Rapid pyrazinamide susceptibility testing of Mycobacterium tuberculosis by flow cytometry

The resurgence of tuberculosis along with the increased resistance of Mycobacterium tuberculosis has emphasized the need for timely susceptibility testing for control of the disease. Previous studies have shown that rapid susceptibility testing can be accomplished for isoniazid, ethambutol, and rifampin using the flow cytometric assays. In this study we compared the flow cytometric susceptibility assay with the BACTEC TB 460 and BACTEC MGIT 960 for pyrazinamide (PZA). There was 93% agreement between the BACTEC MGIT 960 and the flow cytometric methods for 100 microg/mL of PZA. Additionally, there was a 95% and 86% agreement between the BACTEC TB 460 and flow cytometric methods for 50 microg/mL and 100 microg/mL of PZA, respectively. These findings show that susceptibility testing by the flow cytometric assay is accurate. Most importantly, susceptibility results by the flow cytometric assay were available 24 h after initiation of the testing procedure. The advantages of simplicity, speed and accuracy make the flow cytometric susceptibility assay an immediate impact technology to improve patient care.     

Antibiotic susceptibility testing ofCandida albicans by flow cytometry

A flow cytofluorometric susceptibility test (FCST) for in vitro antifungal drug testing ofCandida albicans was developed. Membrane damage was indicated by increased cellular fluorescence owing to propidium iodide or rose bengal uptake. Ketoconazole caused an exponential dose-response effect, best defined by E_max, at therapeutically achievable concentrations (0.02–0.2 µg/ml). This effect was not comparable to the conventional minimum inhibitory concentration (MIC) effect elicited by higher antibiotic concentrations. Amphotericin B, on the other hand, did not elicit E_max, but caused a >1 log dose-response effect which did correspond to the MIC. 5-Fluorocytosine susceptibility was also measurable. Other cytometric data indicating abnormal growth and growth inhibition, as well as conventional growth inhibition testing, confirmed that the 10-h FCST measured useful parameters of in vitro susceptibility.     

In-vitro antimycobacterial drug susceptibility testing of non-tubercular mycobacteria by tetrazolium microplate assay

Background

Signature matching of nucleotide sequences in the V1 and V3 regions 16S rRNA genes using pyrosequencing technology is a powerful tool for typing vaginal Lactobacilli to the species level and has been used for investigating the vaginal microbial niche.

Methods

This study has characterized the normal cultivable vaginal Lactobacillus flora at varying estradiol levels in plasma; the study comprised 17 patients undergoing ovarian stimulation for In Vitro Fertilization (IVF) treatment. The vaginal status of each participant was initially assessed as normal according to Amsel and Nugent criteria.

Results

L. crispatus, L. gasseri and/or L. jensenii were present in 10 of the patients throughout the study period, and little variation among these three species was encountered in individual patients. The flora of three women was dominated by L. delbrüeckii, L. rhamnosus or L. vaginalis. One woman exhibited a dominance of L. iners. The flora of the remaining three women were initially dominated by L. rhamnosus or L. reuteri, but as their estrogen levels rose, their flora composition altered, to become dominated by one of the three species most common in a normal, healthy vagina.

Conclusion

Signature matching of nucleotide sequences in the V1 and V3 regions of 16S rRNA genes is a discriminative tool for the study of vaginal Lactobacilli and can be used to track the Lactobacillus flora under a variety of physiological conditions.     

Amphotericin B susceptibility testing of Candida species by flow cytometry.

We have developed an 8 hr flow cytometry (FCM) method for assessing susceptibility of yeasts to amphotericin B (AmpB). The method detects both high-level and relative-resistance to the drug. Variables found to affect fluorescence of control and AmpB treated cells included pH, presence of glucose, incubation conditions, concentration and length of exposure to both AmpB and ethidium bromide (ETBR), and the degree of resistance to AmpB. The FCM method was optimized based on increased red fluorescence intensity (RF), decreased forward angle light scatter (FALS), and a negative gating technique. A dose response was seen between 0.1 and 10 micrograms AmpB/ml for the susceptible control strain. Greater than 50% of cells from all susceptible strains tested transfer into the negative gate when exposed to 2.5 micrograms Amp B/ml while fewer than 5% of cells of the highly resistant C. tropicalis (ATCC 28707) are affected at concentrations up to 20 micrograms/ml. This method may provide a more accurate assessment of Amp B susceptibility than conventional tube dilution methods.     

Assay to measure CD59 mutations in CHO A(L) cells using flow cytometry.

BACKGROUND: A sensitive mammalian cell mutation assay was developed previously using a Chinese hamster ovary cell line (CHO A(L)) that stably incorporates human chromosome 11. The assay measures mutations in the CD59 gene on chromosome 11 but it requires the use of rabbit complement and colony growth for mutant selection. We have developed a more rapid flow cytometry-based mutation assay with CHO A(L) cells that uses monoclonal antibodies against CD59 to detect mutants and does not require colony formation. METHODS: CHO A(L) cells were treated with gamma-radiation or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and then allowed to grow for various times for mutant expression. Cells were labeled with monoclonal antibodies against CD59 and analyzed by flow cytometry. RESULTS: Negative and positive populations were separated by over 100-fold. Mixing various proportions of CD59-positive and -negative cells demonstrated that the assay is highly linear (r2 = 0.9999) and sensitive (<0.05% background mutants). The yield of CD59-inducible mutants was linearly related to dose for a clastogen (gamma-radiation) and point mutagen (MNNG). The mutant yield was time and treatment specific. CONCLUSIONS: Mutations induced by genotoxic agents can be rapidly and sensitively measured in CHO A(L) cells using flow cytometry.     

Applications of flow cytometry to ecotoxicity testing using microalgae

Flow cytometry is a rapid method for the quantitative measurement of light scattering and fluorescent properties of cells. Although this technique has been widely applied to biomedical and environmental studies, its potential as a tool in ecotoxicological studies has not yet been fully exploited. This article describes the application of flow cytometry to the development of bioassays with marine and freshwater algae for assessing the bioavailability of contaminants in waters and sediments.     

Multi parameter in vitro testing of ratjadone using flow cytometry

Ratjadone, isolated from the myxobacterium Sorangium cellulosum, belongs to the family of so-called orphan ligands, which includes leptomycin, callystatin and other compounds. In previous screening tests, ratjadone revealed a growth inhibitory effect against bacteria, yeast and human cancer cells. Following these first results, ratjadone was tested on several human tumour cell lines (Jurkat, HepG2, U87-MG) and, as a control, on a non-tumour cell line (RLC18) for its mode of action. The cell analysis was carried out by flow cytometry. This comprised cell density measurements, live-dead analysis, cell-cycle analysis and detection of apoptosis. First experiments confirmed the growth inhibitory effect on any chosen tumour cell line. Following these results a dose effect relationship was monitored, confirming the high effectiveness of ratjadone against cell growth at nanomolar concentration. Cell cycle analysis has shown that ratjadone intervenes in the cell cycle by arresting the cells in G1-phase. Biological testing of additional ratjadone derivatives with changed configuration and stereochemistry, identified the pharmacophoric site of the molecule.     

Cytostatic drug testing in human leukemias by means of multiparametric flow cytometry

Human bone marrow cells from 20 patients as well as the permanent human B-cell lines RPMI 1788, Raji, Daudi, T-cell lines Molt, CEM, Jurkat and the promyelocytic line HL 60 were assayed by means of a newly developed in vitro flow cytometric cytostatic drug assay. The cells were exposed to cytosine-arabinoside, L-asparaginase, daunorubicin, prednisone or vincristine. Surviving cells were stained after an incubation period of 2 to 7 days with esterase and pH-indicator dye ADB (1,4-diacetoxy-2,3-dicyanobenzene), dead cells with DNA-dye PI (propidium iodide). Dose-response curves were established using percent surviving cells. It was possible to evaluate bone marrow samples from 16 out of 20 patients. Seven samples were leukemic (acute myeloid leukemia (AML) n = 6, Non-Hodgkin's Lymphoma (NHL) n = 1). Nine samples were from patients either in complete remission or with benign diseases. Daunorubicin and cytosine-arabinoside were cytotoxic in both groups, whereas vincristine was effective mainly in the leukemic group (p less than 0.05). There was significant heterogeneity in the reactivity of AML-marrow cells from different patients to different drugs. The cell lines exhibited different patterns of sensitivity. Vincristine arrested cells in G2/M-phase, cytosine-arabinoside caused an increase of cells in the S-phase.     

Rapid parallel flow cytometry assays of active GTPases using effector beads

We describe a rapid assay for measuring the cellular activity of small guanine triphosphatases (GTPases) in response to a specific stimulus. Effector-functionalized beads are used to quantify in parallel multiple GTP-bound GTPases in the same cell lysate by flow cytometry. In a biologically relevant example, five different Ras family GTPases are shown for the first time to be involved in a concerted signaling cascade downstream of receptor ligation by Sin Nombre hantavirus.     

New possibilities of cytostatic drug testing on patient tumor cells by flow cytometry

A new assay for cytostatic drug testing is described which can be automated. Pleural effusions and ascites are cultured as such for one week. Cells of solid tumors are cultured in the patients own serum for the same time. The cells are then stained with the esterase and intracellular pH-indicator dye 1,4-diacetoxy-2,3-dicyano-benzene (ADB) to label vital cells. They are simultaneously stained with propidium iodide (PI) as an indicator for dead cells. Monosized fluorescent latex particles are added as concentration, volume and fluorescence standard. Inflammatory cells can be distinguished in the assay from tumor cells because of their small cell volume. The number of dead and surviving cells is counted by the flow cytometer and a therapeutic index is calculated as ratio between the surviving inflammatory to surviving tumor cells. An important feature of the assay is that the DNA-distribution of the dead cells (e.g. aneuploidy) as well as the functional state of the surviving tumor cells and inflammatory cells can be judged from intracellular esterase activity and intracellular pH.     

A high-throughput cell-based toxicity analysis of drug metabolites using flow cytometry

The effects of liver enzymes on drug activities are important considerations in the drug discovery process. Frequently, liver microsomes are used to simulate first-pass metabolism in the liver; however, there are significant disadvantages to the microsome system. As an alternative, a simple cell-based, high-throughput system that allows for examination of metabolite activity is described. Using multiparameter flow cytometry and the low-volume, high-sample format of 96-well plates, it is possible to rapidly evaluate a dose-response curve for metabolites based on variables including initial compound concentrations, hepatocyte cell line metabolic activities, and time. Using HepG2 cells as a surrogate for hepatic metabolism of a potential therapeutic, the impact of metabolites on Jurkat cell death was measured by both propidium iodide dye exclusion and cell cycle analysis. While this system is not proposed to supplant liver microsome studies, this alternative assay provides a highly adaptable, low-cost, and high-throughput measure of drug metabolism.     

Comparison between radiometric and proportional methodologies in susceptibility testing of mycobacteria other than tubercle bacilli (MOT)

109 strains of mycobacteria other than tubercle bacilli (MOT) were tested against four primary antituberculous drugs; 27 strains of M. tuberculosis served as controls. While the latter species showed excellent overall agreement with the reference (proportion) method of Canetti (particularly for sensitive strains), MOTT gave such results only if there was a prevalence of sensitive or resistant strains in a species. Since this could not be predicted, the radiometric method cannot substitute for the proportion method for susceptibility testing of MOTT, in spite of early availability of results.     

Rapid identification and antibiotic susceptibility testing of Yersinia pestis using bioluminescent reporter phage

The rapid identification and antibiotic susceptibility testing of Yersinia pestis is paramount for a positive prognosis. We previously engineered a Y. pestis-specific 'bioluminescent' reporter phage for the identification of Y. pestis. In this study, we generated an improved reporter phage and evaluated the ability of this phage to provide direct and rapid susceptibility testing. Compared to the first generation reporter, the second generation reporter exhibited a 100-fold increase in signal strength, leading to a 10-fold increase in assay sensitivity. Y. pestis antimicrobial testing in the presence of the reporter elicited bioluminescent signals that were drug concentration-dependent, and produced susceptibility profiles that mirrored the standard CLSI method. The phage-generated susceptibility profiles, however, were obtained within hours in contrast to days with the conventional method.     

Rapid direct determination using combined separation by prepared immunomagnetic and flow cytometry of Flavobacterium psychrophilum

Flavobacterium psychrophilum, the causative agent of bacterial cold-water disease (BCWD), was originally isolated from coho salmon Oncorhychus kisutch in the USA. Bacterial cold-water disease has since been spreading throughout Japan and has caused serious damage to populations of ayu Plecoglossus altivel in many farms and rivers. The rapid method of detecting for F. psuchrophilum is requested, however, traditional methods are laborious because of complicated assay procedures. In this study, a rapid method of detecting F. psychrophilum was developed using a modified method of flow cytometry (FCM) analysis and immunomagnetic separation (IMS). Magnetic iron, in small particles, was prepared by the reaction of a mixture of ferric and ferrous ions under alkaline conditions. The particles were coated with antiserum against F. psychrophilum by dextran. Polyclonal antibodies (anti-F. psychrophilum) conjugated with fluorescein isothiocyanate (FITC) were reacted with F. psychrophilum, and then prepared immunomagnetic were applied using IMS, followed by FCM determination. A good correlation was observed between the cell numbers determined by the FCM method and the traditional method in the range of 10(2)-10(8) cells ml(-1). The FCM analysis could count cells within 1min, and the total analysis time, including sample preparation, was less than 2 h.     

Addressing the malaria drug resistance challenge using flow cytometry to discover new antimalarials

A new flow cytometry method that uses an optimized DNA and RNA staining strategy to monitor the growth and development of the Plasmodium falciparum strain W2mef has been used in a pilot study and has identified Bay 43-9006 1, SU 11274 2, and TMC 125 5 as compounds that exhibit potent (<1 μM) overall and ring stage in vitro antimalarial activity.     

Androgenesis and homozygous gynogenesis in muskellunge (Esox masquinongy): Evaluation using flow cytometry

The purpose of this work was to study the effects of ultraviolet (UV) irradiation on denucleation of eggs and investigate the heat-shock conditions for diploidization for induction of androgenesis in muskellunge, Esox masquinongy. Several egg incubation media, including saline, Ringer's solution, and Ringer's solution supplemented with bovine serum albumin (BSA), were found suitable to maintain the egg fertility as high as in muskellunge ovarian fluid. The optimal doses of UV radiation were 660–1320 J/m², at which 100% haploid larvae were produced at a hatching rate of 22.5 ± 2.8%. UV irradiation at low doses (165–330 J/m²) generated abnormal larvae, which were morphologically identical to haploids. Using a flow cytometry method, it was found that cellular DNA content of these larvae was close to that of diploids but significantly lower in value and had a wider distribution (expressed as coefficient of variation) than that of control fish. This suggested that a low dose of UV irradiation might cause gene mutations, alteration of chromosomal conformation and fragmentation, but did not prevent maternal DNA from participating in mitotic division. Interference of maternal DNA residues could be another reason for the poor viability of androgenetic fish. A high dose of UV radiation (1980 J/m²) caused development of severely deformed embryos, indicating that UV radiation also damaged molecules in the eggs other than the denucleation. Our results suggest that classic color and allozyme markers might not be sufficient to prove a complete androgenesis. In order to optimize time and duration of shock for induced diploidization, we investigated the heat-shock conditions for inhibiting the first mitotic cleavage through induction of homozygous gynogenesis. We found that heat-shock treatment at 31°C for 9 min starting at 1.4τ₀ (a dimensionless factor describing progress in embryo development) after fertilization produced the highest percentage of diploids at hatching. Mol. Reprod. Dev. 49:10–18, 1998. © 1998 Wiley-Liss, Inc.     

Antimalarial drug susceptibility testing of Plasmodium falciparum in Brazil using a radioisotope method

From March 1996 to August 1997, a study was carried out in a malaria endemic area of the Brazilian Amazon region. In vivo sensitivity evaluation to antimalarial drugs was performed in 129 patients. Blood samples (0.5 ml) were drawn from each patient and cryopreserved to proceed to in vitro studies. In vitro sensitivity evaluation performed using a radioisotope method was carried out with the cryopreserved samples from September to December 1997. Thirty-one samples were tested for chloroquine, mefloquine, halofantrine, quinine, arteether and atovaquone. Resistance was evidenced in 96.6% (29/30) of the samples tested for chloroquine, 3. 3% (1/30) for quinine, none (0/30) for mefloquine and none for halofantrine (0/30). Overall low sensitivity was evidenced in 10% of the samples tested for quinine, 22.5% tested for halofantrine and in 20% tested for mefloquine. Means of IC 50 values were 132.2 (SD: 46. 5) ng/ml for chloroquine, 130.6 (SD: 49.6) ng/ml for quinine, 3.4 (SD: 1.3) ng/ml for mefloquine, 0.7 (SD: 0.3) ng/ml for halofantrine, 1 (SD: 0.6) ng/ml for arteether and 0.4 (SD: 0.2) ng/ml for atovaquone. Means of chloroquine IC 50 of the tested samples were comparable to that of the chloroquine-resistant strain W2 (137.57 ng/ml) and nearly nine times higher than that of the chloroquine-sensitive strain D6 (15.09 ng/ml). Means of quinine IC 50 of the tested samples were 1.7 times higher than that of the low sensitivity strain W2 (74.84 ng/ml) and nearly five times higher than that of the quinine-sensitive strain D6 (27.53 ng/ml). These results disclose in vitro high resistance levels to chloroquine, low sensitivity to quinine and evidence of decreasing sensitivity to mefloquine and halofantrine in the area under evaluation.     

Fluorescent in situ hybridization en suspension (FISHES) using digoxigenin-labeled probes and flow cytometry.

Fluorescent in situ hybridization has become a major technique for visualizing genetic material in fixed cells. Currently, many systems utilize the hybridization of labeled molecular probes to cells that are attached to slides. We have developed a technique that allows for in situ hybridization to be performed using cells in suspension. By using digoxigenin-labeled DNA probes and a fluoresceinated antibody directed against the digoxigenin, we can measure the resulting signal on a flow cytometer and the cells can be attached to microscope slides for visual analysis.     

Uses and future applications of flow cytometry in immunotoxicity testing.

Flow cytometry is an emerging technology that has numerous applications to immunotoxicity testing. The use and development of high-speed single-cell laser-based assays capable of quantitation of fluorescence, light scatter, and electrical impedance measurements can provide important information on xenobiotic-induced toxicity in defined target cell populations. The purpose of this article is to briefly review established and emerging immunotoxicology assays that use flow cytometry. In the coming years it is likely that many new flow cytometry assays will be developed and validated that will improve the sensitivity and perhaps specificity of immunotoxicity testing. Since flow cytometry is readily adaptable to high-throughput screening, it is also likely that this technology will increasingly find its place in the preclinical testing of drugs and chemicals in the pharmaceutical and chemical industries.