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136 |
SOME OPERATIONAL FACTORS INFLUENCING THE RELATIVE
UTILITY OF CULTURE METHOD OF DIAGNOSIS OF PULMONARY TUBERCULOSIS |
K Padmanabha Rao, SS Nair, N Cobbold & N Naganathan:
Indian J TB 1966, 13, 61- 76 & Bull WHO 1966, 34, 589-604. |
Laboratory diagnosis of pulmonary tuberculosis
is based on the presence of tubercle bacilli in sputum by direct
microscopy, culture and/or animal inoculation. Culture examination,
followed by tests for identifying the bacilli, is recognized as
the most accurate and reliable method. Its efficacy depends on the
laboratory techniques employed and its use in different practical
situations such as epidemiological surveys, active community Case-finding,
organization of diagnostic services and evaluation of diagnosis
and treatment in tuberculosis control programmes. But the practicability
of culture method in developing countries must be studied. The present
paper deals with a systematic study of data from four investigations
designed to elucidate the influence of certain operational factors
on the utility of the culture method.
STUDY I: is a longitudinal survey
in a randomly selected population in 134 villages in the three sub-divisions
of Bangalore district. The analysis is based on the material from
the first round, when two samples of sputum, (spot and overnight)
were collected at intervals of 24-48 hours from persons aged 5 years
and above having abnormal x ray shadows. The specimens were collected
in house to house visits, stored after collection in insulated box
with ice container and transported to the main laboratory at the
National Tuberculosis Institute (NTI). The interval between collection
of specimens in the field and culture in the laboratory was 1-7
days. A smear was stained and examined first by fluorescence microscopy
and then by Ziehl-Neelsen (ZN) method. Each specimen was cultured
on two slopes of Lowenstein-Jensen medium. All positive cultures
were submitted to further identification tests; i.e., growth at
room temperature, rate of growth at 37%C, pigment production in
the dark and exposure to light, catalase and peroxidase reactions,
niacin production, and sensitivity to INH, SM and PAS. STUDY
II: relates to a mass Case-finding programme in Tumkur
district when two specimens (spot and overnight) were collected
from individuals aged 20 years and above with symptoms suggestive
of pulmonary tuberculosis and from positive tuberculin reactors
below 20 years voluntarily reporting with symptoms. The specimens
were then treated in the same way as in Study I. STUDY III:
pertains to the technical assessment of microscopy using Ziehl-Neelsen
method performed by the auxiliary health staff of Peripheral
Health Institutions in Bangalore district. A spot specimen was
collected daily by auxiliary staff at each health facility from
patients who were symptomatics. All smears were examined by ZN method
at each centre and the corresponding sputum specimens were transported
to NTI laboratory twice weekly. Duplicate smears were made and reexamined
and culture was also done at NTI. All positive cultures were identified
as in Study I. No refrigeration facilities were available in these
centres and specimens were not transported in an insulated box.
Rest of the procedures were followed as in previous studies. STUDY
IV: is connected with operational and technical assessment of
the District Tuberculosis Programme in Anantapur district one year
after its commencement. A sample was taken from all patients who
started treatment during a particular period but did not collect
their drugs. Spot specimens were collected in the field, stored
without any refrigeration and transported to NTI laboratory, thereafter
the same procedure was followed as above.
An analysis of these four studies brought out certain
operational factors affecting the culture method. (1) The results
showed that an interval of 7 days between collection of sputum
in the field and its processing in the laboratory did not affect
the yield of positive cultures, even though the specimens were
stored and transported under field conditions. (2) A higher proportion
of positive cases were detected by culture than by direct microscopy
but the magnitude of additional yield was dependant upon the procedure
of selecting persons for sputum examination. (3) In service programmes
restricted to persons with symptoms who attend diagnostic centres,
the increase in yield is too small, to justify the introduction
of culture examination.
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KEY WORDS: CULTURE EXAMINATION, DIAGNOSIS, RELATIVE
UTILITY, OPERATIONAL FACTORS. |
137 |
INVITRO STUDY ON SENSITIVITY OF TUBERCLE BACILLI
TO THIOACETAZONE (TB1) |
K Padmanabha Rao, SS Nair, N Naganathan & G Ramanatha
Rao: Indian J TB 1966, 13, 147-57. |
This report is based on the study of 735 cultures
of tubercle bacilli identified as human type. Sputum specimens were
collected from patients attending the Lady Willingdon Tuberculosis
Demonstration and Training Centre (LWTDTC), Bangalore, and from
the mass Case-finding studies in semi-urban areas. Drug sensitivity
tests for streptomycin, isoniazid, PAS and thioacetazone with different
drug concentrations, different size of inoculum and for various
length of incubation were carried out.
No difference was observed in the duration of growth
between sensitive and resitant cultures in their first appearance
on primary diagnostic cultures or sub-cultures on drug free slopes
when innoculated with standard suspension. The primary cultures
took about 3 weeks and sub-cultures 2 weeks to grow on drug free
media. Large sensitive bacillary population required higher concentration
of thioacetazone to inhibit the growth, suggesting standardization
of inoculum size for sensitivity tests. Prolonged incubation
period on drug slopes showed profound influence on the level of
drug inhibiting concentration of thioacetazone; with the increase
in incubation period, fall in growth of sensitive culture
was not observed even on high drug concentration. The reproducibility
of this observation on duplicate specimens from the same patients
after shorter intervals excluded the possibility of experimental
error. A reduction in the inhibition of growth of sensitive organisms
on drug media with time is presumed to be due to either deterioration
of the drugs in the media or due to adaptation by the micro-organisms.
Because of the decrease in inhibition of growth, even sensitive
organisms may be classified as resistant if reading of culture for
drug sensitivity is prolonged beyond 3 weeks of the inoculation
period. It is suggested that a standard inoculum size and a maximum
limit of 3 weeks incubation period should be adopted for finding
out sensitivity to thioacetazone. Cultures classified as sensitive
to the three first line drugs or resistant to one or more, showed
no difference in the pattern of sensitivity to thioacetazone.
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KEY WORDS: INVITRO DRUG SENSITIVITY, M.TUBERCULOSIS,
THIOACETAZONE. |
138 |
A COLD STAINING METHOD FOR TUBERCLE BACILLI USING
CHLOROFORM |
K Padmanabha Rao, N Naganathan & SS Nair: Indian
J TB 1966, 14, 3-9. |
The difficulty in staining tubercle bacilli is
believed to be related to the complex surface structure containing
a large amount of unsaponifiable wax. Any staining technique which
can counteract the influence of this wax could therefore be expected
to give better results. The standard method in vogue is the application
of heat which renders the bacilli permeable to aqueous dyes. Several
attempts have been made to develop a cold staining method for tubercle
bacilli as for other organisms. Since this wax is soluble in chloroform,
a cold staining method using carbol fuchsin containing chloroform
was developed and the results of staining by this new method have
been compared with the conventional Ziehl-Neelsen (ZN) method in
the present study. Triplicate smears were made from 186 specimens
and these were stained by ZN, Cold Staining (CS) and Fluorescent
Microscopy (FM) methods. In addition, single smears of 343 specimens
previously examined by FM were randomly divided into two subgroups
and restained by ZN and CS methods respectively.
The results of examination of duplicate smears
by ZN and CS methods showed a high degree of correlation
with 75%(140/186) showing identical grading and only 8 were positive
by one and negative by the other method. Of the 8 smears positive
by CS alone, 7 were confirmed by culture, whereas 3 were positive
by culture out of the 8 positive by ZN method. This indicates that
those positive by CS alone are likely to be real cases, whereas
those positive by ZN alone may include some false positive cases.
As far as false negatives are concerned, there was no difference
between ZN and CS methods. The reliability of these methods was
judged on the basis of culture results and agreement among themselves.
The cold staining method was found to be as efficient as ZN method
in detecting different gradings of culture positives. In addition,
preparation of stain, training of personnel for CS was also found
to be as simple as ZN method.
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KEY WORDS: STAINING METHODS, COLD STAINING,
TUBERCLE BACILLI, ZIEHL1-NEELSEN, FLUORESCENT. |
139 |
A COMPARATIVE STUDY OF PREVALENCE OF DRUG RESISTANCE
TO MAJOR ANTI TUBERCULOSIS DRUGS |
K Padmanabha Rao: Proceed Natl TB & Chest Dis
Workers Conf, Hyderabad, 1967, 52-62. |
A comparative study to find out the prevalence
of resistance to the major anti-TB drugs, SM, INH and PAS, under
different situations was carried out. The situations considered
were: (i) Sanatoria, (ii) Urban tuberculosis clinic, (iii) Rural
general health dispensaries, (iv) the mass Case-finding among selected
group of population and (v) survey of general population.
The prevalence of drug resistance to INH was 53%,
the highest among sanatoria patients and 11%, the lowest among
patients discovered in tuberculosis surveys i.e., situation
(v). Among newly diagnosed culture positive tuberculosis patients
of urban tuberculosis clinic, rural general health institutions
and selected Case-finding programmes, i.e., situations (ii), (iii)
and (iv), proportion of patients with INH resistant organisms were
26%, 33% and 16% respectively. The resistance rates were the highest
in age group 25 to 44 in all 5 situations, case-wise rates were
not significantly different. The prevalence of INH and streptomycin
resistance among patients found in special mass Case-finding tuberculosis
programme, were significantly lower than among patients attending
special tuberculosis services or general health institutions.
The findings of this study show that prevalence
of drug resistance among patients attending tuberculosis services
and general health institutions is not a true index of the prevalence
of drug resistance in the community and it varies under different
situations.
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KEY WORDS: DRUG RESISTANCE, INH, STREPTOMYCIN,
PAS, GENERAL POPULATION, CLINIC, SANATORIA, PHIs. |
140 |
INTER & INTRA-READER VARIATIONS IN DIRECT MICROSCOPY
AND THEIR INFLUENCE ON SENSITIVITY & SPECIFICITY |
K Padmanabha Rao: Proceed 24th Natl TB & Chest
Dis Workers Conf, Trivandrum, 1969, 99-110. |
A study was done to evaluate inter and intrareader
differences in reading of smears stained by Fluorescent Method (FM)
and Modified ZN Staining (CS) methods and variation in multiple
smears made from the same specimens in order to find out to what
extent sensitivity and specificity are influenced by repeated sputum
smears from same specimens, by change of readers or by repeated
reading.
Eighty sputum specimens with known results, 35
negative and 45 positive with different grades were selected. Ten
smears were prepared from each specimen. All the smears were first
examined by FM and later by CS method. Culture was also done for
these specimens. The findings were: (i) FM was more sensitive
than CS method. The specificity appeared to be equal in both.
(ii) Change of readers influenced the relative sensitivity of both
methods, but repeated examination by the same reader had no effect.
(iii) Both reader as well as reading influenced the specificity
of FM method but not that of CS method. (iv) Repeated sampling from
the same specimen had no effect on the sensitivity of both the methods,
while it had some effect on the specificity of FM method. (v) Consistency
among readers and readings was found to be poor in smears with low
grade positivity. (vi) The relative sensitivity of any method was
influenced by the proportion of low grade positive cultures in the
total pool. (vii) Examination of multiple smears from the same specimen
by more than one reader and more than one reading by the same reader
was more rewarding in CS method.
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KEY WORDS: SPUTUM EXAMINATION, SENSITIVITY,
SPECIFICITY, MODIFIED ZIEHL1-NEELSEN, FLUORESCENT. |
141 |
BACTERIOLOGICAL DIAGNOSIS OF PULMONARY TUBERCULOSIS
SPUTUM MICROSCOPY |
K Padmanabha Rao & DR Nagpaul: Bull IUAT 1970,
44, 67-77. |
Of all the available methods for the diagnosis
of pulmonary tuberculosis, bacteriological examination is the most
reliable. Diagnosis of pulmonary tuberculosis is chiefly done by
sputum microscopy and culture. This paper discusses sputum microscopy
from various points of view. Sputum, which forms the basis of bacteriological
diagnosis, is a variable source material. Type of specimen, its
quality, quantity, bacterial content and viability of organism considerably
influence the sensitivity and the specificity of the methods; and
these in turn would vary under different diagnostic situations.
One of the reasons for the observed variations could be the different
criteria adopted for examination; another might be due to the observed
range of diagnostic situations varying from an epidemiological survey
situation at the one extreme to the other where cases seek treatment
in a comparatively backward community with poor tuberculosis diagnostic
services.
In epidemiological community survey (ICMR
1968), it has been found that culture positives that were also
smear positives varied from 73% to 87%, whereas among patients
attending rural general health institutions for diagnosis, about
82% of the infectious cases found by culture could also be discovered
by microscopy of single spot specimens (Rao, 1966). Sikand (1965)
from New Delhi Tuberculosis Centre, could get 67% of culture positives
by microscopy, whereas Mitchison (1967) found that 35% were smear
positive among the sputum positive patients reporting for the first
time. In the longitudinal epidemiological study carried out in the
Bangalore rural area, it was found that about 40%-48% were positive
by both direct smear and culture and the rest by culture only. Reasons
for these variations could be (i) different criteria adopted for
examination (ii) different situations from where the sputum specimens
were collected (iii) sensitivity and specificity of sputum microscopy
technique adopted and (iv)the experience of the trained technician.
It was observed that over diagnosis by the trained auxiliary staff
in the general health institutions (1.9%) compares favourably with
the over diagnosis of 1.3% by experienced technicians indicating
simplicity of smear examination. Besides these aspects, other factors
like the quality of sputum smear, time spent on examination,
type of sputum specimen, the use of multiple smears, etc., also
influence the results. The cost of bacteriological examination
have also been studied, and the cost ratio between microscopy and
culture have been worked out to be 1:6.6. Cost can become an important
factor in deciding the suitability of bacteriological methods for
diagnosis of pulmonary tuberculosis in various countries and in
different diagnostic situations.
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KEY WORDS: DIAGNOSIS, SPUTUM MICROSCOPY, CULTURE,
DIAGNOSTIC FACTORS. |
145 |
COST OF ESTABLISHING AND OPERATING A TUBERCULOSIS
BACTERIOLOGICAL LABORATORY |
N Naganathan, K Padmanabha Rao & R Rajalakshmi:
Indian J TB 1974, 21, 181-90. |
This paper deals with the cost of establishing
and running a bacteriological laboratory in State Tuberculosis Centres
under the National Tuberculosis Programme, and the cost of various
examinations to be undertaken in such a laboratory. A knowledge
of the cost will enable proper planning and judicious utilization
of the resources. Further, when services are rendered to private
individuals or institutions, the charges for different examinations
can be levied on a rational basis. The place of smear and culture
examinations under the programme, the implications of establishing
a culture laboratory, the limitations of cost worked out, have been
discussed. A plan of the laboratory building is also provided.
The cost has been worked out presuming that about
12,000 specimens per year are likely to be received, of which 25%
might turn out to be positives. Non recurring cost was estimated
to be Rs.1,07,724 and annual recurring cost would be Rs.49,709.
Factors that contribute to the cost structure are overheads,
cost of material and labour. In addition, certain essential
facilities like cold room, incubator room, gas supply, washing and
sterilisation etc., add to the cost. (i) staff-bacteriologist-1,
lab technicians-4, lab attendants-3 and registration clerk-1; their
salaries, (ii) building-rent (iii) electricity (iv) furniture (v)
equipment and supplies (vi) water charges had all been taken into
consideration. The cost of one smear examination was estimated to
be Rs.0.54 and that of culture and sensitivity test Rs.9.43.
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KEY WORDS: COST, LABORATORY, TUBERCLE BACILLI,
ESTABLISHMENT. |
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