First published February 2, 2007 on AIDSmap.com.
A large multicenter study conducted in Honduras and Brazil has shown that a new and relatively simple technique to detect active tuberculosis (TB), the microscopic observation drug susceptibility (MODS) test, is just as sensitive but about three times faster than conventional solid culture, the gold standard for confirming a diagnosis of active TB. MODS yields clear results in a median of seven days, compared to three weeks for solid culture.
In addition, in a smaller subset of patients, MODS performance was similar to that of the automated liquid culture system, the MGIT 960, which is now widely used in industrialized countries. However, MODS’ “low cost, relative to other liquid culture methods, may make it feasible for use in resource-limited countries,” wrote the authors of the study, published in the March 1, 2007 edition of Clinical Infectious Diseases.
Also, although capacity in most peripheral laboratories would need to be upgraded in order to perform MODS, in contrast to concerns expressed in an editorial published with an earlier major study of MODS–which worried about the hazards to laboratory staff handling liquid cultures containing live and infectious micro-organisms–the authors of the current study believe that MODS could be made safer “without complex or expensive measures.”
Background on TB Diagnostics
TB, a generally curable illness, continues to be the leading killer of people with HIV in resource-limited settings. One of the primary reasons for the failure to control the disease is that its diagnosis is dependent upon slow or insensitive techniques that are over 100 years old.
The first laboratory test that a person with suspected TB is likely to have is the smear microscopy test (also called the acid fast bacilli or AFB test). In this test, a biological sample (usually sputum) is smeared onto a glass slide and stained with dyes that specifically bind to the cell wall of Mycobacteria tuberculosis (MTB), the organism that causes TB. When this is magnified under a simple light microscope, the lab technician ought to be able to see the stained microbes on the slide.
However, the method is far from exact, depending upon the relative quantity of the sputum in the specimen and the training, eye, and motivation of the lab technician performing this task day after day. As a result, smear microscopy is notoriously insensitive, and in order reach a diagnosis, a person with suspected TB needs to provide at least two or three specimens (usually on successive days provided the patient can make it back to the laboratory).
Although some researchers are investigating techniques for improving the speed and sensitivity of smear microscopy, in practice it misses about half the cases when performed in resource-limited settings. Among people with HIV, it may miss up to 80 percent of the cases later confirmed to be TB cases by culture. (Such cases are referred to as smear-negative TB).
The insensitivity of smear microscopy has led some activists in resource-limited settings to call for wider access to culture, which essentially involves trying to grow mycobacteria (if there are any in the specimen) in a solid gelatin-like substance (the most commonly used type is called Löwenstein-Jensen [LJ] medium). This can usually only be done at reference laboratories–and in most countries, few laboratories perform culture on any meaningful scale.
Even if a specimen is sent off for culturing, the process takes a few weeks, usually longer in smear-negative cases, and diagnosis often comes too late to save a person’s life.
However, a number of new culturing techniques have been devised which speed the growth of MTB, using new liquid media. Most of these methods are technically sophisticated, automated, and proprietary — increasing the cost of culturing by at least ten fold. For example, the cost of the machine used in the Mycobacterial Growth Indicator Tube (MGIT) 960 system is at least 50,000 euros, and twice as much once transported to Africa or Asia (or if the cost of maintenance is included). Furthermore, preliminary studies in resource-limited settings suggest a much greater susceptibility to bacterial contamination using this system.
MODS is a much simpler, manual, liquid culturing technique that relies on basic laboratory equipment and microscopy skills similar to those used for smear microscopy — and the only aspect of the process that is proprietary is the liquid medium (Middlebrook 7H9). To perform a test (start a culture), enriched liquid medium is placed into a well of a tissue culture plate, which is then inoculated with a patient’s specimen (after it has been decontaminated for other microorganisms). Once a culture has had a chance to grow, MTB should be easily identifiable when examined under a light microscope because it grows in very distinctive cord-like shapes.
A proof-of-concept study by Caviedes et al. had already shown that the MODS assay was highly sensitive for TB. And a recent study in Lima, Peru with a large number of samples demonstrated that MODS had a similar sensitivity and specificity for TB as LJ culture and the MGIT system — and can also be used for faster drug susceptibility testing (Moore et al.).
MODS Study in Honduras and Brazil
The current prospective study demonstrates that the MODS test can be successfully performed in laboratories in two other resource-limited settings: Rio de Janeiro, Brazil and Tegucigalpa, Honduras. The study analyzed data from 1,639 routinely collected respiratory specimens from 854 study participants; 559 from Brazil and 295 from Honduras. The two groups included 102 individuals with HIV.
In this trial, a participant was considered to have TB if at least one culture on LJ medium was positive for MTB. If a specimen was only positive on MODS, the participant from whom it was collected was deemed to have TB, if after 90 days of followup, one of their subsequent specimens was positive on LJ culture, or if they had a clear clinical response to anti-TB treatment (without an alternative diagnosis), or if they died of TB.
A total of 357 participants (41.8%) received a final diagnosis of TB. Of these, 348 participants were positive on the MODS test, while 28 participants who were positive on MODS were diagnosed as negative by LJ. The sensitivity, specificity, positive predictive value, and negative predictive value of the MODS assay were 97.5% (95% CI, 95.7%-98.6%), 94.4% (95% CI, 93.1%-95.2%), 92.6% (95% CI, 90.9%-93.6%), and 98.1% (95% CI, 96.8%-98.9%), respectively.
Not all of the 28 MODS-positive, LJ-negative participants were false positives however: four were later determined to have TB by response to anti-TB treatment. Of the remainder, four were given an alternative diagnosis and eleven were determined to have non-tubercular mycobacteria (NTM) (by biochemical techniques performed on the LJ cultures), while nine were categorized as not- TB because they were lost to follow-up. There is a good chance, however, that some of these were indeed TB because the authors noted, “the clinical suspicion for TB was high.”
Per specimen, there was concordance between MODS and LJ culture in 94.2% (95% CI, 93.1-95.1%). MODS tests were also less prone to contamination than LJ cultures, 62 [3.8%] vs. (95 [5.8%] of 1,639 samples, respectively (P =0.01). Of note, a significantly greater proportion of the LJ cultures were contaminated in the Honduras lab. The performance of the MODS test, however, was consistent across study sites.
The MODS test was also dramatically faster than LJ culture, with a median time to growth of 7 days (interquartile range [IQR] 5-10 days) compared to 21 days (IQR 17-25 days) for LJ cultures (P<0.01). Similarly, a subset of 64 specimens evaluated by the MGIT 960 system had a median time to growth of 8 days [IQR, 6-11.5 days; (P=0.16). MODS detected 90.4% of the positive samples within two weeks, compared to just 16.6% by LJ culture (P<0.01).
A total of 83 individuals were determined to have smear-negative TB. The MODS assay detected 75 of these cases, although the time to detection was slightly longer than for smear-positive patients: a median of ten days for the MODS assay versus 26 days for LJ culture. Nevertheless, MODS compared well to culture–only 2.2% of these were culture positive on LJ medium within two weeks, while the MODS assay detected 73.1% of the positive cultures within two weeks (P< 0.01).
The Promise of MODS
The earlier the diagnosis, the sooner treatment can begin, which is particularly important for people with HIV and smear-negative TB.
One possible drawback, however, could be the inability of laboratory technicians to distinguish between TB and some NTM. This could potentially have clinical impact in settings where NTM prevalence is high and not all mycobacteria respond to anti-TB treatment. This is a problem for other liquid culture techniques as well, so a number of laboratory tests (some based on molecular techniques) are currently in development to improve and speed the identification of NTM.
Another issue is that although the technique can probably be performed in any lab that can currently perform culture, MODS is technically more challenging than smear microscopy. In addition to training technicians, most laboratories would need to upgrade capacity and establish supply chains for reagents and supplies that may not be readily accessible. The process is still manual and requires motivated lab techs. In middle-income countries with a heavy burden of TB (where laboratories must analyze a high number of specimens), it may still be more cost-effective to use an automated system–particularly if countries are receiving assistance from bi- or multilateral funding partners.
And then there is the safety issue–most laboratories where smear microscopy is performed do not have functioning biosafety hoods that are necessary to safely examine live cultures. In this case, the liquid cultures are in plates that must be transported from an incubator to a microscope–and there is a serious risk of spillage and exposure to TB (possibly even MDR- or XDR-TB).
However, the authors of the current study suggest that “modification of the current plate platform into a more secure platform (e.g., the use of a tight-fitting lid to reduce the possibility of spills) would be advantageous and is likely to be feasible.”
Finally, even though this is a low-tech and more rapid way to culture, resource constraints will create logistical problems with transportation of specimens and getting the results back to the clinic. In practice many people will still not receive their diagnosis in time to receive life-saving treatment. Simple, low cost, same-day TB tests that can generate an accurate diagnosis when and where the patient first presents for care are still urgently needed.
- Arias M et al. Clinical evaluation of the microscopic-observation drug-susceptibility assay for detection of tuberculosis. Clin Inf Dis 44: 674-680, 2007.
- Caviedes L et al. Rapid, efficient detection and drug susceptibility testing of mycobacterium tuberculosis in sputum by microscopic observation of broth cultures. The Tuberculosis Working Group in Peru. J Clin Microbiol 38: 1203-1208, 2000.
- Iseman MD et al. Rapid detection of tuberculosis and drug-resistant tuberculosis. N Eng J Med 355: 1606-1608, 2006.
- Moore DAJ et al. Microscopic-observation drug-susceptibility assay for the diagnosis of TB. New Eng J Med 355: 1539-1551, 2006.