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.

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.

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.

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.

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.

This paper brings out certain guidelines to be followed at the time of despatch of specimens for laboratory investigations. Despatch of pathological specimens to laboratories situated away from the place of collection for investigations is quite a common practice. Often those despatching the specimens are not aware of the procedures. Specimens are packed like any other articles sent by post.

There are two important points to be remembered when pathological specimens are sent for investigations. One of them is preservation of the material so that the specimens reach the laboratory in a condition fit for necessary investigations. The other is the proper packing of the specimens to prevent leakage from or breakage of the containers during transit so that they do not become hazardous to persons handling them. For microscopy, it is better to send fixed smears wrapped in a paper and properly labelled. For culture, specimens should always be sent in a sterile container. It is preferable to send them in ice to prevent overgrowth of contaminants and drying. If the transport time is 3-4 days, they can be sent at room temperature. It is advisable to send bulky liquid specimens and more than one specimen through a messenger instead of by post or as an unaccompanied parcel. In case this is not possible, it will be advisable to send them in more than one parcel depending on the number to be sent instead of sending all specimens as a single parcel.

The National Tuberculosis Institute (NTI), Bangalore was established in 1959 and its bacteriological laboratory started functioning from 1961. For the first few years WHO Experts were involved in the establishment and running of the laboratory but since many years the laboratory is being run only by the national staff.

The laboratory has been involved in research and training since its inception. In order to compare the standard of the various tests done in the NTI laboratory with that of a similar laboratory having some standing in tuberculosis research, a series of comparison studies were done between NTI and the laboratory of the Tuberculosis Chemotherapy Centre, Madras. The results were similar except for variations within normal limits. Besides, some cultures isolated in NTI laboratory were sent to the tuberculosis laboratory of the Centre for Disease Control (CDC), Atlanta (Georgia) U.S.A. for purpose of an inter-laboratory comparison. Forty seven cultures were sent to CDC, of which 38 were M.tuberculosis, 1 rapid grower, 1 H37 RV, 3 M.avium, 2 M.bovis, 1 B.C.G and 1 M.phlei. But for some minor variations in a few biochemical tests, the species classification compared well between the two laboratories. Taking all the results into account and making some allowance for unavoidable variations, it was observed that the standard of bacteriological investigations were similar between all the three laboratories.

The issues to be addressed while establishing a tuberculosis culture laboratory are discussed in the paper. Primarily the following questions are to be considered: (1) Is it absolutely essential to have a tuberculosis culture laboratory? (2) Will it be big or small? (3) Are there adequate means to continue work in terms of finance, staff, equipment & specimens and (4) Is there a possibility of taking up any other type of bacteriological work, if necessary? Unlike other bacteriology laboratory, a tuberculosis laboratory has some unique features. Due to the slow growth of the organism, cultures need to be incubated for a long time i.e., 8 10 weeks. So an incubator room is required. More number of glassware are needed. Test tubes with cotton plugs are unsuitable as they are likely to dry up. Hence screw capped tubes or McCartney bottles are required to facilitate long incubation of cultures. For performing identification tests, incubators with varying temperatures, i.e., 23, 37, 44, are to be provided. Plenty of cold storage space is needed to stock cultures, media, etc.

The requirement of staff and organisation of work depends upon the number of specimens handled. If 50 specimens per day are likely to be processed, 5 lab technicians, 3 lab attendants, 1 sweeper and 1 bacteriologist are necessary. Once laboratory is opened, maximum benefit should be derived by getting adequate number of specimens.

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.

The findings of two studies, (i) one on comparison of Ziehl-Neelsen method of staining of acid fast bacilli with and without alcohol decolourisation and use of Gabbet's Methylene blue (in place of decolourisation and counter staining) and (ii) comparison of two different types of Basic Fuchsin dye used in the preparation of Carbol Fuchsin, have been presented. The first study has shown that omission of alcohol decolourisation or the use of Gabbet's Methylene Blue has not influenced the detection of positives, though the latter has more often produced a non- satisfactory background. The second study has brought out the fact that two types of Basic Fuchsin are similar in every respect. However, the findings does not rule out the possibility of a bad dye giving rise to poor results. Need for conducting studies for simplifying the staining procedure has been stressed.

A study was conducted to evaluate the usefulness of pyruvate medium for isolation of M.bovis from human material and additional yield of M. tuberculosis resistant to INH. Specimens from both rural and urban populations were included for this study in order to understand the problem in both the situations. There were two studies in progress at the National Tuberculosis Institute when pyruvate media slopes were introduced for culture purpose. One study was an epidemiological survey; 2518 sputum specimens received from 51 villages covering a population of about 32,300 were used. The specimens were collected from persons aged 5 years and above showing abnormal shadow on X-ray. The other study was conducted in collaboration with the State Tuberculosis Centre, Bangalore; 1204 sputum specimens were received from out patients attending the centre. In addition to LJ medium, pyruvate medium was used for isolation purposes. Identification and sensitivity tests were done on positive cultures as per routine. In all, 129 cultures of tubercle bacilli were isolated from 2118 specimens belonging to study 1 and 398 from 1204 specimens belonging to study 2. The number of cultures contaminated were 253 and 35 respectively. No M.bovis was isolated in either study. There were 24 and 23 cultures resistant to INH among those isolated from LJ and pyruvate medium respectively. Thus, no increase was observed in the isolation of INH resistant strains using pyruvate medium.

Hence, no benefit was derived by using this medium over and above what was obtained from plain Lowenstein Jensen medium in both the situations.

Sputum microscopy is the main casefinding tool in tuberculosis control programmes. The technique of smear preparation is an important step which needs to be simple for wide applicability. It is often stressed that smear should be prepared from the purulent portions of the sputum as they are likely to have more number of bacilli. It may not be possible for the microscopist/paramedical worker at the periphery to strictly follow this procedure. Hence, a study was conducted to compare the sensitivity of 4 methods of sputum smear preparation viz., direct smear prepared (i) blindly without making any selection of portions of sputum specimen, (ii) from portions of sputum material likely to contain the bacilli, (iii) after mixing up the sputum specimens thoroughly, and (iv) from centrifuged deposit after homogenization of sputum with sodium hydroxide and concentration by centrifugation. Culture was also done for Mycobacterium tuberculosis.

A total of 549 specimens were employed. Positivity rates by four methods were: 79.6% by method (i), 80.3% by method (ii), 80.7% by method (iii) and 77.2% by method (iv). There was no statistically significant difference in the number of positives obtained from different methods. Centrifuged deposit smears proved to be in no way better than the direct smears. The differences in the methods lay only in the classification of positive smear as of a low or high grade.

Studies from Madras had shown that the strain of M.tuberculosis isolated from south India were low virulent to guineapigs. The relationship between virulence in guineapigs and pathogenesis in humans could not be established earlier. A study was conducted to investigate the relationship of virulence with the pathogenesis by comparing the virulence of isolates from pulmonary tuberculosis with that from patients with TB meningitis. The strains of bacilli were obtained from three different sources: a) Sputum from rural tuberculosis patients living near Bangalore city, b) sputum of TB patients living in the city and c) from Cerebrospinal fluid (CSF) of patients suffering from tuberculous meningitis and admitted in different institutions in Bangalore city. The specimens were processed by standard recommended procedures and cultured on Lowenstein Jensen medium. The identification of an isolate as M. tuberculosis was based on the niacin test. Albino Guinea pigs of both sexes (who were bred and raised at this Institute) were used for the tests. The virulence assay and the calculation of the root-index of the virulence (RIV) were carried out according to the Mitchison method.

1) As per the RIV method, virulence has been classified into low, moderate and high. The study showed that the percentages of cultures having isolates of low, moderate and high virulence, were the same as that of isolates obtained from patients in Madras, reported by Mitchison et al., in 1960. 2) The distribution of the RIV of virulence of isolates from patients living in the city of Bangalore was significantly different (p < 0.05) from that of isolates from patients living in rural Bangalore. 3) The number of cultures classified as high virulent were significantly greater in isolates from patients with tuberculous meningitis compared with those from patients with pulmonary tuberculosis. However, 36% of the isolates from the meningitis group were of low virulence.