Search

Cytochrome b561 (cytb561) proteins comprise a family of transmembrane oxidoreductases that transfer single electrons across a membrane. Most eukaryotic species, including insects, possess multiple cytb561 homologs. To learn more about this protein family in insects, we carried out a bioinformatics-based investigation of cytb561 family members from nine species representing eight insect orders. We performed a phylogenetic analysis to classify insect cytb561 ortholog groups. We then conducted sequence analyses and analyzed protein models to predict structural elements that may impact the biological functions and localization of these proteins, with a focus on possible ferric reductase activity. Our study revealed three ortholog groups, designated CG1275, Nemy, and CG8399, and a fourth group of less-conserved genes. We found that CG1275 and Nemy proteins are similar to a human ferric reductase, duodenal cytochrome b561 (Dcytb), and have many conserved amino acid residues that function in substrate binding in Dcytb. Notably, CG1275 and Nemy proteins contain a conserved histidine and other residues that play a role in ferric ion reduction by Dcytb. Nemy proteins were distinguished by a novel cysteine-rich cytoplasmic loop sequence. CG8399 orthologs are similar to a putative ferric reductase in humans, stromal cell-derived receptor 2. Like other members of the CYBDOM class of cytb561 proteins, these proteins contain reeler, DOMON, and cytb561 domains. Drosophila melanogaster CG8399 is the only insect cytb561 with known ferric reductase activity. Our investigation of the DOMON domain in CG8399 proteins revealed a probable heme-binding site and a possible site for ferric reduction. The fourth group includes a subgroup of proteins with a conserved "KXXXXKXH" non-cytoplasmic loop motif that may be a substrate binding site and is present in a potential ferric reductase, human tumor suppressor cytochrome b561. This study provides a foundation for future investigations of the biological functions of cytb561 genes in insects., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Holst et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

BACKGROUND: Smoking of illicit drugs may lead to more rapid TB disease progression or late treatment presentation, yet research on this topic is scant. We examined the association between smoked drug use and bacterial burden among patients newly initiated on drug-susceptible TB (DS-TB) therapy. METHODS: Data from 303 participants initiating DS-TB treatment in the Western Cape Province, South Africa, were analyzed. Smoked drug use was defined as self-reported or biologically verified methamphetamine, methaqualone and/or cannabis use. Proportional hazard and logistic regression models (adjusted for age, sex, HIV status and tobacco use) examined associations between smoked drug use and mycobacterial time to culture positivity (TTP), acid-fast bacilli sputum smear positivity and lung cavitation. RESULTS: People who smoked drugs (PWSD) comprised 54.8% ( n = 166) of the cohort. TTP was faster for PWSD (hazard ratio 1.48, 95% CI 1.10-1.97; P = 0.008). Smear positivity was higher among PWSD (OR 2.28, 95% CI 1.22-4.34; P = 0.011). Smoked drug use (OR 1.08, 95% CI 0.62-1.87; P = 0.799) was not associated with increased cavitation. CONCLUSIONS: PWSD had a higher bacterial burden at diagnosis than those who do not smoke drugs. Screening for TB among PWSD in the community may facilitate earlier linkage to TB treatment and reduce community transmission.

Background: Vaccines are a strong public health tool to protect against severe disease, hospitalization, and death from COVID-19. Still, inequities in COVID-19 vaccination rates and health outcomes continue to exist among Black and Latino populations. Boston Medical Center (BMC) has played a significant role in vaccinating medically underserved populations, and organized a series of community-engaged conversations to better understand community concerns regarding the COVID-19 vaccine. This paper describes the themes which resulted from these community-engaged conversations and proposes next steps for healthcare leaders., Methods: We accessed nine publicly available recordings of the community-engaged conversations which were held between March 2021 and September 2021 and ranged from 8 to 122 attendees. Six conversations prioritized specific groups: the Haitian-Creole community, the Cape Verdean community, the Latino community, the Black Christian Faith community, guardians who care for children living with disabilities, and individuals affected by systemic lupus erythematosus. Remaining conversations targeted the general public of the Greater Boston Area. We employed a Consolidated Framework for Implementation Research-driven codebook to code our data. Our analysis utilized a modified version of qualitative rapid analysis methods., Results: Five main themes emerged from these community-engaged conversations: (1) Structural factors are important barriers to COVID-19 vaccination; (2) Mistrust exists due to the negative impact of systemic oppression and perceived motivation of the government; (3) There is a desire to learn more about biological and clinical characteristics of the COVID-19 vaccine as well as the practical implications of being vaccinated; (4) Community leaders emphasize community engagement for delivering COVID-19 information and education and; (5) Community leaders believe that the COVID-19 vaccine is a solution to address the pandemic., Conclusion: This study illustrates a need for community-engaged COVID-19 vaccine messaging which reflects the nuances of the COVID-19 vaccine and pandemic without oversimplifying information. In highlighting common concerns of the Greater Boston Area which contribute to a lack of confidence in the COVID-19 vaccine, we underscore important considerations for public health and healthcare leadership in the development of initiatives which work to advance health equity., (© 2023. The Author(s).)

Background: Throughout the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, healthcare workers (HCWs) have faced risk of infection from within the workplace via patients and staff as well as from the outside community, complicating our ability to resolve transmission chains in order to inform hospital infection control policy. Here we show how the incorporation of sequences from public genomic databases aided genomic surveillance early in the pandemic when circulating viral diversity was limited., Methods: We sequenced a subset of discarded, diagnostic SARS-CoV-2 isolates between March and May 2020 from Boston Medical Center HCWs and combined this data set with publicly available sequences from the surrounding community deposited in GISAID with the goal of inferring specific transmission routes., Results: Contextualizing our data with publicly available sequences reveals that 73% (95% confidence interval, 63%-84%) of coronavirus disease 2019 cases in HCWs are likely novel introductions rather than nosocomial spread., Conclusions: We argue that introductions of SARS-CoV-2 into the hospital environment are frequent and that expanding public genomic surveillance can better aid infection control when determining routes of transmission., Competing Interests: Potential conflicts of interest. W. P. H. is a member of Biobot Analytics’ scientific advisory board. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Transferrin-1 (Tsf1) is an extracellular insect protein with a high affinity for iron. The functions of Tsf1 are still poorly understood; however, Drosophila melanogaster Tsf1 has been shown to influence iron distribution in the fly body and to protect flies against some infections. The goal of this study was to better understand the physiological functions of Tsf1 in D. melanogaster by 1) investigating Tsf1 null phenotypes, 2) determining tissue-specific localization of Tsf1, 3) measuring the concentration of Tsf1 in hemolymph, 4) testing Tsf1 for bacteriostatic activity, and 5) evaluating the effect of metal and paraquat treatments on Tsf1 abundance. Flies lacking Tsf1 had more iron than wild-type flies in specialized midgut cells that take up iron from the diet; however, the absence of Tsf1 had no effect on the iron content of whole midguts, fat body, hemolymph, or heads. Thus, as previous studies have suggested, Tsf1 appears to have a minor role in iron transport. Tsf1 was abundant in hemolymph from larvae (0.4 μM), pupae (1.4 μM), adult females (4.4 μM) and adult males (22 μM). Apo-Tsf1 at 1 μM had bacteriostatic activity whereas holo-Tsf1 did not, suggesting that Tsf1 can inhibit microbial growth by sequestering iron in hemolymph and other extracellular environments. This hypothesis was supported by detection of secreted Tsf1 in tracheae, testes and seminal vesicles. Colocalization of Tsf1 with an endosome marker in oocytes suggested that Tsf1 may provide iron to developing eggs; however, eggs from mothers lacking Tsf1 had the same amount of iron as control eggs, and they hatched at a wild-type rate. Thus, the primary function of Tsf1 uptake by oocytes may be to defend against infection rather than to provide eggs with iron. In beetles, Tsf1 plays a role in protection against oxidative stress. In contrast, we found that flies lacking Tsf1 had a typical life span and greater resistance to paraquat-induced oxidative stress. In addition, Tsf1 abundance remained unchanged in response to ingestion of iron, cadmium or paraquat or to injection of iron. These results suggest that Tsf1 has a limited role in protection against oxidative stress in D. melanogaster., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Objective: To quantify the proportion of people living with HIV (PLWH) with other tuberculosis (TB) risk factors that completed the latent tuberculosis infection (LTBI) care cascade and describe factors associated with attrition. The care cascade was defined as follows: (1) receipt of an LTBI test and result, (2) initiation of LTBI treatment and (3) completion of LTBI treatment., Design: Prospective cohort study., Setting: Reactivation of LTBI remains a large source of active TB disease in the USA. PLWH and those who use substances are at greater risk and are harder to engage and retain in care., Participants: Participants enrolled in a Boston cohort of PLWH from 2012 to 2014., Primary and Secondary Outcome Measures: Our primary outcome was the number and proportion of participants who completed each stage of the cascade and the factors associated with completing each stage. Our secondary outcomes were differences between participants tested with an interferon gamma release assay (IGRA) versus tuberculin skin test and differences between participants who tested positive versus negative for LTBI., Results: Only 189 of 219 (86.3%) participants completed testing. Five of the 11 with LTBI initiated and three completed treatment. Participants tested with an IGRA were more likely to complete testing (OR 3.87, 95% CI 1.05 to 14.30) while among participants successfully tested, being foreign-born was associated with a positive test result (OR 3.95; 95% CI 1.13 to 13.77)., Conclusions: Although the majority completed LTBI testing, our findings warrant further investigation in a larger cohort to better understand factors that lead to suboptimal treatment initiation and completion in a low-burden country., Competing Interests: Competing interests: RS reports that Alkermes provides medication for a comparative effectiveness study funded by NIAAA/NIH for which he is the principal investigator. Authors report no other conflicts., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Biorepositories provide a critical resource for gaining knowledge of emerging infectious diseases and offer a mechanism to rapidly respond to outbreaks; the emergence of the novel coronavirus, SARS-CoV-2, has proved their importance. During the COVID-19 pandemic, the absence of centralized, national biorepository efforts meant that the onus fell on individual institutions to establish sample repositories. As a safety-net hospital, Boston Medical Center (BMC) recognized the importance of creating a COVID-19 biorepository to both support critical science at BMC and ensure representation in research for its urban patient population, most of whom are from underserved communities. This article offers a realistic overview of the authors' experience in establishing this biorepository at the onset of the COVID-19 pandemic during the height of the first surge of cases in Boston, Massachusetts, with the hope that the challenges and solutions described are useful to other institutions. Going forward, funders, policymakers, and infectious disease and public health communities must support biorepository implementation as an essential element of future pandemic preparedness.

Background: Coronavirus disease 2019 (COVID-19) vaccine trials and post-implementation data suggest that vaccination decreases infections. We examine vaccination's impact on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) case rates and viral diversity among health care workers (HCWs) during a high community prevalence period., Methods: In this prospective cohort study, HCW received 2 doses of BNT162b2 or mRNA-1273. We included confirmed cases among HCWs from 9 December 2020 to 23 February 2021. Weekly SARS-CoV-2 rates per 100,000 person-days and by time from first injection (1-14 and ≥15 days) were compared with surrounding community rates. Viral genomes were sequenced., Results: SARS-CoV-2 cases occurred in 1.4% (96/7109) of HCWs given at least a first dose and 0.3% (17/5913) of HCWs given both vaccine doses. Adjusted rate ratios (95% confidence intervals) were 0.73 (.53-1.00) 1-14 days and 0.18 (.10-.32) ≥15 days from first dose. HCW ≥15 days from initial dose compared to 1-14 days were more often older (46 vs 38 years, P = .007), Latinx (10% vs 8%, P = .03), and asymptomatic (48% vs 11%, P = .0002). SARS-CoV-2 rates among HCWs fell below the surrounding community, an 18% vs 11% weekly decrease, respectively ( P = .14). Comparison of 50 genomes from post-first dose cases did not indicate selection pressure toward known spike antibody escape mutations., Conclusions: Our results indicate an early positive impact of vaccines on SARS-CoV-2 case rates. Post-vaccination isolates did not show unusual genetic diversity or selection for mutations of concern., (© The Author(s) 2021. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: A major challenge for prospective, clinical tuberculosis (TB) research is accurately defining a metric for measuring medication adherence., Objective: We aimed to design a method to capture directly observed therapy (DOT) via mobile health carried out by community workers. The program was created specifically to measure TB medication adherence for a prospective TB cohort in Western Cape Province, South Africa., Methods: Community workers collect daily adherence data on mobile smartphones. Participant-level adherence, program-level adherence, and program function are systematically monitored to assess DOT program implementation. A data dashboard allows for regular visualization of indicators. Numerous design elements aim to prevent or limit data falsification and ensure study data integrity., Results: The cohort study is ongoing and data collection is in progress. Enrollment began on May 16, 2017, and as of January 12, 2021, a total of 236 participants were enrolled. Adherence data will be used to analyze the study's primary aims and to investigate adherence as a primary outcome., Conclusions: The DOT program includes a mobile health application for data collection as well as a monitoring framework and dashboard. This approach has potential to be adapted for other settings to improve the capture of medication adherence in clinical TB research., Trial Registration: Clinicaltrials.gov NCT02840877; https://clinicaltrials.gov/ct2/show/NCT02840877., (©Elizabeth J Ragan, Christopher J Gill, Matthew Banos, Tara C Bouton, Jennifer Rooney, Charles R Horsburgh, Robin M Warren, Bronwyn Myers, Karen R Jacobson. Originally published in JMIR Research Protocols (https://www.researchprotocols.org), 16.06.2021.)

Background: South Africa temporarily banned alcohol and tobacco sales for about 20 weeks during the COVID-19 lockdown. We described changes in alcohol and tobacco consumption after implementation of these restrictions among a small number of participants in a tuberculosis treatment cohort., Method: The timeline follow-back procedure and Fägerstrom test for nicotine dependence was used to collect monthly alcohol and tobacco use information. We report changes in heavy drinking days (HDD), average amount of absolute alcohol (AA) consumed per drinking day, and cigarettes smoked daily during the alcohol and tobacco ban compared to use prior to the ban., Results: Of the 61 participants for whom we have pre-ban and within-ban alcohol use information, 17 (27.9%) reported within-ban alcohol use. On average, participants reported one less HDD per fortnight (interquartile range (IQR): -4, 1), but their amount of AA consumed increased by 37.4 g per drinking occasion (IQR: -65.9 g, 71.0 g). Of 53 participants who reported pre-ban tobacco use, 17 (32.1%) stopped smoking during the ban. The number of participants smoking >10 cigarettes per day decreased from 8 to 1., Conclusions: From these observations, we hypothesize that policies restricting alcohol and tobacco availability seem to enable some individuals to reduce their consumption. However, these appear to have little effect on the volume of AA consumed among individuals with more harmful patterns of drinking in the absence of additional behavior change interventions., Competing Interests: The authors declare no conflicts of interest.

Healthcare workers (HCWs) are at an increased risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel virus that causes Coronavirus Disease (COVID-19). We aim to assess the seroprevalence of SARS-CoV-2 IgG among healthcare workers and compare risk-factors between seropositive and seronegative HCWs. In this observational study, serum samples were collected from HCWs between July 13th to 26th, 2020 at Boston Medical Center (BMC). Samples were subsequently tested for SARS-CoV-2 IgG antibody using the Abbott SARS-CoV-2 IgG assay. Participants also answered a questionnaire capturing data on demographics, history of COVID-19 symptoms, occupation, infection prevention and control measures. Overall, 95 of 1743 (5.5%) participants tested positive for SARS-CoV-2 IgG. Of these, 1.8% of the participants had mild or no COVID-19 symptoms and did not require a diagnostic test. Seropositivity was not associated with gender, occupation, hand hygiene and personal protective equipment (PPE) practices amongst HCWs. However, lack of physical distancing among health care workers in work areas and break room was associated with seropositivity (p = 0.05, p = 0.003, respectively). The majority of the HCWs are negative for SARS-CoV-2 IgG. This data highlights the need to promote infection prevention measures, and the importance of distance amongst co-workers to help mitigate infection rates.

In 2011, South Africa implemented a policy to decentralize treatment for rifampin-resistant tuberculosis (TB) to reduce durations of hospitalization and enable local treatment. We assessed policy implementation in Western Cape Province, where services expanded from 6 specialized TB hospitals to 406 facilities, by analyzing National Health Laboratory Service data on TB during 2012-2015. We calculated the percentage of patients who visited a TB hospital <1 year after rifampin-resistant TB diagnosis, the median duration of their hospitalizations, and the total distance between facilities visited. We assessed temporal changes with linear regression and stratified results by location. Of 2,878 patients, 65% were from Cape Town. In Cape Town, 29% visited a TB hospital; elsewhere, 68% visited a TB hospital. We found that hospitalizations and travel distances were shorter in Cape Town than in the surrounding areas.

Setting: Alcohol use increases the risk of tuberculosis (TB) disease and is associated with worse outcomes., Objective: To determine whether alcohol use affects TB severity at diagnosis in a high-burden setting., Design: Participants were smear-positive people living with TB (PLWTB) in India. Disease severity was assessed as 1) high versus low smear grade, 2) time to positivity (TTP) on liquid culture, 3) chest radiograph cavitation, and 4) percent lung affected. Alcohol use and being at-risk for alcohol use disorders (AUD) were assessed using the AUDIT-C. Univariable and multivariable analyses were conducted., Results: Of 1166 PLWTB, 691 (59.3%) were drinkers; of those, 518/691 (75.0%) were at-risk for AUD. Drinkers had more lung affected than non-drinkers (adjusted mean difference 10.8%, p<0.0001); this was not significant for those at-risk for AUD (adjusted mean difference 3.7%, p = 0.11). High smear grade (aOR 1.0, 95%CI: 0.7-1.4), cavitation (aOR 0.8, 95%CI 0.4-1.8), and TTP (mean difference 5.2 hours, p = 0.51) did not differ between drinkers and non-drinkers, nor between those at-risk and not at-risk for AUD., Conclusions: A large proportion of PLWTB were drinkers and were at-risk for AUD. Alcohol drinkers had more lung affected than non-drinkers. Studies are needed to explore mechanisms of this association., Competing Interests: The authors have declared that no competing interests exist.

Alcohol use is associated with increased risk of developing tuberculosis (TB) disease, yet the impact of alcohol use on TB treatment outcomes has not been summarized. We aimed to quantitatively review evidence of the relationship between alcohol use and poor TB treatment outcomes. We conducted a systematic review of PubMed, EMBASE, and Web of Science (January 1980-May 2018). We categorized studies as having a high- or low-quality alcohol use definition and examined poor treatment outcomes individually and as two aggregated definitions (i.e., including or excluding loss to follow-up [LTFU]). We analyzed drug-susceptible (DS-) and multidrug-resistant (MDR-) TB studies separately. Our systematic review yielded 111 studies reporting alcohol use as a predictor of DS- and MDR-TB treatment outcomes. Alcohol use was associated with increased odds of poor treatment outcomes (i.e., death, treatment failure, and LTFU) in DS (OR 1.99, 95% CI 1.57-2.51) and MDR-TB studies (OR 2.00, 95% CI 1.73-2.32). This association persisted for aggregated poor treatment outcomes excluding LTFU, each individual poor outcome, and across sub-group and sensitivity analyses. Only 19% of studies used high-quality alcohol definitions. Alcohol use significantly increased the risk of poor treatment outcomes in both DS- and MDR-TB patients. This study highlights the need for improved assessment of alcohol use in TB outcomes research and potentially modified treatment guidelines for TB patients who consume alcohol.

South Africa led the world with guidelines on bedaquiline (BDQ) use as a single drug substitution to manage rifampin resistant tuberculosis regimen toxicity. We examined reasons for giving BDQ in a retrospective cohort: >75% of patients were switched to BDQ for toxicity (ototoxicity or renal dysfunction) rather than drug resistance., Competing Interests: The authors have declared that no competing interests exist.

Background: In many low and middle-income countries (LMICs), difficulties in patient identification are a major obstacle to the delivery of longitudinal care. In absence of unique identifiers, biometrics have emerged as an attractive solution to the identification problem. We developed an mHealth App for subject identification using pattern recognition around ear morphology (Project SEARCH (Scanning EARS for Child Health). Early field work with the SEARCH App revealed that image stabilization would be required for optimum performance., Methods: To improve image capture, we designed and tested a device (the 'Donut'), which standardizes distance, angle, rotation and lighting. We then ran an experimental trial with 194 participants to measure the impact of the Donut on identification rates. Images of the participant's left ear were taken both with and without use of the Donut, then processed by the SEARCH algorithm, measuring the top one and top ten most likely matches., Results: With the Donut, the top one identification rate and top ten identification rates were 99.5 and 99.5%, respectively, vs. 38.4 and 24.1%, respectively, without the Donut (P < 0.0001 for each comparison). In sensitivity analyses, crop technique during pre-processing of images had a powerful impact on identification rates, but this too was facilitated through the Donut., Conclusions: By standardizing lighting, angle and spatial location of the ear, the Donut achieved near perfect identification rates on a cohort of 194 participants, proving the feasibility and effectiveness of using the ear as a biometric identifier., Trial Registration: This study did not include a medical intervention or assess a medical outcome, and therefore did not meet the definition of a human subjects research study as defined by FDAAA. We did not register our study under clinicaltrials.gov .

Background: Mycobacterium tuberculosis (TB) infection induces systemic inflammation that could impact HIV-1 persistence., Setting: HIV-1-seropositive individuals either with or without pulmonary TB disease were recruited in Kampala, Uganda., Methods: Plasma cytokines, HIV-1 DNA, and cell-associated (ca)-RNA were compared among those coinfected with TB (cases) to those without TB (controls). TB-coinfected cases and controls were compared at presentation (n = 15 and n = 16, respectively) and at around 6 months after HIV-1 treatment initiation among those who had achieved virologic suppression (n = 6 and n = 8, respectively). At follow-up, the TB-coinfected cases had also finished TB treatment., Results: Before treatment, the TB-coinfected cases as compared to the controls had higher levels of soluble(s)-CD163 (P = 0.0002) and interleukin-6 (P = 0.006) but lower levels of macrophage chemoattractant protein-1 (P = 0.04). After treatment, the TB-coinfected cases as compared to controls still had higher plasma s-CD163 levels (P = 0007). Controls as compared to the coinfected cases had higher ca-RNA per DNA template both at baseline (P = 0.03) and at follow-up (P = 0.07). Levels of ca-RNA per DNA copy at follow-up showed a negative correlation with baseline plasma s-CD163 (P = 0.008) and interleukin-6 (P = 0.05) levels., Conclusions: TB disease is associated with inflammation and decreased HIV-1 RNA expression relative to the number of infected cells, both before and after viral suppression. Infections present before antiretroviral initiation impact HIV-1 latency.

Background: An estimated 10% of tuberculosis (TB) deaths are attributable to problematic alcohol use globally, however the causal pathways through which problem alcohol use has an impact on TB treatment outcome is not clear. This study aims to improve understanding of these mechanisms. Specifically, we aim to 1) assess whether poor TB treatment outcomes, measured as delayed time-to-culture conversion, are associated with problem alcohol use after controlling for non-adherence to TB pharmacotherapy; and 2) to determine whether pharmacokinetic (PK) changes in those with problem alcohol use are associated with delayed culture conversion, higher treatment failure/relapse rates or with increased toxicity., Methods: Our longitudinal, repeated measures, prospective cohort study aims to examine the associations between problem alcohol use and TB treatment outcomes and to evaluate the effect of alcohol on the PK and pharmacodynamics (PD) of TB drugs. We will recruit 438 microbiologically confirmed, pulmonary TB patients with evidence of rifampicin susceptibility in Worcester, South Africa with 200 HIV uninfected patients co-enrolled in the PK aim. Participants are followed for the six months of TB treatment and an additional 12 months thereafter, with sputum collected weekly for the first 12 weeks of treatment, alcohol consumption measures repeated monthly in concert with an alcohol biomarker (phosphatidylethanol) measurement at baseline, and in person directly observed therapy (DOT) using real-time mobile phone-based adherence monitoring. The primary outcome is based on time to culture conversion with the second objective to compare PK of first line TB therapy in those with and without problem alcohol use., Discussion: Globally, an urgent need exists to identify modifiable drivers of poor TB treatment outcomes. There is a critical need for more effective TB treatment strategies for patients with a history of problem alcohol use. However, it is not known whether poor treatment outcomes in alcohol using patients are solely attributable to noncompliance. This study will attempt to answer this question and provide guidance for future TB intervention trials., Trial Registration: Clinicaltrials.gov Registration Number: NCT02840877 . Registered on 19 July 2016.

Background: South Africa has the highest tuberculosis incidence globally (781/100,000), with an estimated 4.3% of cases being rifampicin resistant (RR). Control and elimination strategies will require detailed spatial information to understand where drug-resistant tuberculosis exists and why it persists in those communities. We demonstrate a method to enable drug-resistant tuberculosis monitoring by identifying high-burden communities in the Western Cape Province using routinely collected laboratory data., Methods and Findings: We retrospectively identified cases of microbiologically confirmed tuberculosis and RR-tuberculosis from all biological samples submitted for tuberculosis testing (n = 2,219,891) to the Western Cape National Health Laboratory Services (NHLS) between January 1, 2008, and June 30, 2013. Because the NHLS database lacks unique patient identifiers, we performed a series of record-linking processes to match specimen records to individual patients. We counted an individual as having a single disease episode if their positive samples came from within two years of each other. Cases were aggregated by clinic location (n = 302) to estimate the percentage of tuberculosis cases with rifampicin resistance per clinic. We used inverse distance weighting (IDW) to produce heatmaps of the RR-tuberculosis percentage across the province. Regression was used to estimate annual changes in the RR-tuberculosis percentage by clinic, and estimated average size and direction of change was mapped. We identified 799,779 individuals who had specimens submitted from mappable clinics for testing, of whom 222,735 (27.8%) had microbiologically confirmed tuberculosis. The study population was 43% female, the median age was 36 years (IQR 27-44), and 10,255 (4.6%, 95% CI: 4.6-4.7) cases had documented rifampicin resistance. Among individuals with microbiologically confirmed tuberculosis, 8,947 (4.0%) had more than one disease episode during the study period. The percentage of tuberculosis cases with rifampicin resistance documented among these individuals was 11.4% (95% CI: 10.7-12.0). Overall, the percentage of tuberculosis cases that were RR-tuberculosis was spatially heterogeneous, ranging from 0% to 25% across the province. Our maps reveal significant yearly fluctuations in RR-tuberculosis percentages at several locations. Additionally, the directions of change over time in RR-tuberculosis percentage were not uniform. The main limitation of this study is the lack of unique patient identifiers in the NHLS database, rendering findings to be estimates reliant on the accuracy of the person-matching algorithm., Conclusions: Our maps reveal striking spatial and temporal heterogeneity in RR-tuberculosis percentages across this province. We demonstrate the potential to monitor RR-tuberculosis spatially and temporally with routinely collected laboratory data, enabling improved resource targeting and more rapid locally appropriate interventions., Competing Interests: MBM is a member of the Editorial Board of PLOS Medicine. The authors declare no other competing interests exist.

Background.: Xpert MTB/RIF (Xpert) detects rifampicin-resistant tuberculosis (RR-tuberculosis), enabling physicians to rapidly initiate a World Health Organization-recommended 5-drug regimen while awaiting second-line drug-susceptibility test (DST) results. We quantified the second-line DST results time and proportion of patients potentially placed on suboptimal therapy., Methods.: We included RR-tuberculosis patients detected using Xpert at the South African National Health Laboratory Services (NHLS) of the Western Cape between November 2011 and June 2013 and at Eastern Cape, Free State, and Gauteng NHLS between November 2012 and December 2013. We calculated time from specimen collection to phenotypic second-line DST results. We identified isoniazid and ethionamide resistance mutations on line probe assay and performed pyrazinamide sequencing., Results.: Among 1332 RR-tuberculosis patients, only 44.7% (596) had second-line DST for both fluoroquinolones and second-line injectable: 55.8% (466 of 835) in the Western Cape and 26.2% (130 of 497) in the other provinces. Patients with smear negative disease and age ≤10 years were less likely to have a result (risk ratio [RR] = 0.72; 95% CI, 0.64-0.81 and RR = 0.49; 95% CI, 0.26-0.79). Median time to second-line DST was 53 days (range, 8-259). Of the 252 patients with complete second-line DST, 101 (40.1%) potentially initiated a suboptimal regimen: 46.8% in the Western Cape and 25.3% in the other provinces., Conclusions.: Many South Africans diagnosed with RR-tuberculosis by Xpert initiate a suboptimal regimen, with information to adjust therapy available in half of all patients after a median 7 weeks. Algorithm completion and time delays remain challenging., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: One of the greatest public health challenges in low- and middle-income countries (LMICs) is identifying people over time and space. Recent years have seen an explosion of interest in developing electronic approaches to addressing this problem, with mobile technology at the forefront of these efforts. We investigate the possibility of biometrics as a simple, cost-efficient, and portable solution. Common biometrics approaches include fingerprinting, iris scanning and facial recognition, but all are less than ideal due to complexity, infringement on privacy, cost, or portability. Ear biometrics, however, proved to be a unique and viable solution., Methods: We developed an identification algorithm then conducted a cross sectional study in which we photographed left and right ears from 25 consenting adults. We then conducted re-identification and statistical analyses to identify the accuracy and replicability of our approach., Results: Through principal component analysis, we found the curve of the ear helix to be the most reliable anatomical structure and the basis for re-identification. Although an individual ear allowed for high re-identification rate (88.3%), when both left and right ears were paired together, our rate of re-identification amidst the pool of potential matches was 100%., Conclusions: The results of this study have implications on future efforts towards building a biometrics solution for patient identification in LMICs. We provide a conceptual platform for further investigation into the development of an ear biometrics identification mobile application.

The innate immune system of insects include the Toll pathway, which is mediated by an extracellular serine proteinase cascade. In the tobacco hornworm, Manduca sexta, hemolymph proteinase 8 (HP8) promotes the synthesis of antimicrobial proteins by cleaving proSpätzle, the putative ligand of M. sexta Toll. HP8 has been observed to form a complex in hemolymph with M. sexta serpin-1, which has multiple alternative splicing isoforms. To investigate the regulation of HP8 and its processing of proSpätzle, we characterized the interaction of recombinant HP8 with serpin-1 isoform J (serpin-1J). Recombinant serpin-1J formed an SDS-stable complex with HP8 in vitro. The association rate constant of serpin-1J and HP8 was 1.3×10(4)M(-1)s(-1), with a stoichiometry of inhibition of 5.4. Serpin-1J inhibited the cleavage of proSpätzle by HP8. Injection of serpin-1J into M. sexta larvae resulted in decreased bacteria-induced antimicrobial activity in hemolymph and reduced expression of cecropin, attacin and hemolin mRNA in fat body. Altogether, these results suggest that serpin-1J functions to inhibit HP8 and thereby modulates the concentration of active Spätzle to regulate the Toll pathway response in M. sexta., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Leucine-rich repeat containing proteins are involved in immune response in many capacities. In insects, these include Toll-like receptors and the Anopheles gambiae proteins APL1 and LRIM1. Here we describe the identification and characterization of leureptin, a novel extracellular protein with 13 leucine-rich repeats from hemolymph of the insect Manduca sexta. After injection of bacteria, leureptin mRNA level increased in fat body, but protein levels in plasma decreased, an indication that leureptin is consumed during the immune response. Leureptin bound to bacterial lipopolysaccharide (LPS). Microscopy using leureptin antiserum showed that leureptin associates with hemocytes after injection of bacteria, an indication that leureptin is involved in hemocyte responses to bacterial infection. Sequence database searches suggest similar proteins are present in other Lepidopteran species., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Mutually exclusive alternative splicing produces transcripts for 12 serpin-1 isoforms in Manduca sexta that differ only in the region encoding the carboxyl-terminal 36-40-amino acid residues. This variable region includes the reactive center loop, which determines the inhibitory selectivity of the serpin. We investigated mRNA levels of individual serpin-1 isoforms by quantitative PCR. The 12 isoforms were expressed at similar levels in hemocytes, but in fat body isoform B mRNA was present at significantly higher levels than isoforms C, D, E, F, G, J, K, and Z. To investigate the presence of individual serpin-1 isoforms in plasma we used immunoaffinity purification of serpin-1 isoforms from M. sexta plasma, followed by two-dimensional PAGE and identification of protein spots by digestion with a series of proteinases and analysis of the resulting peptides by MALDI-TOF/TOF. We identified nine of the 12 serpin-1 isoforms and, through analysis of putative serpin-1-proteinase complexes, identified three endogenous M. sexta proteinase targets of serpin-1. Our results suggest that M. sexta serpin-1 isoforms A, E, and J can inhibit hemolymph proteinase 8, which activates the cytokine spätzle. At least one isoform of serpin-1 can inhibit hemocyte proteinase 1, another M. sexta blood proteinase. In addition, a complex of serpin-1K in a complex with M. sexta midgut chymotrypsin was identified, suggesting serpin-1 isoforms may also function to protect insect tissues from digestive proteinases that may leak into the hemocoel.

Serine proteinases in insect plasma have been implicated in two types of immune responses; that is, activation of prophenoloxidase (proPO) and activation of cytokine-like proteins. We have identified more than 20 serine proteinases in hemolymph of the tobacco hornworm, Manduca sexta, but functions are known for only a few of them. We report here functions of two additional M. sexta proteinases, hemolymph proteinases 6 and 8 (HP6 and HP8). HP6 and HP8 are each composed of an amino-terminal clip domain and a carboxyl-terminal proteinase domain. HP6 is an apparent ortholog of Drosophila Persephone, whereas HP8 is most similar to Drosophila and Tenebrio spätzle-activating enzymes, all of which activate the Toll pathway. proHP6 and proHP8 are expressed constitutively in fat body and hemocytes and secreted into plasma, where they are activated by proteolytic cleavage in response to infection. To investigate activation and biological activity of HP6 and HP8, we purified recombinant proHP8, proHP6, and mutants of proHP6 in which the catalytic serine was replaced with alanine, and/or the activation site was changed to permit activation by bovine factor Xa. HP6 was found to activate proPO-activating proteinase (proPAP1) in vitro and induce proPO activation in plasma. HP6 was also determined to activate proHP8. Active HP6 or HP8 injected into larvae induced expression of antimicrobial peptides and proteins, including attacin, cecropin, gloverin, moricin, and lysozyme. Our results suggest that proHP6 becomes activated in response to microbial infection and participates in two immune pathways; activation of PAP1, which leads to proPO activation and melanin synthesis, and activation of HP8, which stimulates a Toll-like pathway.