Tuberculosis detection in nonhuman primates is enhanced by use of testing algorithms that include an interferon-γ release assay

OBJECTIVE To develop a testing algorithm that incorporates multiple assays to evaluate host cellular and humoral immunity and antigen detection concerning Mycobacterium tuberculosis complex (MTBC) infection in captive nonhuman primates. ANIMALS Cohorts of captive-bred and wild-caught macaques from 5 different geographic regions. PROCEDURES Macaques were tested for MTBC infection by use of a γ interferon tuberculosis (GIFT) assay, an interferon-γ release assay, and other assays. In the first 2 cohorts (n = 15 and 181), initial validation of the GIFT assay was performed by use of experimentally infected and unexposed control macaques. In the next 3 cohorts (n = 59, 42, and 11), results were obtained for opportunistically collected samples from macaques exposed during spontaneous outbreaks. RESULTS Sensitivity and specificity of the GIFT assay in the control cohorts were 100% and 97%, respectively, and were variable but enhanced by incorporating results from multiple assays in spontaneous outbreaks. CLINICAL RELEVANCE The detection and management of MTBC infection in captive nonhuman primate populations is an ongoing challenge, especially with animal imports and transfers. Despite standardized practices of initial quarantine with regular intradermal tuberculin skin testing, spontaneous outbreaks continue to be reported. Since infection encompasses a range of disease manifestations over time, a testing algorithm that incorporates multiple assays, such as the GIFT assay, to evaluate host cellular and humoral immunity in addition to agent detection is needed. Testing a combination of samples from controlled studies and spontaneous outbreaks of MTBC infection in nonhuman primates would advance the development and validation of a functional algorithm that incorporates promising tools such as the GIFT assay.

Vitamin D concentrations in infancy and the risk of tuberculosis in childhood: A prospective birth cohort in Cape Town, South Africa

Introduction Low vitamin D may increase the risk of tuberculosis; however, previous observational cohort studies have had variable results. We investigated the relationship between vitamin D levels in infancy and subsequent development of tuberculosis throughout childhood. Methods We enrolled pregnant women between 20–28 weeks’ gestation attending antenatal care in a peri-urban South African setting in the Drakenstein Child Health Study. Serum 25(OH)D concentrations were measured in newborn infants between 6–10 weeks of age. Children were followed prospectively for tuberculosis infection and disease using annual tuberculin skin testing, radiographic examinations, and microbiological diagnosis with GeneXpert, culture, and smear testing. Univariable and multivariable Cox regression was performed and hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated. Results Children were followed for tuberculosis for a median of 7.2 years (IQR, 6.2–7.9). Among 744 children (< 1% living with HIV, 21% HIV-exposed living without HIV), those who were vitamin D deficient in early infancy were not at increased risk of developing tuberculosis (AHR, 0.8; 95% CI, 0.4–1.6). Infants in the lowest vitamin D concentration tertile were at similar risk of tuberculosis compared to the highest tertile (AHR, 0.7; 95% CI, 0.4–1.4). Vitamin D deficiency was associated with tuberculin conversion ≤2 years of age at a <30nmol/l (AOR, 1.9; 95% CI, 1.2–3.2), but not <50nmol/l (AOR, 1.5; 95% CI, 0.8–2.9), cutoff. Conclusion In a setting with hyperendemic tuberculosis, vitamin D levels in infancy did not predict tuberculosis at any point in childhood. However, very low vitamin D levels were associated with tuberculin conversion in young children.

Lessons learned from implementation of interferon-gamma release assay to screen for latent tuberculosis infection in a large multicenter observational cohort study in Brazil

Background: Interferon-gamma release assay (IGRA) has emerged as a useful tool in identifying latent tuberculosis infection (LTBI). This assay can be performed through testing platforms, such as QuantiFERON-TB Gold Plus (QFT-Plus). This in vitro test has been incorporated by several guidelines worldwide and has recently been considered for the diagnosis of LTBI by the World Health Organization (WHO). The possibility of systematically implementing IGRAs such as QFT-Plus in centers that perform LTBI screening has been accelerated by the decreased availability of tuberculin skin testing (TST) in several countries. Nevertheless, the process to implement IGRA testing in routine clinical care has many gaps. Methods: The study utilized the expertise acquired by the laboratory teams of the Regional Prospective Observational Research in Tuberculosis (RePORT)-Brazil consortium during study protocol implementation of LTBI screening of TB close contacts. Results: RePORT-Brazil includes clinical research sites from Brazilian cities and is the largest multicenter cohort of TB close contacts to date in the country. Operational and logistical challenges faced during IGRA implementation in all four study laboratories are described, as well as the solutions that were developed and led to the successful establishment of IGRA testing in RePORT-Brazil. Conclusions: The problems identified and resolved in this study can assist laboratories implementing IGRAs, in addition to manufacturers of IGRAs providing effective technical support. This will facilitate the implementation of IGRA testing in countries with a high TB burden, such as Brazil.

"MANTOUX TESTING IN UVEITIS PATIENTS AND TREATMENT OF PRESUMED INTRAOCULAR TUBERCULOSIS IN WESTERN UP"

Tuberculosis is re-emergingasaglobalhealthproblem.Itisaslowlyprogressive,chronic,granulomatous infectioncausedby M.tuberculosiswhichusuallyaffectslungs,butcanalsoaffectotherorganslikeCVS,GI,CNS,SKINandEYES. Choroidal tubercles and tuberculomas are reported to be the most common intraocular manifestations of TB and the most commonintraocularclinicalpresentationappearstobeposterioruveitis. ThediagnosisofocularTBisconsideredinsettingsof1.IsolationofM.tuberculosisfromocularfluidsortissuespecimen.Byamicrobiologicalorhistopathologicalstudy,PCR. 2.AspresumedoculardiseasesuggestiveofTBwithprovensystemicactivedisease. 3.Presumedoculardiseasewithoutevidenceofactivesystemicdisease. DiagnosticCriteriaForPresumedIntraocularTbUveitiswere. 1.Ocular findings consistent with possible intraocular TB with no other cause of uveitis suggested by history of symptoms,or ancillarytestings. 2.StronglypositiveMANTOUX OR TUBERCULINSKINTESTING(>10mmareaofinduration/necrosis) 3.Responsetoantituberculartherapywithabsenceofrecurrences. The aims of our study were to evaluate prevalence of Mantoux positive in newly referred uveitis patients in whom systemic evaluationwasperformedandtoassesstheoutcomeoftreatmentforpresumedintraoculartuberculosisinselectedpatients. MATERIALANDMETHOD The studywas conductedin theRetinaClinic atUpgradeddepartmentof Ophthalmology,LLRM medicalcollege,Meerut,India.Itwasaprospective,noncomparative,interventionalcaseseries. PatientsofocularinflammationreferredtoRetinaClinicwhounderwentsystemicevaluationwereincludedinthestudy A total of patients who satisfy the inclusion criteria , underwent systemic evaluation which include blood tests, chest radiograph,and tuberculin skin testing (0.05 _g purified protein derivative in 0.1 ml,equivalent to 2.5 tuberculin units) Both erythema and induration was measured at 48 hours,and the result were judged to be positive if induration was more than 10 mm Antituberculosis therapyi.eisoniazid300mg/day,rifampin600mg/day,ethambutol 15mg/kg/day,andpyrazinamide25–30 mg /kg/ day for the first 2 months ,thereafter rifampin and isoniazid were used for another 4–7 months was initiated for patients who had clinical findings consistent with possible intraocular tuberculosis,a positive tuberculin skin test result Responsetotherapywasassessedintermsofincreaseordecreaseorresolution OBSERVATIONAND RESULT Ofthe total 32patients 9patients havepositive tubercular contact history and30patientswere mantouxpositive.(94%),ofwhich78%havetheirindurationsizeof>15mmand8patientshavepositivex-rayfindings.(25%) Out of these 32 patients, 25 received antituberculous therapy for 9 months. In addition all of these patients also received systemicprednisone(1mg/kg/day)untilaclinicaleffectwasseenandthenaslowreductionofdosewasdone. 7patientsweredroppedoutfromthestudy. Out of these 25 patients which were started on treatment, 24 patients (96%) showed improvement in their clinical status, 19 patients (76%) showed improvement in their visual acuity after treatment and 35.6 % patients attained visual acuity of 6/9 or better. CONCLUSION Treatment with antitubercular therapy combined with systemic corticosteroids induces resolution of inflammation with no recurrences. So, mantoux testing should remain an integral part of the systemic work-up for uveitis patients.

Intraocular Tuberculosis: A Challenging Case Mimicking Wet Age-Related Macular Degeneration

An otherwise healthy 72-year-old Chinese patient diagnosed with exudative age-related macular degeneration and decreased vision in left eye was fully investigated. The retrospective analysis of past multimodal imaging revealed bilateral severe choroidal neovascularization and choroiditis associated with a positive tuberculin skin testing and interferon-gamma release assay (QuantiFERON-TB Gold – Cellestis^®, Chadstone, VIC, Australia) suggestive of latent ocular tuberculosis. The variable presentation and tests’ results interpretation represent the greatest limitations in understanding and treating intraocular TB (IOTB). This may present without any other systemic symptoms, the intraocular tissues are of limited access to biopsies and other tests, including imaging and immunological tests, are of relative value. This case highlights how variable may be the presentation of IOTB, which can be easily misdiagnosed leading to a delayed treatment and worse prognosis.

A Comparative Study of Tuberculin Reactions to 2 TU and 5 TU of Purified Protein Derivative in a Tertiary Care Center

Introduction: Globally, tuberculosis (TB) is the second leading cause of deaths related to an infectious disease, after HIV. Tuberculin skin testing (TST) which is also called Mantoux test or purified protein derivative (PPD) test remains a useful tool to diagnose TB in children. Current recommendation is to use 2 TU PPD RT23 for all diagnostic purposes. TST or Mantoux test is considered positive if the induration is 10 mm or more. Due to non-availability of 2 TU, unstandardized TST with 5, 10 TU RT23 is in practice which can cause problems in reading of test results. Objectives: The objectives of the study were to compare the tuberculin reaction sizes to 2 TU and 5 TU doses of RT 23 with Tween 80 at 46–48 h and 76–78 h. Methodology: All children who were aged < 18 years and are known case of TB were included in the study. Each child was administered dual intradermal injections with 2 TU and 5 TU doses of tuberculin on the volar aspect of the forearm until a wheal was visible at the injected area. The reactions to both the tests were read at 46–48 h and 70–72 h. Institutional Ethics Committee approval was taken before start of the study. The statistical analysis was performed using SPSS 22 version software. Results A total number of cases included in the study during the study period were 81 cases, out of which 52 (64.19%) were male and 29 (35.8%) were female. Among those 81 cases, the mean reaction to 2 TU at 46–48 h was 12.79 mm and at 76–78 h was 15.33 mm. The mean reaction to 5 TU at 46–48 h was 18.89 mm and at 76–78 h was 22.80 mm. Among the cases, the mean size of induration at 46–48 h was lesser than that of 76–78 h in both 2 TU and 5 TU and it was statistically significant. Reactions to 2 TU and 5 TU PPD can be made comparable using the equation, 5 TU = (2 TU × 0.599) + 11.233 at 46–48 h and 5 TU = (2 TU × 0.074) + 21.66 at 76–78 h. ConclusionThus to conclude, cutaneous hypersensitivity to 2 TU PPD can be made comparison using the equation, 5 TU = (2 TU × 0.

Pulmonary Tuberculosis Outbreak in a High School, China

This study reports an outbreak of Tuberculosis (TB) in a high school in Hunan province, China during January 2017-April 2018. Contact investigation and TB screening were conducted through symptom screening, tuberculin skin testing, chest radiography and smear examination. Identification of positive isolates and drug susceptibility phenotype were assessed by standard method. Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeats (MIRU-VNTR) and Whole Genome Sequencing (WGS) were performed to investigate the relationships among the positive isolates. A total of 90 students and one teacher were diagnosed active pulmonary TB among 2908 students and 188 staff, with an attack rate of 2.94%. Thirteen positive isolates were identified as drug susceptible Beijing family of Mycobacterium tuberculosis. Results of MIRU-VNTR typing and WGS revealed two clones of Mycobacterium tuberculosis circulating during outbreak. One hundred and twenty-nine Single Nucleotide Polymorphisms (SNPs) discriminated the isolates in two clusters; the maximum number of SNPs between any pair of isolates in each cluster was five or fewer. Our findings highlight the importance of early identification and isolation of the TB cases to prevent spread of TB. WGS provides better resolution than MIRU-VNTR to identify recent transmission in TB outbreak.

Temporal dynamics of intradermal cytokine response to tuberculin in Mycobacterium bovis BCG-vaccinated cattle using sampling microneedles

Bovine tuberculosis (bTB) is a disease of livestock with severe and worldwide economic, animal welfare and zoonotic consequences. Application of test-and-slaughter-based control polices reliant on tuberculin skin testing has been the mainstay of bTB control in cattle. However, little is known about the temporal development of the bovine tuberculin skin test response at the dermal sites of antigen injection. To fill this knowledge gap, we applied minimally-invasive sampling microneedles (SMNs) for intradermal sampling of interstitial fluid at the tuberculin skin test sites in Mycobacterium bovis BCG-vaccinated calves and determined the temporal dynamics of a panel of 15 cytokines and chemokines in situ and in the peripheral blood. The results reveal an orchestrated and coordinated cytokine and local chemokine response, identified IL-1RA as a potential soluble biomarker of a positive tuberculin skin response, and confirmed the utility of IFN-γ and IP-10 for bTB detection in blood-based assays. Together, the results highlight the utility of SMNs to identify novel biomarkers and provide mechanistic insights on the intradermal cytokine and chemokine responses associated with the tuberculin skin test in BCG-sensitized cattle.

#54: Tuberculosis in South African Children with Cancer

Background Children with cancer are immunocompromised, with increased susceptibility to infections including infections with Mycobacterium tuberculosis. Method This prospective study, enrolled children one-19 years old hospitalized for cancer treatment. Screening for M. tuberculosis infection was undertaken by tuberculin skin testing (Mantoux method), and ELISPOT (T-SPOT®.TB) (T-spot) on whole blood. Sputum/gastric-washings were investigated for microscopy and culture; and the diagnosis of probable TB was based on existing guidelines. We evaluated the burden of TB in South African children in a setting of high TB-HIV prevalence. Results We enrolled 169 children, 82 (42.5%) with hematological malignancy (HM) and 87 (51.5%) with solid tumors (ST), median age 68.5 months, 10.7% HIV-infected. The majority had severe malnutrition, 78.3% using mid-upper arm circumference and 57.8% using arm muscle anthropometry. Five (2.9%) children with a reactive TST at enrollment, were empirically treated for TB, and were excluded from further analyses. The T-Spot yielded indeterminate/negative results in the first 100 children (including 2.9% who had a reactive TST), and further IGRA testing was terminated. Thirty-four (20.7%) children were diagnosed with TB, (70.6% with HM and 29.4% with ST). At the screening, 14 had indeterminate and 7 negative T-Spot results. Twelve (35.3%) of the 34 TB cases were culture-confirmed. The mean time from initiation of cancer treatment to TB diagnosis was 5.5 months and the time to TB diagnosis from presentation of malignant disease was 6.6 and 4.9 months for culture-confirmed and probable TB, respectively. The spectrum of cancer was acute lymphoblastic leukemia (ALL) (n = 12/32, 37.5%), followed by non-Hodgkin Lymphoma (n = 8/19, 42.1%; seven with HIV infection). The overall incidence (per 100 child-years) of TB was 7.6 (95% CI: 5.3–10.7), 11.3 (95% CI: 7.2–16.8) with HM and 4.3 (95% CI: 2.1–7.9) with ST. HIV-infected children had a higher rate of TB (19.3; 95% CI: 8.3–38) than those not (6.4; 95% CI: 4.2–9.4). Cancer type, HIV-status nor nutritional status was independently associated with risk of TB. Children who developed TB had a higher exposure period to high-dose corticosteroid courses (350 per 100 child-years) than those who did not (29.4 per 100 child-years; P &lt; 0.001). Children with TB compared with those who remained TB-free were more likely to have been treated for septic episodes (SE) associated with profound neutropenia (322.2 vs. 31.3; P &lt; 0.001), profound lymphopenia (288.9 vs. 27.2; P &lt; 0.001) and profound monocytopenia (416.7 vs. 43.0; P &lt; 0.001) prior to the diagnosis of TB. There were 26 deaths (76.5%) in the 34 children with TB and 39/83 (50.0%; P = 0.004) in those who did not develop TB. Eleven had ALL, eight NHL (seven living with HIV), 3 had acute myeloid leukemia, and one each had HIV-related Kaposi Sarcoma, Nephroblastoma, osteosarcoma, and adrenocortical-carcinoma. At the time of death, multiple pathogens were isolated (multi-drug-resistant bacteria, fungi, and respiratory viruses). Conclusion Screening of children undergoing cancer treatment for MTB infection in a setting of high HIV and TB prevalence, was of limited value. The high rate of TB, and poor outcomes, emphasize the need for a high index of suspicion to diagnose TB and consideration for routine prophylaxis.