A doctor who suspects pulmonary tuberculosis (TB) in his patient mustwait a week or more _ sometimes much more _ for a laboratory testto confirm that Mycobacterium tuberculosis is indeed the pathogen,and thus that the disease is indeed TB. It takes that long to cultureenough of the microbe's cells from sputum for light microscopy to pinthe disease definitively on M. tuberculosis. (See BioWorld Today, May7, 1993, p. 1.)That's just the beginning. Then, other lengthy tests must determine towhich of the dozen or so TB-specific antibiotics the particular strain ofthe germ has developed resistance.This is why many major pharmaceutical firms, university researchersand at least one biotechnology company are deploying the mostadvanced tools of molecular bacteriology to blow M. tuberculosis'cover.Pulmonologist Neil Schluger, who directs the Chest Clinic at NewYork's Bellevue Hospital, is enlisting polymerase chain reaction (PCR)to finger the TB pathogen faster. His brief research paper in this week'sLancet is titled: "Amplification of DNA of Mycobacteriumtuberculosis from peripheral blood of patients with pulmonarytuberculosis."He tested eight patients with sputum-confirmed active TB of the lung,and 18 healthy volunteer controls.DNA extracted from their peripheral blood, and PCR-amplified,yielded a final product of 253 base pairs of an insertion elementspecific to the pathogen. All eight known TB patients tested positive;all control samples, negative. The results took two days to acquire,"much faster that sputum culture," Schluger told BioWorld Today."Using PCR," he said, "we could diagnose active pulmonarytuberculosis in all eight cases tested. And there were no false-positivesamong the controls, whether or not they had a history of TB infectionor exposure."To Schluger's knowledge, this is the first TB test of blood rather thansputum. Blood, he pointed out, is easier to obtain, and more reliable,than sputum, which tests positive in only half to three-quarters of cases.Finding mycobacterial DNA in peripheral blood was surprising,because "no patient in our series had clinical signs of TB outside thelungs."He added that AIDS patients with TB often have positive M.tuberculosis blood cultures, but "nearly always in the setting ofextrapulmonary disease." Six of his eight positive patients had AIDS.The Bellevue team will now test "many many more patients than thefew in this preliminary PCR study," Schluger said. His aim is to verifyits continued reliability during and after antibiotic treatment.Jack Crawford, chief of microbacteriology at the Centers for DiseaseControl And Prevention (CDC) in Atlanta, told BioWorld Today thathe knows of some 50 journal papers on PCR tests for TB. At one time,he added, Roche Diagnostic Systems Inc. and SmithKline Beecham plcwere offering in-house testing of samples sent to them, but he is notsure if they are still doing so."Roche Diagnostic," Crawford said, "has a PCR-based assay, which,following clinical trials, is being readied for FDA submission."Christine Aylward, a Roche spokeswomen, confirmed that thecompany will submit its sputum-based PCR test "soon" to FDA. Italready is marketed in Europe, she told BioWorld Today, under thetrade-name Amplicor, and performs a test "in four to six hours."Gen-Probe Inc. of San Diego also has a sputum-based, quasi-PCR TBtest awaiting FDA approval."It can be done in less than five hours," the firm's manager of scientificaffairs, Craig Hill, told BioWorld Today. The test is already marketedin Japan, and in Europe, where its cost to the laboratory is around $30per sample.Clinical specificity of the test, Hill said "has been around 98 percent onaverage; sensitivity around 93 percent."In Gen-Probe's process, Hill explained, "We use a transcription-basedamplification system, where the target is ribosomal RNA. From this weproduce a transcription template, which transcribes many millions ofcopies of the amplicon. This is then detected by chemiluminescence,using an acridinium ester."Chemiluminescence, based on luciferase, is also the detection methoddeveloped by William Jacobs at Albert Einstein College of Medicine inBronx, New York. A year ago , Eli Lilly & Co. and Bristol-MyersSquibb were both dickering to license Jacobs' process. But the licensewent instead to Becton-Dickinson & Co. of Franklin Lakes, N.J.,Jacobs said.That company's director of investor relations, Ronald Jasper, wouldnot comment, beyond saying that the test "is quite a ways fromfruition." n

-- David N. Leff Science Editor

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