Any week now, evaluation of the newest test for mankind'soldest disease will begin at Albert Einstein College of Medicinein New York.
Until lately, pulmonary tuberculosis (TB) seemed to be on theway out, thanks to a growing arsenal of antibiotics specificallyfatal to Mycobacterium tuberculosis. But in recent years, thepathogen has overtaken the drugs ganged up against it, andmultidrug resistance is driving the statistics of TB infection anddeath back up.
TB now infects 8 million people worldwide every year, and 3million people a year die of the disease. There were 28,000cases in the U.S. in 1992. It is the largest cause of death in theworld from a single infectious disease.
In some Third World countries, TB accounts for 40 percent ofall AIDS fatalities, microbial geneticist William Jacobs reportedin today's issue of Science. Jacobs is lead author of a paper inthe journal titled "Rapid Assessment of Drug Susceptibilities ofMycobacterium tuberculosis by Means of Luciferase ReporterPhages"
Jacobs is an associate professor at Albert Einstein, where his"rapid assessment" is about to be checked out, and aninvestigator at its Howard Hughes Medical Institute.
As pulmonary tuberculosis stages its lethal comeback in theFirst World, here is a typical scenario in the deadly duelbetween the thrust of medical science and the mutational parryof Mycobacterium tuberculosis.
A person who has been feeling low for some months, andcoughing more and more, suddenly starts spitting bright redblood and rushes off in alarm to the doctor. The physician takesa sample of sputum and sends it to a clinical microbiology labto test the bacillus' resistance to the 11 TB antibiotics nowavailable. M. tuberculosis is a sluggish bug, which takes 24hours to replicate, so the lab will have to culture the sputumspecimen for three to four weeks to get enough cells to begintesting for drug resistance.
Meanwhile, the doctor starts dosing the patient with a cocktailof the three main antibiotics -- isoniazid, rifampicin andpyrazinamide -- hoping that at least one of them will still beactive against the pathogen.
Jack Crawford, chief of microbacteriology at the Centers forDisease Control and Prevention (CDCP) in Atlanta, explainedthat the typical physician's interim multidrug strategy isintended "to ensure you don't have enough organisms withdouble mutation," which causes multidrug resistance.
"We have one strain from New York City," Crawford toldBioWorld, "that is resistant to six different antibiotics -- sixdifferent mutations. It's caused a real TB outbreak, with wellover 100 cases there." He added, "We'd like to have a muchmore rapid method of knowing before we begin chemotherapywhat the spectrum of drug resistance is, so that we can use twoor preferably three drugs to which the organism is sensitive."CDCP is supporting Jacobs' lab by testing strains for drugsusceptibility.
The upcoming trial of what Jacobs calls his "turn-on-the-lightassay," will begin by taking bacterial cultures of existingsputum samples, grown up for testing by standard methods.Validation should require about a year, he estimated.
"The currency we use for genetics," Jacobs told BioWorld, is thecolony-on-a-plate." The time it takes to grow a colony of M.tuberculosis, which doubles once a day, from a single cellthrough 26 generations to 100 million cells is three weeks orso. That compares with E. coli, which doubles every 20 minutes,and gets you a colony in about eight hours."
In Jacobs' luciferase testing system, sputum samples needculturing for only about a week, to grow up 100,000 bacteria,not 100 million. He infects cultures of known drug sensitivityand known resistance with a bacterial phage vector containingthe FFlux gene that expresses firefly luciferase. This enzyme isthe match that lights luciferin, the molecule that glows in thedark, fueled by adenosine triphosphate.
To the now-transgenic bacilli he adds luciferin. Drug-resistantstrains signaled their viability by lighting up in vitro.Susceptible strains, killed or weakened by the antibiotic,turned off their luciferase expression, and the light went out.
Besides -- and before -- the phage-delivered luciferase systemis developed to accelerate patient testing from weeks to days,Jacobs said, it will go to work for pharmaceutical companies, tospeed up discovery of new drugs against the TB pathogen.
"Now, pharmaceutical companies won't even screen for anti-TBdrugs," Jacobs declared, "because their drugs won't even lastthe three weeks or more while test colonies grow up. We canprovide them with a two-hour assay."
As revealed in Science, Eli Lilly and Co. and Bristol-MyersSquibb are both negotiating with Albert Einstein's industrialliaison office to license the process.
Jacobs added that such use needn't wait for the trial results.Pathologist Sydney Goldfisher, who directs licensure, toldBioWorld, "It's a complex problem; a number of patents areinvolved. But we're very close to putting a package together.We are looking," he said, "for industrial partners with financialand scientific resources, and the capacity for bringing ourtechnology to market as rapidly as possible. Quality partners todevelop a product that meets FDA standards and serves thecommunity."
Meanwhile, Jacobs' co-developer, genetic engineer Graham F.Hatfull at the University of Pittsburgh, has sequenced all52,297 base pairs of a super-phage that delivers a much-amplified dose of FFlux. With it as vector in a future second-generation system, fewer than 100 TB bacillus cells shouldsuffice to test drug resistance by visible light.
"When we get down to that level," Jacobs foresees, "we'll bethinking about a diagnostic test for TB infection that will giveus results from a clinical sample in less than a week."
-- David N. Leff Science Editor
(c) 1997 American Health Consultants. All rights reserved.