Brief Report: Changing Characteristics Among In-Hospital HIV Deaths: An 11-Year Retrospective Review of a Regional Hospital in South Africa.

BACKGROUND: Elevated HIV-associated mortality persists, despite a notable decline with the expansion of antiretroviral therapy (ART). In South Africa, the relative majority of deaths occur in health facilities, providing an opportunity to track decedent characteristics. SETTING: We analyzed data from 14,870 adult patients who died between 2008 and 2018 at Klerksdorp/Tshepong Hospital Complex in South Africa. METHODS: Recorded data included demographics, causes of death, HIV status, ART, and tuberculosis (TB) history. We present summary statistics and results from linear, log-binomial, and multinomial regressions to quantify changes over time. RESULTS: Over the study period, the median age of decedents with HIV in the hospital increased from 39.3 to 43.4 years, and there was a switch to male predominance (46%-54%). Those who died at a younger age (<40 years) remained more likely to be HIV-positive than the older age group, despite the overall proportion of HIV-positivity decreasing over time. The proportion of decedents with HIV ever started on ART increased from 21% to 67%. The proportion of HIV patients dying from TB and AIDS-defining illnesses decreased from 31% to 22%. CONCLUSIONS: We noted a shift in deaths over time to more men and older individuals, whereas the burden of HIV was heaviest on the younger age groups. Advanced HIV disease remained an important cause of mortality. We also observed an increase in less-traditional opportunistic illnesses among those with HIV, including malignancy, cardiovascular disease, and kidney disease. The high proportion of patients on ART who died prematurely requires further research and interventions.

A cross-sectional study of attitudes and behaviors of snuff use and cessation among people with and without HIV in South Africa.

Background: Despite a high (48%) prevalence of snuff use among women with HIV in South Africa, little is known of the attitudes and behaviors of use, strategies for cessation, and potential health risks. Methods: In a cross-sectional study, a questionnaire was administered to adults (≥18 years) with (HIV+) and without HIV (HIV-) who self-reported current snuff use to collect information on demographics, snuff use and cessation attempts, preferred strategies for cessation, other substance use, history of respiratory illness, and mental health. Results: 150 (74 HIV+, 76 HIV-) participants were enrolled; 115 (77%) were daily snuff users, 6 (4%) were current smokers, and 17 (11%) former smokers. Top reasons for current snuff use included improving health (n = 48, 32%), reducing stress (n = 26, 16%), and "being a habit" (n = 38, 25%). Participants believed snuff use to have mostly positive (n = 68, 46%) or no (n = 54, 36%) health impacts, and 57 (38%) participants believed snuff cures headaches. 103 (69%) participants reported a previous quit attempt, and 110 (73%) indicated high interest in quitting snuff. Although 105 (70%) participants indicated that advice from a healthcare provider would aid them in quitting snuff, only 30 (20%) reported ever receiving that advice. A majority of participants (n = 141, 94%) suffer from moderate to high levels of perceived stress, and overall few differences were seen by HIV status. Conclusions: Education on negative impacts of snuff, advice to quit from healthcare providers, and nicotine replacement therapy should be considered in the development of a snuff cessation program.

Introducing a secondary segmentation to construct a radiomics model for pulmonary tuberculosis cavities.

PURPOSE: Accurate segmentation (separating diseased portions of the lung from normal appearing lung) is a challenge in radiomic studies of non-neoplastic diseases, such as pulmonary tuberculosis (PTB). In this study, we developed a segmentation method, applicable to chest X-rays (CXR), that can eliminate the need for precise disease delineation, and that is effective for constructing radiomic models for automatic PTB cavity classification. METHODS: This retrospective study used a dataset of 266 posteroanterior CXR of patients diagnosed with laboratory confirmed PTB. The lungs were segmented using a U-net-based in-house automatic segmentation model. A secondary segmentation was developed using a sliding window, superimposed on the primary lung segmentation. Pyradiomics was used for feature extraction from every window which increased the dimensionality of the data, but this allowed us to accurately capture the spread of the features across the lung. Two separate measures (standard-deviation and variance) were used to consolidate the features. Pearson's correlation analysis (with a 0.8 cut-off value) was then applied for dimensionality reduction followed by the construction of Random Forest radiomic models. RESULTS: Two almost identical radiomic signatures consisting of 10 texture features each (9 were the same plus 1 other feature) were identified using the two separate consolidation measures. Two well performing random forest models were constructed from these signatures. The standard-deviation model (AUC = 0.9444 (95% CI, 0.8762; 0.9814)) performed marginally better than the variance model (AUC = 0.9288 (95% CI, 0.9046; 0.9843)). CONCLUSION: The introduction of the secondary sliding window segmentation on CXR could eliminate the need for disease delineation in pulmonary radiomic studies, and it could improve the accuracy of CXR reporting currently regaining prominence as a high-volume screening tool as the developed radiomic models correctly classify cavities from normal CXR.

A Cross-Sectional Analysis of the Nicotine Metabolite Ratio and Its Association with Sociodemographic and Smoking Characteristics among People with HIV Who Smoke in South Africa.

The nicotine metabolite ratio (NMR) is associated with race/ethnicity but has not been evaluated among smokers in the African region. We conducted a cross-sectional analysis of baseline data from a large randomized, controlled trial for smoking cessation among people with HIV (PWH) in South Africa. Urine samples were analyzed for the NMR and evaluated as a binary variable using a cutoff value of the fourth quartile to determine the fastest metabolizers. The median NMR was 0.31 (IQR: 0.31, 0.32; range: 0.29, 0.57); the cut-point for fast metabolizers was ≥0.3174 ng/mL. A high NMR was not associated with the number of cigarettes per day (OR = 1.10, 95% CI: 0.71, 1.70, p = 0.66) but was associated with 40% lower odds of a quit attempt in the past year (OR = 0.69; 95% CI: 0.44, 1.07, p = 0.09) and alcohol use (OR = 0.59, 95% CI: 0.32, 1.06, p = 0.07). No association was seen with marijuana or HIV clinical characteristics. As we found only minimal variability in the NMR and minimal associations with intensity of smoking, NMR may be of limited clinical value in this population, although it may inform which individuals are less likely to make a quit attempt.

Proteomic Analysis of Mucosal and Systemic Responses to SARS-CoV-2 Antigen.

The mucosal environment of the upper respiratory tract is the first barrier of protection against SARS-CoV-2 transmission. However, the mucosal factors involved in viral transmission and potentially modulating the capacity to prevent such transmission have not fully been identified. In this pilot proteomics study, we compared mucosal and systemic compartments in a South African cohort of vaccinated and unvaccinated individuals undergoing maxillofacial surgery with previous history of COVID-19 or not. Inflammatory profiles were analyzed in plasma, nasopharyngeal swabs, and nasal and oral tissue explant cultures, using Olink and Luminex technologies. SARS-CoV-2-specific antibody levels were measured in serum and tissue explants. An increased pro-inflammatory proteomic profile was measured in the nasal compartment compared to plasma. However, IP-10 and MIG levels were higher in secretions than in nasal tissue, and the opposite was observed for TGF-ß. Nasal anti-SARS-CoV-2 spike IgG correlated with mucosal MIG expression for all participants. A further positive correlation was found with IP-10 in BioNTech/Pfizer-vaccinated individuals. Systemic levels of anti-SARS-CoV-2 spike IgG elicited by this vaccine correlated with plasma IL-10, IL-6 and HBD4. Proteomic profiles measured in mucosal tissues and secretions using combined technologies could reveal correlates of protection at the mucosal portals of viral entry.

Yield of Facility-based Targeted Universal Testing for Tuberculosis With Xpert and Mycobacterial Culture in High-Risk Groups Attending Primary Care Facilities in South Africa.

BACKGROUND: We report the yield of targeted universal tuberculosis (TB) testing of clinic attendees in high-risk groups. METHODS: Clinic attendees in primary healthcare facilities in South Africa with one of the following risk factors underwent sputum testing for TB: human immunodeficiency virus (HIV), contact with a TB patient in the past year, and having had TB in the past 2 years. A single sample was collected for Xpert-Ultra (Xpert) and culture. We report the proportion positive for Mycobacterium tuberculosis. Data were analyzed descriptively. The unadjusted clinical and demographic factors' relative risk of TB detected by culture or Xpert were calculated and concordance between Xpert and culture is described. RESULTS: A total of 30 513 participants had a TB test result. Median age was 39 years, and 11 553 (38%) were men. The majority (n = 21734, 71%) had HIV, 12 492 (41%) reported close contact with a TB patient, and 1573 (5%) reported prior TB. Overall, 8.3% were positive for M. tuberculosis by culture and/or Xpert compared with 6.0% with trace-positive results excluded. In asymptomatic participants, the yield was 6.7% and 10.1% in symptomatic participants (with trace-positives excluded). Only 10% of trace-positive results were culture-positive. We found that 55% of clinic attendees with a sputum result positive for M. tuberculosis did not have a positive TB symptom screen. CONCLUSIONS: A high proportion of clinic attendees with specific risk factors (HIV, close TB contact, history of TB) test positive for M. tuberculosis when universal testing is implemented.

Unmasking Pneumococcal Carriage in a High Human Immunodeficiency Virus (HIV) Prevalence Population in two Community Cohorts in South Africa, 2016-2018: The PHIRST Study.

BACKGROUND: Longitudinal pneumococcus colonization data in high human immunodeficiency virus (HIV) prevalence settings following pneumococcal conjugate vaccine introduction are limited. METHODS: In 327 randomly selected households, 1684 individuals were enrolled and followed-up for 6 to 10 months during 2016 through 2018 from 2 communities. Nasopharyngeal swabs were collected twice weekly and tested for pneumococcus using quantitative lytA real-time polymerase chain reaction. A Markov model was fitted to the data to define the start and end of an episode of colonization. We assessed factors associated with colonization using logistic regression. RESULTS: During the study period, 98% (1655/1684) of participants were colonized with pneumococcus at least once. Younger age (<5 years: adjusted odds ratio [aOR], 14.1; 95% confidence [CI], 1.8-111.3, and 5-24 years: aOR, 4.8, 95% CI, 1.9-11.9, compared with 25-44 years) and HIV infection (aOR, 10.1; 95% CI, 1.3-77.1) were associated with increased odds of colonization. Children aged <5 years had fewer colonization episodes (median, 9) than individuals ≥5 years (median, 18; P < .001) but had a longer episode duration (<5 years: 35.5 days; interquartile range, 17-88) vs. ≥5 years: 5.5 days (4-12). High pneumococcal loads were associated with age (<1 year: aOR 25.4; 95% CI, 7.4-87.6; 1-4 years: aOR 13.5, 95% CI 8.3-22.9; 5-14 years: aOR 3.1, 95% CI, 2.1-4.4 vs. 45-65 year old patients) and HIV infection (aOR 1.7; 95% CI 1.2-2.4). CONCLUSIONS: We observed high levels of pneumococcus colonization across all age groups. Children and people with HIV were more likely to be colonized and had higher pneumococcal loads. Carriage duration decreased with age highlighting that children remain important in pneumococcal transmission.

Household- Versus Incentive-Based Contact Investigation for Tuberculosis in Rural South Africa: A Cluster-Randomized Trial.

BACKGROUND: Household contact investigation for people newly diagnosed with tuberculosis (TB) is poorly implemented, particularly in low- and middle-income countries. Conditional cash incentives may improve uptake. METHODS: We conducted a pragmatic, cluster-randomized, crossover trial of 2 TB contact investigation approaches (household-based and incentive-based) in 28 public primary care clinics in South Africa. Each clinic used 1 approach for 18 months, followed by a 6-month washout period, after which the opposite approach was used. Fourteen clinics were randomized to each approach. In the household-based arm, we conducted TB screening and testing of contacts at the household. In the incentive-based arm, both index patients and ≤10 of their close contacts (either within or outside the household) were given small cash incentives for presenting to study clinics for TB screening. The primary outcome was the number of people with incident TB who were diagnosed and started on treatment at study clinics. RESULTS: From July 2016 to January 2020, we randomized 28 clinics to each study arm, and enrolled 782 index TB patients and 1882 contacts in the household-based arm and 780 index patients and 1940 contacts in the incentive-based arm. A total of 1413 individuals started on TB treatment in the household-based arm and 1510 in the incentive-based arm. The adjusted incidence rate ratio of TB treatment initiation in the incentive- versus household-based arms was 1.05 (95% confidence interval: .97-1.13). CONCLUSIONS: Incentive-based contact investigation for TB has similar effectiveness to traditional household-based approaches and may be a viable alternative or complementary approach to household-based investigation.

Underdiagnosis of iron deficiency anaemia in HIV-infected individuals: a pilot study using soluble transferrin receptors and intensive bone marrow iron stores to improve the diagnosis.

AIM: We compared soluble transferrin receptors (sTfR), serum ferritin, mean cell volume (MCV) of red cells and the sTfR-ferritin index with the intensive method bone marrow trephine (BMT) iron stores in the diagnosis of iron deficiency anaemia (IDA) in Human Immunodeficiency Virus (HIV)-positive hospitalised participants. METHODS: In this cross-sectional study, we recruited hospitalised HIV-positive and coronavirus of 2019 (COVID-19)-negative adults with anaemia who required a bone marrow examination as part of their diagnostic workup. We measured the full blood count, ferritin, sTfR and assessed iron using the intensive method in Haemotoxylin and Eosin (H&E)-stained BMT core biopsies of consenting participants. RESULTS: Of the 60 enrolled participants, 57 were evaluable. Thirteen (22.80%) had IDA on H&E BMT iron stores assessment, and 44 (77.19%) had anaemia of chronic diseases (ACD). The sTfR and the sTfR-ferritin index had sensitivities of 61.54% and 53.85%, respectively, for IDA diagnosis. The sensitivity and specificity of ferritin was 7.69% and 92.31%, respectively. The sTfR and sTfR-ferritin index's diagnostic specificity was relatively low at 46.15% and 38.46%, respectively. CONCLUSION: In this pilot study in HIV-positive participants, the prevalence of iron deficiency using the BMT assessment was low. Both the sTfR and the sTfR-ferritin index had a better quantitative correlation to bone marrow iron stores when compared with the MCV and ferritin and, may be more accurate surrogate markers of IDA.

Household Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 From Adult Index Cases With and Without Human Immunodeficiency Virus in South Africa, 2020-2021: A Case-Ascertained, Prospective, Observational Household Transmission Study.

BACKGROUND: In South Africa, 19% of adults are living with human immunodeficiency virus (HIV; LWH). Few data on the influence of HIV on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) household transmission are available. METHODS: We performed a case-ascertained, prospective household transmission study of symptomatic adult index SARS-CoV-2 cases LWH and not living with HIV (NLWH) and their contacts from October 2020 to September 2021. Households were followed up 3 times a week for 6 weeks to collect nasal swabs for SARS-CoV-2 testing. We estimated household cumulative infection risk (HCIR) and duration of SARS-CoV-2 positivity (at a cycle threshold value <30 as proxy for high viral load). RESULTS: HCIR was 59% (220 of 373), not differing by index HIV status (60% LWH vs 58% NLWH). HCIR increased with index case age (35-59 years: adjusted OR [aOR], 3.4; 95% CI, 1.5-7.8 and ≥60 years: aOR, 3.1; 95% CI, 1.0-10.1) compared with 18-34 years and with contacts' age, 13-17 years (aOR, 7.1; 95% CI, 1.5-33.9) and 18-34 years (aOR, 4.4; 95% CI, 1.0-18.4) compared with <5 years. Mean positivity was longer in cases LWH (adjusted hazard ratio, 0.4; 95% CI, .1-.9). CONCLUSIONS: Index HIV status was not associated with higher HCIR, but cases LWH had longer positivity duration. Adults aged >35 years were more likely to transmit and individuals aged 13-34 to be infected SARS-CoV-2 in the household. As HIV infection may increase transmission, health services must maintain HIV testing and antiretroviral therapy initiation.

Significance of lymph node fine needle aspiration for the diagnosis of HIV-associated lymphoma in a low-resource setting.

OBJECTIVE: Fine needle aspiration (FNA) is an early step in the work-up of lymphadenopathy in people with HIV (PWH). We set out to characterize the FNA cytology in PWH and report on the time to lymphoma diagnosis through the FNA clinics in the public healthcare system in Johannesburg, South Africa. DESIGN: Retrospective review of laboratory database. METHODS: A retrospective chart review of patients undergoing FNA through the department of cytopathology at the National Health Laboratory Service (NHLS) was undertaken. Results of FNAs performed between March and May 2018 were reviewed. Medical record chart abstraction included general demographics, HIV status, site and results of FNA, prior history of malignancy and other laboratory data. RESULTS: Five hundred and thirty-nine lymph node FNAs were performed on PWH. Pathological findings included tuberculosis 47% (252), inadequate sampling 14% (75), reactive adenopathy 13% (71), benign disease 12% (63), suspicious for lymphoproliferative neoplasm 8% (45), other malignancy 4% (21) and inflammation 2% ( n  = 12). Only 53% (24) of lymphomas were confirmed by biopsy. Those not confirmed had a high mortality (57%) and loss to follow-up rate (29%) over the following year. The median diagnostic interval exceeded 8 weeks from time of FNA to lymphoma diagnosis. CONCLUSION: FNA is an important screening modality in this high HIV and tuberculosis (TB) burden region. Patients with cytology suggestive for lymphoma, but without biopsy confirmation, have a high mortality rate suggesting undiagnosed lymphoma. A better understanding of the barriers to appropriate diagnostic triage for lymphoma is needed.

Seroprevalence of Severe Acute Respiratory Syndrome Coronavirus 2 After the Second Wave in South Africa in Human Immunodeficiency Virus-Infected and Uninfected Persons: A Cross-Sectional Household Survey.

BACKGROUND: Seroprevalence studies are important for quantifying the burden of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in resource-constrained countries. METHODS: We conducted a cross-sectional household survey spanning the second pandemic wave (November 2020 to April 2021) in 3 communities. Blood was collected for SARS-CoV-2 antibody (2 enzyme-linked immunosorbent assays targeting spike and nucleocapsid) and human immunodeficiency virus (HIV) testing. An individual was considered seropositive if testing positive on ≥1 assay. Factors associated with infection, and the age-standardized infection case detection rate, infection hospitalization rate, and infection fatality rate were calculated. RESULTS: Overall, 7959 participants were enrolled, with a median age of 34 years and an HIV prevalence of 22.7%. SARS-CoV-2 seroprevalence was 45.2% (95% confidence interval 43.7%-46.7%) and increased from 26.9% among individuals enrolled in December 2020 to 47.1% among those enrolled in April 2021. On multivariable analysis, seropositivity was associated with age, sex, race, being overweight/obese, having respiratory symptoms, and low socioeconomic status. Persons living with HIV with high viral load were less likely to be seropositive than HIV-uninfected individuals. The site-specific infection case detection rate, infection hospitalization rate, and infection fatality rate ranged across sites from 4.4% to 8.2%, 1.2% to 2.5%, and 0.3% to 0.6%, respectively. CONCLUSIONS: South Africa has experienced a large burden of SARS-CoV-2 infections, with <10% of infections diagnosed. Lower seroprevalence among persons living with HIV who are not virally suppressed, likely as a result of inadequate antibody production, highlights the need to prioritize this group for intervention.

Biomarkers of Endothelial Activation in Black South African HIV-Positive Subjects are Associated with Both High Viral Load and Low CD4 Counts.

The prevalence of cardiovascular death in the HIV-infected population is higher than in uninfected individuals. Growing evidence suggests that HIV infection itself is directly linked to endothelial activation and dysfunction. Therefore, the aim of this study was to investigate whether endothelial activation is present in African subjects with HIV infection and identify its possible determinants. Eighty HIV-infected treatment-naive cases, categorized into two groups based on CD4 count (38 subjects with CD4 count ≤350 cells/mm3 and 42 subjects with CD4 count >350 cells/mm3), were compared with 60 HIV-uninfected controls. A small subgroup of the HIV-infected participants (n = 13) were followed up for 18 months following initiation of antiretroviral therapy (ART). Anthropometric data, fasting lipid and glucose levels, viral load, and CD4 counts were measured as were serum levels of intercellular adhesion molecule-1 (ICAM-1), endothelial leukocyte adhesion molecule-1, vascular cell adhesion molecule-1 (VCAM-1), monocyte chemoattractant protein-1, von Willebrand factor (vWF), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8). The HIV-infected low CD4 group had higher levels of ICAM-1 (p < .05), VCAM-1 (p < .0005), TNF-α (p < .005), and vWF (p < .005), compared with the controls. In the HIV-infected cohort, VCAM-1 levels were negatively associated with CD4 counts (ß = -0.474; p < .0005), whereas vWF levels were positively associated with viral load (ß = 0.344; p < .01). Levels of ICAM-1 and VCAM-1 were reduced by ART (p < .05 vs. baseline for both), however, levels of IL-6, IL-8, and TNF-α increased (p < .005 vs. baseline for all). Endothelial activation and inflammation are evident in African ART-naive HIV-infected patients; the former is attenuated, and the latter is increased after 18 months of ART. In HIV-infected subjects, both immunological dysregulation and viral load are associated with biomarkers of endothelial activation.

Conditional Cash Transfers to Incentivize Tuberculosis Screening: Description of a Novel Strategy for Contact Investigation in Rural South Africa.

BACKGROUND: Providing incentives to screen close contacts for tuberculosis (TB) is an alternative to household-based contact investigation. We aimed to characterize patients and contexts where this incentive-based strategy might be preferred. METHODS: This is a secondary analysis of a cluster randomized trial of TB contact investigation in Limpopo District, South Africa, conducted between 2016 and 2020. Twenty-eight clinics were randomly allocated to household-based vs incentive-based contact investigation. In the incentive-based arm, index participants and contacts received transport reimbursement and incentives for TB screening and microbiological diagnosis of contacts. We estimated differences in mean number of contacts per index participant with household-based vs incentive-based contact investigation overall and within subgroups of index participants. RESULTS: A total of 3776 contacts (1903 in the incentive-based and 1873 in the household-based arm) were referred by 2501 index participants. A higher proportion of contacts in the incentive-based than household-based arm were adults (72% vs 59%), reported chronic TB symptoms (25% vs 16%) or ever smoking (23% vs 11%). Index participants who walked or bicycled to a clinic referred 1.03 more contacts per index (95% confidence interval [CI], .48 to 1.57) through incentive-based than household-based investigation. Index participants living with >5 household members referred 0.48 more contacts per index (95% CI, .03 to .94) through household-based than incentive-based investigation. CONCLUSIONS: Relative to household-based investigation, incentive-based investigation identifies contacts likely at higher risk for active TB. Incentive-based investigation may be more appropriate for index participants who can easily access clinics, versus household-based investigation for patients with large households. Clinical Trials Registration. NCT02808507.

Household Contact Tracing With Intensified Tuberculosis and Human Immunodeficiency Virus Screening in South Africa: A Cluster-Randomized Trial.

BACKGROUND: Household contact tracing for tuberculosis (TB) may facilitate diagnosis and access to TB preventive treatment (TPT). We investigated whether household contact tracing and intensive TB/human immunodeficiency virus (HIV) screening would improve TB-free survival. METHODS: Household contacts of index TB patients in 2 South African provinces were randomized to home tracing and intensive HIV/TB screening or standard of care (SOC; clinic referral letters). The primary outcome was incident TB or death at 15 months. Secondary outcomes included tuberculin skin test (TST) positivity in children ≤14 years and undiagnosed HIV. RESULTS: From December 2016 through March 2019, 1032 index patients (4459 contacts) and 1030 (4129 contacts) were randomized to the intervention and SOC arms. Of intervention arm contacts, 3.2% (69 of 2166) had prevalent microbiologically confirmed TB. At 15 months, the cumulative incidence of TB or death did not differ between the intensive screening (93 of 3230, 2.9%) and SOC (80 of 2600, 3.1%) arms (hazard ratio, 0.90; 95% confidence interval [CI], .66-1.24). TST positivity was higher in the intensive screening arm (38 of 845, 4.5%) compared with the SOC arm (15 of 800, 1.9%; odds ratio, 2.25; 95% CI, 1.07-4.72). Undiagnosed HIV was similar between arms (41 of 3185, 1.3% vs 32 of 2543, 1.3%; odds ratio, 1.02; 95% CI, .64-1.64). CONCLUSIONS: Household contact tracing with intensive screening and referral did not reduce incident TB or death. Providing referral letters to household contacts of index patients is an alternative strategy to home visits. CLINICAL TRIALS REGISTRATION: ISRCTN16006202.

CloneRetriever: An Automated Algorithm to Identify Clonal B and T Cell Gene Rearrangements by Next-Generation Sequencing for the Diagnosis of Lymphoid Malignancies.

BACKGROUND: Clonal immunoglobulin and T-cell receptor rearrangements serve as tumor-specific markers that have become mainstays of the diagnosis and monitoring of lymphoid malignancy. Next-generation sequencing (NGS) techniques targeting these loci have been successfully applied to lymphoblastic leukemia and multiple myeloma for minimal residual disease detection. However, adoption of NGS for primary diagnosis remains limited. METHODS: We addressed the bioinformatics challenges associated with immune cell sequencing and clone detection by designing a novel web tool, CloneRetriever (CR), which uses machine-learning principles to generate clone classification schemes that are customizable, and can be applied to large datasets. CR has 2 applications-a "validation" mode to derive a clonality classifier, and a "live" mode to screen for clones by applying a validated and/or customized classifier. In this study, CR-generated multiple classifiers using 2 datasets comprising 106 annotated patient samples. A custom classifier was then applied to 36 unannotated samples. RESULTS: The optimal classifier for clonality required clonal dominance ≥4.5× above background, read representation ≥8% of all reads, and technical replicate agreement. Depending on the dataset and analysis step, the optimal algorithm yielded sensitivities of 81%-90%, specificities of 97%-100%, areas under the curve of 91%-94%, positive predictive values of 92-100%, and negative predictive values of 88%-98%. Customization of the algorithms yielded 95%-100% concordance with gold-standard clonality determination, including rescue of indeterminate samples. Application to a set of unknowns showed concordance rates of 83%-96%. CONCLUSIONS: CR is an out-of-the-box ready and user-friendly software designed to identify clonal rearrangements in large NGS datasets for the diagnosis of lymphoid malignancies.

Systemic DPP4/CD26 is associated with natural HIV-1 control: Implications for COVID-19 susceptibility.

The current intersection of the COVID-19 and HIV-1 pandemics, has raised concerns about the risk for poor COVID-19 outcomes particularly in regions like sub-Saharan Africa, disproportionally affected by HIV. DPP4/CD26 has been suggested to be a potential therapeutic target and a biomarker for risk in COVID-19 patients with high risk co-morbidities. We therefore evaluated soluble DPP4 (sDPP4) levels and activity in plasma of 131 HIV-infected and 20 HIV-uninfected South African individuals. Flow cytometry was performed to compare cell surface expression of DPP4/CD26 and activation markers on peripheral blood mononuclear cells of extreme clinical phenotypes. Progressors had lower specific DPP4 activity and lower frequency of CD3+ T-cells expressing CD26 than HIV-1 controllers, but more activated CD3+CD26+ T-cells. The frequency of CD26-expressing T-cells negatively correlated with HLA-DR+ and CD38+ T-cells. Divergent DPP4/CD26 expression between HIV-1 controllers and progressors may have implications for risk and treatment of COVID-19 in people living with HIV.

Comparing rates of mycobacterial clearance in sputum smear-negative and smear-positive adults living with HIV.

BACKGROUND: Pulmonary tuberculosis (TB) in people living with HIV (PLH) frequently presents as sputum smear-negative. However, clinical trials of TB in adults often use smear-positive individuals to ensure measurable bacterial responses following initiation of treatment, thereby excluding HIV-infected patients from trials. METHODS: In this prospective case cohort study, 118 HIV-seropositive TB patients were assessed prior to initiation of standard four-drug TB therapy and at several time points through 35 days. Sputum bacillary load, as a marker of treatment response, was determined serially by: smear microscopy, Xpert MTB/RIF, liquid culture, and colony counts on agar medium. RESULTS: By all four measures, patients who were baseline smear-positive had higher bacterial loads than those presenting as smear-negative, until day 35. However, most smear-negative PLH had significant bacillary load at enrolment and their mycobacteria were cleared more rapidly than smear-positive patients. Smear-negative patients' decline in bacillary load, determined by colony counts, was linear to day 7 suggesting measurable bactericidal activity. Moreover, the decrease in bacterial counts was comparable to smear-positive individuals. Increasing cycle threshold values (Ct) on the Xpert assay in smear-positive patients to day 14 implied decreasing bacterial load. CONCLUSION: Our data suggest that smear-negative PLH can be included in clinical trials of novel treatment regimens as they contain sufficient viable bacteria, but allowances for late exclusions would have to be made in sample size estimations. We also show that increases in Ct in smear-positive patients to day 14 reflect treatment responses and the Xpert MTB/RIF assay could be used as biomarker for early treatment response.

Feasibility of Cell-Free DNA Collection and Clonal Immunoglobulin Sequencing in South African Patients With HIV-Associated Lymphoma.

PURPOSE: Diagnosis of AIDS lymphoma in low-resource settings, like South Africa, is often delayed, leaving patients with limited treatment options. In tuberculosis (TB) endemic regions, overlapping signs and symptoms often lead to diagnostic delays. Assessment of plasma cell-free DNA (cfDNA) by next-generation sequencing (NGS) may expedite the diagnosis of lymphoma but requires high-quality cfDNA. METHODS: People living with HIV with newly diagnosed aggressive B-cell lymphoma and those with newly diagnosed TB seeking care at Chris Hani Baragwanath Academic Hospital and its surrounding clinics, in Soweto, South Africa, were enrolled in this study. Each participant provided a whole blood specimen collected in cell-stabilizing tubes. Quantity and quality of plasma cfDNA were assessed. NGS of the immunoglobulin heavy chain was performed. RESULTS: Nine HIV+ patients with untreated lymphoma and eight HIV+ patients with TB, but without lymphoma, were enrolled. All cfDNA quantity and quality metrics were similar between the two groups, except that cfDNA accounted for a larger fraction of recovered plasma DNA in patients with lymphoma. The concentration of cfDNA in plasma also trended higher in patients with lymphoma. NGS of immunoglobulin heavy chain showed robust amplification of DNA, with large amplicons (> 250 bp) being more readily detected in patients with lymphoma. Clonal sequences were detected in five of nine patients with lymphoma, and none of the patients with TB. CONCLUSION: This proof-of-principle study demonstrates that whole blood collected for cfDNA in a low-resource setting is suitable for sophisticated sequencing analyses, including clonal immunoglobulin NGS. The detection of clonal sequences in more than half of patients with lymphoma shows promise as a diagnostic marker that may be explored in future studies.

Plasma Kynurenine-to-Tryptophan Ratio, a Highly Sensitive Blood-Based Diagnostic Tool for Tuberculosis in Pregnant Women Living With Human Immunodeficiency Virus (HIV).

BACKGROUND: For pregnant women living with human immunodeficiency virus (HIV), concurrent active tuberculosis (TB) disease increases the risk of maternal mortality and poor pregnancy outcomes. Plasma indoleamine 2,3-dioxygenase (IDO) activity measured by kynurenine-to-tryptophan (K/T) ratio has been proposed as a blood-based TB biomarker. We investigated whether plasma K/T ratio could be used to diagnose active TB among pregnant women with HIV. METHODS: Using an enzyme-linked immunosorbent assay (ELISA), we measured K/T ratio in 72 pregnant women with and active TB and compared them to 117 pregnant women with HIB but without TB, matched by age and gestational age. RESULTS: Plasma K/T ratio was significantly elevated during pregnancy compared to sampling done after pregnancy (P < .0001). Pregnant women who had received isoniazid preventive therapy (IPT) before enrollment had decreased plasma K/T ratio compared to those who had not received IPT (P = .0174). Plasma K/T ratio was elevated in women with active TB at time of diagnosis compared to those without TB (P < .0001). Using a cutoff of 0.100, plasma K/T ratio gave a diagnostic sensitivity of 94% (95% confidence interval [CI]: 82-95), specificity of 90% (95% CI: 80-91), positive predictive value (PPV) 85% and negative predictive value (NPV) 98%. A receiver operating characteristic curve (ROC) gave an area under the curve of 0.95 (95% CI: .92-.97, P < .0001).In conclusion, plasma K/T ratio is a sensitive blood-based diagnostic test for active TB disease in pregnant women living with HIV. Plasma K/T ratio should be further evaluated as an initial TB diagnostic test to determine its impact on patient care.