N-Alkyl-2-[4-(trifluoromethyl)benzoyl]hydrazine-1-carboxamides and Their Analogues: Synthesis and Multitarget Biological Activity.

Based on the isosterism concept, we have designed and synthesized homologous N-alkyl-2-[4-(trifluoromethyl)benzoyl]hydrazine-1-carboxamides (from C1 to C18) as potential antimicrobial agents and enzyme inhibitors. They were obtained from 4-(trifluoromethyl)benzohydrazide by three synthetic approaches and characterized by spectral methods. The derivatives were screened for their inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) via Ellman's method. All the hydrazinecarboxamides revealed a moderate inhibition of both AChE and BuChE, with IC50 values of 27.04-106.75 µM and 58.01-277.48 µM, respectively. Some compounds exhibited lower IC50 for AChE than the clinically used drug rivastigmine. N-Tridecyl/pentadecyl-2-[4-(trifluoromethyl)benzoyl]hydrazine-1-carboxamides were identified as the most potent and selective inhibitors of AChE. For inhibition of BuChE, alkyl chain lengths from C5 to C7 are optimal substituents. Based on molecular docking study, the compounds may work as non-covalent inhibitors that are placed in a close proximity to the active site triad. The compounds were evaluated against Mycobacterium tuberculosis H37Rv and nontuberculous mycobacteria (M. avium, M. kansasii). Reflecting these results, we prepared additional analogues of the most active carboxamide (n-hexyl derivative 2f). N-Hexyl-5-[4-(trifluoromethyl)phenyl]-1,3,4-oxadiazol-2-amine (4) exhibited the lowest minimum inhibitory concentrations within this study (MIC ≥ 62.5 µM), however, this activity is mild. All the compounds avoided cytostatic properties on two eukaryotic cell lines (HepG2, MonoMac6).

Transposon mutagenesis in Mycobacterium kansasii links a small RNA gene to colony morphology and biofilm formation and identifies 9,885 intragenic insertions that do not compromise colony outgrowth.

Mycobacterium kansasii (Mk) is a resilient opportunistic human pathogen that causes tuberculosis-like chronic pulmonary disease and mortality stemming from comorbidities and treatment failure. The standard treatment of Mk infections requires costly, long-term, multidrug courses with adverse side effects. The emergence of drug-resistant isolates further complicates the already challenging drug therapy regimens and threatens to compromise the future control of Mk infections. Despite the increasingly recognized global burden of Mk infections, the biology of this opportunistic pathogen remains essentially unexplored. In particular, studies reporting gene function or generation of defined mutants are scarce. Moreover, no transposon (Tn) mutagenesis tool has been validated for use in Mk, a situation limiting the repertoire of genetic approaches available to accelerate the dissection of gene function and the generation of gene knockout mutants in this poorly characterized pathogen. In this study, we validated the functionality of a powerful Tn mutagenesis tool in Mk and used this tool in conjunction with a forward genetic screen to establish a previously unrecognized role of a conserved mycobacterial small RNA gene of unknown function in colony morphology features and biofilm formation. We also combined Tn mutagenesis with next-generation sequencing to identify 12,071 Tn insertions that do not compromise viability in vitro. Finally, we demonstrated the susceptibility of the Galleria mellonella larva to Mk, setting the stage for further exploration of this simple and economical infection model system to the study of this pathogen.

Bioactivity of Methoxylated and Methylated 1-Hydroxynaphthalene-2-Carboxanilides: Comparative Molecular Surface Analysis.

A series of twenty-six methoxylated and methylated N-aryl-1-hydroxynaphthalene- 2-carboxanilides was prepared and characterized as potential anti-invasive agents. The molecular structure of N-(2,5-dimethylphenyl)-1-hydroxynaphthalene-2-carboxamide as a model compound was determined by single-crystal X-ray diffraction. All the analysed compounds were tested against the reference strain Staphylococcus aureus and three clinical isolates of methicillin-resistant S. aureus as well as against Mycobacterium tuberculosis and M. kansasii. In addition, the inhibitory profile of photosynthetic electron transport in spinach (Spinacia oleracea L.) chloroplasts was specified. In vitro cytotoxicity of the most effective compounds was tested on the human monocytic leukaemia THP-1 cell line. The activities of N-(3,5-dimethylphenyl)-, N-(3-fluoro-5-methoxy-phenyl)- and N-(3,5-dimethoxyphenyl)-1-hydroxynaphthalene-2-carbox- amide were comparable with or even better than the commonly used standards ampicillin and isoniazid. All promising compounds did not show any cytotoxic effect at the concentration >30 µM. Moreover, an in silico evaluation of clogP features was performed for the entire set of the carboxamides using a range of software lipophilicity predictors, and cross-comparison with the experimentally determined lipophilicity (log k), in consensus lipophilicity estimation, was conducted as well. Principal component analysis was employed to illustrate noticeable variations with respect to the molecular lipophilicity (theoretical/experimental) and rule-of-five violations. Additionally, ligand-oriented studies for the assessment of the three-dimensional quantitative structure-activity relationship profile were carried out with the comparative molecular surface analysis to determine electron and/or steric factors that potentially contribute to the biological activities of the investigated compounds.

Clofazimine for the Treatment of Mycobacterium kansasii.

Mycobacterium kansasii pulmonary infection is a global problem. Standard combination therapy consists of isoniazid at 300 mg/day, rifampin at 600 mg/day, and ethambutol at 15 mg/kg of body weight/day for 18 months. Coincubation of M. kansasii with different clofazimine concentrations over 7 days in test tubes resulted in a maximal kill (maximum effect [Emax]) of 2.03 log10 CFU/ml below the day 0 bacterial burden. The concentration associated with Emax was 110 times the MIC. Next, the effects of human-like concentration-time profiles of clofazimine human-equivalent doses ranging from 0 to 200 mg daily for 21 days were examined in the hollow-fiber model of intracellular M. kansasii (HFS-Mkn). On day 14, when the clofazimine microbial effect was maximal, the Emax was 2.57 log10 CFU/ml, while the dose associated with Emax was 100 mg/day. However, no dose killed M. kansasii to levels below the day 0 bacterial burden. Thus, the antimicrobial effect of clofazimine monotherapy in the HFS-Mkn was modest. Human-equivalent concentration-time profiles of standard combination therapy and doses were used as comparators in the HFS-Mkn On day 14, standard therapy killed to a level 2.32 log10 CFU/ml below the day 0 bacterial burden. The effect of standard therapy was consistent with a biexponential decline, with kill rate constants of 1.85 per day (half-life = 0.37 days) and 0.06 per day (half-life = 12.76 days) (r2 > 0.99). This means that standard therapy would take 9.3 to 12 months to completely eliminate M. kansasii in the model, which is consistent with clinical observations. This observation for standard therapy means that the modest to poor effect of clofazimine on M. kansasii identified here is likely to be the same in the clinic.

N-substituted 2-isonicotinoylhydrazinecarboxamides--new antimycobacterial active molecules.

This report presents a new modification of the isoniazid (INH) structure linked with different anilines via a carbonyl group obtained by two synthetic procedures and with N-substituted 5-(pyridine-4-yl)-1,3,4-oxadiazole-2-amines prepared by their cyclisation. All synthesised derivatives were characterised by IR, NMR, MS and elemental analyses and were evaluated in vitro for their antimycobacterial activity against Mycobacterium tuberculosis H37Rv, Mycobacterium avium 330/88, Mycobacterium kansasii 235/80 and one clinical isolated strain of M. kansasii 6509/96. 2-Isonicotinoyl-N-(4-octylphenyl)hydrazinecarboxamide displayed an in vitro efficacy comparable to that of INH for M. tuberculosis with minimum inhibitory concentrations (MICs) of 1-2 µM. Among the halogenated derivatives, the best anti-tuberculosis activity was found for 2-isonicotinoyl-N-(2,4,6-trichlorophenyl)hydrazinecarboxamide (MIC=4 µM). In silico modelling on the enoyl-acyl carrier protein reductase InhA confirmed that longer alkyl substituents are advantageous for the interactions and affinity to InhA. Most of the hydrazinecarboxamides, especially those derived from 4-alkylanilines, exhibited significant activity against INH-resistant nontuberculous mycobacteria.

Lithium inhibits growth of intracellular Mycobacterium kansasii through enhancement of macrophage apoptosis.

Mycobacterium kansasii (Mk) is an emerging pathogen that causes a pulmonary disease similar to tuberculosis. Macrophage apoptosis contributes to innate host defense against mycobacterial infection. Recent studies have suggested that lithium significantly enhances the cytotoxic activity of death stimuli in many cell types. We examined the effect of lithium on the viability of host cells and intracellular Mk in infected macrophages. Lithium treatment resulted in a substantial reduction in the viability of intracellular Mk in macrophages. Macrophage cell death was significantly enhanced after adding lithium to Mk-infected cells but not after adding to uninfected macrophages. Lithium-enhanced cell death was due to an apoptotic response, as evidenced by augmented DNA fragmentation and caspase activation. Reactive oxygen species were essential for lithium-induced apoptosis. Intracellular scavenging by N-acetylcysteine abrogated the lithium-mediated decrease in intracellular Mk growth as well as apoptosis. These data suggest that lithium is associated with control of intracellular Mk growth through modulation of the apoptotic response in infected macrophages.

Non-tuberculous slow-growing mycobacterial pulmonary infections in non-HIV-infected patients in south London.

BACKGROUND: UK data on slow-growing non-tuberculous mycobacterial (NTM) pulmonary infections are sparse and there is little consensus on optimal treatment regimens. METHODS: This was a retrospective study of NTM pulmonary infections in a London teaching hospital. Inclusion criteria were culture of slow-growing mycobacteria between 2000 and 2007, age > 18 y, HIV-negative, and meeting American Thoracic Society criteria. RESULTS: Fifty-seven patients were included; 68% were males and the median age was 61 y. Predisposing factors were smoking (70%), alcohol abuse (28%), and chronic obstructive pulmonary disease (37%). Cavitation (56%) and infiltrates (42%) were common radiological findings. The predominant organism was Mycobacterium kansasii (70%). Ninety-three percent of patients with M. kansasii, 63% with Mycobacterium avium intracellulare, 60% with Mycobacterium malmoense, and 25% with Mycobacterium xenopi had clinical disease. Of the 57 patients, 37 were treated and had follow-up data available. Most patients received 3 drugs: rifampicin, ethambutol, and clarithromycin or ciprofloxacin for at least 9 months. Thirty percent experienced drug side effects. M. kansasii treatment had a 100% cure and 10% relapse rate, but 15% died. CONCLUSIONS: M. kansasii was the most common NTM and its isolation was predictive of clinical disease. Compared with other studies, treatment with 3 agents had a similar rate of cure and did not appear to reduce the relapse rate of disease, but did increase the risk of side effects.

Cutaneous infection by Mycobacterium haemophilum and kansasii in an IgA-deficient man.

BACKGROUND: The prevalence of infections by nontuberculous mycobacteria (NTM) has steadily increased over the past decades, especially in immunocompromised patients. CASE PRESENTATION: We present a patient with IgA-deficiency and mixed cutaneous infection by two slowly growing mycobacteria, Mycobacterium (M.) haemophilum and M. kansasii. CONCLUSIONS: Cutaneous M. haemophilum infections most often result from HIV or transplantation-associated immunosuppression. Rarely, M. haemophilum may also infect healthy patients or iatrogenically immunosuppressed patients without transplantation. M. kansasii is one of the most frequent NTM and large awareness exists about its involvement in human diseases. Mycobacterial diagnosis of cutaneous infections should be considered in long-lasting skin lesions.

Natural occurrence of horizontal transfer of Mycobacterium avium- specific insertion sequence IS1245 to Mycobacterium kansasii.

Mycobacterium kansasii carrying IS1245, a highly prevalent insertion sequence among Mycobacterium avium isolates, was detected in a mixed culture of M. avium and M. kansasii. The insertion sequence was stable and able to transpose by a replicative mechanism in M. kansasii. These findings may have significant implications for molecular diagnosis and treatment outcome.

Induction of macrophage death by clinical strains of Mycobacterium kansasii.

Mycobacterium kansasii is a facultative intracellular pathogen causing pulmonary disease in immunocompetent patients. Little is known about the host defense against M. kansasii and its intracellular survival strategy inside macrophages. In the present study, we obtained six clinical isolates from patients with M. kansasii pulmonary disease and investigated the intracellular growth and cytotoxic effects of M. kansasii inside mouse bone marrow-derived macrophages (BMDM) as well as cytokine secretion from BMDM. Interestingly, two isolates, SM-1 and 2693-20, displayed faster growth rates and higher levels of TNF-alpha secretion from macrophages when compared to the other strains. In addition, SM-1 and 2693-20 also induced massive cell death in BMDM and THP-1 acute monocytic leukemia cells, while the slow growing strains induced significantly lower levels of cell death. This cytotoxicity was mainly caused by necrosis, not apoptosis and it was TNF-alpha-independent. Caspase inhibitors failed to block M. kansasii-induced macrophage death. In addition, necrosis caused by the fast growing strains was accompanied by the loss of mitochondrial membrane potential (DeltaPsi(m)). When dissipation of DeltaPsi(m) was inhibited by the classical mitochondrial permeability transition (MPT) inhibitor cyclosporine A (CsA), macrophage necrosis was reduced. These results suggest that clinical isolates of M. kansasii that grow faster in macrophages induce higher levels of necrosis in a DeltaPsi(m) loss-dependent manner.

The Neff strain of Acanthamoeba castellanii, a tool for testing the virulence of Mycobacterium kansasii.

Virulent Mycobacterium kansasii (mainly subtype 1) may cause lung infections, whereas certain other strains (essentially subtype 3) are commonly non-pathogenic mycobacteria colonizing the human lower respiratory tract of patients. Determining the clinical significance of a strain isolated from a respiratory sample represents a major challenge for clinicians. Since some mycobacteria may use free-living amoebae as a training ground to select virulence traits, we wondered whether the Acanthamoeba castellanii amoeba could be used to determine the virulence of these intracellular bacteria. We investigated whether the growth and cytopathic effect of M. kansasii in A. castellanii correlate with the virulence of M. kansasii determined clinically and by subtyping. Pathogenic subtype 1 M. kansasii strains grew better in A. castellanii than non-pathogenic subtype 3 strains when considering both the number of bacteria per amoeba and the percentage of infected amoebae. Moreover, a subtype 3 M. kansasii strain isolated from blood culture, and thus considered pathogenic, was revealed to grow in A. castellanii similarly to pathogenic subtype 1 strains. These results suggest that amoebae may represent useful tools for testing the virulence of intracellular mycobacteria and other amoeba-resisting bacteria. This is important, since identification of novel bacterial virulence factors relies largely on in vitro assessment of virulence.

Synthesis and antimycobacterial activity of 1,2,4-triazole 3-benzylsulfanyl derivatives.

Series of 3-benzylsulfanyl derivatives of 1,2,4-triazole and 4-methyl-1,2,4-triazole were synthesized by alkylation of starting triazole-3-thiol with appropriately substituted benzyl halide. All members of the set were evaluated for in vitro antimycobacterial activity against Mycobacterium tuberculosis, M. avium, and two strains of M. kansasii. The activities were expressed as the minimum inhibitory concentration. The compounds exhibited only a moderate or slight antimycobacterial activity. Minimum inhibitory concentrations fall into a range of 32->1000 micromol/l. The most active substances bear two nitro groups or a thioamide group on the benzyl moiety. As regards the cytotoxicity effect, the evaluated compounds can be considered as moderately toxic.

Microscopic morphology in smears prepared from MGIT broth medium for rapid presumptive identification of Mycobacterium tuberculosis complex, Mycobacterium avium complex and Mycobacterium kansasii.

Mycobacterium species has a specific morphology when grown in liquid medium. Mycobacterium tuberculosis complex (MTB) often exhibits serpentine cording, which is different from the dot and cross-barring morphology observed in Mycobacterium avium complex (MAC) and Mycobacterium kansasii (MK), respectively. These characteristic morphologies can be used as a cost-effective method for rapid, presumptive identification of mycobacterial isolates cultured from the MGIT 960 system. By using Kinyoun acid-fast stain, serpentine cording was found in 840 of 904 (92.1%) samples positive for MTB; dot or loose aggregation was observed in 112 of 136 (82.3%) samples positive for MAC; and the cross-barring, ladder-like, morphology was observed in 45 of 56 (80.5%) samples positive for MK. The sensitivity and specificity were 92.9% and 96.4% for MTB; 82.4% and 94.5% for MAC; and 80.4% and 94.6% for MK, respectively. Using growth rate selection to exclude rapid growers, the positive and negative predictive values were 98% and 87.6% for MTB; 78.3% and 98% for MAC; and 78.9% and 99.1% for MK, respectively. Twenty-eight (93.3%) of 30 strains with ball morphology were rapid growers. Microscopic morphology can be used for rapid, presumptive identification of M. tuberculosis complex, M. kansasii, and M. avium complex and act as a guide for appropriate selection of initial probes to reduce costs.

Comparison of the BACTEC MGIT 960 with Löwenstein-Jensen medium for recovery of mycobacteria from clinical specimens.

SETTING: Taiwan Provincial Chronic Disease Control Bureau. OBJECTIVE: To evaluate the rate of recovery and the mean time to detection (TTD) of mycobacteria in clinical specimens with two culture systems, the BACTEC MGIT 960 and Löwenstein-Jensen (LJ) medium. DESIGN: We studied 365 specimens, collected from 166 patients. Specimens were processed with standard N-acetyl-L-cysteine (NALC)-NaOH method, then inoculated onto BACTEC MGIT 960 and onto LJ slants. RESULTS: A total of 124 mycobacterial isolates (114 Mycobacterium tuberculosis and 10 non-tuberculous mycobacteria) were detected. The recovery rates were 94% (117/124) with BACTEC MGIT 960 and 75.8% (94/124) with LJ. The rates of contamination for each of the systems were 5.5% with BACTEC MGIT 960 and 4.1% with LJ. The TTDs for mycobacteria were 10.7 days with BACTEC MGIT 960 and 30.6 days with LJ. Excluding the non-tuberculous mycobacteria, the TTDs for M. tuberculosis were 11.1 days with BACTEC MGIT 960 and 30.7 days with LJ. The difference in TTD between smear-positive and smear-negative specimens for either mycobacteria (10.0 vs 12.6 days; P = 0.06) or M. tuberculosis (10.1 vs 12.7 days; P = 0.06) with BACTEC MGIT 960 was not statistically significant. CONCLUSION: The BACTEC MGIT 960 system can expedite the recovery of mycobacteria in culture. Combined with conventional solid medium, it also increases the overall recovery of mycobacteria in culture.

Assessment of morphology for rapid presumptive identification of Mycobacterium tuberculosis and Mycobacterium kansasii.

Mycobacterium tuberculosis often exhibits serpentine cording when grown in liquid medium, whereas Mycobacterium kansasii can be larger and cross-barred. We assessed the use of these morphologic characteristics as a cost-effective method for rapid presumptive identification of isolates from BACTEC bottles. Without specific training, using the Kinyoun acid-fast stain, definitive cording was found in 237 of 373 specimens positive for M. tuberculosis (64%) and cross-barring was recognized within 63 of 76 (83%) of the specimens positive for M. kansasii, giving sensitivities specificities, positive predictive values, and negative predictive values of 63.5, 96, 92, and 79%, respectively, for M. tuberculosis and 83, 95, 59, and 98%, respectively, for M. kansasii. With training and experience, these results improved to 74.5, 98, 96, and 84% and 93, 98, 79, and 98%, respectively. The major improvements were in distinguishing the pseudocording, or loose aggregation of Mycobacterium avium complex from M. tuberculosis and the long beaded forms of Mycobacterium gordonae from M. kansasii. Mycobacterium asiaticum and Mycobacterium szulgai, which rarely occur, are genetically related to M. kansasii and morphologically difficult to distinguish. In defined circumstances, serpentine cording and cross-barring can be used for rapid presumptive identification of M. tuberculosis and M. kansasii, respectively, and as guides for initial probe selection to reduce costs.

[Negative effect of the components of the lysis-centrifugation system in the growth of mycobacteria in MGIT and Septi-Chek AFB liquid media]. / Efecto negativo de los componentes del sistema lisis-centrifugación en el crecimiento de micobacterias en los medios líquidos MGIT y Septi-Chek AFB.

BACKGROUND: The lysis-centrifugation system (Isolator system) is a technique with excellent results in the recovery of mycobacteria from blood specimens. This system consists mainly of saponin (SAP), polypropylenglycol (PPG), and sodium polianthol sulfonate (SPS). The objective of this work was to determine the effect of SAP, PPG, and SPS on the growth of Mycobacterium avium, M. kansasii, M. tuberculosis, and M. xenopi in fluid culture media MGIT and Septi-Chek AFB. METHODS: Two concentrations each of SAP, PPG, and SPS were prepared, and were added in 0.1 ml amounts (alone, in pairs and in combination) to fluid media MGIT and Septi-Chek AFB. Fluid culture media were then in individually inoculated with two different concentrations (10(3) and 10(5) CFU/ml) of each of the four mycobacterial strains used in this study. Culture media were incubated at 37 degrees C and were checked for growth daily. RESULTS: SAP, PPG, and SPS did not inhibit growth of mycobacteria but growth of these strains was indeed retarded (a lengthier time was required for detection of bacterial growth compared with the positive control). Final concentrations of SAP, PPG, and SPS which retarded mycobacterial growth varied, depending upon species, mycobacterial inoculum size, and fluid culture media used. CONCLUSIONS: Components included in the lysy-centrifugation system (SAP, PPG, and SPS), either alone or in combination retarded growth of M. avium, M. kansasii, M. tuberculosis, and M. xenopi in 10(3) and 10(5) CFU/ml concentrations in fluid culture media MGIT and Septi-Chek AFB. These results suggest that strategies should be adopted to decrease the concentrations of these three components, present in the sediment of the processed blood by the Isolator System, which eventually are going to be added to fluid media MGIT and Septi-Check AFB.

In vitro susceptibility of Mycobacterium kansasii to the difluorinated quinolone sparfloxacin using a broth microdilution and macrodilution MIC system.

OBJECTIVES: To study the minimum inhibitory concentrations (MIC) of the difluorinated quinolone sparfloxacin against 32 clinical isolates of Mycobacterium kansasii from 23 patients, all of whom had clinically significant infections due to M. kansasii, and 11 the acquired immune-deficiency syndrome (AIDS). To study the correlation between the microdilution and macrodilution techniques in M7H9 broth. DESIGN: The MICs were determined by two methods: broth microdilution in microplates and broth macrodilution in tubes. The isolates were inoculated into two-fold drug dilutions (ranging from 0.063 to 8 microg/ml) in Middlebrook 7H9 broth and then incubated at 37 degrees C for 21 days. RESULTS: All 32 strains were susceptible, with identical MIC results in both methods, 96.9% of them showing an MIC of 0.25 microg/ml. CONCLUSION: These MIC studies suggest that sparfloxacin may be useful for drug treatment of slow-growing nontuberculous mycobacteria such as M. kansasii. The microdilution method appears to be a reliable method for routine susceptibility testing of M. kansasii, and is easy to interpret and to carry out.

Comparison of the MB/BacT system with a revised antibiotic supplement kit to the BACTEC 460 system for detection of mycobacteria in clinical specimens.

The MB/BacT system (MB/BacT) with a revised antibiotic supplement kit was compared with the BACTEC 460 system (BACTEC 460) in a test of 488 specimens submitted for mycobacterial culture from 302 patients. Twenty-four Mycobacterium tuberculosis isolates were detected by the BACTEC 460 versus 23 isolates by the MB/BacT. Mean time until detection of M. tuberculosis isolates identified by both systems was 11.9 days for the BACTEC 460 versus 13.7 days for the MB/BacT (P = 0.046). M. avium complex was detected in 12 specimens by the MB/BacT versus 10 specimens by the BACTEC 460. Only 8 of 14 (57%) M. avium isolates were detected by both systems, with a mean time until detection of 10.1 days for the BACTEC 460 and 14.2 days for the MB/BacT (P = 0.009). The BACTEC 460 and the MB/BacT detected M. gordonae in four specimens, but only a single specimen was positive by both systems. One M. fortuitum isolate and one of five M. kansasii isolates were recovered only by the BACTEC 460. The bacterial overgrowth rate was 7.0% for the MB/BacT versus 4.1% for the BACTEC 460. We found the MB/BacT to be comparable to the BACTEC 460 for mycobacterial detection. Even though time until detection with the MB/BacT was slightly longer (1.8 days longer for M. tuberculosis and 4.1 days for M. avium [mean values]) and the bacterial overgrowth rate was somewhat higher, the decreased labor, the availability of a computerized data management system, and the noninvasive, nonradiometric aspects of the MB/BacT offset these relative disadvantages and make it an acceptable alternative for use in the diagnostic laboratory.