRs. Separate comparisons were performed among patients prescribed third agents and backbones to explicitly explore the benefits derived from each individual component. Our study found that patients who were treated with regimens including an FTC/TAF backbone had higher persistence compared to regimens with ABC/3TC and FTC/TDF, providing one of the first real world studies assessing persistence of the FTC/TAF backbone, which was approved in 2016. While reasons for persistence could not be assessed in this analysis, the higher persistence of FTC/TAF likely relates to clinical trial data showing high efficacy and bone and renal safety advantages compared to prior regimens, and the 2018 IAS-USA guidelines recommended TAF but not TDF as a component of initial suggested regimens , 44.The findings from our study should be viewed in the context of study and claims data limitations. While claims data are extremely valuable for the efficient and effective examination of health care outcomes, they are primarily collected for business purposes rather than research purposes. Therefore, analyses may be subject to inherent limitations of the source administrative claims data, such as coding errors or diagnoses entered for administrative processing rather than clinical completeness. Moreover, the presence of a claim for a filled prescription does not indicate that the medication was consumed or taken as prescribed. Also, medications filled over-the-counter or provided as samples by the physician are not observed in claims data. Certain information that could influence study outcomes is not readily available in claims data, such as clinical and disease-specific parameters including HIV viral load, which could be used as a surrogate for adherence. In addition, although the study adjusted for observed baseline characteristics in the multivariable analyses, some unobserved confounders may remain. Further, given the study timeframe, newly-available HIV regimens were not included. Lastly, this study was limited in that health claims may be subject to error; however, given that ART is only used for HIV and that at least two ART claims were required for inclusion, it is reasonable to conclude that patients identified in the Truven Medicaid population by ART use are HIV patients.In this analysis of real world claims data associated with US Medicaid recipients newly initiating ART, adherence and persistence was greater among patients initiating STRs than those initiating MTRs. In fact, the proportion of patients who were adherent was almost double for those initiating STRs compared to MTRs. Additionally, median treatment persistence was also higher among STR initiators. Regimens containing EVG/COBI as a third agent and FTC/TAF as a backbone showed higher persistence than other third agents and backbones, respectively. In addition, both adherence and persistence were greater with single-tablet EVG/COBI/FTC/TAF than with other STRs. As new regimens with improved safety and tolerability profiles become available, continued research on persistence and adherence for HIV treatments is warranted to inform better therapeutic management.Additional file 1: Figure S1. Proportion of Adherent Patients on Individual STRs. 3TC Lamivudine, ABC Abacavir, COBI Cobicistat, DTG Dolutegravir, EVG Elvitegravir, FTC Emtricitabine, RPV Rilpivirine, STR single tablet regimen, TAF Tenofovir Alafenamide Fumarate, TDF Tenofovir Disoproxil Fumarate; Adherence ≤ 5-day gap between successive fills.Additional file 2: Figure S2. Proportion of Adherent Patients on Individual MTRs. ATV/c Atazanavir boosted with cobicistat, DRV/c Darunavir boosted with cobicistat, DRV/r Darunavir boosted with ritonavir, DTG Dolutegravir, FTC Emtricitabine, TDF Tenofovir Disoproxil Fumarate; Adherence: ≤ 5 days gap in fill for one or more drugs in the regimen.