Circulating levels of GDF15 in patients with myalgic encephalomyelitis/chronic fatigue syndrome.

BACKGROUND: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating condition characterised by fatigue and post-exertional malaise. Its pathogenesis is poorly understood. GDF15 is a circulating protein secreted by cells in response to a variety of stressors. The receptor for GDF15 is expressed in the brain, where its activation results in a range of responses. Among the conditions in which circulating GDF15 levels are highly elevated are mitochondrial disorders, where early skeletal muscle fatigue is a key symptom. We hypothesised that GDF15 may represent a marker of cellular stress in ME/CFS. METHODS: GDF15 was measured in serum from patients with ME/CFS (n = 150; 100 with mild/moderate and 50 with severe symptoms), "healthy volunteers" (n = 150) and a cohort of patients with multiple sclerosis (n = 50). RESULTS: Circulating GDF15 remained stable in a subset of ME/CFS patients when sampled on two occasions ~ 7 months (IQR 6.7-8.8) apart, 720 pg/ml (95% CI 625-816) vs 670 pg/ml (95% CI 598-796), P = 0.5. GDF15 levels were 491 pg/ml in controls (95% CI 429-553), 546 pg/ml (95% CI 478-614) in MS patients, 560 pg/ml (95% CI 502-617) in mild/moderate ME/CFS patients and 602 pg/ml (95% CI 531-674) in severely affected ME/CFS patients. Accounting for potential confounders, severely affected ME/CFS patients had GDF15 concentrations that were significantly increased compared to healthy controls (P = 0.01). GDF15 levels were positively correlated (P = 0.026) with fatigue scores in ME/CFS. CONCLUSIONS: Severe ME/CFS is associated with increased levels of GDF15, a circulating biomarker of cellular stress that appears which stable over several months.

Addressing diversity in tuberculosis using multidimensional approaches.

Tuberculosis is a complex disease, which can affect many organs other than the lungs. Initial infection may be cleared without inducing immunological memory, or progress directly to primary disease. Alternatively, the infection may be controlled as latent TB infection, that may progress to active tuberculosis at a later stage. There is now a greater understanding that these infection states are part of a continuum, and studies using PET/CT imaging have shown that individual lung granulomas may respond to infection independently, in an un-synchronized manner. In addition, the Mycobacterium tuberculosis organisms themselves can exist in different states: as nonculturable forms, as 'persisters', as rapidly growing bacteria and a biofilm-forming cording phenotype. The 'omics' approaches of transcriptomics, metabolomics and proteomics can help reveal the mechanisms underlying these different infection states in the host, and identify biosignatures with diagnostic potential, that can predict the development of disease, in 'progressors' as early as 12-18 months before it can be detected clinically, or that can monitor the success of anti-TB therapy. Further insights can be obtained from studies of BCG vaccination and new TB vaccines. For example, epigenetic changes associated with trained immunity and a stronger immune responses following BCG vaccination can be identified. These omics approaches may be particularly valuable when linked to studies of mycobacterial growth inhibition, as a direct read-out of the ability to control mycobacterial growth. The second generation of omics studies is identifying much smaller signatures based on as few as 3 or 4 genes. Thus, narrowing down omics-derived biosignatures to a manageable set of markers now opens the way to field-friendly point of care assays.

Differential transcriptomic and metabolic profiles of M. africanum- and M. tuberculosis-infected patients after, but not before, drug treatment.

The epidemiology of Mycobacterium tuberculosis (Mtb) and M. africanum (Maf) suggests differences in their virulence, but the host immune profile to better understand the pathogenesis of tuberculosis (TB) have not been studied. We compared the transcriptomic and metabolic profiles between Mtb- and Maf-infected TB cases to identify host biomarkers associated with lineages-specific pathogenesis and response to anti-TB chemotherapy. Venous blood samples from Mtb- and Maf-infected patients obtained before and after anti-TB treatment were analyzed for cell composition, gene expression and metabolic profiles. Prior to treatment, similar transcriptomic profiles were seen in Maf- and Mtb-infected patients. In contrast, post treatment, over 1600 genes related to immune responses and metabolic diseases were differentially expressed between the groups. Notably, the upstream regulator hepatocyte nuclear factor 4-alpha (HNF4α), which regulated 15% of these genes, was markedly enriched. Serum metabolic profiles were similar in both group pre-treatment, but the decline in pro-inflammatory metabolites post treatment were most pronounced in Mtb-infected patients. Together, the differences in both peripheral blood transcriptomic and serum metabolic profiles between Maf- and Mtb-infected patients observed over the treatment period, might be indicative of intrinsic host factors related to susceptibility to TB and/or differential efficacy of the standard anti-TB treatment on the two lineages.

Diabetes characteristics and long-term management needs in diabetic TB patients.

BACKGROUND: Diabetes mellitus (DM) is common among patients with TB. We assessed DM characteristics and long-term needs of DM-TB patients after completing TB treatment.METHODS: Newly diagnosed TB patients with DM were recruited for screening in a randomised clinical trial evaluating a simple algorithm to improve glycaemic control during TB treatment. DM characteristics, lifestyle and medication were compared before and after TB treatment and 6 months later. Risk of cardiovascular disease (CVD), albuminuria and neuropathy were assessed after TB treatment.RESULTS: Of 218 TB-DM patients identified, 170 (78%) were followed up. Half were males, the mean age was 53 years, 26.5% were newly diagnosed DM. High glycated haemoglobin at TB diagnosis (median 11.2%) decreased during TB treatment (to 7.4% with intensified management and 8.4% with standard care), but this effect was lost 6 months later (9.3%). Hypertension and dyslipidemia contributed to a high 10-year CVD risk (32.9% at month 6 and 35.5% at month 12). Neuropathy (33.8%) and albuminuria (61.3%) were common. After TB treatment, few patients used CVD-mitigating drugs.CONCLUSION: DM in TB-DM patients is characterised by poor glycaemic control, high CVD risk, and nephropathy. TB treatment provides opportunities for better DM management, but effort is needed to improve long-term care.

Potential business model for a European vaccine R&D infrastructure and its estimated socio-economic impact.

Background: Research infrastructures are facilities or resources that have proven fundamental for supporting scientific research and innovation. However, they are also known to be very expensive in their establishment, operation and maintenance. As by far the biggest share of these costs is always borne by public funders, there is a strong interest and indeed a necessity to develop alternative business models for such infrastructures that allow them to function in a more sustainable manner that is less dependent on public financing. Methods: In this article, we describe a feasibility study we have undertaken to develop a potentially sustainable business model for a vaccine research and development (R&D) infrastructure. The model we have developed integrates two different types of business models that would provide the infrastructure with two different types of revenue streams which would facilitate its establishment and would be a measure of risk reduction. For the business model we are proposing, we have undertaken an ex ante impact assessment that estimates the expected impact for a vaccine R&D infrastructure based on the proposed models along three different dimensions: health, society and economy. Results: Our impact assessment demonstrates that such a vaccine R&D infrastructure could achieve a very significant socio-economic impact, and so its establishment is therefore considered worthwhile pursuing. Conclusions: The business model we have developed, the impact assessment and the overall process we have followed might also be of interest to other research infrastructure initiatives in the biomedical field.

Antituberculosis BCG vaccination: more reasons for varying innate and adaptive immune responses.

Bacillus Calmette-Guérin (BCG) vaccination induces variable protection against pulmonary tuberculosis (TB), and a more effective TB vaccine is needed. The potential for BCG to provide protection against heterologous infections, by induction of innate immune memory, is increasingly recognized. These nonspecific responses may substantially benefit public health, but are also variable. In this issue of the JCI, Koeken and de Bree et al. report that BCG reduces circulating inflammatory markers in males but not in females, while de Bree and Mouritis et al. describe how diurnal rhythms affect the degree of BCG-induced innate memory. These studies further delineate factors that influence the magnitude of responses to BCG and may be crucial to harnessing its potential benefits.

Point of care HbA1c level for diabetes mellitus management and its accuracy among tuberculosis patients: a study in four countries.

BACKGROUND: Diabetes mellitus (DM) is common among tuberculosis (TB) patients and often undiagnosed or poorly controlled. We compared point of care (POC) with laboratory glycated haemoglobin (HbA1c) testing among newly diagnosed TB patients to assess POC test accuracy, safety and acceptability in settings in which immediate access to DM services may be difficult. METHODS: We measured POC and accredited laboratory HbA1c (using high-performance liquid chromatography) in 1942 TB patients aged 18 years recruited from Peru, Romania, Indonesia and South Africa. We calculated overall agreement and individual variation (mean ± 2 standard deviations) stratified by country, age, sex, body mass index (BMI), HbA1c level and comorbidities (anaemia, human immunodeficiency virus [HIV]). We used an error grid approach to identify disagreement that could raise significant concerns. RESULTS: Overall mean POC HbA1c values were modestly higher than laboratory HbA1c levels by 0.1% units (95%CI 0.1-0.2); however, there was a substantial discrepancy for those with severe anaemia (1.1% HbA1c, 95%CI 0.7-1.5). For 89.6% of 1942 patients, both values indicated the same DM status (no DM, HbA1c <6.5%) or had acceptable deviation (relative difference <6%). Individual agreement was variable, with POC values up to 1.8% units higher or 1.6% lower. For a minority, use of POC HbA1c alone could result in error leading to potential overtreatment (n = 40, 2.1%) or undertreatment (n = 1, 0.1%). The remainder had moderate disagreement, which was less likely to influence clinical decisions. CONCLUSION: POC HbA1c is pragmatic and sufficiently accurate to screen for hyperglycaemia and DM risk among TB patients.

Isolation and Potential for Transmission of Mycobacterium bovis at Human-livestock-wildlife Interface of the Serengeti Ecosystem, Northern Tanzania.

Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), is a multihost pathogen of public health and veterinary importance. We characterized the M. bovis isolated at the human-livestock-wildlife interface of the Serengeti ecosystem to determine the epidemiology and risk of cross-species transmission between interacting hosts species. DNA was extracted from mycobacterial cultures obtained from sputum samples of 472 tuberculosis (TB) suspected patients and tissue samples from 606 livestock and wild animal species. M. bovis isolates were characterized using spoligotyping and Mycobacterial Interspersed Repetitive Units-Variable Tandem Repeats (MIRU-VNTR) on 24 loci. Only 5 M. bovis were isolated from the cultured samples. Spoligotyping results revealed that three M. bovis isolates from two buffaloes (Syncerus caffer) and 1 African civet (Civettictis civetta) belonged to SB0133 spoligotype. The two novel strains (AR1 and AR2) assigned as spoligotype SB2290 and SB2289, respectively, were identified from indigenous cattle (Bos indicus). No M. bovis was detected from patients with clinical signs consistent with TB. Of the 606 animal tissue specimens and sputa of 472 TB-suspected patients 43 (7.09%) and 12 (2.9%), respectively, yielded non-tuberculous mycobacteria (NTM), of which 20 isolates were M. intracellulare. No M. avium was identified. M. bovis isolates from wildlife had 45.2% and 96.8% spoligotype pattern agreement with AR1 and AR2 strains, respectively. This finding indicates that bTB infections in wild animals and cattle were epidemiologically related. Of the 24 MIRU-VNTR loci, QUB 11b showed the highest discrimination among the M. bovis strains. The novel strains obtained in this study have not been previously reported in the area, but no clear evidence for recent cross-species transmission of M. bovis was found between human, livestock and wild animals.

Association of slow recovery of Mycobacterium africanum-infected patients posttreatment with high content of Persister-Like bacilli in pretreatment sputum.

OBJECTIVES/BACKGROUND: Mycobacterium africanum that causes 40% of tuberculosis (TB) in West Africa grows more slowly in culture and has similar transmission capacity compared with Mycobacterium tuberculosis, but M. africanum-exposed contacts progress more slowly to active disease. The presence of lipid body (LB) containing M. tuberculosis complex (MTBC) cells in sputum samples has been associated with mycobacterial transcriptomes indicating slow or no growth and persister-like antibiotic tolerance. Slow-growing bacilli have been found to display a persister-like phenotype with the accumulation of LBs and drug tolerance. Our previous study showed that the body mass index and lung damage resolution on chest X-ray were significantly improved slower in M. africanum-infected patients posttreatment than in M. tuberculosis-infected patients; however, the reason for this remains unclear. Therefore, we hypothesized that these differences could be either due to significant differences in drug resistance between the MTBC lineages or a difference in their content of persisters, as indicated by the percentage of LP-positive bacilli in sputum. METHODS: Sputum isolates collected before treatment from patients with TB were subjected to drug susceptibility testing using the BD BACTEC MGIT 960 SIRE kit. The percentage of acid-fast bacilli (AFB) and LB-positive bacilli in pretreatment sputum was determined by a dual staining procedure using Auramine O and LipidTOX Red neutral lipid stain, respectively, and fluorescence microscopy imaging. RESULTS: Out of the 77 isolates tested, 9 showed resistance to at least one drug and only 2 showed multidrug (rifampicin and isoniazid) resistance among M. tuberculosis-infected patients. The percentage of AFB-positive smears was similar between the two groups (p=0.821), whereas that of LP-positive bacilli was significantly higher (p=0.0059) in M. africanum-infected patients' sputa (n=24) than in M. tuberculosis-infected patients' sputa (n=36). In addition, the bacillary lengths were significantly higher in M. africanum-infected patients' sputa than in M. tuberculosis-infected patients' sputa (p=0.0007). A high frequency of LP-positive bacilli in pretreatment sputum was associated with a poor body mass index and lung damage on chest X-ray improvement following anti-TB treatment in both the groups (r2=0.022; p=0.017). CONCLUSION: The slow clinical recovery of M. africanum-infected patients compared with M. tuberculosis-infected patients posttreatment may be at least partially associated with the persistence of drug-tolerant "fat and lazy" bacilli.

Interferon-gamma responses to ESAT-6 in tuberculosis patients early into and after anti-tuberculosis treatment.

OBJECTIVE: To investigate T-cell responses to ESAT-6 by an interferon-gamma (IFN-gamma) ex vivo enzyme-linked immunospot (ELISpot) assay in tuberculosis (TB) patients early during treatment and in patients who have completed a course of anti-tuberculosis chemotherapy. DESIGN: T-cell responses following overnight stimulation with 6-kD early secretory antigenic target (ESAT-6) antigen were compared to responses obtained using cells cultured with ESAT-6 for 6 days, using an ELISpot assay. RESULTS: In the ex vivo ELISpot assay, the median IFN-gamma responses in TB patients, irrespective of treatment status, were significantly higher than in healthy BCG-vaccinated controls. In the 6-day ELISpot assay, median IFN-gamma responses were significantly higher in TB patients who had completed treatment than in patients early during therapy. There was considerable individual variability in the degree of expansion of ESAT-6 specific T-cells from day 1 to day 6 in both treatment groups. CONCLUSION: Further studies are required to assess which type of assay provides the best indicator of a memory T-cell response and how ESAT-6 specific T-cells relate to protective immunity in TB infection.

High level expression of genes cloned in phage lambda gt11.

Plasmid cloning vectors have been constructed which allow genes originally cloned in lambda gt11 to be expressed at a high level in Escherichia coli. They are based on the pEMBL and pUC vectors, with the genes transcribed from the lac promoter. The EcoRI site in the vector has been altered to be in the same reading frame as the site used for cloning in lambda gt11. Cloned proteins are expressed fused to a 2-kDa leader sequence containing a run of six Aparagine residues which considerably improves the stability of the recombinant proteins, but does not interfere with immunological assays. Using these vectors, the Mycobacterium leprae 18-kDa protein was expressed at 20 mg per litre of culture and constituted 15% of total cell protein.

Vaccinia expression of Mycobacterium tuberculosis-secreted proteins: tissue plasminogen activator signal sequence enhances expression and immunogenicity of M. tuberculosis Ag85.

There is increasing evidence to implicate a role for CD8(+) T cells in protective immunity against tuberculosis. Recombinant vaccinia (rVV) expressing Mycobacterium tuberculosis (MTB) proteins can be used both as tools to dissect CD8(+) T-cell responses and, in attenuated form, as candidate vaccines capable of inducing a balanced CD4(+)/CD8(+) T-cell response. A panel of rVV was constructed to express four immunodominant secreted proteins of MTB: 85A, 85B and 85C and ESAT-6. A parallel group of rVV was constructed to include the heterologous eukaryotic tissue plasminogen activator (tPA) signal sequence to assess if this would enhance expression and immunogenicity. Clear expression was obtained for 85A, 85B and ESAT-6 and the addition of tPA resulted in N-glycosylation and a 4-10-fold increase in expression. Female C57BL/6 mice were immunised using the rVV-Ag85 constructs, and interleukin-2 and gamma-interferon were assayed using a co-culture of immune splenocytes and recall antigen. There was a marked increase in cytokine production in mice immunised with the tPA-containing constructs. We report the first data demonstrating enhanced immunogenicity of rVV using a tPA signal sequence, which has significant implications for future vaccine design.

A simple method for the solubilisation of reduced NBT, and its use as a colorimetric assay for activation of human macrophages by gamma-interferon.

We describe a simple method by which the insoluble blue formazan dye produced by the reduction of nitro blue tetrazolium can be dissolved without heating using potassium hydroxide and dimethyl sulphoxide. This modification enhances the sensitivity and increases the applications of tests performed using the microELISA method and removes variations caused by uneven cell monolayers. It also allows quantification of NBT reduced by cells adherent to coverslips or in larger wells or Petri dishes, and can be used as a sensitive assay for macrophage activation by gamma-interferon.

Development of a whole blood assay to measure T cell responses to leprosy: a new tool for immuno-epidemiological field studies of leprosy immunity.

A whole blood assay is described to measure T cell mediated immune responses to leprosy and provide an alternative to the conventional lymphocyte transformation test. Optimal conditions were defined for the whole blood assay, and interferon-gamma measurement was found to be a more sensitive way of measuring responses than tritiated thymidine incorporation. The assay was shown to be useful for investigating responses to a range of leprosy antigens. A whole blood assay has the advantages of being quick, simple and requiring only a small volume of blood, making it more appropriate as an immuno-epidemiological field test in leprosy endemic areas.

Decreased IFN- gamma and increased IL-4 production by human CD8(+) T cells in response to Mycobacterium tuberculosis in tuberculosis patients.

To investigate the role of MHC class I restricted CD8(+) T cells in host defense to M. tuberculosis, peripheral blood mononuclear cells (PBMC) from healthy BCG-vaccinated donors and untreated pulmonary tuberculosis (TB) patients in The Gambia were stimulated for 6 days with M. bovis BCG or M. tuberculosis and the CD8(+) T cell response analyzed. Intracellular FACS analysis of cytokine production by CD8(+) T cells showed that IFN- gamma and TNF- alpha production were greatly reduced in TB patients compared to healthy controls. IL-4-producing CD8(+) T cells were detected in TB patients, a phenotype absent in controls. Collectively, these data suggest that an alteration in the type 1/type 2 cytokine balance occurs in CD8(+) T cells during clinical tuberculosis, and that this may provide a surrogate marker for disease.

The immune response to Mycobacterium tuberculosis in HIV-infected and uninfected adults in Uganda: application of a whole blood cytokine assay in an epidemiological study.

SETTING: Out-patient clinic, Entebbe, Uganda. BACKGROUND: It has been proposed that 'type 1' cytokines are essential in protective immunity to Mycobacterium tuberculosis and that suppression of 'type 1' or a switch to a 'type 2' profile is deleterious. We employed a simple assay to examine whether the dependence of the immunological responses to mycobacterial antigens on a range of explanatory factors could be determined in a population where tuberculosis is endemic. OBJECTIVE: To determine the relationship between the tuberculin skin test response and cytokine profile, and the effect of human immunodeficiency virus (HIV) infection. DESIGN: A cross-sectional study of 97 Ugandan adults (22 HIV-positive, 75 HIV-negative). Whole blood was stimulated in vitro using mycobacterial antigens (purified protein derivative [PPD] and culture filtrate proteins [CFP]). 'Type 1' cytokines (gamma interferon [IFN-gamma] and interleukin-2 [IL-2]), 'type 2' cytokines (IL-5 and IL-10) and tumour necrosis factor alpha (TNF-alpha) were measured in culture supernatants. RESULTS: Among HIV-negative subjects, a positive tuberculin skin test was associated with type 1 or mixed (type 1 + type 2) cytokine production, but a positive IFN-gamma response also occurred in a proportion of tuberculin skin test negative subjects (36% for PPD, 17% for CFP). In association with HIV infection, IFN-gamma responses to mycobacterial antigens were profoundly impaired (odds ratio [OR] 0.10 for PPD, 0.06 for CFP, P< or =0.001), but production of IL-2, IL-5 and TNF-alpha was relatively sustained, and IL-10 increased or sustained (OR 3.97 for PPD, P = 0.01, 1.14 for CFP, P = 0.99). CONCLUSION: The type 1/type 2 cytokine balance was not defined by the tuberculin skin test response, and may have a closer relation to protective immunity. IFN-gamma production was strikingly impaired in association with HIV infection, while production of type 2 cytokines was sustained or increased. Use of a simple assay allowed a large sample of subjects to be examined, producing epidemiologically meaningful results.

Relationship between IFN-gamma and skin test responsiveness to Mycobacterium tuberculosis PPD in healthy, non-BCG-vaccinated young adults in Northern Malawi.

SETTING: Rural northern Malawi, where vaccination with BCG Glaxo (1077) provides protection against leprosy but not against pulmonary tuberculosis. OBJECTIVE: To evaluate the patterns of responsiveness to purified protein derivative of Mycobacterium tuberculosis (PPD) in terms of delayed type hypersensitivity (DTH) and interferon-gamma (IFN-gamma) production. DESIGN: IFN-gamma was measured in 6 day whole blood cultures diluted 1 in 10, stimulated with PPD RT48, and the results compared to the DTH response to PPD RT23. A total of 633 individuals aged 12 to 28 years, without prior BCG vaccination, were recruited. RESULTS: Overall, 63% of subjects made a positive IFN-gamma response (defined as >62 pg/ml), and 37% gave a DTH induration of >5 mm. A strong correlation between skin test and IFN-gamma responses was observed, although with interesting exceptions: 13/270 individuals with zero DTH showed IFN-gamma responses >500 pg/ml, and 7/53 individuals with >10 mm induration showed IFN-gamma responses < or = 62 pg/ml. The prevalence of skin test responsiveness increased with age, and was higher among older males than females; age-sex patterns were less clear for IFN-gamma production. CONCLUSION: The 6 day IFN-gamma response to PPD correlates well with Mantoux skin test induration. The discordant individuals may represent important subsets in terms of protective immunity and risk of clinical tuberculosis.

Detection of high titres of Toxoplasma gondii antibodies in sera of patients with leprosy in Pakistan.

Untreated and treated leprosy patients and their household contacts were screened for antibody to Toxoplasma gondii using antigen-coated latex particles. A significantly high level of seroprevalence (29.6%) was observed in the untreated leprosy patients compared to endemic controls (P < 0.01) with a mean reciprocal antibody titre of 20,007 +/- 3580 (n = 98) in seropositive patients. In treated patients seroprevalence dropped to 13.5%. Seroprevalence in a group of household contacts of leprosy patients was similar to that of control subjects from an endemic area but not exposed to leprosy (7.8% and 6.1% respectively), indicating that the increased seroprevalence in leprosy patients was not merely due to increased exposure related to socioeconomic factors. Antigenic cross-reactivity between T. gondii and Mycobacterium leprae antigens was ruled out by cross inhibition experiments carried out with soluble antigens from each of the organisms. We believe these antibodies may be induced by an increase in T. gondii load in leprosy due to a transient reactivation of latent T. gondii infections, as the antibodies in these leprosy patients were not associated with any sign of eye or lymphatic pathology related to toxoplasmosis.

Whole blood assays for interferon-gamma: practicalities and potential for use as diagnostic tests in the field.

Recent years have seen the introduction of a number of whole-blood assays, in which unseparated heparinized blood is stimulated with antigen either overnight or for as long as 6 days, and cytokine production is measured in the plasma or supernatant. These assays have potential for use in the field as immunodiagnostic assays, as they require only a small blood sample and basic laboratory facilities. Use of these assays in a large study of the immunological effects of BCG vaccination in Malawi has shown that the diluted blood, 6-day whole-blood assays is robust, and can be used to assess T-cell responses to both crude and recombinant antigens. If used with antigens specific to Mycobacterium leprae, these assays could be used to measure exposure of M. leprae within communities or populations, or to aid the early diagnosis of leprosy.

Use of a whole blood assay to monitor the immune response to mycobacterial antigens in leprosy patients: a predictor for type 1 reaction onset?

Longitudinal studies are more appropriate than cross-sectional studies for investigating changes in the immune response to Mycobacterium leprae during leprosy, such as occur in type 1 (reversal) reactions. A test for predicting the onset of reactions in leprosy would greatly reduce disability associated with leprosy. Whole blood assays are appropriate for longitudinal studies of the in vitro T-cell response, as they are robust and reproducible, and require only a small volume of blood. Whole blood assays were used to assess the natural variation in the 'normal' T-cell response to mycobacterial antigens in healthy UK donors, and healthy Nepali donors, tested over 6 months. This was compared with variation in T-cell responses measured over 6 months in 22 leprosy patients in Nepal, including eight who developed type 1 reactions during this time. The in vitro T-cell response to M. leprae sonicate, M. tuberculosis PPD, the mitogen PHA, and (in the UK study) recombinant mycobacterial antigens (70 kD and 30/31 kD proteins) was measured by lymphoproliferation and interferon-gamma (IFN gamma) responses, and variation in responses over time in each subject calculated as a coefficient of variation (CV). The baseline high, low or non-responder status of the healthy UK donors remained stable. The magnitude of IFN gamma responses varied by mean CV ranging from 26% (to PPD) to 63% (to Mtb 70 kD); proliferation responses showed less variation, ranging from mean CV of 18% (to PHA) to 47% (to Mtb 70 kD). Response variation was independent of lymphocyte number in culture. Similar variation in lymphoproliferation responses to MLS, PPD and PHA was observed in the group of healthy Nepali subjects, and in Nepali leprosy patients who did not experience reactions during the study. Of the eight leprosy patients who developed type 1 reactions, four (two BT, one BB, one BL) showed significantly increased proliferation to MLS at the time of reaction (74-300% above baseline); four (one BB, two BL, one LL) remained low or non-responders to MLS throughout. An alternative marker of immune response--anti-phenolic glycolipid-1 (PGL-1) antibody titre--was not predictive of reaction onset in these patients. This study demonstrated that whole blood assays provide reproducible in vitro measurements that can be used to monitor changes in T-cell responses to M. leprae antigens; their practical use as a diagnostic marker of type 1 reaction onset is discussed.