EPIDEMIOLOGY <<Back
 
 
015
EXAMINATION OF MULTIPLE SPUTUM SPECIMENS IN A TUBERCULOSIS SURVEY
P Chandrasekhar, SS Nair, K Padmanabha Rao, G Ramanatha Rao & Pyare Lal: Tubercle, 1970, 51, 255-62.

Prevalence surveys are useful for estimating the tuberculosis problem in different countries. Three techniques are commonly used in surveys, tuberculin test, mass miniature radiography and sputum examination. Each has its own limitations. A limitation of sputum examination is that all the sputum positive cases in the community cannot be diagnosed when only one sample of sputum is examined from each eligible person. Multiple sputum examinations are not often possible under field conditions of surveys covering the whole community. It would be worthwhile to have some idea of the extent of under-diagnosis in sputum examination. For this purpose, during an epidemiological survey, four specimens of sputum were collected within seven days of X-ray examination from each person with an abnormal chest X-ray in 30 villages of a district of south India. Each specimen was examined by Fluorescent Microscopy (FM), Ziehl Neelson (ZN) technique and culture.

There were 34 culture positive cases among 2,164 persons for whom all the four culture examination results were available. Of them, 21 (62%) were found positive on one specimen. The second specimen increased the positivity to 32 (95%). Thus, for detecting both smear and culture positive cases two specimens are adequate. A third specimen is helpful for detecting cases positive by culture alone. An estimate of prevalence obtained from one sputum specimen can be estimated for the prevalence obtained from many specimens by applying correction factor of 1.67 and estimates based on two specimens by applying 1.26. Of the remaining 37 smear positive cases detected by one specimen, 20 were smear positive and culture negative. Of the remaining 17 smear positive and culture positive, 14(82%) were detected by one smear examination only.

ZN positives not confirmed by culture (mostly with less than four bacilli reported in the smear) increased from 7 from the first specimen to 18 from all four specimens, while positives confirmed by culture method showed only a marginal increase from 13 to 15. FM did not have this disadvantage as only two were culture negative among the 18 smear positive results by FM method. Examination of two specimens by FM detected about 95% of cases demonstrable by this method. But with the ZN technique additional specimens may add more “false positives”. Thus, for detecting cases both smear and culture-positive two specimens appear adequate. A third specimen is helpful for detecting cases positive on culture only.

KEY WORDS: SPUTUM EXAMINATION, MULTIPLE SPUTUM SPECIMEN, SURVEY, RURAL, ZIEHL NEELSON, FLUORESCENT, CULTURE.

025
PRECISION OF ESTIMATES OF PREVALENCE OF BACTERIOLOGICALLY CONFIRMED PULMONARY TUBERCULOSIS IN GENERAL POPULATION
SS Nair, GD Gothi, N Naganathan, K Padmanabha Rao, GC Banerjee & R Rajalakshmi: Indian J TB 1976, 23, 152-59.

This paper reports on a study conducted in the year 1975 to estimate yield of tuberculosis cases from multiple sputum specimens, and work out correction factors to be applied to estimates based on small number of specimens. Eight sputum specimens were collected within a fortnight from each person with an abnormal chest X-ray during an epidemiological survey in 77 villages in a district of south India. Each specimen was examined by Ziehl-Neelsen technique of microscopy and culture. In all, 3,199 persons were referred for sputum examination and results of all the eight specimens were available for 1,652. Of the latter, 64 were culture positive.

The first specimen detected 58% of the culture positives and the additional positives by later specimens generally decreased. The contribution from the first specimen was 71% for cultures showing good growth and 19% for cultures with scanty growth. Similarly for positives on both culture and microscopy, first specimen detected 87% whereas the corresponding proportion was 32% for those positive only on culture. The type of specimen (viz., spot or overnight) and age or sex of the case did not influence the yield from multiple examinations. The precision of an estimate of prevalence will depend on the number of specimens on which it is based and the coverage obtained in the collection and examination of specimens. Correction factors to be applied to such estimates based on one or two specimens, for various levels of coverage have been presented. For example, an estimate of prevalence based on one sputum specimen with 90% coverage will have to be nearly doubled to get a more precise estimate. Using these correction factors, revised estimates of prevalence have been presented for a number of prevalence surveys conducted in India. It has been estimated that the total number of infectious cases in India at present may be at least 3 million, as against 2 million according to earlier estimates.

KEY WORDS: PREVALENCE, CASE, RURAL POPULATION, MULTIPLE SPUTUM SPECIMEN, ESTIMATES, SPUTUM EXAMINATION.
 

 
  OPERATIONS RESEARCH  
 
A : Problem Definition
 
082
ASSESSMENT OF DIAGNOSIS OF PULMONARY TUBERCULOSIS BY SPUTUM MICROSCOPY IN A DISTRICT TUBERCULOSIS PROGRAMME
K Padmanabha Rao, SS Nair, N Naganathan & R Rajalakshmi: Indian J TB 1971, 18, 10-25.

In the District Tuberculosis Programme (DTP) the diagnosis is based on sputum microscopy. Majority of health institutions in the district are provided with microscopes for this purpose. In the Peripheral Health Institutions, the programme activities have to be carried out by its staff after a short period of training given by District TB Centre personnel on the spot. So the microscopy work in the PHIs is likely to be carried out by any paramedical personnel and not necessarily by a qualified laboratory technician. It is therefore, necessary to know whether the standard of microscopy carried out by these paramedical personnel after a short training will be upto the mark. To assess the efficiency of smear examination done by these individuals, a study was conducted in Bangalore district covering nine microscopy centres in various types of health institutions, a few months after the implementation of the programme. Under the DTP a spot specimen is collected from every chest symptomatic attending the health institutions and a smear is made and examined for the presence of AFB and all positive cases are put under treatment. The sputum specimens and the smears examined in these nine centres were brought to National TB Institute laboratory. The smears were examined by an experienced laboratory technician. Duplicate smears were also prepared from these specimens and their results compared with results of re examination and centre's examination. All specimens were cultured by swab method and all positive cultures were subjected to sensitivity and identification tests.

Analysis of the results based on culture showed that barring a few centres where the performance was poor, the standard of examination was fairly good. The under and over diagnosis based on culture were 38.2% and 2.6% respectively, and these were within the limits observed generally. Comparison of results on re examination of centre smears and duplicate smears indicated that both reading variation and defective smear preparations and staining could have influenced under diagnosis in these centres. The study has also thrown some light on methodology of assessment of sputum examination that could be adopted wherever a tuberculosis control programme is functioning.

KEY WORDS: CONTROL PROGRAMME, ASSESSMENT, DIAGNOSIS, SPUTUM MICROSCOPY.
 

  B : Programme Development  
 
094
COLLECTION AND CONSUMPTION OF SELF ADMINISTERED ANTI-TUBERCULOSIS DRUGS UNDER PROGRAMME CONDITION
GD Gothi, D Savic, GVJ Baily, K Padmanabha Rao, SS Nair & GE Rupert Samuel: Indian J TB 1971, 18, 107-13.

This investigation was to find out the drug consumption among tuberculosis patients put on domiciliary self administered chemotherapy, in terms of proportion of patients that make various levels of drug collections and proportion among them that consume drugs at different points of time during the course of treatment. In all, 816 tuberculosis patients aged 5 years and above residing in Bangalore city were admitted to the study. They were randomly divided into 6 groups at the time of inclusion into the study, for examination of urine samples for the presence of INH and PAS. One surprise urine sample was collected from each patient at the pre determined time after the drug collection. The samples of urine were collected from one group at first month, another at second month, third at fourth month, fourth at sixth month, fifth at ninth month and sixth at twelfth month of treatment. Urine samples were collected within 33 days of drug collection for the month because the drugs were supplied at a time for the said period. Urine specimens were examined for the presence of drugs or their metabolites. For INH, NM test & acetyl INH test and for PAS, ferriechloride and case test were performed. The drug collection was judged on the basis of treatment record and its consumption on the basis of results of urine examination.

Of the total patients included in the study, 54% made 10 or more drug collections over a period of 15 months. The initial radiological or bacteriological status or severity of disease did not influence the drug collection; however smaller proportion of old persons in both sexes collected the drugs for 10 months or more. Urine specimens of 71% of patients who had collected drugs were positive for INH on any one day. Bacteriological quiescence was obtained among the 82% INH sensitive patients who had made 10 or more collections. The above findings suggest that the patients who collect drugs also consume with fair amount of regularity and achieve a high degree of bacteriological quiescence.

KEY WORDS: SELF ADMINISTERED REGIMEN, DRUG COLLECTION LEVEL, DRUG CONSUMPTION, CONTROL PROGRAMME, COMPLIANCE.
 

 
  BACTERIOLOGY  
 
 
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.

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.

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.

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.

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.

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.

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.

KEY WORDS: COST, LABORATORY, TUBERCLE BACILLI, ESTABLISHMENT.
 
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