Mixed results on ELISPOT TB testing in resource-limited settings

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Ex-vivo enzyme linked immunospot (ELISPOT) tests for TB could prove useful for detecting latent TB infections in resource-limited settings with a high prevalence of HIV according to two studies presented on Monday at the World AIDS Conference in Toronto. The studies, conducted in Zimbabwe and South Africa, demonstrated that ELISPOT results are less likely than skin testing to be confounded by exposure to environmental mybobacteria, the BCG vaccination, or by anergy in people with HIV.

However, in the South African study, it was not always clear whether the ELISPOT added much to diagnosis when the HIV status of an adult was known and the skin test interpreted accordingly. Furthermore, issues of logistics and cost effectiveness need to be overcome before this test can be scaled up for more widespread use in resource-limited settings.

Diagnosing TB exposure

For the past hundred years, skin tests have been used to determine whether a person has been exposed to TB.

There are essentially two variations of TB skin test, the tuberculin skin test (TST) or the purified protein derivative (PPD or Mantoux) test, which both involve the injection of a sample of non-infectious mycobacterium tuberculosis (Mtb) proteins under the skin (the Mantoux test, which uses a syringe, is the more precise test).

Glossary

mycobacteria

Family of bacteria that includes the causes of tuberculosis and MAI.

immune system

The body's mechanisms for fighting infections and eradicating dysfunctional cells.

confounding

Confounding exists if the true association between one factor (Factor A) and an outcome is obscured because there is a second factor (Factor B) which is associated with both Factor A and the outcome. Confounding is often a problem in observational studies when the characteristics of people in one group differ from the characteristics of people in another group. When confounding factors are known they can be measured and controlled for (see ‘multivariable analysis’), but some confounding factors are likely to be unknown or unmeasured. This can lead to biased results. Confounding is not usually a problem in randomised controlled trials. 

culture

In a bacteria culture test, a sample of urine, blood, sputum or another substance is taken from the patient. The cells are put in a specific environment in a laboratory to encourage cell growth and to allow the specific type of bacteria to be identified. Culture can be used to identify the TB bacteria, but is a more complex, slow and expensive method than others.

smear

A specimen of tissue or other material taken from part of the body and smeared onto a microscope slide for examination. A Pap smear is a specimen of material scraped from the cervix (neck of the uterus) examined for precancerous changes.

If someone has been previously exposed to TB, the immune system should ‘recognise’ the injected proteins, and within a few days, a raised bump or induration should form at the site of the injection — like an allergic reaction, although not the same mechanism. This bump is measured by a healthcare worker and if it is large enough, it is considered a positive result.

However, TB skin tests have a couple of shortcomings. Firstly, reactions may occur whether someone has been exposed to Mtb or other environmental mycobacteria. Since the BCG vaccine is derived from a usually harmless bovine mycobacteria, it can also cause a reaction to a TB skin test, especially in children or adolescents who have been recently vaccinated.

Another shortcoming of the TB skin tests is that, when HIV disease progresses, the immune system can become so weak (anergic) that it no longer mounts a clearly recognisable reaction to the skin test — making it hard to tell whether someone has been exposed to TB or not. Thus exact size of the induration that is considered positive really depends upon the person being tested: if they are HIV-negative, it should generally be 10 to 15mm or larger; but in many HIV-positive people, an induration about 5mm can be considered positive. This also illustrates another problem with skin tests — they have to be measured by someone which creates the potential for miscalculations or misinterpretation.

Zimbabwean study

To answer this question, researchers in Zimbabwe led by Dr Junior Mutsvangwa, compared responses to both tuberculin skin tests and ELISPOT in 129 factory workers with TB (diagnosed by smear microscopy, or culture) and 149 randomly selected factory workers without evidence of TB. The study also compared the TB test results in the household contacts (older than ten years of age) of the TB cases (n=226) and of the controls (188).

In this study, an induration of at least 10mm was considered positive, while on the ELISPOT (which was read manually rather than with an expensive “reader” machine), more than five spots seen in the background (or negative control) and more than twice the number of spots as in the background were considered positive.

Using the 10mm cut-off, less than 50% of the TB cases tested positive by skin test compared to over 65% on the ELISPOT. In contrast, responses on TST were more likely to be positive than the ELISPOT in the controls and household contacts (and couldn’t really distinguish between those with TB from the controls).

However, almost 80% of the TB cases (and ~28% of their contacts) were also HIV-positive, compared to between 10-20% for the controls and their contacts. HIV status had a clear effect on TST responses, with only 37% of those with HIV and TB testing TST positive compared to 80% of the TB cases without HIV (p<0.001). Similar effects were observed in the HIV-positive controls and contacts.

HIV’s effect on TSTs was more marked in smear negative culture negative patients with HIV — but it is not clear whether this is due to immunosuppression or diagnostic uncertainty.

One interesting finding that Dr. Mutsvangwa noted was that on ELISPOT, there appeared to be “little evidence of transmission to household contacts from patients with HIV-related TB.”

Dr. Mutsvangwa concluded that “the ELISPOT is a more appropriate test for MTB infection in for HIV prevalent settings" though issues of logistics and cost still must be sorted out before the test can be introduced more widely. However, according to Dr Mutsvangwa, lab technicians can read the test manually (rather than with a machine reader) — which reduces the cost substantially.

But the use of the 10mm cut-off throws some of these conclusions into doubt because a number of studies show that 5mm is a better cut-off for people with HIV, while 15mm may be more appropriate (with fewer false negatives due to BCG vaccination and background mycobacteria) in people with healthier immune systems.

The ELISPOT test

The ELISPOT represents one of the first significant advances in TB diagnostics in decades. The ELISPOT TB test also looks for an immune responses for TB but in the blood test. ELISPOT looks for an immune reaction (the secretion of gamma interferon) in a blood sample exposed to fragments of two proteins (ESAT-6 and CFP10) that are unique to the human form of Mtb. The test is less subjective and should thus be able to distinguish between exposure to TB and other mycobacteria or the BCG vaccine.

Preliminary data also seem to indicate that the ELISPOT is less affected by advancing HIV disease. As a result, the test has been approved for use in industrialised nations, but it hasn’t been clear how useful the test would be in resource-limited settings.

The South Africa study

In contrast, the South African study looked at three different cut-offs for TB skin tests, comparing the more precise PPD (Mantoux) test to two commercial ELISPOT tests, the T-Spot TB and QuantiFERON-TB Gold tests.

160 patients without active TB referred by an HIV testing clinic to the study, which was conducted at the Ubuntu Clinic in Khayelitsha, Cape Town. The mean age was 30 and the male female ratio was 1:5. 74 of the participants were HIV-positive, and 86 were uninfected. The median CD4 cell count (in those who were HIV positive) was 392. Other baseline characteristics were similar between the HIV infected and uninfected group.

The results are listed in the following table.

Effect of HIV infection on tests

 

HIV positive

(n=74) %

HIV negative

(n=86) %

p-value

TST

     

5 mm cut-off positive

52

86

<0.0001

10 mm cut-off

49

83

<0.0001

15 mm cut-off

37

58

0.01

T-Spot TB

     

Positive

53

59

0.41

Uninterpretable

1

0

 

QFT-Gold

     

Positive

46

48

0.89

Uninterpretable

7

2

 

Positive results with the Quantiferon-Gold and the PPD tests (any cut-off) were lower in the people with HIV with a baseline CD4 cell count below 250. However, the T-Spot TB test appeared to be less affected by CD4 cell count.

Likewise the T-Spot TB test tended to have better agreement with TSTs in people with HIV, though not in HIV-negative patients. However, in the HIV-negative group, this result was due “most likely confounding of the TST by the sensitisation by environmental mycobacteria or BCG,” according to Dr. Molebogeng Rangaka, who presented the study.

One interesting finding is that, using the lower cut-off for a positive skin test, there wasn’t a major difference in the number of HIV-positive patients who tested positive between the skin test and the T-Spot TB test. However, the fact that the proportion of HIV-positive people with lower CD4 cell counts who scored positive on the T-Spot TB test indicates that these were not always the same patients testing positive.

Further study needed

So although these studies demonstrate that the ELISPOT tests can be performed at a couple of research sites in resource limited settings, it is still not clear how dramatic an advantage they will truly offer in HIV-positive people beyond PPD skin testing, or whether it would be more cost-effective.

However, the test does have one key advantage in that it does not require a delayed second clinical contact to evaluate the test result — which may partly compensate for the increased cost.

Furthermore, these studies didn’t really address the population in which ELISPOT could be most important — recently vaccinated children.

References

Mutsvangwa J et al. ELISPOTs in the detection of Mycobacterium tuberculosis infection in a population with high prevalence of HIV and high exposure to BCG and environmental mycobacteria. Sixteenth International AIDS Conference, Toronto, abstract MoAB0101, 2006.

Rangaka M. Blood based methods of identifying tuberculous infections in high HIV/TB incidence areas. Sixteenth International AIDS Conference, Toronto, abstract MoAB0106, 2006.