Clinical and Immune Impact of Mycobacterium bovis BCG Vaccination Scarring

Study population.During three periods in 1997 and 1998, we enrolled all 497 febrile (oral temperature ≥ 38°C) persons ≥13 years old and all 245 acutely ill persons <13 years old admitted to the Lilongwe Central Hospital, a regional public hospital in Malawi into a study of bloodstream infections (16). All hospitalized children were included because infected children often do not present with fever. For each patient at admission, blood samples, epidemiologic data, and a medical history were obtained and a physical examination was performed by one of the investigators. A random subset of participants had immune studies done; this subset was comparable to the general study population (16). Currently, in Malawi, BCG vaccination with 0.05 ml of Pasteur Mérieux Connaught BCG vaccine, derived from strain 1077 and containing between 0.4 × 10⁶ and 1.6 × 10⁶ culturable particles per newborn dose, is offered within the first 3 days of life. If not given then, it is again offered at each subsequent routine health care visit.

The study protocol was approved by the institutional review boards of the Centers for Disease Control and Prevention and the Malawian Health Sciences Research Committee; informed consent was obtained from all participants and/or their guardians.

Clinical definitions.An acute pulmonary history was positive if the patient or caretaker reported either cough or shortness of breath. Respiratory findings were considered positive when any of the following were present on physical examination: a respiratory rate ≥20 (adults) or ≥60 breaths/min (children), lung crackles, or auscultatory findings suggesting pulmonary consolidation. Diarrhea was defined as any quantity or frequency of watery stool. Sepsis was proven by a positive blood culture or suspected on the basis of leukocytosis, abnormal temperature, and organomegally. Medical history included the chronic (>1 month) presence or absence of fever or cough.

Laboratory procedures.Blood cultures were performed as previously described (1); thick and thin malaria smears were done at admission and were considered positive if any Plasmodium falciparum asexual parasites were seen. HIV antibody testing was done at study enrollment by using enzyme-linked immunosorbent assay test kits (Murex Diagnostics Inc., Norcross, Ga.). HIV type 2 (HIV-2) has not been reported in Malawi. Plasma HIV-1 RNA levels for 81 participants were assessed (Monitor, version 1.5; Roche Diagnostics, Indianapolis, Ind.). Those with viral studies done tended to be younger than other participants but did not differ from other participants in regard to BCG scarring, infections, or mortality. Heparinized whole blood was either stimulated for 5 h at 37°C with phorbol 12-myristate 13-acetate (PMA; 200 ng/ml; Sigma Chemical Co., St. Louis, Mo.) and ionomycin (4 μg/ml) (Sigma) in the presence of brefeldin A (40 μg/ml) (Sigma) and RPMI 1640 with l-glutamine (induced or stimulated cytokine expression) or was retained in identical media without PMA and ionomycin but with brefeldin A (spontaneous or unstimulated cytokine expression) (16). No serum was added to the cultures. Cells were permeabilized and fixed with Permeafix (Ortho Diagnostic Systems, Inc., Raritan, N.J.) and then shipped at 4 to 8°C to the Centers for Disease Control and Prevention, stained with conjugated monoclonal antibodies, and processed for four-color flow-cytometric assessment by using a FACSort or FACSCalibur flow cytometer and CellQuest software (Becton Dickinson Immunochemistry Systems [BD], San Jose, Calif.). Isotype controls were obtained from BD. All antibodies used were pretested for stability to the permeabilization/fixation protocol. Cell identification and gating were done as previously described (16). Between 50,000 and 80,000 ungated events were collected from each tube in the panel.

Analytic techniques.For each participant, analyses were done for all lymphocytes, CD3⁺ (T) lymphocytes, CD3⁺ CD8⁺ lymphocytes, CD3⁺ CD8⁻ lymphocytes, CD3⁺ CD16/56⁺ lymphocytes (NT cells), CD3⁻ CD16/56⁺ lymphocytes (NK cells), CD19⁺ (B) lymphocytes, and monocytes, depending on the tube configuration. Cytokine profiles were represented by the ratios of the percentages of CD3⁺ lymphocytes producing interleukin-10 (IL-10) or IL-4 to the percentages producing each of the other cytokines. IL-10 and IL-4 are both anti-inflammatory, regulatory, and type 2 (humoral) cytokines; they induce antibody maturation and production. IL-4 also induces the production of other type 2 cytokines (e.g., IL-5 and IL-13) and thus might also be considered “pro-type 2.” IL-10 inhibits the production of type 1 cytokines (e.g., gamma interferon [IFN-γ] and IL-12) and thus is “anti-type 1.” Tumor necrosis factor alpha (TNF-α), IL-6, and IL-8 are proinflammatory cytokines; IL-2, IFN-γ, and, possibly, TNF-α are type 1 cytokines, inducing and involved in cellular immunity, which is essential for immunity to intracellular organisms. We calculated similar ratios for monocytes in regard to IL-10 and IL-6, IL-8, and TNF-α.

Memory and activation markers.CD45RO (a memory cell marker), CD45RA (a “naive” cell marker), HLA-DR (major histocompatibility complex antigen receptor that is induced with activation), CD38 (also induced with activation), and the percentages of cells expressing CD4 were assessed by using unstimulated cells. CD62L, often used to differentiate memory and naive cells, is not stable to our permeabilization/fixation protocol and therefore was not assessed.

Statistical techniques.Analyses were done with statistical software from SAS Institute, Inc. (Cary, N.C.). Comparisons of continuous data between dichotomized categories were made by using Wilcoxon rank sum and, in some instances, also logistic regression analyses; trichotomous data were analyzed by using Kruskal-Wallis tests. For HIV⁺ persons with undetectable plasma HIV RNA levels, the analysis values were set at 200 copies/ml, half the lower detection limit of the assay. Proportions were compared by using Fisher's exact tests or chi-square tests. P values less than 0.05 were considered significant.

Clinical and demographic characteristics of all participants.Of the patients enrolled, 474 adults (95% of adult participants) and 226 children (92%) had data concerning the presence or absence of BCG scarring recorded. Of those with scarring and clinical data recorded, 32 adults had M. tuberculosis isolated from their blood and 10 of these had immune studies done at admission; no child had mycobacteremia. No patient had active measles infection. Of the children <6 months old, 48 had BCG site scarring/inflammation status and clinical data recorded; 19 of these had immune studies done.

The proportion of patients with BCG scarring increased over the first 6 months of life; the proportion was stable for those ≥6 months through 50 years of age and was lower in the few participants 51 to 61 years of age (n = 13 with clinical data and n = 5 with immune data also) (Fig. 1). The trend seen in Fig. 1 is consistent with persons in Malawi being vaccinated in infancy and some persons in the oldest age stratum having been missed by the vaccination campaigns in previous decades. None of those in the two extreme-age strata (<6 months and 51 to 61 years of age) had mycobacteria isolated from their blood.

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FIG. 1.

Median percentages of participants with BCG scars by age group. Numbers of participants in the different subgroups are as follows: <0.5 years old, 48; 0.5 to <1 year old, 42; 1 to <5 years old, 96; 5 to <13 years old, 40; 13 to <21 years old, 64; 21 to <31 years old, 181; 31 to <41 years old, 150; 41 to 51 years old, 53; and 51 to 61 years old, 13.

For those ≥6 months old, gender and age were not associated with BCG scarring (Table 1). Those with and without BCG scarring had similar rates of mycobacteremia, blood culture positivity, malaria smear positivity, HIV seropositivity, and mortality (Table 1). For HIV-seropositive persons, HIV-1 plasma viral titers did not vary by BCG scarring status and the percentages of lymphocytes expressing CD4 were low irrespective of scarring status. The distributions of bloodstream pathogens for both groups were similar and included M. tuberculosis (present in 23 of 479 participants with scarring and 9 of 173 without scarring), other Mycobacteria spp. (n = 3, all in persons with vaccine scarring), fungi or candida (present in 1.8% of those with scarring and 1.7% of those without scarring), gram-positive organisms (5.0 and 5.2% of those with and without scarring, respectively), and gram-negative organisms (11.2 and 11.6% of those with and without scarring, respectively). The proportions of participants with or without scarring who had positive acute pulmonary histories, pulmonary findings, or diarrhea were similar, as were the proportions who had chronic fever or cough.

Participants with M. tuberculosis bloodstream infections.Of these 32 patients, those with or without BCG scarring did not differ in demographic characteristics, vital signs, pulmonary symptoms or histories, malaria smear positivity, or mortality rates (data not shown). No mycobacteremic person with BCG scarring reported acute diarrhea, compared to three of nine without scarring (P = 0.004). Of the 10 patients with immune studies done, those with BCG scarring had higher proportions of memory T cells (CD45RO⁺), activated CD8⁺ T cells (CD38⁺ and/or HLA-DR⁺), T and B cells making the pro-type 2 cytokine IL-4, and NT cells (a small, immunoregulatory cell population) making both TNF-α and IFN-γ (Table 2). There was very little overlap in the distributions of these parameters for the two scarring groups, and the distributions for those with scarring were extremely skewed toward higher levels (Table 2). Scarring was not associated with differences in the proportions of non-NT cells making type 1 (IL-2 and IFN-γ) or proinflammatory cytokines (TNF-α and IL-8) (data not shown). Those with scarring had T-cell cytokine production skewed toward an anti-inflammatory profile (ratios of the percentages producing IL-4 or IL-10 to the percentages producing IL-8) (Table 2). None of these scar-related immune differences were found in those without M. tuberculosis bloodstream infections or, specifically, in the HIV⁺ participants without M. tuberculosis bloodstream infections (data not shown).

Participants <6 months old.The age-related trend in Fig. 1 is consistent with children in Malawi being vaccinated in infancy. Therefore, we analyzed this age group separately to assess the possible effects of recent vaccination and also compared those with scarred lesions to those with inflamed BCG lesions, i.e., suppuration at the site of the inoculation occurring within the first few weeks postvaccination (Tables 3 and 4). Ten of 48 children <6 months old had a BCG vaccine scar, five had inflammation at the vaccine site, and 33 had no sign of vaccination (Table 3). Only one other person had inflammation at a BCG vaccination site: a 2.3-year-old HIV⁺ female with pneumonia and a negative blood culture and malaria smear who survived. In this age group, it is likely that the vast majority of those without any sign of scarring or inflammation had not been vaccinated, since full vaccination healing takes weeks. Supporting this probability, ages were lowest in those without scarring, followed by those with inflammation at the vaccination site, followed by those with scarring. Therefore, we examined three groups of infants: those with scarring postvaccination; those with inflammation at the site of vaccination, presumably representing recent or poorly healing vaccination; and those without evidence of vaccination (Table 3).

None of these 48 infants had mycobacteremia. Two-thirds of all these infants were male; fewer males than females had BCG scarring (P = 0.022). Males tended to be younger than females, but the difference was not significant (0.1 versus 0.3 years). In a logistic model excluding those with inflammation, the interaction between age and gender made it impossible to determine which of these two variables, if either, had a predominant relationship with BCG scarring (only the interaction variable was significant). Sex was not related to HIV seropositivity rates (males, 22%; females, 25%), nor did males and females differ in admitting diagnoses, symptoms, blood culture positivity rates, or mortality rates.

The proportion of infants with inflammation at the vaccine site that were HIV seropositive was higher than the proportion of those without a lesion or with scarring (Table 3). Three of 5 infants with inflammation at the vaccine site were HIV seropositive (versus 8 of 40 with scarring or no visible BCG lesion); the HIV status of 3 infants was unknown. When age, gender, and the interaction between age and gender were taken into account, the relationship between HIV serostatus and inflammation was of borderline significance (P = 0.050), suggesting that those with HIV infection might have had difficulty with localized healing at the vaccination site. Consistent with this, those with BCG site inflammation had the highest rate of localized infections, followed by those with BCG scarring (Table 3). These localized infections included otitis media with suspected meningitis, submandibular cellulitis, bronchiolitis with possible pneumonia, and HIV-associated thrush. Of the 19 infants <6 month old with immune testing done, the percentages of lymphocytes expressing CD4 for those with scarring, with inflammation, and without scarring were not different (Table 4), nor were the percentages expressing any memory or activation markers (data not shown).

Relationships with either sign of vaccination (scarring or inflammation).None of those with signs of vaccination (i.e., BCG scarring or inflammation) had documented sepsis or symptoms suggesting sepsis (Table 3). Eight infants without BCG scarring or inflammation had positive blood cultures (all Salmonella spp.), but of these, six were ≤1 month old. One 3-month-old male without a scar had a 4+ positive malaria smear (on a scale of 1+ to 4+). Those with either type of BCG lesion, scarring or inflammation, had lower median percentages of lymphocytes spontaneously making IL-4 or TNF-α and a lower ratio of the percentage of T cells spontaneously making IL-4 to the percentage of those spontaneously making IL-6. In logistic regression analyses with age as an additional independent variable, the percentages of lymphocytes spontaneously making IL-4 and the percentages spontaneously making TNF-α remained significantly related to vaccination status (for the former, beta coefficient [β] = −1.6, conditional odds ratio [OR] = 0.19, 95% confidence interval [CI] = 0.03 to 0.64, and P = 0.016; for the latter, β = −2.8, OR = 0.06, CI = 0.00 to 0.50, and P = 0.033).

Relationships with BCG vaccination site scarring.Scarring per se, compared to inflammation at the vaccine site or absence of a BCG-associated lesion, appeared to be associated with lower median proportions of lymphocytes making induced IL-4, a heightened median ratio of T cells making induced IL-10 to those making induced IL-4 (2.5 versus 0.6; P = 0.007), and a heightened median ratio of monocytes making induced IL-10 to those making IL-6 (Fig. 2). In logistic regression analyses with age as an additional independent variable, the T-cell and monocyte ratios remained significantly related to BCG scarring (for the former, β = +2.4, OR = 10.7, CI = 2 to 89, and P = 0.002; for the latter, β = +5.9, OR = 353, CI = 2 to 999, and P = 0.007).

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FIG. 2.

Peripheral blood immune findings significantly associated with the presence or absence of a BCG vaccine scar in participants <6 months old. (A) Percentages of lymphocytes making IL-4 with stimulation; (B) ratios of the percentages of CD3⁺ cells making IL-10 to the percentages of those making IL-4 with stimulation; (C) ratios of the percentages of monocytes making IL-10 to the percentages of those making IL-6 with stimulation. Note that y axis scales differ among the panels. One outlier is excluded from the graph: for one child without a BCG scar, 23% of lymphocytes made IL-4 with stimulation. When this value is excluded from statistical calculations, results remain significant. Solid circles, individuals in the BCG scar-negative group who had inflammation at the BCG vaccination site but no scarring.