Russell R. Kempker

Plasma Metabolomics in Human Pulmonary Tuberculosis Disease: A Pilot Study

We aimed to characterize metabolites during tuberculosis (TB) disease and identify new pathophysiologic pathways involved in infection as well as biomarkers of TB onset, progression and resolution. Such data may inform development of new anti-tuberculosis drugs. Plasma samples from adults with newly diagnosed pulmonary TB disease and their matched, asymptomatic, sputum culture-negative household contacts were analyzed using liquid chromatography high-resolution mass spectrometry (LC-MS) to identify metabolites. Statistical and bioinformatics methods were used to select accurate mass/charge (m/z) ions that were significantly different between the two groups at a false discovery rate (FDR) of q<0.05. Two-way hierarchical cluster analysis (HCA) was used to identify clusters of ions contributing to separation of cases and controls, and metabolomics databases were used to match these ions to known metabolites. Identity of specific D-series resolvins, glutamate and Mycobacterium tuberculosis (Mtb)-derived trehalose-6-mycolate was confirmed using LC-MS/MS analysis. Over 23,000 metabolites were detected in untargeted metabolomic analysis and 61 metabolites were significantly different between the two groups. HCA revealed 8 metabolite clusters containing metabolites largely upregulated in patients with TB disease, including anti-TB drugs, glutamate, choline derivatives, Mycobacterium tuberculosis-derived cell wall glycolipids (trehalose-6-mycolate and phosphatidylinositol) and pro-resolving lipid mediators of inflammation, known to stimulate resolution, efferocytosis and microbial killing. The resolvins were confirmed to be RvD1, aspirin-triggered RvD1, and RvD2. This study shows that high-resolution metabolomic analysis can differentiate patients with active TB disease from their asymptomatic household contacts. Specific metabolites upregulated in the plasma of patients with active TB disease, including Mtb-derived glycolipids and resolvins, have potential as biomarkers and may reveal pathways involved in TB disease pathogenesis and resolution.

Surgical treatment of drug-resistant tuberculosis

The global emergence and spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis has led to the re-examination of surgery as a possible adjunctive treatment. We present the case of a 26-year-old HIV-seronegative patient with XDR pulmonary tuberculosis refractory to medical therapy. Surgical resection of the patients solitary cavitary lesion was done as adjunctive treatment, and a successful outcome with a combination of surgery and drug therapy was achieved. We review the history of surgical therapy for tuberculosis and reports of its role in treatment of MDR and XDR tuberculosis. 26 case series and cohort studies were included, and together showed that surgical resection is beneficial in the treatment of drug-resistant tuberculosis. However, the results might not be applicable in all settings because investigations were observational and typically included patients with less severe disease, and all surgeries were done at specialised thoracic-surgery centres. Well designed studies are needed to establish the efficacy of surgery in treatment of drug-resistant tuberculosis.

Beware of the pet dog: a case of Staphylococcus intermedius infection.

Staphylococcus intermedius is a common commensal and pathogen in dogs and cats and only rarely has been identified as causing human infection. We report a human case of postoperative sinus infection caused by methicillin-resistant S. intermedius. A 28-year-old woman with a history of endoscopic pituitary adenoma resection presented with 3 weeks of foul smelling nasal discharge. Nasal endoscopy revealed purulent sinus drainage. Cultures, initially misidentified as coagulase-negative Staphylococcus and then as Staphylococcus aureus, revealed the presence of S. intermedius. Cultures from the patients pet dog also grew S. intermedius strains that were confirmed to be identical to those of the patients by pulse field gel electrophoresis analysis. The patient was successfully treated with endoscopic debridement and a prolonged antibiotic regimen with vancomycin and linezolid. Our case illustrates the possibility of transmission of antibiotic-resistant bacteria causing infection from pets to humans.

Use of a Molecular Diagnostic Test in AFB Smear Positive Tuberculosis Suspects Greatly Reduces Time to Detection of Multidrug Resistant Tuberculosis

Background The WHO has recommended the implementation of rapid diagnostic tests to detect and help combat M/XDR tuberculosis (TB). There are limited data on the performance and impact of these tests in field settings. Methods The performance of the commercially available Genotype MTBDRplus molecular assay was compared to conventional methods including AFB smear, culture and drug susceptibility testing (DST) using both an absolute concentration method on Löwenstein-Jensen media and broth-based method using the MGIT 960 system. Sputum specimens were obtained from TB suspects in the country of Georgia who received care through the National TB Program. Results Among 500 AFB smear-positive sputum specimens, 458 (91.6%) had both a positive sputum culture for Mycobacterium tuberculosis and a valid MTBDRplus assay result. The MTBDRplus assay detected isoniazid (INH) resistance directly from the sputum specimen in 159 (89.8%) of 177 specimens and MDR-TB in 109 (95.6%) of 114 specimens compared to conventional methods. There was high agreement between the MTBDRplus assay and conventional DST results in detecting MDR-TB (kappa = 0.95, p<0.01). The most prevalent INH resistance mutation was S315T (78%) in the katG codon and the most common rifampicin resistance mutation was S531L (68%) in the rpoB codon. Among 13 specimens from TB suspects with negative sputum cultures, 7 had a positive MTBDRplus assay (3 with MDR-TB). The time to detection of MDR-TB was significantly less using the MTBDRplus assay (4.2 days) compared to the use of standard phenotypic tests (67.3 days with solid media and 21.6 days with broth-based media). Conclusions Compared to conventional methods, the MTBDRplus assay had high accuracy and significantly reduced time to detection of MDR-TB in an area with high MDR-TB prevalence. The use of rapid molecular diagnostic tests for TB and drug resistance should increase the proportion of patients promptly placed on appropriate therapy.

High-dose vitamin D3 in adults with pulmonary tuberculosis: a double-blind randomized controlled trial.

BACKGROUND Tuberculosis, including multidrug-resistant tuberculosis (MDR-TB), is a major global health problem. Individuals with tuberculosis disease commonly exhibit vitamin D deficiency, which may adversely affect immunity and the response to therapy. OBJECTIVE We determined whether adjunctive high-dose vitamin D3 supplementation improves outcomes in individuals with pulmonary tuberculosis disease. DESIGN The study was a double-blind, randomized, placebo-controlled, intent-to-treat trial in 199 individuals with pulmonary tuberculosis disease in Tbilisi, Georgia. Subjects were randomly assigned to receive oral vitamin D3 [50,000 IUs (1.25 mg) thrice weekly for 8 wk and 50,000 IU every other week for 8 wk] or a placebo concomitant with standard first-line antituberculosis drugs. The primary outcome was the time for the conversion of a Mycobacterium tuberculosis (Mtb) sputum culture to negative. RESULTS Baseline characteristics between groups were similar. Most subjects (74%) were vitamin D deficient (plasma 25-hydroxyvitamin D [25(OH)D] concentration <50 nmol/L). With vitamin D3, plasma 25(OH)D concentrations peaked at ∼250 nmol/L by 8 wk and decreased to ∼125 nmol/L at week 16. Adverse events and plasma calcium concentrations were similar between groups. In 192 subjects with culture-confirmed tuberculosis, an adjusted efficacy analysis showed similar median culture-conversion times between vitamin D3 and placebo groups [29 and 27 d, respectively; HR: 0.86; 95% CI: 0.63, 1.18; P = 0.33). Eight-week culture-conversion rates were also similar (84.0% and 82.1% for vitamin D3 and placebo, respectively; P = 0.99). CONCLUSION A high-dose vitamin D3 regimen safely corrected vitamin D deficiency but did not improve the rate of sputum Mtb clearance over 16 wk in this pulmonary tuberculosis cohort. This trial was registered at clinicaltrials.gov at NCT00918086.

High prevalence of Cryptococcal antigenemia among HIV-infected patients receiving antiretroviral therapy in Ethiopia.

Background Cryptococcal disease is estimated to be responsible for significant mortality in Sub-Saharan Africa; however, only scarce epidemiology data exists. We sought to evaluate the prevalence of and risk factors for cryptococcal antigenemia in Ethiopia. Methods Consecutive adult HIV-infected patients from two public HIV clinics in Addis Ababa, Ethiopia were enrolled into the study. A CD4 count ≤200 cells/μl was required for study participation. Patients receiving anti-retroviral therapy (ART) were not excluded. A cryptococcal antigen test was performed for all patients along with an interview, physical exam, and medical chart abstraction. Logistic regression analysis was used to assess risk factors for cryptococcal antigenemia. Results 369 HIV-infected patients were enrolled; mean CD4 123 cells/μl and 74% receiving ART. The overall prevalence of cryptococcal antigenemia was 8.4%; 11% in patients with a CD4 count <100 cells/μl, 8.9% with CD4 100 to 150 cells/μl and 5.7% with CD4150-200 cell/μl. 84% of patients with cryptococcal antigenemia were receiving ART. In multivariable analysis, increasing age, self reported fever, CD4 count <100 cells/μl, and site of screening were associated with an increased risk of cryptococcal antigenemia. No individual or combination of clinical symptoms had optimal sensitivity or specificity for cryptococcal antigenemia. Conclusion Cryptococcal antigenemia is high in Ethiopia and rapid scale up of screening programs is needed. Screening should be implemented for HIV-infected patients with low CD4 counts regardless of symptoms or receipt of ART. Further study into the effect of location and environment on cryptococcal disease is warranted.

Adjunctive surgery improves treatment outcomes among patients with multidrug-resistant and extensively drug-resistant tuberculosis.

OBJECTIVES To determine risk factors for poor outcomes among patients with pulmonary multidrug- or extensively drug-resistant (M/XDR) tuberculosis (TB) in Georgia. METHODS This was a prospective, population-based observational cohort study. RESULTS Among 380M/XDR-TB patients (mean age 38 years), 179 (47%) had a poor outcome: 59 (16%) died, 37 (10%) failed, and 83 (22%) defaulted. Newly diagnosed M/XDR-TB cases were significantly more likely to have a favorable outcome than retreatment cases (odds ratio (OR) 4.26, 95% confidence interval (CI) 1.99-9.10, p<0.001). In the multivariable analysis, independent risk factors for a poor treatment outcome included previous treatment history (OR 2.92, 95% CI 1.29-6.58), bilateral disease (OR 1.90, 95% CI 1.20-3.01), body mass index (BMI, kg/m(2)) ≤18.5 (OR 1.91, 95% CI 1.11-3.29), and XDR-TB (OR 2.28, 95% CI 1.11-4.71). Patients who underwent surgical resection (OR 0.27, 95% CI 0.11-0.64) and had sputum culture conversion by 4 months (OR 0.33, 95% CI 0.21-0.52) were significantly less likely to have poor treatment outcomes. CONCLUSIONS Adjunctive surgery appeared to be beneficial in treating patients with M/XDR-TB. Retreatment cases, XDR-TB, bilateral disease, and low BMI were associated with a poor outcome. Additional studies are needed to further define the apparent beneficial role of surgery in the treatment of M/XDR-TB.

Additional drug resistance in Mycobacterium tuberculosis isolates from resected cavities among patients with multidrug-resistant or extensively drug-resistant pulmonary tuberculosis.

The pathogenesis of increasing drug resistance among patients with multidrug-resistant or extensively drug-resistant tuberculosis undergoing treatment is poorly understood. Increasing drug resistance found among Mycobacterium tuberculosis recovered from cavitary isolates compared with paired sputum isolates suggests pulmonary cavities may play a role in the development of worsening tuberculosis drug resistance.

Prevalence and Incidence of Latent Tuberculosis Infection in Georgian Healthcare Workers

Background Tuberculosis is a major occupational hazard in low and middle-income countries. Limited data exist on serial testing of healthcare workers (HCWs) with interferon-γ release assays (IGRAs) for latent tuberculosis infection (LTBI), especially in low and middle-income countries. We sought to evaluate the rates of and risk factors for LTBI prevalence and LTBI test conversion among HCWs using the tuberculin skin test (TST) and QuantiFERON-TB Gold In-tube assay (QFT-GIT). Methods A prospective longitudinal study was conducted among HCWs in the country of Georgia. Subjects completed a questionnaire, and TST and QFT-GIT tests were performed. LTBI testing was repeated 6-26 months after baseline testing. Results Among 319 HCWs enrolled, 89% reported prior BCG vaccination, and 60% worked in TB healthcare facilities (HCFs). HCWs from TB HCFs had higher prevalence of positive QFT-GIT and TST than those from non-TB HCFs: 107/194 (55%) vs. 30/125 (31%) QFT-GIT positive (p<0.0001) and 128/189 (69%) vs. 64/119 (54%) TST positive (p = 0.01). There was fair agreement between TST and QFT-GIT (kappa = 0.42, 95% CI 0.31–0.52). In multivariate analysis, frequent contact with TB patients was associated with increased risk of positive QFT-GIT (aOR 3.04, 95% CI 1.79–5.14) but not positive TST. Increasing age was associated with increased risk of positive QFT-GIT (aOR 1.05, 95% CI 1.01–1.09) and TST (aOR 1.05, 95% CI 1.01–1.10). High rates of HCW conversion were seen: the QFT-GIT conversion rate was 22.8/100 person-years, and TST conversion rate was 17.1/100 person-years. In multivariate analysis, female HCWs had decreased risk of TST conversion (aOR 0.05, 95% CI 0.01–0.43), and older HCWs had increased risk of QFT-GIT conversion (aOR 1.07 per year, 95% CI 1.01–1.13). Conclusion LTBI prevalence and LTBI test conversion rates were high among Georgian HCWs, especially among those working at TB HCFs. These data highlight the need for increased implementation of TB infection control measures.

Hepatitis C virus co-infection increases the risk of anti-tuberculosis drug-induced hepatotoxicity among patients with pulmonary tuberculosis.

Background The country of Georgia has a high prevalence of tuberculosis (TB) and hepatitis C virus (HCV) infection. Purpose To determine whether HCV co-infection increases the risk of incident drug-induced hepatitis among patients on first-line anti-TB drug therapy. Methods Prospective cohort study; HCV serology was obtained on all study subjects at the time of TB diagnosis; hepatic enzyme tests (serum alanine aminotransferase [ALT] activity) were obtained at baseline and monthly during treatment. Results Among 326 study patients with culture-confirmed TB, 68 (21%) were HCV co-infected, 14 (4.3%) had chronic hepatitis B virus (HBV) infection (hepatitis B virus surface antigen positive [HBsAg+]), and 6 (1.8%) were HIV co-infected. Overall, 19% of TB patients developed mild to moderate incident hepatotoxicity. In multi-variable analysis, HCV co-infection (adjusted Hazards Ratio [aHR]=3.2, 95% CI=1.6-6.5) was found to be an independent risk factor for incident anti-TB drug-induced hepatotoxicity. Survival analysis showed that HCV co-infected patients developed hepatitis more quickly compared to HCV seronegative patients with TB. Conclusion A high prevalence of HCV co-infection was found among patients with TB in Georgia. Drug-induced hepatotoxicity was significantly associated with HCV co-infection but severe drug-induced hepatotoxicity (WHO grade III or IV) was rare.