M MOSSMAN ASSOCIATES

MycoDot
TM

For the Detection of Antimycobacterial Antibodies as an Aid

in the Diagnosis of Active Tuberculosis

Background TB cases are extrapulmonary, for which diagnosis is made

Tuberculosis (TB) has been a cause of significant morbidity on the basis of clinical presentation and detection of the
and mortality for mankind throughout history (1). Currently, bacteria in biopsy specimens. For these reasons, a test ca-
there are 20 million cases of TB worldwide with 8 million pable of detecting active TB irrespective of its focus of infec-
new cases each year. Three million deaths annually are di- tion in the body would be a valuable addition to effective
rectly attributable to tuberculosis (2,3). TB continues to be a diagnosis. Mossman Associates’ MycoDot test meets this
significant problem in the developing world. In addition, need.
there has been a dramatic increase over the last five years in
the incidence of tuberculosis in developed countries. This Test Description
increase can be attributed to increases in the incidence of MycoDot is a simple, rapid (20 minutes) and reliable test
Acquired Immunodeficiency Syndrome (AIDS) (3), immigra- that can detect antimycobacterial antibodies in serum or
tion, and the other primary risk factors for the disease such blood. The test offers a low cost, single visit aid in the diag-
as poverty, malnutrition and overcrowded living conditions nosis of tuberculosis with good sensitivity and-excellent
(8). Tuberculosis usually presents itself as an infection of the specificity. A positive reaction will only occur in patients
lungs but it can affect virtually any organ system in the body with active mycobacterial diseases like TB. Healthy, infected
(1,3). It is caused by the bacterium Mycobacterium tubercu- (PPD-positive) patients and/or BCG-vaccinated individuals
losis and is primarily spread by bacteria present in aerosols react negatively.
generated by coughing. Since TB is curable with the use of
antibiotics, early diagnosis will dramatically reduce patient The MycoDot test employs lipoarabinomannan (LAM) anti-
morbidity and mortality if diagnosis is followed by appro- gen, bound to plastic combs. When the combs are incu-
priate intervention. bated in diluted serum/blood, specific anti-LAM antibodies
from the sample, if present, bind to the antigen. The combs
Currently, the diagnosis of pulmonary TB is based largely on are then washed to remove non-specific antibody, and in-
the microscopic detection of the causative organism in pa- cubated in a suspension of colored particles which bind to
tient sputum, pleural fluid, and bronchoalveolar lavage. This the bound anti-LAM antibodies. If enough of the specific
method of detection, the acid fast bacillus (AFB) smear, is antibodies are present in the serum sample, a colored spot
error prone. It has a sensitivity of, at best, 20-40% in devel- will form where the antigen is attached to the plastic comb.
oping countries and 40-60% in developed countries. Cul- The sensitivity of the test is calibrated so that only cases of
turing the bacteria on solid media yields significantly better active mycobacterial disease such as tuberculosis will gen-
results. However, this procedure is much more expensive erate a colored spot which is as strong or stronger than the
and, due to the slow rate of growth of the bacteria, results weakest positive spot on the reference comb that is provided
are seldom obtained before 4 weeks. Detection of bacteria as a guide to interpret results.
by AFB smear or culture is only possible for bacillary cases
of pulmonary TB (when bacteria are present in the body Sample Preparation
fluids) and for each case of pulmonary TB that is bacillary, Whole blood, serum or heparin derived plasma can be used.
there are 2-3 that are non-bacillary (bacteria are not detected Plasma derived by the addition of divalent cation chelators
in the specimen by either of these two methods). While the such as sodium citrate or EDTA must not be used. Speci-
chest X-ray is used in the diagnosis of pulmonary TB, it is mens can be kept at 2-80C for short-term storage. However,
often inadequate due to the non-specific nature of the find- they must be stored frozen (-200C or lower) for long-term
ings (4). Also, the new problem of HIV-infected TB patients storage. It is recommended that whole or heparinized blood
has created a complication in the use of this method, since be centrifuged to remove red blood cells, prior to freezing.
HIV infection can change the classical appearance of the Grossly hemolyzed and/or contaminated samples must not
chest X-ray (8,9). In addition, an appreciable number of all be used.
Materials provided 6. In one test run, do not combine antigen combs and sig-
The kit components for tests include nal generating reagent from boxes with different lot num-
— Twelve antigen coated combs bers.
— One dropper bottle of Signal generating reagent 7. Use only microtiter plate wells which have not been
previously used.
— One bottle of 5X Concentrated rinse buffer
8. Do not let diluted serum sit in microplate wells for longer
— One dropper bottle of sample diluent
than twenty minutes without testing.
— One bottle of negative control serum
— One bottle of positive control serum
Limitations
— One reference comb Since the test detects antimycobacterial lgG antibodies, pa-
— One wash tray tients who have had active TB in the past may continue to
— Two microtiter test plate(s) have antibody titers above the cut-off after they have been
cured of TB. The attending physician should therefore inter-
view the patient regarding any history of tuberculosis.
Materials required but not provided
— Pipette and disposable tips capable of delivering 40 to If a TB patient is immunocompromised with lowered CD4
80 microliters (µls) counts, such as in certain AIDS patients, the MycoDot test
— 100 ml graduated cylinder may not provide a positive result since antimycobacterial
— Distilled Water antibodies may not be present at levels indicative of active
disease.
— Timer capable of timing 6 and 10 minutes
— Paper towels or other absorbent pad A positive reaction may be obtained with MycoDot for ac-
tive mycobacterial diseases other than TB. However, in gen-
Storage eral, non-tuberculous active mycobacterioses seldom occur
When stored between 2-80C, all kit components are stable except when the immune system is compromised; in those
until the expiration date shown on the label. The pouch con- circumstances the antibody response is curtailed and the
taining the antigen coated combs should be brought to room MycoDot reaction is likely to be negative. Leprosy patients
temperature before opening to prevent condensation. Un- without TB will react positively by the MycoDot, however;
used antigen combs should be stored in the aluminum the clinical presentation of leprosy cannot be confused with
pouches along with the silica gel bag and tightly closed in that for TB. Therefore, for all practical purposes, when a
the zipper seal bag, to protect from moisture during storage. patient presents with symptoms suggestive of TB, a positive
Once diluted, the rinse buffer is stable for one week if stored reaction by MycoDot, is an indicator of active tuberculosis.
between 2-80C.
Set Up for the Test
Warning: All samples for analysis should be considered Dilute the concentrated rinse buffer 1:5 with distilled water.
potentially infectious and handled with care. Positive and Add 1 2mL of the concentrated rinse buffer to 48 ml of wa-
negative control sera have been serotested for HIV and ter. Fill the wash tray with the rinse buffer taking care to
HBsAg and are negative. They have also been filtered avoid foaming when pouring. Use the resulting 60 ml of
through a 0.2 µm filter to remove bacteria. Nevertheless, rinse buffer to wash no more than 2 combs. Bring all samples
all samples should be handled with appropriate precautions. and kit components to room temperature prior to testing.

Precautions Test Procedure

1. Upon assay completion, collect remnants of samples, 1. While holding the dropper bottle at a 450 angle, deliver
buffers and aspirated reagents to be disposed in a con- 5 drops of sample diluent to each well of alternating 8
tainer specifically kept for this purpose and autoclave well columns (1,3,5,7,9 and 11) on the microtiter plate
for 1 hour at 1210C or treat with sodium hypochlorite for each sample to be tested as well as for the positive
(10% final concentration) for 30 minutes. and negative control sera. Positive and negative assay
2. Wear gloves while performing the test and wash hands controls, supplied in the kit, are to be routinely tested
thoroughly after completion of test. each day the test is performed, or as lab protocol dic-
tates.
3. Do not smoke, eat or drink in areas where biological
samples are handled. 2. While holding the dropper bottle at a 450 angle, deliver
4 drops of signal generating reagent to the correspond-
4. Avoid splashing or spilling of reagents.
ing wells in adjacent columns (2,4,6,8,10 and 12) on
5. Extreme care should be taken to avoid microbial con- the plate for each sample/control serum to be tested.
tamination of reagents.
3. Add 20 µl of serum or plasma (or 35 µl of heparinized much clinical information as possible to aid in making the
or whole blood) to each sample diluent well. Mix thor- diagnosis.
oughly by pipetting back and forth in the well.
4. Remove a test comb from a foil pouch and label it on Performance Characteristics
the dull side in the space above the teeth with the ap- For the MycoDot test, a total of 1,602 samples were evalu-
propriate test sample number for each sample/control ated in three countries. Of the 564 pulmonary TB patients
to be tested. tested, 396 reacted positively in the MycoDot test for an
overall sensitivity of 70.2%. Of the 564 pulmonary TB
5. Set a timer to 6 minutes, and holding the comb verti- samples, 328 were tested for the presence of anti-HIV anti-
cally with the teeth pointing down, place the comb, dull bodies. Of 28 HIV-positive TB patients, 21 reacted positively
side facing the user, into the first row of diluted samples in the MycoDot test for a sensitivity of 75% in this popula-
and begin timing. Gently rock the comb back and forth tion. Of the 1,038 TB negative individuals tested, 51 reacted
8–10 times. Repeat the rocking step at least one more positively, for an overall specificity of 95.1% It should be
time approximately halfway through the incubation step. noted that among the false positive” reactors, active TB or
6. After 6 minutes, remove the comb and touch the tips of other active mycobacterial disease could not be conclusively
the teeth to a paper towel to remove excess liquid. Blot ruled out. There were 120 patients in the negative control
only the tips of the teeth. Rinse the teeth of the comb in group that were directly under the care of a physician in-
the diluted rinse buffer by moving the comb back and volved in the evaluation of the Mycodot test, and in his clini-
forth across the wash tray 10 times. Again, touch the cal judgment, these patients did not have tuberculosis. Only
tips of the teeth to the paper towel to remove excess four patients in this group reacted as false positive, for a
liquid. specificity of 96.7% Among the pulmonary TB negative group
7. Set the timer to 10 minutes, place the teeth of the comb 182 had been vaccinated with the BCG Vaccine. In this group
into the signal generating reagent and begin timing. Gen- only 5 reacted as false positive for a specificity in this popu-
tly rock the comb back and forth 8-10 times. lation of 97.3%.
8. At the end of 10 minutes, remove the comb and blot
In general, the diagnosis of TB was made by a combination
and rinse as in step 6.
of AFB smear examination result, culture result, and/or a
Note: The diluted rinse buffer in the tray must be combination of chest X-ray finding and clinical
changed for every two combs that have been washed. symptomology. In some tuberculosis patients with active
9. Touch off excess liquid and allow the comb to air dry. disease, antimycobacterial antibodies are not detectable and
To determine the result of each sample, place the comb these individuals will react as false negative. There is some
on a flat solid surface with a white background under evidence that the presence of circulating antigen-antibody
good lighting and read the reaction perpendicular to the complexes may reduce the detectable antibody titer (15).
comb. Incandescent light may make it difficult to inter-
pret faintly positive reactions. Compare the intensities It should be noted that the results of the MycoDot test must
of the test reactions to those on the reference comb pro- be evaluated in light of all available clinical information in-
vided with each kit. Note: Too much light on the comb cluding results of AFB smear, chest X-ray, and clinical symp-
or viewing the comb at an angle can make the interpre- toms such as fever, malaise, weight loss, and cough.
tation of results difficult. Placing the comb against a white
background allows for easier interpretation. Precision studies of the MycoDot test were performed as
follows. Eight serum samples were tested a total of nineteen
10. Record the results as positive (+) or negative (–)
times each, by four different analysts on six different days.
11. Once dried, the comb may be attached with transparent Of the eight specimens, there were three moderate to strong
tape to a patient file or notebook to serve as a perma- reactors, four were borderline positive reactors, and one was
nent record. negative. Except for one borderline positive reactor, all of
the samples were tested and interpreted with one hundred
Interpretation of Results percent accuracy by each of the four analysts. The border-
To interpret the reaction generated by the MycoDot test, one line sample was interpreted differently by the same analysts.
must use the reference comb provided in the kit. A colored on different occasions. In addition, four samples were tested
spot as intense, or more intense, than the weakest positive by the MycoDot test, sixteen times each, by one analyst.
spot on the reference comb is to be considered a positive The results for each of the four samples was identical for
reaction. A spot less intense than the weakest positive spot every determination
on the reference comb, or no spot at all, is a negative reac-
tion. For borderline reactions, it is recommended that a fresh References
sample be drawn after 2-4 weeks and that it be retested. 1. Reichman, L. B. and Hershfield, E. S., Tuberculosis,
During this period, the attending physician should obtain as Marcel Dekker, Inc., New York-Basel-Hong Kong (1993).
2. Kochi, A., The Global Tuberculosis Situation and the New 9. Snider,D. E., and Roper, W. L., The New Tuberculosis.
Control Strategy of the World Health Organization, Tu- New EngI. J. Med., 326 : 703-705 (1992).
bercle 72:1 (1991). 10. Sada, E., Herrera, T., Torres, M., and Carvajal, G., Evalu-
3. Collins, F.M., Tuberculosis: The Return of an Old En- ation of Lipoarabinomannan (LAM) from Mycobaterium
emy, Critical Reviews in Microbiol. 19:1-16 (1993). tuberculosis for the Serological Diagnosis of Tuberculo-
4. Tsao, T. C. Y., Juang, Y. C, Tsai, Y. H, Lan, R. S., and Lee, sis, Abstract from the Annual Meeting of the American
C. H., Whole Lung Tuberculosis: A Disease with High Society for Microbiology, p.160 (1989).
Mortality which is Frequently Misdiagnosed, Chest 11. Wilkins, E.G.L., and Ivanyi, J., Potential value of Serol-
101:1309-1311 (1992). ogy for Diagnosis of Extrapulmonary Tuberculosis, The
5. Sada, E., Brennan, P. J., Herrera, T., and Torres, M., Evalu- Lancet, 336:641-644 (1990).
ation of Lipoarabinomannan for the Serological Diag- 12. Chattenee, D., Bozic, C.M., McNeil, M., Brennan, P.J.,
nosis of Tuberculosis, J. Clin. Microbiol., 28:2587-2590 Structural Features of the Arabinan Component of the
(1990). Lipoarabinomannan of Mycobacterrium tuberculosis,
6. Brennan, P. J., Structure of Mycobacteria: Recent De- J.Biol. Chem., 266:9652-9660 (1991).
velopments in Defining Cell Wall Carbohydrantes and 13. Hunter, SW., and Brennan, P.J., Evidence for the Pres-
Proteins. Rev. Inf. Dis., Vol.2, Suppl. 2, p. S420-S-430 ence of a Phosphatidylinositol Anchor on the Lipoarabin-
(1989). omannan and Lipomannan of Mycobacterium tubercu-
7. Strumpf, I. J., Tsang, A. Y., and Sayre, J. W., Re-Evalua- losis. J.Biol. Chem., 265: 9272-9279 (1990).
tion of Sputum Staining for the Diagnosis of Pulmonary 14. Brennan, P. J. and Chatterjee, D., Purified LAM and Syn-
Tuberculosis, Am. Rev. Resp. Dis., 119:599-602(1979). thetic Analogs Thereof.
8. Bass, J. B., Farer, L. S., Hopewell, P. C., Jacobs, R. F., and 15. Sada, E., Aguilar, D., Torres, M., and Herrera, T., Detec-
Snider, D. E., Diagnostic Standards and Classification of tion of Lipoarabininomannan as a Diagnostic Test for
Tuberculosis, Joint Statement of the American Thoracic Tuberculosis, J.Clin. Microbiol., 30:2415-2418 (1992).
Society and The Centers for Disease Control, Am. Rev.
of Respir, Dis., 142:725-735 (1990).

MycoDot was Codeveloped by DynaGen, Inc., and Program for Appropriate Technology
in Health with support from the United States Agency for International Development
under the Technologies for Child Health Program.

MycoDot is a trademark of Mossman Associates.

M
MOSSMAN ASSOCIATES
30 Liberty Hill Drive, Blackstone, MA 01504 USA
Tel 508-883-4722 • Fax 508-883-4993 • [email protected]