Timing of geological events in the lunar highlands recorded in shocked zircon-bearing clasts from Apollo 16.

Apollo 16 soil-like regolith breccia 65745,7 contains two zircon-bearing clasts. One of these clasts is a thermally annealed silica-rich rock, which mineralogically has affinities with the High Alkali Suite (Clast 1), and yields zircon dates ranging from 4.08 to 3.38 Ga. The other clast is a KREEP-rich impact melt breccia (Clast 2) and yields zircon dates ranging from 3.97 to 3.91 Ga. The crystalline cores of both grains, which yield dates of ca 3.9 Ga, have undergone shock pressure modification at less than 20 GPa. We interpret that the U-Pb chronometer in these zircon grains has been partially reset by the Imbrium basin-forming event when the clasts were incorporated into the Cayley Plains ejecta blanket deposit. The zircon grains in Clast 1 have been partially decomposed, resulting in a breakdown polymineralic texture, with elevated U, Pb and Th abundances compared with those in the crystalline zircon. These decomposed areas exhibit younger dates around 3.4 Ga, suggesting a secondary high-pressure, high-temperature event, probably caused by an impact in the local Apollo 16 highlands area.

Assessing the shock state of the lunar highlands: Implications for the petrogenesis and chronology of crustal anorthosites.

Our understanding of the formation and evolution of the primary lunar crust is based on geochemical systematics from the lunar ferroan anorthosite (FAN) suite. Recently, much effort has been made to understand this suite's petrologic history to constrain the timing of crystallisation and to interpret FAN chemical diversity. We investigate the shock histories of lunar anorthosites by combining Optical Microscope (OM) 'cold' cathodoluminescence (CL)-imaging and Fourier Transform Infrared (FTIR) spectroscopy analyses. In the first combined study of its kind, this study demonstrates that over ~4.5 Ga of impact processing, plagioclase is on average weakly shocked (<15 GPa) and examples of high shock states (>30 GPa; maskelynite) are uncommon. To investigate how plagioclase trace-element systematics are affected by moderate to weak shock (~5 to 30 GPa) we couple REE+Y abundances with FTIR analyses for FAN clasts from lunar meteorite Northwest Africa (NWA) 2995. We observe weak correlations between plagioclase shock state and some REE+Y systematics (e.g., La/Y and Sm/Nd ratios). This observation could prove significant to our understanding of how crystallisation ages are evaluated (e.g., plagioclase-whole rock Sm-Nd isochrons) and for what trace-elements can be used to differentiate between lunar lithologies and assess magma source compositional differences.

[Prosthetic hip infection due to Listeria monocytogenes: review of the literature and case report]. / Infection prothétique de hanche à Listeria monocytogenes.

With the ageing of the population, articular prosthetic replacements are becoming more and more frequent. One of the most feared complications is prosthetic infection, mostly due to bacteria of the cutaneous flora. Listeria monocytogenes is rarely the cause. This paper describes the management of a hip prosthetic infection due to Listeria monocytogenes. The patient was cured with antimicrobial therapy and a two-stage exchange. This case report creates an opportunity to review the literature in the aim of determining the risk factors and the optimal care.

Beta-lactam resistance in Staphylococcus aureus: the adaptive resistance of a plastic genome.

Staphylococci have two mechanisms for resistance to beta-lactam antibiotics. One is the production of beta-lactamases, enzymes that hydrolytically destroy beta-lactams. The other is the expression of penicillin-binding protein 2a (PBP 2a), which is not susceptible to inhibition by beta-lactam antibiotics. Strains of S. aureus exhibiting either beta-lactamase or PBP 2a-directed resistance (or both) have established a considerable ecological niche among human pathogens. The emergence and subsequent spread of bacterial strains designated as methicillin-resistant S. aureus (MRSA), from the 1960s to the present, has created clinical difficulties for nosocomial treatment on a global scale. The recent variants of MRSA that are resistant to glycopeptide antibiotics (such as vancomycin) have ushered in a new and disconcerting chapter in the evolution of this organism.

Efficacy of a single intravenous dose of amphotericin B for Candida urinary tract infections: further favorable experience.

Studies in experimental animals and humans have shown that Amphotericin B (AmB) persists in urine for days to weeks after a single IV dose in levels that should inhibit candidal organisms and thereby obviate the need for frequent dosing. Including data from four previously described patients, we have now treated a total of 11 patients (12 episodes) with Candida urinary tract infections with single-dose AmB (six, Candida albicans; two, C. tropicalis; four, other nonalbicans Candida). The duration of candiduria prior to entry ranged from 18 to 180 days. Predisposing conditions included renal transplantation (1), diabetes mellitus (8), genitourinary stones (1) or anomalies (4), catheterization (2), and antibacterial therapy (11). A single patient was intolerant of AmB. Out of 11 evaluable candiduric episodes, eight resolved. Failure occurred in one patient with a chronic indwelling bladder catheter and in the allograft recipient. The data suggest that the sustained urinary excretion of AmB may permit successful single- or paucidose therapy of Candida urinary tract infections in some patients with a minimum of toxicity.

Unusual steroid specificity of the cell surface progesterone receptor on human sperm.

The steroid specificity of the cell surface progesterone receptor in human sperm was examined with the use of progesterone, testosterone, and androstane analogues. Many compounds were shown to be more effective than progesterone at increasing intracellular free calcium concentration, e.g., 2 alpha-methyl-17beta-methoxy-5 alpha-androstan-3-one. Several testosterone analogues were demonstrated to be antagonists of progesterone, e.g., 9(11)-dehydro-2 alpha,17alpha-dimethyltestosterone. The synthetic potent progestigens, norethynodrel, cyproterone acetate, norethindrone, and megestrol acetate, were found to be only weak stimulators of the sperm cell surface receptor. Furthermore, these compounds were shown to antagonize the effect of progesterone to elevate intracellular free calcium concentration in sperm. It is known that progesterone and some of its analogues bind to the intracellular progesterone nuclear receptor via the alpha-face of the steroid molecule. In stark contrast, it was concluded from the analysis of the steroid analogues examined on human sperm in this study that intimate contact exists between the effective progesterone analogues and the sperm cell surface progesterone receptor across the beta-face of the steroid C/D-ring "upper" edge (C11, C12, and C17). Positioning of the C21 methyl group is also critical for efficacy, and recognition of the steroid A-ring seems not to be involved.

Kinetics and inhibition of lipid exchange catalyzed by plasma cholesteryl ester transfer protein (lipid transfer protein).

The cholesteryl ester transfer protein-catalyzed cholesteryl ester transfer is inhibited by two compounds identified by a large-scale screening of cholesterol backbone-containing molecules. Kinetic analysis shows that U-95,594, an amino steroid, inhibits competitively the cholesteryl ester transfer protein-catalyzed transfer of both cholesteryl esters and triglycerides, as well from high-density lipoproteins as from synthetic microemulsions. In contrast, U-617, an organomercurial derivative of cholesterol, inhibits competitively the transfer of cholesteryl ester from either donor but is without any effect on triglyceride transfer. In addition to the rapid, competitive inhibition of cholesteryl ester transfer, U-617 also slowly and reversibly reacts with cholesteryl ester transfer protein to produce an additional 10-fold decrease in cholesteryl ester transfer activity but, again, without effect on triglyceride transfer.

Method for measuring the activities of cholesteryl ester transfer protein (lipid transfer protein).

A continuous recording fluorescence assay was developed for cholesteryl ester transfer protein (CETP). The assay measures the increase in fluorescence accompanying the relocation of fluorescent lipids, cholesteryl esters and triglycerides, from a donor emulsion to an acceptor emulsion. In the absence of CETP, the quantum yields of the fluorescent lipids is low because their high concentrations in the donor emulsions result in self-quenching. CETP catalyzes the redistribution of the fluorescent lipids from the donor to the acceptor emulsions and fluorescence increases substantially. Efficient sonication and incorporation of apolipoproteins from human HDL into the emulsions significantly increased the transfer rates. Under optimal conditions, the redistribution of fluorescent compounds reaches equilibrium within < 30 min and the kinetics of this process are consistent with a simple, first-order reaction pathway. The redistribution kinetics support a mechanism of adsorption --> exchange --> desorption --> diffusion.

A convenient synthesis of 7 alpha-hydroxycholest-4-en-3-one by the hydroxypropyl-beta-cyclodextrin-facilitated cholesterol oxidase oxidation of 3 beta,7 alpha-cholest-5-ene-3,7-diol.

The initial biosynthetic conversions of cholesterol to the bile acids involve sequential 7 alpha-hydroxylation (catalyzed by cholesterol 7 alpha-hydroxylase) followed by C-3 oxidation and concomitant double bond migration (to a delta 4-configuration, catalyzed by 3 beta-delta 5-C27-steroid oxidoreductase) to provide 7 alpha-hydroxycholest-4-en-3-one. A straightforward, and economical, preparation (on a 0.1 g scale) of this pivotal biosynthetic intermediate has been devised. Reduction of 3 beta-(benzoyloxy)-cholest-5-en-7-one with LiB(sec-butyl)3H provided a 4:1 mixture, respectively, of the 7 alpha- and 7 beta-hydroxy diastereomers, which were separated chromatographically. Solvolytic removal of the C-3 benzoyl group gave 3 beta,7 alpha-cholest-5-ene-3,7-diol. A suspension of the 1:1 (v/v) complex (formed by mutual dissolution in MeOH, followed by evaporation of the solvent) of this diol with hydroxypropyl-beta-cyclodextrin, at a concentration of 1 mg mL-1 (in neutral phosphate buffer), was converted by Brevibacterium sp cholesterol oxidase (0.25 U mg-1 of substrate) and catalase (70 U mg-1 of substrate, to recover O2 from the H2O2 produced by the enzymatic oxidation) to a suspension of 7 alpha-hydroxycholest-4-en-3-one and the hydroxypropyl-beta-cyclodextrin. The yield for the enzymatic conversion was in excess of 90%. A much poorer and less reproducible yield (< 20%) was seen in the absence of the hydroxypropyl-beta-cyclodextrin. Routine extraction of this aqueous suspension, and chromatographic purification (85:15 CHCl3/acetone v/v on silica) of the residue, gave pure 7 alpha-hydroxycholest-4-en-3-one in 68% isolated yield. This route is a significant improvement, in terms of reaction scale and convenience, over the previous procedures for the preparation of this steroid.

Yeast in the urine: solutions for a budding problem.

The significance of candiduria ranges from simple procurement-related contamination to disseminated candidiasis. Ensuring that a valid urine specimen is collected and carefully assessing patients for risk factors predisposing to disseminated candidiasis permit the stratification of cases into three clinical categories: (1) asymptomatic candiduria in a previously healthy patient; (2) candiduria in a high-risk patient in whom disseminated candidiasis is unlikely; and (3) candiduria in a high-risk patient with a potential for disseminated candidiasis. Strategies for management are tailored to the individual patient. Appropriate management of anatomic genitourinary abnormalities and removal of bladder catheters may result in the resolution of candiduria, although some patients require systemic antifungal therapy. All patients with candiduria should be evaluated for evidence of deep-seated tissue infection or candidemia before therapy is instituted. Fluconazole appears to be a safe and effective agent for the management of candidal urinary tract infection. Both its safety and its ease of administration make it superior to amphotericin B for this purpose.

Appraisal of a glycopeptide cloaking strategy for a therapeutic oligopeptide: glycopeptide analogs of the renin inhibitor ditekiren.

Among the limitations to the practical therapeutic oligopeptide are low oral availability, indifferent aqueous solubility, and an astonishing efficient sequestration and biliary elimination by a multi-capacity liver transporter. Given the purposed use of N- and O- linked saccharides as functional appendages of eukaryotic peptides and proteins, a strategy of glycopeptide mimicry was examined for the oligopeptide renin inhibitor, ditekiren. The anticipation was that the saccharide would impart significant aqueous solubility, and might impact beneficially on the remaining two limitations. Execution of this approach was achieved by the removal of the (dimethylethoxy)carbonyl amino terminus of ditekiren, and its substitution by Boc-L-asparagine N-linked mono- and disaccharides. Potent hypotensive activity, as measured by a human renin-infused rat assay, is observed for virtually all of these structures (N-linked beta-pyranose D-N-acetyglucosaminyl, D-glucosaminyl, D-N-acetylgalactosaminyl, D-mannosyl, D-galactosyl, D-maltosyl, D-cellobiosyl, D-chitobiosyl, but not L-fucosyl). The basis for this dramatic improvement (relative to ditekiren in the same assay) is the diversion of the peptide clearance from rapid liver biliary clearance to slower urinary clearance (Fisher, J. F.; Harrison, A. W.; Wilkinson, K. F.; Rush, B. R.; Ruwart, M. J. J. Med. Chem. 1991, 34, 3140). Guided by the human renin-infused rat hypertension assay, an evaluation of the linker-saccharide pairing was made. Loss of hypotensive activity is observed upon substitution of the Boc-L-asn by Boc-D-asn, and by removal of the Boc amino terminus of the glycopeptide. Potent hypotensive activity is preserved by replacement of the Boc-L-asn linker by succinate, malate, tartrate, and adipate linkers. With the longer adipate spacer, attachment of the saccharide to the P-3 phenylalanine--with omission of the P-4 proline--retains activity. These data suggest value to the glycopeptide guise for preserving the in vivo activity, and for the beneficial manipulation of pharmacodynamics, of this renin inhibitory oligopeptide. This strategy may have general applicability.

Evaluation of a vitamin-cloaking strategy for oligopeptide therapeutics: biotinylated HIV-1 protease inhibitors.

The outstanding limitations to the oligopeptide as a therapeutic agent are poor oral availability and rapid biliary clearance. To address these concerns a series of eight peptidic HIV-1 protease inhibitors containing the structural segment of the vitamin biotin have been prepared. These have been evaluated with regard to the hypothesis that this vitamin would cloak the peptidic character of these oligopeptides, and thus impart to these inhibitors the potential for absorption and distribution via biotin transporters and receptors. By iterative optimization about a -Cha psi[CH-(OH)CH(OH)]Val- core inhibitory insert, three particularly potent inhibitors (K(i) < or = 10 nM) of the HIV-1 protease were obtained. Although excellent cell culture antiviral activity is observed for other peptidic protease inhibitors of comparable affinity, none in this series exhibited satisfactory antiviral activity. This failure is attributed to the incompatibility of the hydrophilic and hydrogen-bonding biotin segment, with the facile membrane permeability and intracellular access presumably required for antiviral activity. The ability of the biotin to cloak the peptide, and thus render the overall appearance of the conjugate as that of a vitamin, was evaluated. Four of this series were evaluated for recognition by the Caco-2 cell intestinal biotin transporter. None inhibited competitively biotin uptake, indicating a lack of recognition. A vitamin may bind to a specific protein carrier, and thus attain an improved serum profile (by resistance to biliary clearance) and advantageous delivery to cells. Therefore, the serum concentrations of three were evaluated following an iv bolus in a rat model for serum clearance. One of the three protease inhibitors (L-idonamide, 6-cyclohexyl-2,5,6-trideoxy-2-(1-methylethyl)-5-[[3-methyl-1-oxo-2-[[5- (hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-4-yl)-1- oxopentyl]amino]butyl]amino]-N-[2-methyl-1-[[(2- pyridinylmethyl)amino]carbonyl]butyl]-, [3aS-[3a alpha, 4 beta (1R*,2R*,3R*),6 alpha]]-) sustained a more than 5-fold increase in serum concentration at all time points relative to the benchmark structure. The remaining two had serum concentrations at least equal to the benchmark, suggestive of improved resistance to clearance. One (L-idonamide,6-cyclohexyl-2,5,6-trideoxy-5-[[2-[[5-(hexahydro-2-ox o-1H- thieno-[3,4-d]imidazol-4-yl)pentyl]thio]benzoyl]amino]-2-(1- methylethyl)-N-[2-methyl-1-[[(2-pyridinyl- methyl)amino]carbonyl]butyl]-, [3aS-[3a alpha, 4 beta(1R*,2R*),6a alpha]]-) was prepared as a complex with the biotin-binding protein avidin. Avidin may resemble an endogenous serum biotin carrier protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Tissue penetration of clindamycin in diabetic foot infections.

Serum and tissue samples were obtained during surgery from four diabetics with neuropathy who underwent debridement or amputation for foot infections while receiving clindamycin 600 or 900 mg iv. Clindamycin concentrations were assayed by radioimmunoassay. Clindamycin was detected in all serum and tissue samples (range: 0.04-2.8 mg/kg in tissues and 1.1-11.1 mg/L in serum). In nine of the eleven tissue samples the clindamycin concentration exceeded the MICs reported for many pathogens commonly involved in such infections. In only a single instance was the ratio of tissue to serum concentration < 0.13.

Evaluation of the bile acid transporter in enhancing intestinal permeability to renin-inhibitory peptides.

To evaluate the bile acid transporter as a means of enhancing the ability of renin-inhibitory peptides (RIPs) to penetrate the intestinal mucosa, two RIP-cholic acid conjugates and an RIP-taurocholic acid conjugate were synthesized. Conjugation was through the N-terminus of an RIP and the 3-position of the bile acid, via a six-carbon spacer. An RIP derivative containing the spacer without the bile acid moiety was also synthesized. The bile acid-RIP conjugates and the RIP derivative were shown to be potent inhibitors of human renin in vivo and to have in vivo hypotensive activity equivalent to that of the parent RIP (ditekiren) in a human renin-infused rat model. The ability of these RIP derivatives to bind to the bile acid transporter and be transported across an epithelial cell monolayer was evaluated in an in vitro model of the intestinal mucosa consisting of Caco-2 cell monolayers grown on microporous membranes. One of the RIP-cholic acid conjugate (KI = 60 +/- 10 microM) and the RIP-taurocholic acid conjugate (KI = 19 +/- 5 microM), but not the RIP derivative, were shown to be potent inhibitors of the apical (AP) to basolateral (BL) transport of [14C]-taurocholic acid ([14C]-TA). At concentrations up to 250 microM these RIP-bile acid conjugates had no effect on the diffusion of [3H]-PEG (800-1000), which is a marker of the paracellular pathway. The permeability coefficients of the RIP-bile acid conjugates, determined using Caco-2 cell monolayers, were shown to be six times less than that of [3H]-PEG (800-1000). In addition, the transport of one of the RIP-cholic acid conjugates was investigated in perfused rat ileum in which the mesenteric vein was cannulated. The conjugate was not detected in blood samples taken from the mesenteric vein, while its concentration in intestinal perfusate remained almost constant during the perfusion experiment. These results suggest that while the peptide-bile acid conjugates retain binding affinity for the intestinal bile acid transporter, the molecules are not themselves transported.

Spectral characterization of environment-sensitive adducts of interleukin-1 beta.

We have determined the fluorescence properties of two covalently attached acrylodan derivatives of recombinant human interleukin-1 beta, namely the Cys-8 and Lys-103 adducts. The emission and excitation maxima indicated the presence of two operationally distinct conformers for each probe. The iodide quenching and the kinetics of fluorescence changes associated with guanidinium chloride-induced denaturation show that each covalent adduct exists both in hydrated and dehydrated environments. Furthermore, fluorescence changes associated with the binding of the adducts to a recombinant soluble murine receptor indicated that only the conformers with the label in the hydrophobic environment are competent toward the soluble murine interleukin receptor and that the hydrated and dehydrated conformers are in a dynamic equilibrium on the time scale of the binding experiments.

Comparison of amphotericin B with fluconazole in the treatment of acute AIDS-associated cryptococcal meningitis. The NIAID Mycoses Study Group and the AIDS Clinical Trials Group.

BACKGROUND: Intravenous amphotericin B, with or without flucytosine, is usually standard therapy for cryptococcal meningitis in patients with the acquired immunodeficiency syndrome (AIDS). Fluconazole, an oral triazole agent, represents a promising new approach to the treatment of cryptococcal disease. METHODS: In a randomized multicenter trial, we compared intravenous amphotericin B with oral fluconazole as primary therapy for AIDS-associated acute cryptococcal meningitis. Eligible patients, in all of whom the diagnosis had been confirmed by culture, were randomly assigned in a 2:1 ratio to receive either fluconazole (200 mg per day) or amphotericin B. Treatment was considered successful if the patient had had two consecutive negative cerebrospinal fluid cultures by the end of the 10-week treatment period. RESULTS: Of the 194 eligible patients, 131 received fluconazole and 63 received amphotericin B (mean daily dose, 0.4 mg per kilogram of body weight in patients with successful treatment and 0.5 mg per kilogram in patients with treatment failure; P = 0.34). Treatment was successful in 25 of the 63 amphotericin B recipients (40 percent; 95 percent confidence interval, 26 percent to 53 percent) and in 44 of the 131 fluconazole recipients (34 percent; 95 percent confidence interval, 25 percent to 42 percent) (P = 0.40). There was no significant difference between the groups in overall mortality due to cryptococcosis (amphotericin vs. fluconazole, 9 of 63 [14 percent] vs. 24 of 131 [18 percent]; P = 0.48); however, mortality during the first two weeks of therapy was higher in the fluconazole group (15 percent vs. 8 percent; P = 0.25). The median length of time to the first negative cerebrospinal fluid culture was 42 days (95 percent confidence interval, 28 to 71) in the amphotericin B group and 64 days (95 percent confidence interval, 53 to 67) in the fluconazole group (P = 0.25). Multivariate analyses identified abnormal mental status (lethargy, somnolence, or obtundation) as the most important predictive factor of a high risk of death during therapy (P less than 0.0001). CONCLUSIONS: Fluconazole is an effective alternative to amphotericin B as primary treatment of cryptococcal meningitis in patients with AIDS. Single-drug therapy with either drug is most effective in patients who are at low risk for treatment failure. The optimal therapy for patients at high risk remains to be determined.

Infection due to the fungus Acremonium (cephalosporium).

Human infections due to fungi belonging to the genus Acremonium occur uncommonly, but unlike infections due to other filamentous fungi, usually affect immunocompetent individuals. Mycetoma, which usually develops following trauma, is the most common infection caused by Acremonium spp. Other sites of infection include the eye (generally following abrogation of ocular defenses), colonizing disease of the lung and gastrointestinal tract, as well as locally invasive infections such as osteomyelitis, sinusitis, arthritis, and peritonitis. Pneumonia and disseminated infections including meningitis, endocarditis, and cerebritis rarely have been reported. Optimal treatment of acremonium infections is not well defined both because infections due to these organisms are rare, and because many reports antedate effective antifungal therapy. In addition, susceptibility testing of filamentous fungi is poorly standardized, and in vitro sensitivity may not correlate with clinical response. Based on anecdotal reports, treatment of most invasive acremonium infections requires a combination of surgical intervention, when possible, and a regimen of amphotericin B. Some azoles also display inhibitory activity. Until more details are available regarding susceptibility of these organisms to antifungal agents, amphotericin B is recommended as initial therapy with the addition of either ketoconazole or fluconazole in infections of a life-threatening nature.

Development of an immunoassay for endogenous digitalislike factor.

Recently, attempts to purify and identify a circulating inhibitor of the sodium pump have been successful. Based on the outcome of mass spectral analysis of purified inhibitor, we raised in rabbits antibodies to conjugates of the commercially available cardenolide ouabain and used them in the development of an indirect enzyme-linked immunosorbent assay (ELISA) for endogenous digitalislike factor (EDLF). Antisera obtained were of high antibody titer (1:2 x 10(6)) and showed full cross-reactivity with purified EDLF. The antisera were highly specific for ouabain and structurally related cardenolides but showed no cross-reactivity with numerous endogenous steroids and peptides. At each step in the purification of EDLF, inhibition of the sodium pump and immunologic cross-reactivity were inseparable. The ELISA as developed had a working range of 5-2,000 fmol, with an IC50 of 80 fmol/well. Using solid-phase extraction and the ELISA, we determined the circulating level of EDLF in plasma from normal human volunteers to be 138 +/- 43 fmol/ml, whereas patients on total parenteral nutrition for at least 1 week had a circulating level of 108 +/- 17 fmol/ml, suggesting that the circulating factor was of endogenous origin. The ELISA developed appears to measure a naturally occurring counterpart to the cardenolides that could play a role in modulating the sodium pump and thereby cellular electrolyte homeostasis.

Utility of Gram's and Giemsa stains in the diagnosis of pulmonary tuberculosis.

Conflicting information in the literature regarding the staining properties of Mycobacterium tuberculosis using Gram's stain and experience in two patients with pulmonary tuberculosis in whom the diagnosis was suspected after staining with non-acid-fast bacillus stains prompted the study of Gram's stain in this disease. The main finding was that mycobacteria appear as refractile, gram-neutral, or faintly gram-positive bacilli after Gram's stain, depending upon the plane of focus in which the organisms are regarded. It is concluded that the diagnosis of mycobacterial disease may be suggested by Giemsa- and Gram-stained smears of clinical specimens. M. tuberculosis may be either gram-neutral or gram-positive.