Subcutaneously administered interferon-gamma for the treatment of multidrug-resistant pulmonary tuberculosis.

OBJECTIVE: We evaluated the clinical and laboratory effects of subcutaneously administered interferon-gamma (IFN-gamma) in the treatment of chronic and advanced multidrug-resistant tuberculosis (MDR-TB). DESIGN: Eight patients with sputum smear and culture persistently positive MDR-TB were subcutaneously administered 2 million international units of recombinant human IFN-gamma three times a week for 24 weeks (72 doses total) between December 2002 and May 2003. Subjects also received a customized drug regimen containing second- and third-line antituberculosis agents based upon drug susceptibility testing and previous treatment history. RESULTS: Body weight remained stable or slightly decreased in all subjects during the study period, and none displayed radiographic improvement on serial chest computed tomography scanning. Sputum smears and cultures remained positive for all patients, and there was no increase in the mean time to yield a positive culture (from 16.5+/-6.4 to 11.8+/-4.9 days). There was no enhancement of cell-mediated immune responses in terms of production of IFN-gamma or IL-10, or of composition of lymphocytes among peripheral blood mononuclear cells. In four patients, therapy was discontinued because of adverse reactions. CONCLUSION: In patients with chronic and advanced MDR-TB, subcutaneous IFN-gamma treatment did not result in improvement in clinical, radiologic, microbiologic, or immunologic parameters.     

Clarithromycin Susceptibility Testing of Mycobacterium avium Complex Using 2,3-Diphenyl-5-thienyl-(2)-tetrazolium Chloride Microplate Assay with Middlebrook 7H9 Broth

A series of 119 Mycobacterium avium complex isolates were subjected to clarithromycin susceptibility testing using microplates containing 2,3-diphenyl-5-thienyl-(2)-tetrazolium chloride (STC). Among 119 isolates, 114 (95.8%) were susceptible to clarithromycin and 5 were resistant according to the new and the standard method. STC counts the low cost and reduces the number of procedures needed for susceptibility testing.     

Interferon gamma mRNA quantitative real-time polymerase chain reaction for the diagnosis of latent tuberculosis: a novel interferon gamma release assay

The interferon gamma (IFN-γ) release assay (IGRA) is widely used as a diagnostic method for latent tuberculosis infection (LTBI). The QuantiFERON-TB Gold and QuantiFERON-TB Gold In-tube (QFT-IT) tests measure plasma IFN-γ levels using enzyme-linked immunosorbent assay (ELISA), and T-SPOT.TB counts IFN-γ–producing cells using enzyme-linked immunosorbent spot assay. IFN-γ mRNA was evaluated as an indicator of IGRA in comparison with QFT-IT IFN-γ ELISA in 46 subjects with active TB and in 73 at low risk for TB. Significant IFN-γ mRNA expression was detected from 30 min and peaked 4 h after stimulation with MTB antigens or mitogen. This was defined as the optimal time point for IFN-γ mRNA real-time polymerase chain reaction (PCR). The sensitivities of IFN-γ mRNA real-time PCR and IFN-γ ELISA were 84.8% (39/46) and 89.1% (41/46), respectively (no significant difference). Although the specificities of IFN-γ ELISA was 4.1% higher than that of IFN-γ mRNA real-time PCR (60.3% versus 56.2%), the difference was not statistically significant. The overall agreement between IFN-γ mRNA real-time PCR and IFN-γ ELISA was 79.8% (kappa = 0.475). Whilst there was no difference in the performance of IFN-γ mRNA real-time PCR and IFN-γ ELISA, IFN-γ mRNA real-time PCR was superior to IFN-γ ELISA in terms of the time required for detection of MTB infection.     

Efficacy and Safety of Metronidazole for Pulmonary Multidrug-Resistant Tuberculosis

Pulmonary lesions from active tuberculosis patients are thought to contain persistent, nonreplicating bacilli that arise from hypoxic stress. Metronidazole, approved for anaerobic infections, has antituberculosis activity against anoxic bacilli in vitro and in some animal models and may target persistent, nonreplicating bacilli. In this double-blind, placebo-controlled trial, pulmonary multidrug-resistant tuberculosis subjects were randomly assigned to receive metronidazole (500 mg thrice daily) or placebo for 8 weeks in addition to an individualized background regimen. Outcomes were measured radiologically (change on high-resolution computed tomography [HRCT]), microbiologically (time to sputum smear and culture conversion), and clinically (status 6 months after stopping therapy). Enrollment was stopped early due to excessive peripheral neuropathies in the metronidazole arm. Among 35 randomized subjects, 31 (15 metronidazole, 16 placebo) were included in the modified intent-to-treat analysis. There were no significant differences by arm in improvement of HRCT lesions from baseline to 2 or 6 months. More subjects in the metronidazole arm converted their sputum smear (P = 0.04) and liquid culture (P = 0.04) to negative at 1 month, but these differences were lost by 2 months. Overall, 81% showed clinical success 6 months after stopping therapy, with no differences by arm. However, 8/16 (50%) of subjects in the metronidazole group and 2/17 (12%) of those in the placebo group developed peripheral neuropathy. Subjects who received metronidazole were 4.3-fold (95% confidence interval [CI], 1.1 to 17.1) more likely to develop peripheral neuropathies than subjects who received placebo. Metronidazole may have increased early sputum smear and culture conversion but was too neurotoxic to use over the longer term. Newer nitroimidazoles with both aerobic and anaerobic activity, now in clinical trials, may increase the sterilizing potency of future treatment regimens.     

Enhanced immunogenicity and protective efficacy with the use of interleukin-12-encapsulated microspheres plus AS01B in tuberculosis subunit vaccination

Tuberculosis subunit vaccines codelivered with interleukin-12 (IL-12)-encapsulated microspheres (IL-12EM) are designed for a sustained release of IL-12 and could induce strong Th1 immune responses specific to Ag85A and ESAT-6. The adjuvant combination of IL-12EM plus AS01B was a more efficient way to induce a sustained Th1 immunity and protection against Mycobacterium tuberculosis.     

Isoniazid Could Be Used for Antibiotic-loaded Bone Cement for Musculoskeletal Tuberculosis: An In Vitro Study

Background

Antibiotic-loaded bone cement (ALBC) has been used in serious cases of musculoskeletal tuberculosis, but the type and amount of antibiotic that should be used in ALBC have not been determined.

Questions/purposes

We therefore determined the (1) elution characteristics and (2) antimycobacterial activity of isoniazid- and rifampicin-loaded bone cement.

Methods

A total of 240 elution samples of each of three discs from 40 g bone cement mixed with one of eight dosages: 1 g, 2 g, and 4 g isoniazid, 1 g, 2 g, and 4 g rifampicin, and a combination of 1 + 1 g or 2 + 2 g of isoniazid and rifampicin. The polymerization of rifampicin-loaded bone cement was delayed to mean 122.5 ± 31.1 minutes. We measured the quantity of isoniazid and rifampicin and the antimycobacterial activity on Days 1, 3, 7, 14, and 30.

Results

Isoniazid eluted in almost all the samples while rifampicin was detected only on Day 1 with 2 g (0.7 ± 0.4 ug/mL/day), and until Day 14 with 4 g (0.1 ± 0.0u g/mL/day). Most of the samples containing isoniazid showed antimycobacterial activity while the samples containing rifampicin showed antimycobacterial activity only on Day 1 with 1 g (0.52 ± 0.18 ug/mL), until Day 14 with 2 g (0.03 ± 0.00 ug/mL), and until Day 30 with 4 g (1.84 ± 1.90 ug/mL).

Conclusion

Rifampicin was unsuitable for ALBC because of its delayed polymerization. Isoniazid eluted and showed antimycobacterial activity for 30 days.

Clinical Relevance

The data suggest isoniazid could be considered for use in ALBC for musculoskeletal tuberculosis if used with systemic treatment. For preventing resistance and systemic toxicity, a combination with a second-line drug and an in vivo study would be needed.     

Sensititre MYCOTB MIC Plate for Testing Mycobacterium tuberculosis Susceptibility to First- and Second-Line Drugs

For Mycobacterium tuberculosis, phenotypic methods for drug susceptibility testing of second-line drugs are poorly standardized and technically challenging. The Sensititre MYCOTB MIC plate (MYCOTB) is a microtiter plate containing lyophilized antibiotics and configured for determination of MICs to first- and second-line antituberculosis drugs. To evaluate the performance of MYCOTB for M. tuberculosis drug susceptibility testing using the Middlebrook 7H10 agar proportion method (APM) as the comparator, we conducted a two-site study using archived M. tuberculosis isolates from Uganda and the Republic of Korea. Thawed isolates were subcultured, and dilutions were inoculated into MYCOTB wells and onto 7H10 agar. MYCOTB results were read at days 7, 10, 14, and 21; APM results were read at 21 days. A total of 222 isolates provided results on both platforms. By APM, 106/222 (47.7%) of isolates were resistant to at least isoniazid and rifampin. Agreement between MYCOTB and APM with respect to susceptibility or resistance was ≥92% for 7 of 12 drugs when a strict definition was used and ≥96% for 10 of 12 drugs when agreement was defined by allowing a ± one-well range of dilutions around the APM critical concentration. For ethambutol, agreement was 80% to 81%. For moxifloxacin, agreement was 83% to 85%; incorporating existing DNA sequencing information for discrepant analysis raised agreement to 91% to 96%. For MYCOTB, the median time to plate interpretation was 10 days and interreader agreement was ≥95% for all drugs. MYCOTB provided reliable results for M. tuberculosis susceptibility testing of first- and second-line drugs except ethambutol, and results were available sooner than those determined by APM.     

Population-Based Molecular Epidemiology of Leprosy in Cebu,Philippines

To address the persisting problem of leprosy in Cebu, Philippines, we compiled a database of more than 200 patients who attend an established referral skin clinic. We described the patient characteristics in conventional demographic parameters and also applied multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) and single nucleotide polymorphism (SNP) typing for Mycobacterium leprae in biopsied skin lesion samples. These combined approaches revealed that transmission is ongoing, with the affected including the young Cebuano population under 40 years of age in both crowded cities and rural areas of the island. The emergence of multicase families (MCF) is indicative of infection unconstrained by standard care measures. For the SNPs, we designed a low-cost PCR-restriction fragment length polymorphism typing method. MLVA in M. leprae was highly discriminatory in this population yet could retain broad groups, as defined by the more stable SNPs, implying temporal marker stability suitable for interpreting population structures and evolution. The majority of isolates belong to an Asian lineage (SNP type 1), and the rest belong to a putative postcolonial lineage (SNP type 3). Specific alleles at two VNTR loci, (GGT)5 and 21-3, were highly associated with SNP type 3 in this population. MLVA identified M. leprae genotype associations for patients with known epidemiological links such as in MCFs and in some villages. These methods provide a molecular database and a rational framework for targeted approaches to search and confirm leprosy transmission in various scenarios.During the last 4 to 5 years, genetic variation in Mycobacterium leprae has been investigated for the purpose of strain typing. Although the M. leprae genome (4) has undergone reductive evolution and is highly mutated, limited genome variability has been found between global isolates, and except for loci prone to mutation, such as variable-number tandem repeats (VNTR) (7, 8, 12, 13, 16, 19, 22, 24, 25, 26), M. leprae strains are highly clonal species. Three single nucleotide polymorphisms (SNPs) were subsequently discovered by further genome sequencing efforts that allowed the separation of global isolates into four subtypes (14).Formal, systematic study of M. leprae diversity by the application of the known polymorphic markers in defined endemic settings for studying extant population structures and leprosy transmission is limited. Previously, we presented the outcome of a focused study in Qiubei County in Yunnan Province, South West China (22). Using VNTR loci, we discovered subgroups within a major lineage. A differential geographical distribution of these subgroups was seen across the county. Furthermore, we noted the conservation of M. leprae genotypes carried by patients of multicase families (MCFs), which is indicative of localized transmission from shared sources.We now extend such approaches to Cebu, Philippines, an island in the Central Visayas where leprosy is still in existence. From a case detection rate of 5.1 in 2001 to the current rate of 4.19, with an actual rate of about 300 registered cases for the entire island of Cebu, multibacillary cases have consistently comprised 85% to 90% of the total number of registered cases. We describe observations from conventional epidemiological and novel molecular studies. Currently, we can routinely map up to 15 VNTR loci using multiplex PCR and fragment length analysis (FLA) methods (8). In this study, we demonstrate the development, feasibility, and applicability of a low-cost technology, a PCR-restriction fragment length polymorphism (RFLP) scheme, for rapid SNP subtyping of M. leprae DNA (14).     

Development and evaluation of an automated stainer for acid-fast bacilli

The current strategy for the control of tuberculosis (TB) relies on early diagnosis, and smear microscopy is an essential component of the laboratory diagnosis of TB in most countries with a high prevalence of the disease. However, even simple smear microscopy examination is far from satisfactory because staining results can vary among individual technicians. In an effort to minimize variations in manual staining procedures, we developed an automated stainer for AFB and evaluated its usefulness in comparison with manual staining. The key feature of our automated stainer is a heating apparatus required for fixation and carbol-fuchsin staining. After smear slides are placed into the machine, the entire staining process is fully automated, from fixation to final washing and drying. With the automated methods, five slides can be fixed and stained in 21 min at consistent high quality. Using sputum samples from 91 TB patients, the staining results of the automated stainer were compared blindly with those of manual staining. The concordance rate between the two methods was 94.5%. In addition, there was no significant difference in the rate of detection of AFB in the sputum samples. Although further optimization of the auto staining procedures is required, the results indicate that the automated AFB stainer developed in this study looks promising for use in clinical mycobacteriology laboratory in order to minimize personal variation during AFB staining.