Two advances in TB diagnostics at South African HIV conference

Diagnosing active TB in people with HIV can be a challenge, particularly in the developing world where resources, laboratory facilities and technical expertise are often limited. Most of TB’s symptoms and X-ray findings are indistinguishable from those caused by other respiratory conditions.

Thus, diagnosis is dependent upon laboratory tests. Evidence of TB — acid fast bacilli (AFBs) — can sometimes be detected under a microscope after applying a special stain (dye) to a specimen (smear) of the patient’s sputum, bronchial fluid or other biological samples. A positive result is proof that the patient has an active TB infection, but smear results often come back negative even when the patient turns out to have active TB.

The diagnosis can be confirmed by culturing (growing) the mycobacterium from the patient’s specimen but this is costly and takes several weeks to perform — far too long to postpone therapy or to subject an uninfected person to unnecessary and potentially harmful treatment. Such late diagnosis is not only bad for the patient, it represents a threat to the public health since an infected individual is likely to spread the infection to others in his or her community.

“By 2005,” said Dr. Douglas Wilson, formerly of the University of Cape Town and currently of Grey’s Hospital in Pietermaritzburg, “45% of TB cases in South Africa will be sputum smear-negative disease in HIV-infected adults. New diagnostic tools are urgently needed,” said Dr. Wilson, “particularly in countries with a high prevalence of HIV.”

One such potential tool, the FastPlaqueTB^TM , made by Biotec Laboratories in Ipswich, is a relatively simple test that requires only basic laboratory facilities, and equipment and skills. Results are produced within 48-72 hours, can be read by eye and are easy to interpret.

Dr. Wilson presented findings from a study of the FastPlaqueTB test during an oral session at the 1st South African Conference on AIDS on Wednesday morning. The study enrolled 143 HIV-positive adults who were receiving care from a resource-limited facility, GF Jooste Hospital in Mannenburg, which treats patients from peri-urban and informal settlements in and around Cape Town.

The study enrolled 143 consenting patients who were suspected of having TB but whose cases could not be diagnosed by sputum smears (defined as either two negative results or an inability to produce sputum). Suspicion of TB was based upon chest X-ray findings and/or symptoms that failed to respond to a course of antibiotics.

Sputum was induced from patients with hypertonic saline by using an ultrasonic nebuliser, an inexpensive but very effective device for this purpose. Urine samples were also collected from all patients, and other clinically relevant specimens were obtained when deemed necessary (e.g., in cases of suspected extrapulmonary TB). Specimens were sent for both culturing and FastPlaque tests.

The vast majority of participants (93%) gave their consent in Xhosa. The average age was ~33 years old, and 65 percent were women, and 80 percent were unemployed. The average CD4 cell count was 103. At study entry, 77 (54%) of the participants were smear negative, while 66 (46%) were unable to produce sputum. TB was eventually diagnosed in 125 of the patients. Culture proven TB was confirmed in 103.

The FastPlaqueTB test detected TB in 64% of the specimens that were culture positive, and produced no false positives. Induced sputum samples from patients who could not previously produce sputum were also submitted for smear testing for AFB. A percentage came back positive or “scanty” (barely) positive. Positive and scanty positive smears were highly correlated with positive results on FastPlaqueTB, although there were two smear positive specimens that FastPlaqueTB did not detect.

Dr. Wilson noted that these specimens came from the same patient who may have had something unusual in his lungs that interfered with the assay. “The FastPlaqueTB consistently picks up smear positive cases,” he said.

In the cases of pulmonary TB, FastPlaqueTB consistently detected significantly more cases than induced smear tests (p

TB can be very difficult to diagnose in smears from other bodily specimens such as urine or lymph node biopsies. FastPlaqueTB detected only 38% of TB culture positive urine samples, and performed poorly when other bodily fluids (e.g., pleural or ascitic) were investigated. However, the test picked up 63% of the culture positive needle core lymph node biopsy specimens, and 10 out of 10 of the cold abscess aspirates.

“The FastPlaqueTB^TM assay cannot replace mycobacterial culture as the ‘gold standard for the diagnosis of TB, but it is better than induced sputum smear and may have a role in diagnosing smear-negative tuberculosis,” Dr. Wilson concluded.

Dr. Wilson noted that the test is commercially available and relatively cheap. When asked how cheap, Dr. Wilson replied that it would depend upon the scale of the purchase. If South African provincial health departments or other governments decide to use the test on a large scale, “the cost could be quite low,” he said.

At the same oral session, Dr. Inraan Mahomed of Wits University in South Africa reported on his efforts to develop an even faster, more sensitive and specific approach to diagnosing TB. Essentially, the test is a modified PCR technique that utilises a “Light Cycler” a fluorometric device that can continuously monitor very rapid amplification of TB gene sequences in small volumes.

In Dr. Mahomed’s hands, the test generates results in less than two hours. Better yet, by incorporating probes for the gene mutations responsible for drug resistance, the assay can concurrently differentiate between wild type and drug-resistant TB. In other words, drug susceptibility testing, which used to take months by culturing, could soon be done in real time.

Unfortunately, unlike the FastPlaqueTB assay, this technology requires expensive, sophisticated laboratory equipment and most importantly, skilled technicians trained to perform it. It will likely be confined to the research laboratory for the time being. However, as multidrug-resistant TB (MDR-TB) grows more common in Sub-Saharan Africa, the speed at which the Light Cycler TB PCR performs drug susceptibility testing could make it cost-effective enough to introduce into regional reference labs.