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We showed previously that susceptibility testing for Mycobacterium tuberculosis labeled with fluorescein diacetate could be accomplished rapidly by using flow cytometry. However, safety was a major concern because mycobacteria were not killed prior to flow cytometric analysis. In this study, we developed a biologically safe flow cytometric susceptibility test that depends on detection and enumeration of actively growing M. tuberculosis organisms in drug-free and antimycobacterial agent-containing medium. The susceptibilities of 17 clinical isolates of M. tuberculosis to ethambutol, isoniazid, and rifampin were tested by the agar proportion and flow cytometric methods. Subsequently, all flow cytometric susceptibility test samples were inactivated by exposure to paraformaldehyde before analysis with a flow cytometer. Agreement between the results from the two methods was 98%. In addition, the flow cytometric results were available 72 h after the initiation of testing. The flow cytometric susceptibility assay is safe, simple to perform, and more rapid than conventional test methods, such as the BACTEC system and the proportion method.

Susceptibility testing of Mycobacterium tuberculosis is seriously limited by the time required to obtain results. We show that susceptibility testing of clinical isolates of M. tuberculosis can be accomplished rapidly with acceptable accuracy by using flow cytometry. The susceptibilities of 35 clinical isolates of M. tuberculosis to various concentrations of isoniazid, rifampin, and ethambutol were tested by the agar proportion method and by flow cytometry. Agreement between the results from the two methods was 95, 92, and 83% for isoniazid, ethambutol, and rifampin, respectively. Only 11 discrepancies were detected among 155 total tests. The results of flow cytometric susceptibility tests were available within 24 h of inoculation of drug-containing medium, while the proportion method required 3 weeks to complete. The flow cytometric method is also simple to perform.

Summary These results demonstrate that sus-ceptibility testing of M. tuberculosis can be accomplished rapidly by using a flow cytometer. Results of tests were readily available within 24 h after M. tuberculosis organisms were incubated with antimycobacterial agents. Our method also does not require multiplication of M. tuberculosis cells. These findings are important and greatly decrease the time for obtaining sus-ceptibility test results.

The emerging magnitude of human fungal infections has renewed interest in developing rapid and standardized methods for susceptibility testing. We demonstrated that susceptibility testing of Candida albicans can be accomplished rapidly by using flow cytometry. Test results were available within 8 to 24 h after C. albicans isolates were incubated with amphotericin B, itraconazole, and flucytosine. This is an improvement of 24 to 60 h in the time to availability of susceptibility test results compared to the time to availability of National Committee for Clinical Laboratory Standards-recommended broth macrodilution test results. In addition, the flow cytometric endpoints, mean channel fluorescence, and number of fluorescence-labeled C. albicans cells were easy to interpret for greater sensitivity and reliability. Flow cytometry provides a more accurate means of obtaining antifungal susceptibility test results.

Here is another unit with clinical relevance. Tuberculosis remains a major health problem throughout the world, with approximately one-quarter of the population being infected. Rapid and accurate susceptibility testing for the tubercle bacillus is essential for control of the disease. Such testing can be accomplished by flow cytometry within twenty-four hours, instead of the days to weeks required by traditional methods. The use of flow cytometry both improves the quality of susceptibility testing and advances public health measures for the prevention and control of this ancient scourge.

Elevated blood plasma cholesterol (hypercholesterolemia) is a major risk factor for coronary artery disease (CAD) in humans. Genetic dissection of polygenic lipid and lipoprotein disorders in swine, a key animal model for the study of familial hypercholesterolemia (FH) and CAD, led to the isolation of a monogenic subphenotype (FH-r), that is inherited in the recessive (r) manner. A genome scan mapped the FH-r locus close to the centromere of chromosome 2. Comparative mapping showed that this region shares homology with a part of human chromosome 19 that harbors the low density lipoprotein receptor (LDLR) locus, and therefore suggested LDLR as the prime candidate gene for FH-r. Cloning and sequencing of hepatic LDLR cDNA from two FH-r/r and one normal (N/N) animals disclosed a single missense mutation (R84C) in a region that corresponds to human exon 4. The C84 mutation cosegregates invariantly with hypercholesterolemia, which strongly suggests that this mutation is responsible for the observed hyperlipidemia.

DNA sequence information underpins genetic research, enabling discoveries of important biological or medical benefit. Sequencing projects have traditionally used long (400-800 base pair) reads, but the existence of reference sequences for the human and many other genomes makes it possible to develop new, fast approaches to re-sequencing, whereby shorter reads are compared to a reference to identify intraspecies genetic variation. Here we report an approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost. Single molecules of DNA are attached to a flat surface, amplified in situ and used as templates for synthetic sequencing with fluorescent reversible terminator deoxyribonucleotides. Images of the surface are analysed to generate high-quality sequence. We demonstrate application of this approach to human genome sequencing on flow-sorted X chromosomes and then scale the approach to determine the genome sequence of a male Yoruba from Ibadan, Nigeria. We build an accurate consensus sequence from >30x average depth of paired 35-base reads. We characterize four million single-nucleotide polymorphisms and four hundred thousand structural variants, many of which were previously unknown. Our approach is effective for accurate, rapid and economical whole-genome re-sequencing and many other biomedical applications.