Abstract
SUMMARY
An experimental comparative study was conducted in order to compare the positivity pattern of sputum microscopy using the direct and overnight sedimentation Ziehl-Neelsen (ZN) smears, direct and NaOCl pretreated centrifuged ZN smears and overnight sedimentation and NaOC1 pretreated centrifuged ZN smears methods. We examined 660 sputum smeared slides for the presence of acid-fast bacillus from 220 sputum samples. Compared with the direct microscopy method, positivity increased by 91.1% after centrifugation (P < 0.001, χ2 = 18.27) and by 71.1% after overnight sedimentation (P < 0.001, χ2 = 11.61). No statistical significant difference was observed between the results of two different concentration techniques (P > 0.05, χ2 = 0.789).
The application of overnight sedimentation (in a laboratory without electricity) and/or the centrifugation technique could make a positive impact on the effectiveness of national tuberculosis control programmes.
Introduction
More than 90% of tuberculosis (TB) cases and 98% of TB deaths occur in the developing world. The annual risk of TB infection in sub-Saharan Africa is more than 50 times the rate for Western Europe. 1 In Ethiopia, TB is one of the leading causes of outpatient morbidity, ranking eighth in 2000 and fourth in 2003/2004. 2 Human immunodeficiency virus is the most important factor fuelling the TB epidemic. 3
Most developed nations use molecular, serological and rapid drug sensitivity testing methods. 4 However, in developing countries, these techniques are used for research purposes and not for general use. Cultures are not available in most health institutions in Ethiopia, 5 and they are therefore cannot be used for primary diagnostic methods. As a result, Ziehl-Neelsen (ZN) staining is often the only primary diagnostic technique available.
Sputum smear microscopy is the most cost-effective technique, 2 but has only a 30%–40% sensitivity rate using a single sample and 65%–75% with repeated smear examinations. 6 Different methods were designed to increase the positivity of direct smear microscopy. Liquefaction of sputa with NaOCl followed by centrifugation has recently been evaluated in several developing countries. 7
The aim of this study aim was to compare the positivity pattern of sputum microscopy using three different methods – the direct and overnight sedimentation ZN smears method, the direct and NaOCl pretreated centrifuged ZN smears method and the overnight sedimentation and NaOC1 pretreated centrifuged ZN smears method.
Materials and methods
Sample collection
An experimental comparative study was conducted for the microscopic examination of sputum of clinically suspected pulmonary TB patients, who visited the Jimma Health Center, Jimma, Ethiopia, during the study period March to June 2006. A convenient sampling technique was used. The standard sample collection procedures were followed strictly. This study is based only on morning sputum samples.
Laboratory procedures
Direct smears were prepared, air dried, heat fixed and stained by the ZN method. 8 One millilitre of sputum from the same sample was added to two labelled 10 mL screw-capped centrifuge tubes, mixed with 1 mL of 5% sodium hypochlorite, and incubated at room temperature for 10 min which were shaken at regular intervals. Eight millilitres of distilled water was then added and one tube was centrifuged for 15 min at relative centrifugal force (RCT) 3000 g. Another tube was left overnight at room temperature. The supernatant was discarded and the pellet was suspended in a few drops of the remaining fluid. Smears were prepared from the suspended sediment for each method and then dried, fixed and stained with ZN stain.
The results were reported based on the 1999 National Tuberculosis and Leprosy Control programme. 8
three or more acid fast bacilli (AFB)/100 field-----Reported as positive,
one to two AFB/100 field-----Reported exact number,
No AFB/100 field-----Reported as ‘no AFB seen’.
Patients with one to two bacilli per 100 fields are not included under data analysis and interpretation.
Analysis
A comparison was made between results obtained with direct and overnight sedimentation ZN smears, direct and NaOCl pretreated centrifuged ZN smears, and overnight sedimentation and NaOCl pretreated centrifuged ZN smears. The results were analysed statistically using χ2 testing and P value of ≤0.05 was regarded as significant.
Ethical considerations
The purpose of the test was explained to the patients, and they were included only after verbal consent was reached.
Results
A total of 660 sputum-smeared slides were examined for the presence of AFB from 220 sputum samples. A comparison of the results of three methods is listed in Table 1. High positive results were obtained in two concentration techniques in contrast to the direct smear microscopy.
Results obtained from direct smear microscopy, NaOCl pretreated sputum samples plus overnight sedimentation and NaOCl pretreated sputum sample plus centrifugation
Discussion
According to the results of this study, there was a pronounced increase in the relative efficiency of the concentration technique over the direct sputum smear microscopy. The number of positive result was increased by 91.1% after using the NaOCl pretreated plus centrifugation technique (P < 0.001, χ2 = 18.27) and by 71.1% after the NaOCl pretreated sputum sample plus overnight sedimentation technique (P < 0.001, χ2 = 11.61). However, there was no statistical significance difference between the results of the NaOCl pretreated plus overnight sedimentation and the NaOCl pretreated plus centrifugation techniques (P > 0.05, χ2 = 0.789) (Table 2).
The results obtained from three different techniques
Df, degree of freedom
A study compared centrifugation with the overnight sedimentation of NaOCl treated sputum. The results were almost identical. 9 This could make the technique very effective for the diagnosis of pulmonary TB in rural laboratories where there is a shortage of centrifuge and electricity.
The sample size of the study conducted in Jimma was 500 (Mulugeta M, unpublished data) and that of the Jimma Health Center was 100. 4 However, our results were similar to their results.
Due to the unavailability of bacteriological facilities and also to time constraints, the concentration method was not evaluated against the reference standard method.
In general, highly improved sensitivity results were obtained using both concentration methods, but not when using the direct method. This may have been due to the change in the surface properties of the mycobacterium and the subsequent increase in the mycobacteria sediment rate. Another advantage of using concentrating sputum is the reduction in time spent on sputum examination. As a potent disinfectant, NaOCl also has the advantage of limiting the risk of laboratory infection. 7 However, the sample preparation is more laborious than for the direct method.
The immediate application of the concentration method could make a positive impact on the effectiveness of the National TB Control programme in sub-Saharan Africa as the technique is inexpensive, has a better yield, and is readily available in the form of household bleach. 7