A.S. BAGGA, M.S. KRISHNA MURTHY, K.R. RANGASWAMY AND M.S. KRISHNA MURTHY (From Tuberculosis Prevention Trial, Bangalore). surveys.

Transcription

1 PREVALENCE OF TUBERCULOSIS DISEASE AND INFECTION IN KADAMBATHUR PANCHAYAT UNION,* CHINGLEPUT DISTRICT, SOUTH INDIA A.S. BAGGA, M.S. KRISHNA MURTHY, K.R. RANGASWAMY AND M.S. KRISHNA MURTHY (From Tuberculosis Prevention Trial, Bangalore). Introduction In connection with a controlled trial of the protective effect of BCG vaccination, currently under survey in a part of Chingleput District, a base line survey was carried out, with two skin tests to all (aged one year and above), X- ray examination of all except children below 10 years, and sputum examination (smear and culture) on the basis of the X-ray readings. The earliest results are presented here as a contribution to the knowledge about tuberculosis prevalence in various parts of India. Several prevalence surveys for tuberculous infection and disease have been carried out in India. National Sample Survey 1 revealed for the first time the widespread nature of tuberculous disease. Raj Narain et al 2, Frimodt-Moller 3 and Sikand and Raj Narain 4 confirmed the findings of National Sample Survey regarding prevalence of disease. Edwards 5, Palmer and Edwards 6, and Nyboe 7 observed that there is a high degree of prevalence of non-specific sensitivity in various parts of India. Two tuberculosis prevalence surveys carried out in Kancheepuram and in Ponneri Taluks of Chingleput District also confirmed both these findings. As first shown by Palmer and Strange Petersen 10, some reactions to tuberculin are nonspecific, that is, not caused by infection with M. tuberculosis. Non-specific reactions tend to be weaker than specific reactions. Nyboe 7 has shown that in temperate and subtropical countries almost all tuberculin reactions are either clearly positive or clearly negative indicating that the test is highly efficient. In tropical regions, on the other hand, a large proportion of reactions are of intermediate size and distinction between positive and negative reactions is therefore difficult and a clear cut distinction between tuberculous infected and uninfected evidently cannot be made by means of the present tuberculin test. He further observed that it is not known whether strong sensitivity to tuberculin can be carried by agents other than the tubercle bacilli. It is generally believed not to be the case, but the possibility cannot be ruled out. Palmer and Edwards 11 on the basis of enormous experience in the laboratory and among naval recruits recommended using a sensitin prepared from an atypical mycobacterium, in addition to the tuberculin in order to distinguish human beings infected with mycobacterium tuberculosis from those infected with other mycobacteria. By the use of dual test Edwards and co-workers 12 have shown that the studies carried out in tuberculosis patients using tuberculin and PPD-B (prepared from the Battey organism) were promising. Almost all patients had more reactions to tuberculin than to the other antigen given at the same time. False positive findings as shown by Roelsgaard and Co-workers 13 in X-ray readings and with regards to the examination of smears by microscopy as shown by Raj Narain and coworkers 14 can also be a problem in prevalence surveys. Material and Methods : The complete census of Kadambathur Panchayat Union consisting of 42 panchayats was taken (on pre-numbered cards) moving from house-to-house. The total number of persons registered was 71,685. A centre for examinations was established in each village (a group of villages makes a panchayat). Presence or absence of old scare due to BCG was recorded after verifying the identity of every person. Persons with old scars have been excluded from the analysis. Only the de jure population have been included in the present analysis. Each person aged one year and more was tested intradermally with 0.1 ml each of both PPD-S (5 IU)* and PPD-B (10 Units)+ on the * PPD-S is a batch of purified protein derivative of Mammalian tuberculin prepared by Dr Florence Seibert and Glenn 15. A part of this batch is now the international standard of PPD. + Palmer and Edwards 11 have shown that when both PPD-S and PPD-B are used in equal concentrations (by weight) the human tuberculin appears to be more potent than PPD-B in persons who had tuberculous infection and less potent in persons who have had nonspecific type of reactions. The two preparations are certainly not identical (qualitatively as well as quantitatively) nor is the one merely more concentrated than the other. Further they argue that when sensitising agent is not known, its identity can only be inferred by testing with qualitatively and quantitatively different tuberculins.

2 PREVALENCE OF TUBERCULOSIS DISEASE AND INFECTION IN KADAMBATHUR PANCHAYAT UNION 7 upper dorsal surface of either forearm depending upon the last digit of the individual number, in order to avoid bias at reading. In the event of last digit being an even number, PPD-S was injected on the right fore-arm and PPD-B on the left fore-arm but in case the last digit was an odd number, the injections were given in the reverse order. The reactions were read on the third day moving from house-tohouse. A clerk was provided to record the reactions. (Whenever freeze dried PPD-S and PPD-B were used, these were reconstituted with normal saline). Every day syringes were sterilised by autoclaving. Biologicals were stored at refrigerator temperature until use. Ampoules and vials once used on a working day and the contents if any, left over after a day s work, were discarded. All individuals aged 10 years and more were X-rayed by mobile mass miniature radiography on 70x70 roll films. The films were read independently by two readers who had no knowledge of the results of tuberculin testing or the bacteriological examination. The readers classified shadows indicating lung pathology in order of severity as infiltrate with cavity, infiltrate with doubtful cavity, Infiltrate without cavity, Hydropneumothorax, Pleurisy with effusion, Glandular lesion in and around mediastinum, consolidations, fibrotic and calcified lesions. Cardiac, diaphragmatic and thoracic cage abnormalities if present were also recorded. The extent of the lesion and whether unilateral or bilateral was also recorded using codes. The readers also recorded their interpretation according to the following categories in order of severity: D Probably active tuberculosis C Possibly active tuberculosis B Inactive tuberculosis A Non-tuberculous. Where more than one lesion was seen, the classification was determined by the most severe finding. Persons with pulmonary or pleural lesions irrespective of their interpretation by either reader were eligible for sputum collection, the exceptions being healed pleural scar, single calcification in either lung less than 1.5 mm and a pulmonary scar less than 1.5 mm only in one lung (on the 70 mm film). In addition, the persons who volunteered presence of symptoms suggestive of tuberculosis and those who had undergone treatment for tuberculosis were also eligible for sputum collection. A Spot and an Overnight sputum samples were collected by a house-to-house visit. Sputum samples were sent to the Tuberculosis Research Unit Laboratory at Madanapalle, where these were examined independently by microscopy and culture. Strains positive on culture were examined for drug sensitivity. (The results of drug sensitivity are not presented in this paper). An attempt was made to classify the cultures as typical or atypical for mycobacteria. Results Sensitivity to Tuberculin : Tuberculin reactions to PPD-S in general population in the age group 1 9 years, were compared with those of contacts of the same age of culture positive cases and shown in Fig. 1. Distribution of reactions among contacts was bimodal, one mode being at 4-5 mm and another at mm. On the other hand in general population the distribution has a pronounced mode at 4-5 mm. Another mode of reactions on the right hand which is just discernible corresponds closely in position and shape with the right hand distribution of reactions seen in the contacts of culture positive cases. It seems reasonable to assume that the child population contains a small group whose tuberculin sensitivity closely resembles that of the contacts. The reactions to PPD-S in the general population both in males and females in the age group years are shown in Fig. 2. The reactions are of bimodal distribution both in males and females. The distribution on the left hand with a mode at 4-5 mm not only contains negative reactors but fairly large number of intermediate reactors. The right hand distribution with a mode at mm in both males and females corresponds in its position and shape to the distribution in the culture positive cases shown later. Tuberculin reactions to PPD-S in general population in the age group years and in the age group 40 years and above both in males and females along with the corresponding reactions of the same sex and age group of culture positive cases are shown in Fig. 3. Reactions in general population in both the age groups and in either sex are of bimodal distribution. The reactions among culture positive cases are of unimodal distribution, the mode being at mm in the males and at mm in the females. Over 95 percent of the culture positive cases, 20 years or more of

3 8 A.S. BAGGA, M.S. KRISHNA MURTHY, K.R. RANGASWAMY AND M.S. KRISHNA MURTHY FIG. 1 DISTRIBUTION OF REACTIONS TO PPD-S IN AGE GROUPCl-9) YEARS BY SEX AMONG HOUSE-HOLD CONTACTS OF CULTURE POSITIVE CASES AND IN GENERAL POPULATION Frequency Polygon refers to General Population (G.P.) Histogram refers to Contacts of Culture positive cases age in both sexes had reactions more than 13 mm. The right hand distribution of the frequency polygon corresponds to the reactions among culture positive cases, and represent specific reactions. The left hand distribution with a mode at 6-7 mm consists as shown previously not only of negative reactors, but includes a fairly large number of intermediate reactors. Based upon these findings it is not unreasonable to assume that those having reactions more than 13 mm to PPD-S were infected with tubercle bacilli and that they were showing specific reactions with a minimum of overlapping. Prevalence of Infection : Prevalence of infection (reaction to PPD-S more than 13 mm) in Kadambathur Panchayat Union by age and sex is shown in Fig. 4. It may be seen from this figure that the prevalence rates rise sharply among younger age groups and reach maximum by the age of 40 years both in males and in females. In males almost 80 per cent and among females almost 65 per cent were tuberculin reactors by that age. Thereafter prevalence rates remain near the peak level. There may be an additional number of infected whose sensitivety has waned. Panchayat wise prevalence of infection rates in the age group years (the age group

4 PREVALENCE OF TUBERCULOSIS DISEASE AND INFECTION IN KADAMBATHUR PANCHAYAT UNION 9 FIG. 3 DISTRIBUTION OF REACTIONS TO PPD-S BY SEX AND TWO AGE GROUPS (20-39) AND (40 + YEARS) IN PATIENTS AND IN GENERAL POPULATION homogenous in order to demonstrate the prevalence of infection rates) in both sexes were studied. Infection rates (reaction size more than or equal to 14 mm to PPD-S has been used to distinguish between infected and uninfected) range between 13 percent to 46 per cent. The variation between panchayats was highly significant statistically (P<0.01) Distribution of panchayats according to infection rates in the age group years are shown in Fig 5. Also shown in the figure are the prevalence rates of culture positive cases among X-rayed persons of both sexes. It may be seen from Fig. 5, that the Panchayats with low infection rates (^20 per cent) are distributed in the South West and Panchayats with high infection rates O40 per cent infected) are distributed in the North East of Kadambathur Panchayat Union and are very near to Tiruvallur Town. Panchayats with infection rates between 20 per cent to 40 per cent are scattered all over the Kadambathur Panchayat Union. _ Size of population and the percentage infected were studied in each Panchayat (not shown). No correlation was observed between these two factors. Prevalence of Non-Specific Sensitivity : Distribution of mean reactions PPD-B according to age and sex among these reacting < 14 mm to PPD-S are shown in Fig 6 The mean reactions rise sharply among younger age groups and reaches maximum by 20 years of age. Thereafter it remains near peak level except beyond 60 years of age when a slight tall is observed. No sex difference was observed in the distribution of mean reactions. The correlation of reactions between PPD-S

5 10 A.S. BAGGA, M.S. KRISHNA MURTHY, K.R. RANGASWAMY AND M.S. KRISHNA MURTHY F I G. 4 and PPD-B for the age group years (the age group was considered to be large enough PREVALENCE OF INFECTION (REACTION as well as reasonably homogenous in order to TO PPD-S >I4mm) BY AGE AND SEX demonstrate the presence of non-specific sensitivity) is shown in Table I. The reactions are distributed in an inverted-l shaped pattern. The distribution of reactions to PPD-B is unimodal with a mode mm. The distribution of reactions to PPD-S is unimodal, one mode is at 4-5 mm and another mode at mm. The latter mode corresponds in its position and shape to the mode observed in culture positive cases. Prevalence of Disease : Definitions of some of the terms used in this paper are listed below : Smear Positive case : A person was called a Smear positive case if at least one sputum smear was found to be positive for >4 tubercle bacilli. (Smears showing 1-3 bacilli were not reported by the laboratory as those were probably regarded as artefacts). Culture Positive case : A person was TABLE 1 Correlation between reactions to PPD-S and to PPD-B in age group (10-14) years Reaction to PPD-B (Induration in mm)

6 PREVALENCE OF TUBERCULOSIS DISEASE AND INFECTION IN KADAMBATHUR PANCHAYAT UNION 11 Fig. 5 MAP SHOWING PREVALENCE OF INFECTION 0 IN AGE GROUP(10-19) YEARS AND PREVALENCE OF DISEASE 00 IN AGE GROUP (1O+) YEARS IN EACH PANCHAYAT Criterion for Infection: Reaction to PPD-S > 14mm Percentage to test read. Disease: Culture positive: Per thousand X-Rayed persons KADAMBATHUR PANCHAYAT UNION called a sputum culture positive case if on at least one sputum culture, colonies of mycobacterium tuberculosis were grown. Sputum Positive case : A person was called a sputum positive case if at least one sputum specimen was found to be positive by either smear or by culture. X-ray case : A person was called a X-ray case if both X-ray readers classified his radiological shadows as probably or possibly active tuberculosis (X-ray alphabet codes C or D) and whose sputum was negative. The distribution of tuberculin reactions among randomly selected controls from the general population (matched for age and sex with the X-ray cases), among the X-ray cases and among culture positive cases were studied separately for males and females and are shown in Fig 7. Reactions to the tuberculin in the controls are of bimodal distribution in males as well as in females very similar to the pattern Ind. J. Tub. Vol. XXI, No. 1

7 12 A.S. BAGGA, M.S. KRISHNA MURTHY, K.R. RANGASWAMY AND M.S. KRISHNA MURTHY seen in the general population. The reactions in the X-ray cases both in males and females FIG.6 DISTRIBUTION OF MEAN REACTION TO PPD-B FOR NEGATIVE REACTORS TO PPD-S (PPD-S<14mm) BY AGE AND SEX are also distributed bimodally with a pronounced mode at mm and a small mode at 6-7 mm. The distribution of reactions in culture positive cases is unimodal (a normal distribution curve with a mode at mm in males and at mm in females). Mean tuberculin reactions in the culture positive cases were studied further in two sub-groups, viz. (a) Scanty positive on culture i.e. in whom only 1-3 colonies of mycobacterium tuberculosis were grown; (b) highly positive on culture i.e. in whom >4 colonies of mycobacterium tuberculosis were grown. No statistically significant difference was observed in the mean reactions in these two sub-groups i.e. the mean reaction in terms of level of tuberculin sensitivity. It may be seen from the histogram in Fig. 7 that the culture positive cases displayed a distinct pattern. The bimodal distribution seen FIG. 7 DISTRIBUTION OF REACTIONS TO PPD-S AMONG CONTROLS TO X-RAY CASES. X-RAY CASES AND CULTURE POSITIVE CASES CONTROLS CULTURE POSITIVE Reaction to PPD-S(in mm)

8 PREVALENCE OF TUBERCULOSIS DISEASE AND INFECTION IN KADAMBATHUR PANCHAYAT UNION 13 FIG.8 PREVALENCE OF CULTURE POSITIVES AND X-RAY POSITIVES BY AGE AND SEX IN KADAMBATHUR PANCHAYAT UNION in the X-ray cases may mean that active tuberculosis was diagnosed by X-ray in some of the sputum negative persons who were probably not infected with mycobacteriom tuberculosis. Scanty and highly positive cases on culture were further studied regarding their X-ray status. X-ray reading by two readers are shown in Table II (a) and II (b) in terms of correlation table between the two readings. Cases scanty positive on culture were compared with randomly selected controls from the general population (matched for age and sex with twice the number of cases scanty positive on culture, excluding sputum positive cases and also those with technically inadequate X-rays). X-ray readings between two readers among cases highly positive on culture were also studied similarly and are shown in Table II (c). It may be seen from Table II (b) that as few as one third (35 per cent) were read as possibly or probably active (X-ray alphabet codes C or D) tuberculosis by both X-ray readers in cases scanty positive on culture while as many as one fourth (26 per cent) were missed by X-ray by both X-ray readers. Reading of POSSIBLY or PROBABLY ACTIVE Tuberculosis by at least one of two X-Ray readers. The correlation between smear and culture results is shown in Table III. It may be seen that as many as 129 (44 per cent) were culture positive but negative on smear while only 166 (56 per cent) were positive on culture and were also positive on smear. TABLE II (a) Correlation between two x-ray readings of controls* to culture scanty positives I Reading 0** A B C D Others(i) Total 0** A II Reading B C D Others(i) Total * Matched for age (by 5 years age groups) and sex (excluding sputum positive cases and those with technically inadequate X-rays) with twice the number of scanty positive cases. ** No abnormality. (i) No code recorded.

9 14 A.S. BAGGA, M.S. KRISHNA MURTHY, K.R. RANGASWAMY AND M.S. KRISHNA MURTHY TABLE II (b) Correlation between two x-ray readings of culture scanty positives I Reading 0* A B C D Other** Total 0* II Reading A B C D *No abnormality ** No code recorded Others** Total TABLE II (c) Correlation between two x-ray readings of cases highly positive on culture I Reading 0* A B C D Other** Total 0* II Reading A B C D Others** 1 1 * No abnormality ** No code recorded Total Smear Positive Cases : 16 cases were found to be positive on smear only but were negative on culture. Could these be atypical mycobacteria or dead bacilli in recently treated cases or were they artefacts? At 6-12 months follow-up, 2 of the 14 followed up were found to be positive on culture and remaining were found to be negative on smear and also on culture. Sex and age distribution of culture positive cases (in 5 years age groups) was studied and is shown in Table IV. Only over the age of 39 years, a significant difference was seen between sexes, males having a higher rate of highly positive cases on culture than females (not shown in this table). Prevalence of X-ray cases (>10 years) in males was 2.1 per cent and in females was 1.1% per cent. The prevalence of culture positive

10 PREVALENCE OF TUBERCULOSIS DISEASE AND INFECTION IN KADAMBATHUR PANCHAYAT UNION 15 TABLE III Correlation between smear and culture results of sputum positive cases Culture Negative (No bacilli) (1-3) colonies 4 or more colonies Total Smear Negative (No bacilli) or more bacilli Total TABLE IV Age and sex distribution of sputum positive and x-ray cases in 5 year age groups Total Sputum Positive Cases Males Females Both Sexes Males X-ray Females Cases Both Sexes cases (> 10 years) in males was 10.9 per cent and in females 2.8 per cent. Prevalence of X-ray and sputum positive cases by age and sex has been shown in Fig. 8. (for absolute figures see Table IV). It may be seen from Fig. 8 that the prevalence of X-ray cases both in males and in females rises gradually upto the age of 35 years and thereafter it rises very sharply. On the other hand, the prevalence of culture positive cases in males rises gradually upto the age of 30 years. The peak is reached by about 40 years of age and thereafter it remains near the peak level. However, in females no such phenomenon was observed. Discussion : Many authors (BMRC trial 16, Raj Narain et al 2, Edwards and Smith 17, Hsu et al 18 ) in the past have adopted different sizes of reaction to tuberculin to classify reactors and non-reactors; the chief aim in such a classification has been to have a minimum of over lapping. Edwards and Edwards 12 suggested that tuberculin reactions above 12 mm only should be considered without any doubt as due to tuberculous origin while reactions between 6-12 mm are likely to be of non-specific origin. Based upon the material presented, a reaction size to tuberculin of 14 mm or more (irrespective of reaction size to PPD-B) was considered as indicative of specific reaction. Frimodt-Moller 3 has shown in South Indian villages that beyond the age of 10 years, nonspecific sensitivity reactions are almost universal. Our findings are more or less similar to those findings. Palmer and Edwards 11 have demonstrated among naval recruits by dual testing that those infected with M. tuberculosis and those infected with atypical mycobacteria run different kinds of risks. The controlled BCG trial population is

11 16 A.S. BAGGA, M.S. KRISHNA MURTHY, K.R. RANGASWAMY AND M.S. KRISHNA MURTHY TABLE V Comparative prevalence of radiological and bacteriological disease in three surveys Sex Radiological prevalence in percentage (%) Bacteriological prevalence per thousand (%) * National Sample Survey (Madanapalle Zone) Males Females Both Sexes Males Tumkur Survey Females Both Seves 1.9** 4.1** Kadambathur Panchayat Union Males Females Both Sexes * In the National Sample Survey the average for all the zones of radiological prevalence was 1.8 per cent and the bacteriological prevalence was 4.0 per cent. ** For the villages the average radiological prevalence was 1.8 per cent and bacteriological prevalence was 3.9 per cent being followed now for many years and it might become possible to arrive at an accurate definition to distinguish infected and uninfected and the risks involved. Prevalence of Disease : The comparative prevalence of radiological and bacteriological disease observed in the National Sample Survey, in the Madanapalle zone of National Sample Survey, in the Tumkur District Survey and in the Kadambathur Panchayat Union are shown in Table V. The Madanapalle zone in South India examined during National Sample Survey lies not quite far off from Kadambathur Panchayat Union. The prevalences of radiological disease in these areas do not differ much. The prevalences of bacteriological disease in the National Sample Survey and in the Tumkur district were of the same order. Comparatively higher prevalences were observed in the Madanapalle zone of National Sample Survey and in the Kadambathur Panchayat Union. However, the highest prevalence was observed in the Kadambathur Panchayat Union. The prevalence of bacteriological disease in the males was observed to be the highest in the Kadambathur Panchayat Union. ACKNOWLEDGEMENTS The authors are grateful to Dr. Raj Narain, Deputy Director General of Health Services, Government of India, on deputation as Project Director, Tuberculosis Prevention Trial, Bangalore, for his constructive criticism. The authors are also deeply indebted to Dr. J. Guld, Medical Officer, Tuberculosis Unit World Health Organisation, Geneva, for his very useful suggestions and criticism. Our thanks are also due to Dr S. Mayurnath, Medical Officer, for his suggestions and to the field teams, for their hard work. We wish to thank Mr M. Haridas, Statistical Assistant and the other staff of the Statistical Section for their help in the analysis and Mr P.R. Krishna Moorthy and Miss Vaidehi for their Secretarial help.

12 PREVALENCE OF TUBERCULOSIS DISEASE AND INFECTION IN K.ADAMBATHUR PANCHAYAT UNION Indian Council of Medical Research (1959) Tuberculosis in India. A sample survey , New Delhi (Special Report Series No. 34). 2. Raj Narain, Geser, A., Jambunathan, M.V., and Subramanian, M. (1963) Bull. Wld. Hlth. Org. 29, Frimodt-Moller, J (1960) Bull. Wld. Hlth. Org., 22, Sikand, B.K., and Raj Narain (1958) Radio logical problems of tuberculosis as revealed by the national survey. In : Tuberculosis Associa tion of India, Proceedings of the 14th All India Tuberculosis and Chest Diseases Workers Conference, held in Madras under the auspices of Tuberculosis Association of India, pp Edwards LB., Palmer, C.E., and Edwards, P.Q., (1955) Bull. Wld. Hlth. Org. 12, Palmer, C.E., and Edwards, P.Q., (1955) Bull. Wld. Hlth. Org. 12, Nyboe, J (1960) The Efficiency of the Tuber culin Test, an Analysisbased on results from 33 countries. Bull. Wld. Hlth. Org. 22, Raj Narain, S. Mayurnath, A.S. Bagga, K. Naganna, M.S, Subba Rao and K.R. Rangaswamy (1969) Proceedings of the 24th TB and Chest Diseases Workers Conference, Trivandrum, January Raj Narain, A.S. Bagga, S. Mayurnath, K. Naganna, M.S. Subba Rao and K.R. Rangaswamy (1969) Proceedings of the 24th TB and Chest Diseases Workers Conference, Trivandrum, January, REFERENCES 10. Palmer, C.E., and Strange Peterson (1950). Pub. Hlth. Rep. (Wash) 65, Palmer, C.E. and Edwards, L.B.. (1968) Identi fying the Tuberculous infected. J. Amer. Med. Ass. 205, Edwards, L.B., Edwards, P.Q., and Palmer, C.E. (1959) Ada Tuber. Scand. Supplement, 47, p Roelsgaard, E, Iverson, E and Blocher, C (1964) Bull. Wld. Hlth. Org. 30, Raj Narain, Subba Rao, M.S., Chandrasekhar, P. and Pyarelal (1971) Microscopy Positive and Microscopy Negative cases of Pulmonary Tuber culosis, Amer. Rev. Resp. Dis. 103, Seibert, F.B., and Glenn, J.T., (1941) Tuber culin purified Protein derivative: Preparation and analysis of a large quantity for standard. Amer. Rev. Tubcrc. 44, Medical Research Council, Tuberculosis Vaccines Clinical Trial Committee (1959) II Report, Brit. Med. J. 2, Edwards, L.B., and Smith, D.T. (1955) Amer. Rev. Resp. Dis., 92, Hsu, Katharine H.K., Fongee Jeu and Jenkins, Daniel B.C., (1961) Amer. Rev. Resp. Dis. 90, 36. Ind. J. Tub.. Vol. XXI, No. 1