B and T Cell Responses to SARS-CoV-2 Vaccination in Kidney and Liver Transplant Recipients with and without Previous COVID-19.

(1) Background: Vulnerable populations including transplant recipients are jeopardised by COVID-19. Herein, we report on B and T cell responses among liver and kidney organ recipients at our centre. (2) Methods: 23 liver and 45 kidney (14 thereof combined kidney/pancreas) transplanted patients were vaccinated with two doses of BNT162b2 followed by a booster dose of mRNA-1273 in 28 non-responders 4 months thereafter. Anti-SARS-CoV-2-Ig was measured by specific ELISA and virus neutralisation assay; T cell responses were measured by a spike protein-specific IFN-γ release assay. (3) Results: Compared to controls, B and T cell responses were weak in transplant recipients, particularly in those without prior exposure to SARS-CoV-2. Within this group, only 15% after the first and 58.3% after the second vaccination achieved seroconversion. A total of 14 out of 28 vaccination non-responders achieved a seroconversion after a third dose. Vaccination side effects were more frequent in healthy controls. The use of mycophenolate was associated with reduced anti-SARS-CoV-2-Ig production. (4) Conclusions: Our data confirm that vaccination responses are insufficient after standard vaccination in liver and kidney transplant recipients and are affected to a variable degree by specific immunosuppressants, particularly mycophenolate. Monitoring vaccination success and re-vaccinating those who are unresponsive seems prudent to achieve sufficient titres. Overall, prospective large-scale, multinational, multicentre studies or high-quality meta-analyses will be needed to generate personalised vaccination strategies in order to achieve protective immunity in high-risk, hard-to-immunize populations.

Transcriptomic Profiling Identifies CD8+ T Cells in the Brain of Aged and Alzheimer's Disease Transgenic Mice as Tissue-Resident Memory T Cells.

Peripheral immune cell infiltration into the brain is a prominent feature in aging and various neurodegenerative diseases such as Alzheimer's disease (AD). As AD progresses, CD8+ T cells infiltrate into the brain parenchyma, where they tightly associate with neurons and microglia. The functional properties of CD8+ T cells in the brain are largely unknown. To gain further insights into the putative functions of CD8+ T cells in the brain, we explored and compared the transcriptomic profile of CD8+ T cells isolated from the brain and blood of transgenic AD (APPswe/PSEN1dE9, line 85 [APP-PS1]) and age-matched wild-type (WT) mice. Brain CD8+ T cells of APP-PS1 and WT animals had similar transcriptomic profiles and substantially differed from blood circulating CD8+ T cells. The gene signature of brain CD8+ T cells identified them as tissue-resident memory (Trm) T cells. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analysis on the significantly upregulated genes revealed overrepresentation of biological processes involved in IFN-ß signaling and the response to viral infections. Furthermore, brain CD8+ T cells of APP-PS1 and aged WT mice showed similar differentially regulated genes as brain Trm CD8+ T cells in mouse models with acute virus infection, chronic parasite infection, and tumor growth. In conclusion, our profiling of brain CD8+ T cells suggests that in AD, these cells exhibit similar adaptive immune responses as in other inflammatory diseases of the CNS, potentially opening the door for immunotherapy in AD.

Identification of IL-10 competent B cells in swine.

Progress in the phenotypic characterisation of porcine B cells is ongoing, with recent advances in the identification of B1 cell subsets and plasma cells. However, regulatory B cells, commonly identified by interleukin (IL)-10 production, have not been studied in pigs so far. Here we investigate IL-10 expression in B cell subsets in response to CpG-oligodeoxynucleotides, phorbol 12-myristate 13-acetate and ionomycin stimulation in vitro. Our results reflect similar findings in human and mice. We identify a small subset of IL-10 competent B cells, present within both porcine B1 and B2 cell subsets across blood, spleen, mediastinal lymph nodes and lung tissue, with varied differentiation statuses. The capacity for IL-10 production coincided with CD95 expression, suggesting an activated phenotype of IL-10 competent B cells. These findings support the emerging paradigm that B cell IL-10 production is a function of various B cell subsets influenced by activation history and microenvironmental factors.

Identification of CD4+ T cells with T follicular helper cell characteristics in the pig.

T follicular helper (Tfh) cells provide help to germinal center B cells for affinity maturation, class switch and memory formation. Despite these important functions, this subset has not been studied in detail in pigs due to a lack of species-specific antibodies. We investigated putative Tfh cells from lymphoid tissues and blood of healthy pigs by using cross-reactive antibodies for inducible T-cell costimulator (ICOS) and B-cell lymphoma 6 (Bcl-6). In lymph nodes, we identified a CD4+ T cell population with an ICOS+Bcl-6+CD8α+ phenotype, reminiscent of human and murine germinal center Tfh cells. Within blood-derived CD4+ T cells, sorted ICOShiCD25- and ICOSdimCD25dim cells were able to induce the differentiation of CD21+IgM+ B cells into Ig-secreting plasmablasts. Compared to naïve CD4+ T cells, these two phenotypes were 3- to 7-fold enriched for cells expressing the Tfh-related transcripts CD28, CD40LG, IL6R and MAF, as identified by single-cell RNA sequencing. These results provide a first characterization of Tfh cells in swine and confirm their ability to provide B-cell help.

Paired nicking-mediated COL17A1 reframing for junctional epidermolysis bullosa.

Junctional epidermolysis bullosa (JEB) is a debilitating hereditary skin disorder caused by mutations in genes encoding laminin-332, type XVII collagen (C17), and integrin-α6ß4, which maintain stability between the dermis and epidermis. We designed patient-specific Cas9-nuclease- and -nickase-based targeting strategies for reframing a common homozygous deletion in exon 52 of COL17A1 associated with a lack of full-length C17 expression. Subsequent characterization of protein restoration, indel composition, and divergence of DNA and mRNA outcomes after treatment revealed auspicious efficiency, safety, and precision profiles for paired nicking-based COL17A1 editing. Almost 46% of treated primary JEB keratinocytes expressed reframed C17. Reframed COL17A1 transcripts predominantly featured 25- and 37-nt deletions, accounting for >42% of all edits and encoding C17 protein variants that localized accurately to the cell membrane. Furthermore, corrected cells showed accurate shedding of the extracellular 120-kDa C17 domain and improved adhesion capabilities to laminin-332 compared with untreated JEB cells. Three-dimensional (3D) skin equivalents demonstrated accurate and continuous deposition of C17 within the basal membrane zone between epidermis and dermis. Our findings constitute, for the first time, gene-editing-based correction of a COL17A1 mutation and demonstrate the superiority of proximal paired nicking strategies based on Cas9 D10A nickase over wild-type Cas9-based strategies for gene reframing in a clinical context.

A non-viral and selection-free COL7A1 HDR approach with improved safety profile for dystrophic epidermolysis bullosa.

Gene editing via homology-directed repair (HDR) currently comprises the best strategy to obtain perfect corrections for pathogenic mutations of monogenic diseases, such as the severe recessive dystrophic form of the blistering skin disease epidermolysis bullosa (RDEB). Limitations of this strategy, in particular low efficiencies and off-target effects, hinder progress toward clinical applications. However, the severity of RDEB necessitates the development of efficient and safe gene-editing therapies based on perfect repair. To this end, we sought to assess the corrective efficiencies following optimal Cas9 nuclease and nickase-based COL7A1-targeting strategies in combination with single- or double-stranded donor templates for HDR at the COL7A1 mutation site. We achieved HDR-mediated correction efficiencies of up to 21% and 10% in primary RDEB keratinocytes and fibroblasts, respectively, as analyzed by next-generation sequencing, leading to full-length type VII collagen restoration and accurate deposition within engineered three-dimensional (3D) skin equivalents (SEs). Extensive on- and off-target analyses confirmed that the combined treatment of paired nicking and single-stranded oligonucleotides constituted a highly efficient COL7A1-editing strategy, associated with a significantly improved safety profile. Our findings, therefore, represent a further advancement in the field of traceless genome editing for genodermatoses.

Self-assembly of differentiated progenitor cells facilitates spheroid human skin organoid formation and planar skin regeneration.

Self-assembly of solid organs from single cells would greatly expand applicability of regenerative medicine. Stem/progenitor cells can self-organize into micro-sized organ units, termed organoids, partially modelling tissue function and regeneration. Here we demonstrated 3D self-assembly of adult and induced pluripotent stem cell (iPSC)-derived fibroblasts, keratinocytes and endothelial progenitors into both, planar human skin in vivo and a novel type of spheroid-shaped skin organoids in vitro, under the aegis of human platelet lysate. Methods: Primary endothelial colony forming cells (ECFCs), skin fibroblasts (FBs) and keratinocytes (KCs) were isolated from human tissues and polyclonally propagated under 2D xeno-free conditions. Human tissue-derived iPSCs were differentiated into endothelial cells (hiPSC-ECs), fibroblasts (hiPSC-FBs) and keratinocytes (hiPSC-KCs) according to efficiency-optimized protocols. Cell identity and purity were confirmed by flow cytometry and clonogenicity indicated their stem/progenitor potential. Triple cell type floating spheroids formation was promoted by human platelet-derived growth factors containing culture conditions, using nanoparticle cell labelling for monitoring the organization process. Planar human skin regeneration was assessed in full-thickness wounds of immune-deficient mice upon transplantation of hiPSC-derived single cell suspensions. Results: Organoids displayed a distinct architecture with surface-anchored keratinocytes surrounding a stromal core, and specific signaling patterns in response to inflammatory stimuli. FGF-7 mRNA transfection was required to accelerate keratinocyte long-term fitness. Stratified human skin also self-assembled within two weeks after either adult- or iPSC-derived skin cell-suspension liquid-transplantation, healing deep wounds of mice. Transplant vascularization significantly accelerated in the presence of co-transplanted endothelial progenitors. Mechanistically, extracellular vesicles mediated the multifactorial platelet-derived trophic effects. No tumorigenesis occurred upon xenografting. Conclusion: This illustrates the superordinate progenitor self-organization principle and permits novel rapid 3D skin-related pharmaceutical high-content testing opportunities with floating spheroid skin organoids. Multi-cell transplant self-organization facilitates development of iPSC-based organ regeneration strategies using cell suspension transplantation supported by human platelet factors.

Expression of CD9 on porcine lymphocytes and its relation to T cell differentiation and cytokine production.

In this work, we report on two novel monoclonal antibodies, specific for porcine CD9. CD9 is a tetraspanin that is expressed on a wide variety of cells. We phenotyped porcine immune cell subsets and found that CD9 was expressed on all monocytes as well as a subset of B cells. CD9 was variably expressed on T cells, with CD4 T cells containing the highest frequency of CD9+ cells. CD9 expression positively correlated with the frequency of central memory CD4 T cells in ex vivo PBMC. Therefore, we proceeded to explore CD9 as a marker of T cell function. Here we observed that CD9 was expressed on the vast majority of long-lived influenza A virus-specific effector cells that retained the capacity for cytokine production in response to in vitro recall antigen. Therefore, the new antibodies enable the detection of a cell surface molecule with functional relevance to T cells. Considering the importance of CD9 in membrane remodelling across many cell types, they will also benefit the wider field of swine biomedical research.

Deoxynivalenol Has the Capacity to Increase Transcription Factor Expression and Cytokine Production in Porcine T Cells.

Deoxynivalenol (DON) is a Fusarium mycotoxin that frequently contaminates the feed of farm animals. Pigs with their monogastric digestive system are in particular sensitive to DON-contaminated feed. At high concentrations, DON causes acute toxic effects, whereas lower concentrations lead to more subtle changes in the metabolism. This applies in particular to the immune system, for which immunosuppressive but also immunostimulatory phenomena have been described. Research in human and rodent cell lines indicates that this may be partially explained by a binding of DON to the ribosome and subsequent influences on cell signaling molecules like mitogen-activated protein kinases. However, a detailed understanding of the influence of DON on functional traits of porcine immune cells is still lacking. In this study, we investigated the influence of DON on transcription factor expression and cytokine production within CD4+, CD8+, and γδ T cells in vitro. At a DON concentration, that already negatively affects proliferation after Concanavalin A stimulation (0.8 µM) an increase of T-bet expression in CD4+ and CD8+ T cells was observed. This increase in T-bet expression coincided with elevated levels of IFN-γ and TNF-α producing T-cell populations. Increases in T-bet expression and cytokine production were found in proliferating and non-proliferating T cells, although increases were more prominent in proliferating cell subsets. Differently, IL-17A production by CD4+ T cells was not influenced by DON. In addition, frequencies of regulatory T cells and their expression of Foxp3 were not affected. In γδ T cells, GATA-3 expression was slightly reduced by DON, whereas T-bet levels were only slightly modulated and hence IFN-γ, TNF-α, or IL-17A production were not affected. Our results show for the single-cell level that DON has the capacity to modulate the expression of transcription factors and related cytokines. In particular, they suggest that for CD4+ and CD8+ T cells, DON can drive T-cell differentiation into a pro-inflammatory type-1 direction, probably depending on the already prevailing cytokine milieu. This could have beneficial or detrimental effects in ongoing immune responses to infection or vaccination.

Predictable CRISPR/Cas9-Mediated COL7A1 Reframing for Dystrophic Epidermolysis Bullosa.

End-joining‒based gene editing is frequently used for efficient reframing and knockout of target genes. However, the associated random, unpredictable, and often heterogeneous repair outcomes limit its applicability for therapeutic approaches. This study revealed more precise and predictable outcomes simply on the basis of the sequence context at the CRISPR/Cas9 target site. The severe dystrophic form of the blistering skin disease epidermolysis bullosa (DEB) represents a suitable model platform to test these recent developments for the disruption and reframing of dominant and recessive alleles, respectively, both frequently seen in DEB. We delivered a CRISPR/Cas9 nuclease as ribonucleoprotein into primary wild-type and recessive DEB keratinocytes to introduce a precise predictable single adenine sense-strand insertion at the target site. We achieved type VII collagen knockout in more than 40% of ribonucleoprotein-treated primary wild-type keratinocytes and type VII collagen restoration in more than 70% of ribonucleoprotein-treated recessive DEB keratinocytes. Next-generation sequencing of the on-target site revealed the presence of the precise adenine insertion upstream of the pathogenic mutation in at least 17% of all analyzed COL7A1 alleles. This demonstrates that COL7A1 editing based on precise end-joining‒mediated DNA repair is an efficient strategy to revert the disease-associated nature of DEB regardless of the mutational inheritance.

The Incidence of Tuberculosis in Infants, Siaya District, Western Kenya.

BACKGROUND: Infants are a target population for new tuberculosis (TB) vaccines. TB incidence estimates are needed to guide the design of trials. To determine the TB incidence and cohort retention among young children using comprehensive diagnostic methods in a high burden area. METHODS: Infants 0-42 days were enrolled. Through 4 monthly follow-up and unscheduled (sick) visits up to the age of 2 years, infants with presumptive TB based on a history of contact, TB symptoms or pre-determined hospitalization criteria were admitted to a case verification ward. Two induced sputa and gastric aspirates were collected for culture and GeneXpert. Mantoux and HIV tests were done. Clinical management was based on the Keith Edwards score. Cases were classified into microbiologically confirmed or radiologic, diagnosed by blinded expert assessment. Cox regression was used to identify risk factors for incident TB and study retention. RESULTS: Of 2900 infants enrolled, 927 (32%) developed presumptive TB, 737/927 (80%) were investigated. Sixty-nine TB cases were diagnosed (bacteriologic and radiologic). All TB incidence was 2/100 person-years of observation (pyo) (95% CI: 1.65-2.65). Nine were bacteriologic cases, incidence 0.3/100 pyo. The radiologic TB incidence was 1.82/100 pyo. Bacteriologic TB was associated with infant HIV infection, higher Keith Edwards scores. Completeness of 4-month vaccinations and HIV infection were positively associated with retention. CONCLUSIONS: TB incidence was high. An all TB endpoint would require a sample size of a few thousand children, but tens of thousands, when limited to bacteriologic TB.

A study to better understand under-utilization of laboratory tests for antenatal care in Senegal.

OBJECTIVE: To better understand factors contributing to underutilization of laboratory services for health care delivery in sub-Saharan Africa, we conducted a study in Senegalese Antenatal Care clinics (ANC) and laboratories to determine the extent of underutilization, contributing factors, and bottlenecks in the cascade of care from first ANC visit, test uptake, to availability of test results and appropriate clinical management. METHODS: At 16 health facilities, pregnant women attending for their first ANC visit were consecutively recruited and information was prospectively collected on the request, execution, results and clinical management of seven nationally recommended laboratory screening tests for normal pregnancy: hemoglobin concentration (Hb), syphilis serology, HIV serology, determination of proteinuria (PU), determination of blood group and Rhesus factor, Emmel test to detect sickle cell disease, and glycaemia. Health facility staff were interviewed on human resource capacity, management of the ANC and the laboratory, and availability and use of guidelines. RESULTS: Of 1246 ANC attendants, 400 (32%) had complete results. Completeness varied between facilities from 0-99%. In multilevel logistic regression analysis of women nested in facilities, complete uptake was lower if women started ANC later in pregnancy; very low in rural ANC attendants who ever delivered compared to urban primigravidae (OR 0.064; 95%CI 0.00-0.52); and higher if the facility routinely recommended all seven tests. In the cascade from test request to clinical management, the most frequent bottleneck was non-execution of requested tests, while unavailability of results for executed test was uncommon (<2%). Overall, of 525 abnormal test results 97(18%) had a record of adequate clinical management. CONCLUSION: Our study illustrates challenges to test uptake even when laboratory testing capacity is in place, with large differences between facilities, and underscores the importance of management, policy, and the importance of considering local context in order to improve service delivery to expectant mothers.

Estimating the annual risk of infection with Mycobacterium tuberculosis among adolescents in Western Kenya in preparation for TB vaccine trials.

BACKGROUND: Adolescents are a prime target group for tuberculosis (TB) vaccine trials that include prevention of infection (POI). The BCG vaccine is given at birth and does not prevent TB infection. TB infection, a critical endpoint for POI vaccine trials would need to be documented to estimate sample sizes in target populations. METHODS: Adolescents aged 12-18 years of age were enrolled in an area under continuous demographic surveillance. A tuberculin skin test (TST) survey was conducted as part of a study on TB prevalence and incidence. All adolescents got TSTs at enrolment and returned after 72 h for reading. A TST of ≥10 mm if HIV negative or ≥ 5 mm if HIV positive, was considered positive. RESULTS: Of 4808 adolescents returning for TST readings (96% of those enrolled), mean age was 14.4 (SD 1.9), 4518(94%) were enrolled in school and 21(0.4%) gave a previous history of tuberculosis. Among adolescents with TST reactivity, the mean TST induration was 13.2 mm (SD 5.4). The overall prevalence of latent TB infection was 1544/4808 (32.1, 95% CI 29.2-35.1) with a corresponding annual risk of TB infection (ARTI) of 2.6% (95% CI 2.2-3.1). Risk factors for a positive TST included being male (OR 1.3, 95% CI 1.2,1.5), history of having a household TB contact (OR 1.5, 95% CI 1.2,1.8), having a BCG scar (OR 1.5,95% CI 1.2,1.8), living in a rural area (OR 1.4, 95% CI 1.1,1.9), and being out of school (OR 1.8, 95% CI 1.4,2.3). CONCLUSION: We conclude that the high TB transmission rates we found in this study, suggest that adolescents in this region may be an appropriate target group for TB vaccine trials including TB vaccine trials aiming to prevent infection.

Incidence of Active Tuberculosis and Cohort Retention Among Adolescents in Western Kenya.

SETTING: Siaya County, with the highest tuberculosis notification rates in Kenya. OBJECTIVES: To determine the incidence of active tuberculosis and 1-year cohort retention in 12-18-year-old adolescents, in preparation for phase III tuberculosis vaccine trials. METHODS: Adolescents were enrolled and followed up for 1-2 years to determine tuberculosis incidence. Adolescents with a positive tuberculin skin test, history of cohabitation with a tuberculosis case or at least 1 tuberculosis symptom received clinical and sputum examination and a chest radiograph. Definite tuberculosis cases were bacteriologically confirmed and clinical cases diagnosed by a clinician based on a suggestive chest radiograph and having clinical symptoms. Risk factors were explored using Poisson regression. RESULTS: Among 4934 adolescents without tuberculosis at baseline, 26 tuberculosis cases were identified during follow-up with a corresponding incidence density of 4.4 [95% confidence interval (CI): 3.0-6.4] events per 1000 person-years of observation, 12 definite tuberculosis cases; incidence density of 2.0 (95% CI: 0.9-3.1). Having previous tuberculosis (rate ratio: 12.5; CI: 1.8-100) and presence of tuberculin skin test conversion (rate ratio: 3.4; CI: 1.5-7.7) were significantly associated with higher risk of tuberculosis. Overall (4086/4925), 83.0% of adolescents were retained in the study after 1 year of follow-up. Being female, older, out of school and being orphaned were significant risk factors for loss to follow-up. CONCLUSION: The tuberculosis incidence in adolescents will help inform future tuberculosis vaccine trial sample size calculations for this setting. The predictive factors for tuberculosis and retention can be further explored in future trials.

Use of Xpert MTB/RIF in Decentralized Public Health Settings and Its Effect on Pulmonary TB and DR-TB Case Finding in India.

BACKGROUND: Xpert MTB/RIF, the first automated molecular test for tuberculosis, is transforming the diagnostic landscape in high-burden settings. This study assessed the impact of up-front Xpert MTB/RIF testing on detection of pulmonary tuberculosis (PTB) and rifampicin-resistant PTB (DR-TB) cases in India. METHODS: This demonstration study was implemented in 18 sub-district level TB programme units (TUs) in India in diverse geographic and demographic settings covering a population of 8.8 million. A baseline phase in 14 TUs captured programmatic baseline data, and an intervention phase in 18 TUs had Xpert MTB/RIF offered to all presumptive TB patients. We estimated changes in detection of TB and DR-TB, the former using binomial regression models to adjust for clustering and covariates. RESULTS: In the 14 study TUs, which participated in both phases, 10,675 and 70,556 presumptive TB patients were enrolled in the baseline and intervention phase, respectively, and 1,532 (14.4%) and 14,299 (20.3%) bacteriologically confirmed PTB cases were detected. The implementation of Xpert MTB/RIF was associated with increases in both notification rates of bacteriologically confirmed TB cases (adjusted incidence rate ratio [aIRR] 1.39; CI 1.18-1.64), and proportion of bacteriological confirmed TB cases among presumptive TB cases (adjusted risk ratio (aRR) 1.33; CI 1.6-1.52). Compared with the baseline strategy of selective drug-susceptibility testing only for PTB cases at high risk of drug-resistant TB, Xpert MTB/RIF implementation increased rifampicin resistant TB case detection by over fivefold. Among, 2765 rifampicin resistance cases detected, 1055 were retested with conventional drug susceptibility testing (DST). Positive predictive value (PPV) of rifampicin resistance detected by Xpert MTB/RIF was 94.7% (CI 91.3-98.1), in comparison to conventional DST. CONCLUSION: Introduction of Xpert MTB/RIF as initial diagnostic test for TB in public health facilities significantly increased case-notification rates of all bacteriologically confirmed TB by 39% and rifampicin-resistant TB case notification by fivefold.

Prevalence of tuberculosis in adolescents, western Kenya: implications for control programs.

OBJECTIVE: The aim of this study was to determine the prevalence of tuberculosis (TB) in adolescents in western Kenya. METHODS: A cohort study of 5004 adolescents aged 12-18 years was conducted. Adolescents were screened for prevalent TB using clinical criteria, history of TB contact, and a Mantoux test. Cases of suspected TB were investigated through two sputum examinations (microscopy and liquid culture) and chest radiography. RESULTS: Out of 5004 adolescents enrolled, 1960 (39.2%) were identified with suspected TB, including 1544 with a positive Mantoux (prevalence 1544/4808, 32.1%), 515 with symptoms suggestive of TB (10.3%), and 144 (2.9%) with household TB contact. Sixteen culture-confirmed (definite) and 18 probable pulmonary TB (PTB) cases were identified, reflecting a prevalence estimate of 3.2/1000 (definite) and 6.8/1000 all PTB, respectively. Only one smear-positive case was detected. The case notification rate among 12-18-year-old adolescents for all TB was 101/100000, yielding a patient diagnostic rate of 0.13 (95% confidence interval 0.03-3.7) cases detected per person-year for all TB. CONCLUSION: The prevalence of PTB among adolescents is high, with the majority of cases not detected routinely. Innovative active case finding including the wider use of Xpert MTB/RIF is needed to detect smear-negative TB among adolescents.

Choosing algorithms for TB screening: a modelling study to compare yield, predictive value and diagnostic burden.

BACKGROUND: To inform the choice of an appropriate screening and diagnostic algorithm for tuberculosis (TB) screening initiatives in different epidemiological settings, we compare algorithms composed of currently available methods. METHODS: Of twelve algorithms composed of screening for symptoms (prolonged cough or any TB symptom) and/or chest radiography abnormalities, and either sputum-smear microscopy (SSM) or Xpert MTB/RIF (XP) as confirmatory test we model algorithm outcomes and summarize the yield, number needed to screen (NNS) and positive predictive value (PPV) for different levels of TB prevalence. RESULTS: Screening for prolonged cough has low yield, 22% if confirmatory testing is by SSM and 32% if XP, and a high NNS, exceeding 1000 if TB prevalence is ≤0.5%. Due to low specificity the PPV of screening for any TB symptom followed by SSM is less than 50%, even if TB prevalence is 2%. CXR screening for TB abnormalities followed by XP has the highest case detection (87%) and lowest NNS, but is resource intensive. CXR as a second screen for symptom screen positives improves efficiency. CONCLUSIONS: The ideal algorithm does not exist. The choice will be setting specific, for which this study provides guidance. Generally an algorithm composed of CXR screening followed by confirmatory testing with XP can achieve the lowest NNS and highest PPV, and is the least amenable to setting-specific variation. However resource requirements for tests and equipment may be prohibitive in some settings and a reason to opt for symptom screening and SSM. To better inform disease control programs we need empirical data to confirm the modeled yield, cost-effectiveness studies, transmission models and a better screening test.

Optimal triage test characteristics to improve the cost-effectiveness of the Xpert MTB/RIF assay for TB diagnosis: a decision analysis.

BACKGROUND: High costs are a limitation to scaling up the Xpert MTB/RIF assay (Xpert) for the diagnosis of tuberculosis in resource-constrained settings. A triaging strategy in which a sensitive but not necessarily highly specific rapid test is used to select patients for Xpert may result in a more affordable diagnostic algorithm. To inform the selection and development of particular diagnostics as a triage test we explored combinations of sensitivity, specificity and cost at which a hypothetical triage test will improve affordability of the Xpert assay. METHODS: In a decision analytical model parameterized for Uganda, India and South Africa, we compared a diagnostic algorithm in which a cohort of patients with presumptive TB received Xpert to a triage algorithm whereby only those with a positive triage test were tested by Xpert. FINDINGS: A triage test with sensitivity equal to Xpert, 75% specificity, and costs of US$5 per patient tested reduced total diagnostic costs by 42% in the Uganda setting, and by 34% and 39% respectively in the India and South Africa settings. When exploring triage algorithms with lower sensitivity, the use of an example triage test with 95% sensitivity relative to Xpert, 75% specificity and test costs $5 resulted in similar cost reduction, and was cost-effective by the WHO willingness-to-pay threshold compared to Xpert for all in Uganda, but not in India and South Africa. The gain in affordability of the examined triage algorithms increased with decreasing prevalence of tuberculosis among the cohort. CONCLUSIONS: A triage test strategy could potentially improve the affordability of Xpert for TB diagnosis, particularly in low-income countries and with enhanced case-finding. Tests and markers with lower accuracy than desired of a diagnostic test may fall within the ranges of sensitivity, specificity and cost required for triage tests and be developed as such.

Risk factors for inadequate TB case finding in Rural Western Kenya: a comparison of actively and passively identified TB patients.

BACKGROUND: The findings of a prevalence survey conducted in western Kenya, in a population with 14.9% HIV prevalence suggested inadequate case finding. We found a high burden of infectious and largely undiagnosed pulmonary tuberculosis (PTB), that a quarter of the prevalent cases had not yet sought care, and a low case detection rate. OBJECTIVE AND METHODS: We aimed to identify factors associated with inadequate case finding among adults with PTB in this population by comparing characteristics of 194 PTB patients diagnosed in a health facility after self-report, i.e., through passive case detection, with 88 patients identified through active case detection during the prevalence survey. We examined associations between method of case detection and patient characteristics, including HIV-status, socio-demographic variables and disease severity in univariable and multivariable logistic regression analyses. FINDINGS: HIV-infection was associated with faster passive case detection in univariable analysis (crude OR 3.5, 95% confidence interval (CI) 2.0-5.9), but in multivariable logistic regression this was largely explained by the presence of cough, illness and clinically diagnosed smear-negative TB (adjusted OR (aOR) HIV 1.8, 95% CI 0.85-3.7). Among the HIV-uninfected passive case detection was less successful in older patients aOR 0.76, 95%CI 0.60-0.97 per 10 years increase), and women (aOR 0.27, 95%CI 0.10-0.73). Reported current or past alcohol use reduced passive case detection in both groups (0.42, 95% CI 0.23-0.79). Among smear-positive patients median durations of cough were 4.0 and 6.9 months in HIV-infected and uninfected patients, respectively. CONCLUSION: HIV-uninfected patients with infectious TB who were older, female, relatively less ill, or had a cough of a shorter duration were less likely found through passive case detection. In addition to intensified case finding in HIV-infected persons, increasing the suspicion of TB among HIV-uninfected women and the elderly are needed to improve TB case detection in Kenya.

Screening strategies for tuberculosis prevalence surveys: the value of chest radiography and symptoms.

BACKGROUND: We conducted a tuberculosis (TB) prevalence survey and evaluated the screening methods used in our survey, to assess if screening in TB prevalence surveys could be simplified, and to assess the accuracy of screening algorithms that may be applicable for active case finding. METHODS: All participants with a positive screen on either a symptom questionnaire, chest radiography (CXR) and/or sputum smear microscopy submitted sputum for culture. HIV status was obtained from prevalent cases. We estimated the accuracy of modified screening strategies with bacteriologically confirmed TB as the gold standard, and compared these with other survey reports. We also assessed whether sequential rather than parallel application of symptom, CXR and HIV screening would substantially reduce the number of participants requiring CXR and/or sputum culture. RESULTS: Presence of any abnormality on CXR had 94% (95%CI 88-98) sensitivity (92% in HIV-infected and 100% in HIV-uninfected) and 73% (95%CI 68-77) specificity. Symptom screening combinations had significantly lower sensitivity than CXR except for 'any TB symptom' which had 90% (95%CI 84-95) sensitivity (96% in HIV-infected and 82% in HIV-uninfected) and 32% (95%CI 30-34) specificity. Smear microscopy did not yield additional suspects, thus the combined symptom/CXR screen applied in the survey had 100% (95%CI 97-100) sensitivity. Specificity was 65% (95%CI 61-68). Sequential application of first a symptom screen for 'any symptom', followed by CXR-evaluation and different suspect criteria depending on HIV status would result in the largest reduction of the need for CXR and sputum culture, approximately 36%, but would underestimate prevalence by 11%. CONCLUSION: CXR screening alone had higher accuracy compared to symptom screening alone. Combined CXR and symptom screening had the highest sensitivity and remains important for suspect identification in TB prevalence surveys in settings where bacteriological sputum examination of all participants is not feasible.