Tuberculin conversion and leukocyte migration inhibition test after BCG vaccination in newborn infants

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1 Research paper 5:10, ; October 2009; 2009 Landes Bioscience Tuberculin conversion and leukocyte migration inhibition test after BCG vaccination in newborn infants M.M.A. Faridi,1,* Sarvpreet Kaur,1 Sriram Krishnamurthy1 and Pramod Kumari2 Division of Neonatology; Departments of 1Pediatrics and 2Pharmacology; University College of Medical Sciences; University of Delhi and Guru Tegh Bahadur Hospital; New Delhi, India Keywords: mantoux test, leukocyte migration inhibition test, BCG vaccination, low birth weight, preterm baby Background: There are reports that BCG vaccine take-up may be delayed or poor among preterm and low birth weight. Objective: The present study was conducted with the objective of (a) studying BCG take-up through tuberculin conversion by Mantoux test at 12 and 6 months of age in vaccinated with BCG at birth and (b) evaluating the Leukocyte Migration Inhibition Test (LMIT) in BCG vaccinated who did not react to Mantoux test. Design: Prospective observational study. Results: Local BCG reaction was exhibited by 95.5% at 8 and scar formed in 47.2% of them by 12(+1). The tuberculin conversion rate after 12 of BCG vaccination was 42.7%. The rate of tuberculin conversion was significantly more in with birth weight 2,500 g as compared to low birth weight. Also, the rate of tuberculin conversion was significantly more in term infants as compared to preterm. Six months after BCG vaccination, 41.5% of previously Mantoux negative showed tuberculin conversion. Overall, 66.4% reacted to PPD after 6 months. LMIT was positive in 84.1% who had a negative Mantoux test at 12. Six months after BCG vaccination, 53.8% of who had LMIT negative at 3 months became LMIT positive. Thus out of 143, 95.8% had either a reactive Mantoux test or positive LMIT 6 months after BCG vaccination. There were only 6 (4.2%) who did not show adequate responses to BCG vaccination. Methods: BCG vaccine was administered to 143 neonates of gestation within 7 days of birth. At 12 of age, Purified Protein Derivative (1 TU PPD-RT 23 with Tween 80) was injected to all the and induration was measured at the injection site after 48 hours. Mantoux negative were subjected to the LMIT. All the Mantoux test and LMIT negative were followed up and given another Mantoux test at 6 months. Those who showed a negative Mantoux test at 6 months and negative LMIT at 12 were subjected to another LMIT at 6 months. Conclusion: Preterm of 31 to 36 of gestation and low birth weight weighing <2,500 gram at birth can be effectively vaccinated with BCG vaccine in the early neonatal period. However, tuberculin conversion is not a reliable indicator to assess responses induced by BCG vaccination either 12 or 6 months after vaccination because inspite of negative Mantoux test, the vaccinated infant may still be having an adequate response to BCG vaccine. The local BCG reaction at 8 corresponds to cell mediated immunity conferred by BCG vaccination. Introduction Bacille Calmette-Guérin (BCG) vaccine is one of the six vaccines given under the Universal Immunization Programme (UIP).1 The vaccine is credited to reduce the incidence of disseminated tuberculosis and tubercular meningitis in BCG vaccinated children.2 A successful vaccination results in a sequence of local changes after a correct intradermal injection of a potent BCG vaccine.3 Two to three after vaccination, a papule develops at the site of vaccination which slowly increases in size and reaches a diameter of approximately 4 8 mm in about 5 6. It then ulcerates and healing occurs spontaneously within 6 12 leaving a permanent tiny round scar. The onset and completion of local BCG reaction may be delayed in low birth and scar formation may not occur by 12 of vaccination.4 Recently it has been reported that it may take 6 months or longer to form a BCG scar even among term and normal birth weight and therefore, it may not necessarily imply absent cell mediated immunity (CMI).5 The immunity conferred by BCG can be detected both in-vivo and in-vitro. The in-vivo tests, commonest of which is the tuberculin test, indicate delayed hypersensitivity, while the in-vitro tests like Leukocyte Migration Inhibition Test (LMIT), Lymphocyte Transformation Test and T-cell interferon gamma (IFNγ) Release Assays (IGRAs) are markers of CMI. Other in vitro tests such as enzyme linked immunospot (ELISPOT) and *Correspondence to: M.M.A. Faridi; drmmafaridi@gmail.com, mmafaridi@yahoo.co.in, drsriramk@yahoo.com Submitted: 03/28/09; Revised: 06/14/09; Accepted: 06/22/09 Previously published online: Volume 5 Issue 10

2 Research Paper Research paper enzyme linked immunosorbent assay (ELISA), though useful, require large laboratory set up and cost. Mantoux test is the most commonly used tuberculin test. There are conflicting reports on tuberculin sensitivity patterns among BCG vaccinated children with tuberculin conversion rates ranging from 50 80%.6,7 However, a negative tuberculin skin test does not imply absence of CMI. LMIT is a relatively simple and economic investigation that has been found to be positive in tuberculin skin test negative subjects8 and BCG scar negative who were vaccinated at birth.9 The present study was therefore, conducted with the aim of evaluating the responses to BCG vaccination in pre-term and low birth weight newborn infants vaccinated within the first week of life. The specific objectives were (1) to study Mantoux Test reaction in BCG vaccinated at 3 and 6 months of age and (2) to evaluate Leukocyte Migration Inhibition Test in BCG vaccinated who do not react to Mantoux test. Table 1. Mantoux test 12 and 6 months after BCG vaccination according to birth weight No. of subjects with size of Mantoux test (after 12 of BCG vaccination) Weight (grams) <5 mm* 5 9 mm* <5 mm** Negative < (69.2%) 24 (30.8%) 38 (70.4%) 16 (29.6%) (43.1%) 37 (56.9%) 10 (35.7%) 18 (64.3%) 82 (57.3%) *p = 0.016; **p = (42.7%) 48 (58.5%) 34 (41.5%) mm** Table 2. Mantoux test 12 and 6 months after BCG vaccination according to gestation No. of subjects with size of Mantoux test(after 12 of BCG vaccination) Results A total of 143 neonates between of gestational age were recruited. There were 90 term and 53 preterm ; the male to female ratio was 1.16:1. According to birth weight, 78 were LBW which comprised of 64.1% preterm infants. Out of the 90 term, 54 were AGA and 36 were SGA. Similarly, among preterm, 32 and 21 newborns were AGA and SGA respectively. At 8 BCG reaction was observed in 93.6% preterm, 97.3% term, 94% LBW and 98.1% infants weighing more than 2,500 g at birth. Scar was formed in 47.2% infants at 12(+1) including 45.3% in preterm and 48.9% among term infants respectively. There were no significant differences between LBW and those weighing >2,500 g with regard to local BCG reactions (p = 0.9). Similarly, there were no significant differences between preterm (<37 gestation) and term with respect to local BCG reactions (p = 0.235). The overall tuberculin conversion rate after 12(+1) of BCG vaccination was 42.7%. The rate of tuberculin conversion was significantly more in with birth weight 2,500 g (p = 0.016) as compared to LBW. After 6 months of BCG vaccination, 41.5% (n = 34) of previously Mantoux negative (n = 82) showed tuberculin conversion. Babies weighing = 2,500 g at birth had a significantly higher (p = 0.02) incidence of tuberculin conversion (64.3%) as compared to weighing <2,500 g at birth (29.6%) (Table 1). Similarly, the rate of tuberculin conversion was significantly more among term infants (p = 0.02) than preterms (Table 2). The maximum incidence (56%) of tuberculin conversion was seen in the gestational age group. However, this was not significantly different from other groups of gestation (p = 0.19). LMIT was positive in 84.1% who had a negative Mantoux test at 12 (Table 3) that increased to 92.6% at the end of 6 months. LMIT in Mantoux negative at 12(+1) after BCG vaccination was not significantly different between term and preterm (Table 4). No. of subjects with size of Mantoux text (at 6 months in who were Mantoux test negative at 12 ) No. of subjects with size of Mantoux test (at 6 months in who were Mantoux test negative at 12 ) Gestational age <5 mm Negative 5 9 mm <5 mm Negative 5 9 mm (75%) 2 (25%) 5 (83.3%) 1 (16.7%) (73.3%) 12 (26.7%) 22 (66.7%) 11 (33.3%) All preterm 39 (73.58%)* 14 (26.42)* 27 (69.23%)** 12 (30.76%)** (48.1%) 27 (51.9%) 11 (44%) 14 (56%) (47.4%) 20 (52.6%) 10 (55.6%) 8 (44.4%) All term 43 (48.86%)* 47 (53.40%)* 21 (48.83%)** 22 (51.16%)** 82 (57.3%) 61 (42.7%) *p value = 0.02; **p value = (58.5%) 34 (41.5%) Table 3. LMIT in Mantoux negative at 12(+1) after BCG vaccination according to birth weight Birth weight Number of subjects No. of subjects with positive LMI No. of subjects with negative LMI <2500 grams * 54 45(83.3%) 9(16.7%) 2500 grams * 28 24(85.7%) 4(14.3%) (84.1%) 13 (15.9%) * p value= 0.45 At 12(+1) and 6 months 130 (90.9%) and 137 (95.8%) were either Mantoux test or LMIT positive. There were only 6 (4.2%) who did not show adequate responses to BCG vaccination at the end of six months (Tables 5 and 6). The results are summarized in Figure

3 Table 4. LMIT in Mantoux negative at 12(+1) after BCG vaccination according to gestation Discussion In the present study, 42.7% of showed a positive Mantoux test at 12(+1). This is comparable to the Mantoux test positivity of 50 80% obtained in India by various authors6,7,10,11 after neonatal BCG vaccination and tuberculin testing at 3 months of (83.3%) 1(16.7%) age. Others like Thapar et al.12 observed a 100% Mantoux test (n=8) reactivity (75.8%) 8(24.2%) The objective of the present study was to evaluate the take-up (n=45) of BCG vaccine in preterm and low birth weight. Either All preterm 39 30(76.9%) 9(23.1%) a positive Mantoux test or positive LMIT following vaccina* tion was considered as suitable evidences for BCG take-up. (n=53) Neonates showing a positive Mantoux test were not subjected (95.8%) 1(4.2%) to LMIT as they were already demonstrating evidence of BCG (n=52) take-up (83.3) 3(16.7%) When BCG vaccination is administered, the live attenuated (n=38) M. bovis BCG mycobacteria multiply in the skin and are engulfed All term * 43 39(90.7%) 4(9.3%) by macrophages. The macrophages present the mycobacteria to = T-helper cells (CD4 cells) through MHC-II. The CD4 cells then * p value=0.2 secrete cytokines and IFNγ, which activate the macrophages to kill the mycobacteria.13 Few T-helper cells are converted to memory T cells. In this way the infant is sensitized to BCG antigen. When an antigen challenge is given subsequently in the form of Table 5. LMIT at 6 months in who were LMIT negative at 3 tuberculin skin test, the sensitized T cells come in contact with months after BCG vaccination the specific antigen, secrete lymphokines which in turn recruit Period of LMIT negative LMIT positive LMIT negative non-sensitized lymphoid cells to the site of inflammation, retain gestation at 3 months (n) at 6 months (n) at 6 months (n) them at the site where they become activated.14 The lymphokines (100%) 0 also cause increased vascular permeability causing erythema and (50%) 4 induration at the site of testing. It takes about for the cell mediated immunity to develop and, therefore, Mantoux (100%) 0 test is generally done after 12 of BCG vaccination to see (33.3%) 2 whether the infant has been sensitized to the BCG vaccine. At 13 7 (53.8%) 6 (46.2%)* hours of giving PPD, the test is read by measuring the *In three no local reaction; 3 were SGA; 3 had transverse diameter of the induration. An induration of less than Abortive Reaction; scar not formed in all of them. 5 mm is taken as a negative test. Induration of 5 9 mm occurs after recent vaccination with BCG.15 An induration of more than 10 mm requires further investigation for the presence of the Table 6. Local BCG reaction, mantoux test and LMIT in different groups of tubercular disease. Profile of No. of subjects in percentage The variation in the tuberculin conversion following BCG vaccibcg Other Mantoux test LMIT in Overall BCG nation could be due to various Scar stages case of Mantoux uptake at 6 factors like difference in the vaccine present present negative at months on used, strength of tuberculin used 12 basis of positive for testing, the technique of placing Mantoux and LMIT the Mantoux skin test, age groups examined, time of tuberculin 12 6 reaction months testing after BCG vaccination, Preterm % environmental mycobacteria,7,15 defective storage and transport of Term % vaccine and technique of vaccination.7 The Mantoux test is read by LBW % measuring the size of the induraabw % tion after hours commonly Overall %* by the palpation method or more precisely by the ballpoint pen techlbw = Low birth weight; ABW = Appropriate birth weight; *Does not include abortive reactors. Gestational age ( subjects) 692 Mantoux negative subjects No. of subjects with positive LMIT No. of subjects with negative LMIT Volume 5 Issue 10

4 Figure 1. Flow chart showing recruitment of subjects and BCG vaccination take-up. nique. In a study by Pouchot et al.16 it was found that the area of imprecision was 38% less broad for the ballpoint method than the palpation technique. In the present study, weighing <2,500 g showed lower Mantoux test positivity as compared to weighing 2,500 g. Ferguson et al.17 and Chandra18 have stated that in IUGR, there are low levels of lgg, low numbers of B cells and low percentage as well as number of T cells in the cord blood. Severe intrauterine growth retardation (IUGR) also interferes with the humoral immune responses.19 The tuberculin test is affected in LBW in general and in SGA in particular as it indicates delayed hypersensitivity to tuberculoprotein, which is dependent on CMI. The lowest rate of Mantoux test reactivity was observed in gestation age group in whom 6 out of 8 i.e., 75% were Mantoux test negative. This can be explained by the fact that cell mediated immunity, chemotactic ability of polymorphs and complement system are not fully developed in the preterm infants.20 Sedaghatian et al.21 also found only 31% tuberculin conversion in preterm following BCG vaccination at birth. However, the number of Mantoux test converters in our study increased from 42.7% after 12(+1) of BCG vaccination to 66.4% after 6 months. The immunological system of newborn baby although adequate, is not fully mature and takes more time to respond to antigenic stimuli. Tuberculin reactivity and Leucocyte Migration Inhibition continue to increase from 3 months to 6 months. It is apparent that a major health intervention like BCG vaccination to control tuberculosis in the community will not pass the test of cost benefit relationship if only about two third show immunity against a disease. But the fact is that many studies speak otherwise. Several authors8,22-24 from India and abroad have reported that LMIT, which is more specific test for cell mediated immunity (CMI) against a specific antigen, has been found to be positive in Mantoux non-converters. This means that an absent skin test response does not necessarily mean absent CMI. In fact, Surekha et al.9 compared LMIT with the presence or absence of scar and found that out of 41 scar negative children, 88.2%, 94.7% and 80% of the children given BCG at 0 1 day, 2 30 days and days of life, had a positive LMI test. Leukocyte migration inhibition test is one of the in-vitro tests used to detect CMI.25 When BCG is given to a non-infected individual, naive T lymphocytes travel to T-cell areas of secondary lymphoid organs in search of antigen presented by dendritic cells. Once activated, they proliferate vigorously, generating effector cells that can migrate to B-cell areas or to inflamed tissues. A fraction of primed T lymphocytes persists as circulating memory cells that can confer protection.26 On in-vitro challenge to these T cells with PPD, the T cells secrete lymphokines which inhibit the migration of leukocytes and macrophages thus forming the basis of the LMIT. We observed an overall LMIT positivity of 84.1% in Mantoux test negative subjects after 12(+1) of BCG vaccination which increased to 92.6% after 6 months. We performed LMIT within 7 days of administration of the Mantoux test. There was a chance that infants immune responses might have been boosted by the inoculation of the PPD. This may constitute a limitation of the study. Nevertheless, 6 did not react to LMIT. They were Mantoux test negative and did not develop scar. Out of them 3 infants were SGA and 3 showed Abortive Reaction; a papule or pustule developed in them 6 to 8 after BCG vaccination, but disappeared at 12 and did not leave any scar at the vaccination site.4,5 IUGR is known to be associated with poor take-up of BCG.19 We chose LMIT to measure cell mediated immunity in Mantoux test negative individuals because it was available in the hospital, simple to perform and economic. Other tests such as T-cell interferon gamma (IFNγ) Release Assays (IGRAs) are now available for measuring cell mediated immunity following BCG vaccination.27,28 They measure IFNγ production by sensitized T-cells in response to stimulation by relatively specific M. tuberculosis antigens. IGRAs differ from each other mainly with respect to the technique of IFNγ detection (enzyme linked immunospot; ELISPOT vs. enzyme linked immunosorbent assay; ELISA) and have high specificity. Two randomized controlled trials were 693

5 conducted by Black et al.28 to study BCG-induced increase in IFNγ response to mycobacterial antigens and efficacy of BCG vaccination in Malawi and the UK. In these trials, 483 adolescents and young adults in Malawi and 180 adolescents in the UK were tested for interferon-gamma (IFNg) response to M. tuberculosis purified protein derivative (PPD) in a whole blood assay, and for delayed type hypersensitivity (DTH) skin test response to tuberculin PPD, before and 1 year after receiving BCG vaccination or placebo or no vaccine. The IFNg and DTH responses were strongly associated, except among vaccinees in Malawi. However, a major limitation of IFN-assays in comparison to Mantoux test and LMIT are their higher material costs and the need for laboratory infrastructure.29,30 We found that LMIT was positive in significantly large number of Mantoux test negative irrespective of their birth weight, gestation or presence of the scar suggesting that preterm (>31 ) and low birth weight ( 1,200 g) take the BCG vaccination as efficiently as term and appropriate birth weight neonates when vaccinated in the early neonatal period. We also found that local BCG reaction is a good indicator of immunity as LMIT was negative in scar failed infants and that as against 95.5% having local BCG reaction, 95.8% reacted to LMIT or Mantoux test. Our results also indicate that tuberculin conversion is not a reliable indicator to assess BCG vaccine take-up both 12 and 6 months after vaccination because in spite of negative Mantoux test and scar failure (in the presence of pre-scar stages), the vaccinated children may still be having an adequate BCG vaccine uptake and in turn possess an adequate cell mediated immunity against tuberculosis. Babies should be observed for local BCG reaction at 8. If local BCG reaction is present at 8, it is an indicator that cell mediated immunity has developed. Material and Methods Design. Prospective observational study. Subjects. After meeting inclusion criteria and obtaining parental informed consent, 143 consecutive delivered newborns of both sexes were selected from the NICU or Post-natal ward. The gestational age was calculated by Naegele s rule31 and/or Dubowitz criteria.32 Babies between 37 to 41 completed gestation were defined as term, whereas those of 36 gestation or less were classified as preterm. Newborns weighing <2,500 g were defined as low-birth-weight (LBW). Babies weighing between 10th to 90th centile for weight at birth for a given gestational age were classified as appropriate-for-gestational age (AGA) and those weighing less than 10th centile for a given gestational age at birth were classified as small-for-gestational age (SGA).33 Babies born to mothers suffering from tuberculosis or receiving anti-tubercular drugs, ecclampsia, chronic infection, severe anemia (Hb < 7 g%), HIV positive, Hepatitis B positive, having clinical evidence of intrauterine infection, congenital malformations, undergoing exchange transfusion, severe birth asphyxia (Apgar score <3 at 1 min) and on prolonged medications were excluded from the study. Informed parental consent was obtained in all 694 cases. The study was approved by the Ethical Committee of the institution. Methods. All the were given 0.1 ml of BCG vaccine (Danish 1331 strain, prepared at Guindy, Chennai, India) intradermally on the left arm just above the insertion of the deltoid muscle with a 26G needle within 7 days of birth with a tuberculin syringe to produce a wheal of 5 mm. The freeze dried vaccine was reconstituted with normal saline preserved at 4 to 8 C and the left over reconstituted vaccine was discarded after 3 hours. The first follow-up was done in the out patient department (OPD) at the age of 4 and subsequent follow-ups took place fortnightly to see the BCG reaction and detect any complications. At 12(+1) of age, Purified Protein Derivative (PPD) was given to all the. A dose of 1TU of PPD-RT 23 with Tween 80 (0.1 ml) was injected intradermally with a tuberculin syringe fitted with a 26 G needle on the upper one-third of the flexor aspect of the left forearm to produce a wheal of 5 mm. Both the BCG and PPD were administered by two staff nurses only, who had more than 5 years experience of the procedure, and were working in the Immunization Clinic. The test was read at 48 (±4) hours. The area of induration was measured at its largest transverse diameter by the ball-pen method.34 An induration of less than 5 mm was taken as a negative Mantoux test. Babies showing tuberculin conversion by an induration of 5 9 mm were considered as having good BCG uptake.35 All the Mantoux test negative were subjected to the leukocyte migration inhibition test (LMIT) within a week following Mantoux test. Mantoux test negative who showed a positive LMIT were regarded as having adequate BCG uptake. All the Mantoux test negative and LMIT negative were followed up and given another Mantoux test at 6 months. Those who showed a negative Mantoux test at 6 months and negative LMIT at 12 were subjected to another LMIT at 6 months. Babies who became Mantoux test positive or LMIT positive at 6 months were regarded as having adequate BCG uptake. The LMIT was carried out by the method described by Mustafa25 with some modifications. About 5 6 ml of blood was drawn in a heparinized syringe containing 250 units of heparin/ ml of blood under sterile conditions. The blood was then added to a siliconized round bottom tube containing 2 ml of 3% dextran. The tube was kept in a slanting position at 37 C for 45 minutes and then vertically for 15 minutes. The leukocyte rich plasma was taken out in a siliconized tube and centrifuged at 900 rpm for 5 minutes. The pellet was washed thrice with lactalbumin hydrolysate medium with HBSS (Hi-media) with 1 IU/ml of heparin and final cell suspension was filled in microlitre capillaries and plugged at one end with plasticine and centrifuged at 500 rpm for 5 minutes. The capillaries were cut at the cell fluid interphase and affixed with silicon wax in pyrogen free Perspex chamber plates. The chambers were immediately filled with lactalbumin hydrolysate medium enriched with 5% fetal calf serum. Few chambers were filled with enriched medium to which antigen (PPD, 5 TU/ml) was added. All chambers were closed with cover slips, ensuring that there were no air bubbles. The chamber plates Volume 5 Issue 10

6 thus prepared were incubated in humid atmosphere at 37 C for 20 hours and the area of migration in control as well as antigen chambers was recorded on a centimeter graph paper fixed on a screen with the aid of an overhead projector. Distance between the overhead projector and screen was kept constant. The image of the plate was focused on the graph paper and the area of migration was drawn and calculated by counting the number of squares in the marked area. While counting squares, those that were less than half were omitted, whereas those more than half were counted as one. The area of migration was then calculated as: Percentage migration inhibition (%MI) = [(Area of migration in antigen added chambers/area of migration in control chambers) x 100] % MI more than 20% was taken as significant. Statistics. Continuous variables with normal distribution were compared using t test, whereas continuous variables not normally distributed were analyzed using Mann Whitney U test. All proportionate data were analyzed with Pearson Chi-square test. Key messages. (1) Tuberculin conversion is not a reliable indicator to assess BCG vaccine take-up both 3 months and 6 months after vaccination. References Chadha VK, Jaganath PS, Kumar P. Tuberculin sensitivity among children vaccinated with BCG under universal immunization programme. Indian J Pediatr 2004; 71: Rodrigues LC, Diwan VK, Wheeler JG. Protective effect of BCG against tuberculous meningitis and miliary tuberculosis: a meta-analysis. Int J Epidemiol 1993; 22: Munoz FM, Starke JR. Tuberculosis (Mycobacterium tuberculosis). In: Berhrman RE, Kleigman RM, Jenson HB, editors. Nelson Textbook of Pediatrics. 17th edition. Philadelphia, Pennsylvania: Saunders 2004; Kaur S, Faridi MMA, Agarwal KN. BCG vaccination reaction in low birth weight infants. Indian J Med Res 2002; 116:64-9. Faridi MMA, Krishnamurthy S. Abortive reaction and time of scar formation after BCG vaccination. Vaccine 2008; 26: Das SK, Gautam KD, Mehrotra ML, Rajan RD, Sharma JP. Timing of BCG vaccination in infants. Indian J Tuberc 1980; 27:63-4. Aggarwal A, Dutta AK. Timing and dose of BCG vaccination in infants as assessed by Postvaccination tuberculin sensitivity. Indian Pediatr 1995; 32: Rajaje S, Narayanan PR. Immune response to BCG vaccination in children. J Trop Pediatr 1985; 31:1858. Surekha RH, Vijayalakshmi V, Kumar S, Lakshmi KA, Sumalatha G, Murthy K. CMI in children with scar failure following BCG vaccination. Indian Pediatr 1998; 35: Mehta NR, Mahajan BH, Phadia EG, Swadia PK. Size of local reaction after BCG vaccination: Its relation with conversion rate, size of allergy and its duration. Indian J Med Res 1964; 52:10. Ghosh S, Bhargava V, Bhargava SK, Batra S. Tuberculin conversion after BCG vaccination in newborn. Indian J Med Res 1971; 59: Thapar RK, Mehrotra ML, Rajan JD, Verma JK, Dayal RS, Parshad R. Mantoux conversion in infants after BCG vaccination in the neonatal period. Indian Pediatr 1971; 8: Munk ME, Emoto M. Functions of T-cellsubsets and cytokines in mycobacterial infections. Eur Respir J Suppl 1995; 20: (2) The evaluation and interpretation of BCG take-up on the basis of Mantoux test and LMI test should be done 6 months after BCG vaccination. (3) Preterm of >31 gestation and weighing more than 1,200 g take the BCG vaccination as efficiently as term and appropriate birth weight neonates when vaccinated in the early neonatal period. (4) Local BCG reaction is a reliable sign of ensuing immunity following vaccination. Acknowledgements M.M.A. Faridi conceptualized the study, supervised the work, critically reviewed the manuscript and shall act as guarantor of the paper. Sarvpreet Kaur collected the data, searched literature and prepared initial draft. Sriram Krishnamurthy reviewed the literature and drafted the manuscript. Pramod Kumari carried out LMIT. 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New Delhi, CBS publishers 1992; Sallusto F, Lenig D, Förster R, Lipp M, Lanzavecchia A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 1999; 401: Weir RE, Fine PE, Floyd S, Stenson S, Stanley C, Branson K, et al. Comparison of IFNgamma responses to mycobacterial antigens as markers of response to BCG vaccination. Tuberculosis (Edinb) 2008; 88: Black GF, Weir RE, Floyd S, Bliss L, WarndorffDK, Crampin AC, et al. BCG-induced increase in interferon-gamma response to mycobacterial antigens and efficacy of BCG vaccination in Malawi and the UK: two randomized controlled studies. Lancet 2002; 359: Pai M, Riley LW, Colford JM. Interferon-g assays in the immunodiagnosis of tuberculosis: a systematic review. Lancet Infect Dis 2004; 4: Pai M. Alternatives to the tuberculin skin test: interferon-gamma assays in the diagnosis of Mycobacterium tuberculosis infection. 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