Optimizing Tuberculosis Testing for Basic Laboratories

Outline

Introduction
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95% of tuberculosis deaths occur in resource-limited settings

Obstacles to application of tuberculosis testing in resource-limited settings

o Low sensitivity of culture without centrifuge concentration
o Centrifugation may create biohazardous vapor that can risk laboratory
tuberculosis transmission
o Equipment for centrifugation is very expensive and difficult to maintain
in resource-limited settings
o Difficulties detecting mycobacterium growth in broth cultures

Recently microscopic-observation drug-susceptibility (MODS) technique has

been proven to be an inexpensive broth-culture technique that can diagnose
tuberculosis in 1-3 weeks
o However, MODS is time consuming and relies upon the technicians
thoroughness when searching under the microscope

Methods
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Clinical Samples
o Saliva samples were collected from outpatients with
suspected/diagnosed tuberculosis in health centers in Peru.
Sample Processing and Decontamination
o A direct smear for Ziehl-Neelson staining was prepared from each
sample
Used to detect acid-fast molecules such as mycobacteria
Post-Decontamination Concentration
o The samples were subjected to two concentration methods
Centrifugation and filtration
Mycobacterial Culture
o Cultures were separated and placed into Middlebrook 7H9 broth
A liquid growth medium specially used for culture of
mycobacterium species
Culture Assessment
o Cultures were subjected to two culture assessment methods
Naked-eye and Microscopic observation
o Cultures were examined 3 times a week
First by naked-eye for colorimetric evidence of microbial growth
Determining the concentration of a chemical
element/compound in a solution with the aid of a color
reagent
Then under a light microscope with 40x magnification
Statistical Analysis

The rate of culture positivity was calculated as the number of positive

cultures by either centrifugation and/or filtration as well as naked-eye
and/or microscopic observation as a proportion of the total number of
samples tested
Data was summarized and compared through statistical analysis

Results
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Study Population
o 51% of saliva collected was positive
Detection Sensitivity
o 56% were culture positive by centrifugation
o 55% were culture positive by filtration
o Centrifugation and filtration test results yielded with 94% agreement
Contamination
o Partial contamination did not completely obscure tuberculosis growth
Time to Positivity
o Culture of centrifuge test and filtration test had similar times to
positivity
o The median time to culture positivity was 14 days by all techniques
Colorimetric Growth Detection
o The colorimetric indicator changed color in all cases of tuberculosis
growth.
o No false negatives with the naked-eye/microscopic screenings
Workload
o Considered all time required to process saliva samples
o Centrifugation required 30minutes, filtration required 10 minutes
Filters reduced the processing time compared to centrifugation
o Light microscope examination required 20 minutes and less than 10
seconds was required for naked eye examination
Naked-eye colorimetric detection of microbial growth
considerably reduced workload compared with microscopy.

Why did the scientists do this research or what were they trying to determine?Did they apply
the scientific method?What did they predict would happen?What experiments did they do?
What techniques did they use?What were the results?What did the results mean?