bacillus calmette guerin ( bcg ) vaccine , derived from multiple passages of wild - type mycobacterium bovis , has been used in humans since 1921 and is still the only vaccine available against tuberculosis ( 1 ) .
approximately , 100 million children routinely receive one or more bcg vaccines in most countries of the world every year and in iran yearly , 1.4 million neonates receive one dose at birth as per the expanded immunization program ( 2 ) . although bcg immunization is relatively safe , it may be associated with adverse events , such as self - limited injection site lesions , regional adenitis , and in rare cases , life threatening disseminated bcg infection ( 3 ) .
the incidence of disseminated bcg infection has previously been reported to be 0.19 - 1.56 cases per one million vaccinated infants and such disease has been mainly seen in severe immune deficiency , with high mortality rate ( 3 , 4 ) .
diagnosing disseminated bcg infection that affects mainly infants and young children and is characterized by fever , weight loss , lymphadenopathy , and hepatosplenomegaly requires a high index of physicians suspicion ( 5 ) .
a wide variety of differential diagnosis should be considered including other mycobacterial , some bacterial , viral , parasitic , and fungal infections as well as neoplastic and connective tissue diseases , and also metabolic or hematologic diseases with superimposed infections ( 3 , 5 ) . to confirm the diagnosis , differentiation of isolated mycobacterium bcg sub - strain from atypical mycobacteria and other members of mycobacterium tuberculosis complex ( mtc ) such as
m. tuberculosis , wild - type m. bovis , and m. africanum by various methods is mandatory ( 5 ) .
although isolation by culture and then identification are the optimal confirmatory steps in diagnosis , polymerase chain reaction ( pcr ) is directly applicable for smear - positive clinical specimens , indicating that it is a rapid and specific diagnostic test . a precise and rapid diagnosis of disseminated bcg infection is crucial in starting a prompt aggressive anti - mycobacterial treatment , which needs to be continued for a prolonged period of time ( 3 , 5 ) .
the present study aimed to report on the various manifestations , laboratory findings and outcomes of this rare life - threatening complication in a considerable number of infants and young children managed at a single center and to propose pcr as an efficient confirmatory diagnostic method applied directly on pathology specimens .
we retrospectively reviewed the medical files of all children hospitalized for disseminated bcg infection at nemazee teaching hospital , a referral hospital in southern iran , between 1990 and 2007 .
all of the 34 individuals included in this study were diagnosed and treated as disseminated bcg infection , by a pediatric infectious diseases specialist .
the presumptive diagnosis was based on ( a ) the presence of systemic syndromes compatible with disseminated mycobacterial disease , ( b ) a positive history of bcg vaccination , ( c ) a temporal relationship between bcg vaccination and disease , ( d ) the presence of disease in the region of vaccination , ( e ) histopathologic evidence of mycobacterial infection including the presence of acid fast bacilli and/or granuloma at two or more anatomic sites other than the site of vaccination , and ( f ) having inconclusive results for the work - ups of other diseases .
the pathologic reports of 59 samples were available in the patients medical files , including 24 bone marrow aspirations or biopsies , 14 liver biopsies , 13 distant lymph node aspirations or biopsies , three skin rash biopsies , two bone biopsies , two lung biopsies and one gastric washing .
the competency of the immune system was evaluated by different tests including nitroblue - tetrazolium test , t - cell and b - cell counts by flow cytometry ( facscalibur , bd . us ) , human immunodeficiency virus ( hiv ) test by enzyme - linked immunosorbent assay ( elisa ) and evaluation of serum immunoglobulins and ch50 .
twenty - eight samples from 21 patients , including nine smears from bone marrow specimens and 19 formalin - fixed and paraffin - embedded ( ffpe ) tissue specimens ( i.e. , six lymph node , nine liver , one subcutaneous nodule , one lung , one rib , and one smear of intra - abdominal abscess ) were available .
nucleic acid extraction was performed on one to five sections ( 5 m ) of these specimens .
( fermentas co. , lithuania ) lysis and phenol - chloroform ( sina gene co. , iran ) extraction , followed by ethanol ( merck co. , germany ) precipitation to isolate deoxyribonucleic acid ( dna ) from the smears and tissues . for differentiating m. bovis bcg from the other members of the m. tuberculosis complex , we performed an in - house multiplex pcr method to confirm the identity of the isolates , as described by magdalena et al .
the following primers were used : spacer region - specific primers for m. bovis bcg , spacer region 33 specific ( 5acaccgacatgacggcgg3 ) and spacer region 34 specific ( 5cgacggtgtgggcgagg3 ) ; the insertion sequence is6110 , mtc - specific primers , tb284 ( 5ggacaacgccgaattgcg3 ) , tb850 ( 5taggcgtcggtgacaaaggccac3 ) ; and mycobacterium genus - specific ( 65-kda antigen gene ) primers , tb11 ( 5accaacgatggtgtgtccat3 ) and tb12 ( 5cttgtcgaaccgcataccct3 ) .
the pcr conditions were 95c for three minutes ; 30 cycles at 95c for 20 seconds , 65c for 30 seconds , and 72c for 30 seconds ; and 72c for seven minutes .
the products were analyzed by electrophoresis on a 2% ( wt / vol ) agarose gel ( 8) .
negative controls contained the pcr mixture without a dna template and positive controls contained mycobacterial dna extracted from the bcg vaccine .
the patients were classified into two categories , definite and probable cases , according to the working definition of disseminated bcg disease described by talbot et al . in 1997 ( 5 ) .
the definite disseminated bcg infections met all three criteria , including : ( i ) identification of m. bovis bcg substrain by pcr , ( ii ) evidence of infection ( including a positive culture , pcr or histopathologic demonstration of acid - fast bacilli ) at two or more anatomic sites beyond the site of vaccination , and ( iii ) the presence of a systemic syndrome compatible with mycobacterial infection ( such as fever , weight loss , and anemia ) .
the probable case was defined based on all the criteria for a definite case except a positive pcr for m. bovis bcg ( 9 ) . in the present study , the pcr result for m. bovis bcg was either negative or not performed in probable cases .
the study design , conduct and assessments were all in accordance with ethical considerations , established and approved by the ethics committee of shiraz university of medical sciences .
data were analyzed using spss , version 11.5 ( spss , chicago , il ) .
there were 34 consecutive patients , 21 definite ( tables 1 ) and 13 probable ( table 2 ) cases , reported from 1990 to 2007 .
all patients were healthy at birth and vaccinated routinely with the pasteur 1173 m. bovis bcg substrain at the insertion of the deltoid .
the patients were one to 60 months old with a mean (  sd ) of 10.2 (  12.5 ) .
abbreviations : a indicates alive ; bm , bone marrow ; cgd , chronic granulomatous disease ; c , clarithromycin ; cmi , unidentified cell - mediated immune defect ; d , died ; dln , distant lymphadenopathy ; e , ethambutol ; f , fever ; gln , generalized lymphadenopathy ; hsm , hepatosplenomegaly ; i , isoniazid ; lad , lymphadenopathy ; mo , months , o , ofloxacin ; p , pneumonia ; r , rifampin ; scid , severe combined immunodeficiency ; s , streptomycin .
evidence of dissemination included the histopathologic demonstration of acid - fast bacilli and/or typical histopathologic changes with granulomatous inflammation and/or the identification of mycobacterium substrains , by standard polymerase chain reaction .
evidence of dissemination included the histopathologic demonstration of acid - fast bacilli and/or typical histopathologic changes with granulomatous inflammation and/or the identification of mycobacterium substrains , by standard polymerase chain reaction .
fever in 31 ( 91.2% ) , axillary lymphadenopathy in 26 ( 76.5% ) with fistulization in 50% , hepatosplenomegaly in 25 ( 73.5% ) , stunted growth in 21 ( 61.8% ) , distant lymphadenopathy in 16 ( 47.1% ) , cough ( longer than three weeks ) in 15 ( 44.1% ) , skin rash in 9 ( 26.5% ) and vomiting in 9 ( 26.5% ) were the most commonly reported signs and symptoms .
disseminated bcg infection was diagnosed in three of the cases after laparotomy for bowel obstruction ( cases 13 and 18 , table 1 ; case 5 , table 2 ) .
specific pathologic findings ( presence of granuloma and/or acid fast bacilli in tissue specimens ) were found in 12 of 13 ( 92.3% ) biopsies or aspirations of distant lymph nodes , 12 of 14 ( 85.7% ) liver biopsies and 15 of 24 ( 62.5% ) bone marrow aspirations and biopsies . in the two lung biopsies and one of the two bone biopsy specimens ,
one gastric washing , done for a patient with pneumonia , and two of three biopsies of skin rashes revealed acid fast bacilli ( tables 1 and 2 ) .
the most common abnormal findings in blood profiles were as follows : anemia in 30 ( 88.2% ) , leukopenia ( white blood counts less than 4000/l ) in 16 ( 47.1% ) , thrombocytopenia ( platelet counts less than 150000/l ) in 15 ( 44.1% ) , and leukocytosis ( leukocyte count that was two standard deviations above the mean count for a particular age ) in 15 ( 44.1% ) .
severe combined immunodeficiency ( scid ) was identified in 12 ( 35.3% ) , followed by unidentified cell mediated immune defect in 4 ( 11.8% ) and chronic granulomatous disease in 1 ( 2.9% ) .
disseminated bcg infection developed in two brothers ( case 8 , table 1 ; case 13 , table 2 ) and a brother and sister ( cases 7 and 19 , table 1 ) among the patients .
response to therapy was poor , with an overall mortality rate of 58.8% ( 20 of 34 ) .
of the 20 patients who died of disseminated bcg infection , 15 ( 75% ) had an immune defect . in contrast , of the fourteen patients who recovered , only two ( 14% ) had an identified immunodeficiency .
all 12 infants with severe combined immunodeficiency ( scid ) died despite the received treatment .
different treatment protocols were used in our patients , i.e. the patients were mostly treated with isoniazid and rifampin up to 2004 and afterwards by three or more anti - tuberculosis drugs ( tables 1 and 2 ) . disseminated bcg infection was confirmed in 21 patients by pcr , which was directly applied to the 28 available clinical specimens in pathology specimen bank and one intraabdominal abscess specimen ( tables 1 and 2 ) ( 10 ) .
pcr results were positive on 26 specimens for m. bovis bcg including 9 of 9 ( 100% ) bone marrow smears slides , 16 of 19 ( 84.2% ) of ffpe tissue specimens and one intra - abdominal abscess specimen .
as for 16 ffpe tissue specimens , pcr results on 3 specimens from skin rash , bone and lung were positive for m. bovis bcg ; and pcr positivity was 100% for distant lymph nodes ( 6 of 6 ) and 77.8% for liver ( 7 of 9 ) .
herein , we on report 34 patients with disseminated bcg infection , 21 definite and 13 probable cases and to the best of our knowledge , this is the largest study on children with definite disseminated bcg infection , reported from iran . disseminated bcg infection is a rare consequence of bcg vaccination , with a reported frequency of less than five per million vaccines and has mainly been seen in children with severe immune deficiencies ( 3 ) .
adverse reactions to bcg vaccine appear to be grossly underreported ( 11 ) . approximately , 1.4 million children receive bcg vaccines every year in iran and according to recent reports from iran , the number of disseminated bcg infections is estimated to be higher than the expected rate ( 12 - 19 ) .
the greater reactogenicity of pasteur 1173p2 strain vaccines , used in the national immunization program or higher prevalence of primary immunodeficiency may be possible explanations , which need further investigations ( 20 , 21 ) .
the majority of our patients ( 91.2% ) were infants , consistent with previous reports in which 76 -100% of the patients were under two years of age ( 5 , 13 , 17 - 19 , 22 ) .
the most clinical presentations in our series were fever found in 31 ( 91.2% ) , axillary s lymphadenopathy in 26 ( 76.5% ) with fistulization in 50% , hepatosplenomegaly in 25 ( 73.5% ) , and stunted growth in 21 ( 61.8% ) , in agreement with previous reports ( 5 , 13 , 17 - 19 , 22 ) . unusual presentation of disseminated bcg infection could be accompanied by gastrointestinal manifestations .
of the 34 patients , two cases presented bowel obstruction ( cases 17 and 26 , table 1 ) and one with intussusception ( case 5 , table 2 ) .
one patient with retroperitoneal abscess presented an abdominal mass , as reported previously ( case 11 , table 1 ) ( 10 ) .
the histopathologic evidence of mycobacterial infection including the presence of acid fast bacilli and/or granuloma in tissue specimens removed by the liver and distant lymph node biopsy had very high yield of diagnosis ( 85.7% and 92.3% , respectively ) , so in children with suspected disseminated bcg infection in the absence of distant lymphadenopathy , liver biopsy could be suggested for early diagnosis . besides
, biopsy or smear of other organs such as the skin nodules , bone and lungs may serve as diagnostic tools , as previously reported ( 5 , 23 , 24 ) .
the identification of m. bovis bcg substrain in tissue specimens of patients with suspected disseminated bcg infection is required for definite diagnosis . in this study , disseminated bcg infection was confirmed in 21 patients by pcr , which was directly applied to the 28 available clinical specimens with high rate of positivity ( 92.8% , 26 out of 28 ) ( tables 1 and 2 ) ( 10 ) .
in fact , the spread of bcg is a normal sequela of bcg vaccination , yet the identification and speciation of m. bovis bcg substrain by standard pcr in a patient with clinical findings consistent with disseminated bcg infection , could serve as a well - established criteria for the diagnosis of disseminated bcg infection ( 5 , 9 ) .
as for our patients , response to therapy was poor , with an overall mortality rate of 58.8% , which differ significantly in immunocompromised cases and patients without an identifiable immunodeficiency ( 75% vs. 14% , respectively ; p = 0.0003 ) . the overall mortality rate of 71% was reported previously , despite aggressive management , and it ranged between 0% in patients without an identifiable immunodeficiency to 83% in immunocompromised counterparts ( 5 ) .
chemotherapy seems to be complicated by the inherent resistance of all m. bovis strains to pyrazinamide , the inherent intermediate resistance of some bcg strains to isoniazid , and the emergence of additional resistance during inappropriate therapy ( 25 , 26 ) . in a review in 1993
, most m. bovis isolates from 73 patients were sensitive to isoniazid and rifampin , so during 1991 - 2004 , the majority of our patients were treated with a combination of isoniazid and rifampin .
concern for the selection of antibiotic - resistance , led to a change from the two - drug combined therapy to three or more drugs since about 2005 in our cases ( 27 ) .
it has been suggested that four or more anti - tuberculosis drugs should be used for longer than one year in children with disseminated bcg disease until full recovery .
the mortality in our patients who received different regimens of anti - tuberculosis was not significantly different ( tables 1 and 2 ) .
it seems that the patient s outcome is determined by their immunity state and/or progression of infectious process rather than anti - tuberculosis regimens .
several reports suggest that early bone marrow transplantation together with appropriate antimicrobials in immunocompromised patients is effective in restoring immunity and curing the infection ( 27 , 28 ) . in the present study ,
about half of the patients were with identified with immunodeficiency . despite a few cases of disseminated bcg infection reported in normal hosts , immunodeficiency has been detected in the majority of patients i.e. , 86% in the review of talbot et al .
although , in our study , the majority of immunologic defects predisposed patients to mycobacterial infections , in our study , the majority of immunologic deficiencies except the defects of inretleukin-12-interferon- ( il-12-inf- ) axis predisposed patients to mycobacterial infections were investigated .
the major defects of il 12- inf- axis leading to mendelian susceptibility to mycobacterial diseases ( msmd ) includes ifn  r1 , il-12r  1/il-12 p40 , and signal transducers and activator of transcription ( stat ) 1 deficiency ( 31 , 32 ) .
the major defects of the il-12-inf - g axis include ifn  r1 , il-12r  1/il-12 p40 , and signal transducers and activator of transcription ( stat ) 1 deficiency ( 31 , 32 ) .
four patients with sibling relationship in our series were immunodeficient and all except one died despite aggressive treatment ( cases no 7 and 8 , table 1 ; no 4 and 13 , table 2 ) . since most of the cases with disseminated bcg infection had immunodeficiency , mostly inherited as autosomal recessive , we suggest that all the siblings of cases with disseminated bcg infection should not be vaccinated at birth until complete evaluation and immunological work ups are performed . in conclusion
, infants and young children with prolonged fever , hepatosplenomegaly , lymphadenopathy and a history of bcg vaccination should be examined for disseminated bcg infection .
polymerase chain reaction specific for m. bovis bcg in tissue specimens has a high rate of positivity in such cases .
nevertheless , further studies and clinical trials are required to assess the most appropriate anti - tuberculosis regimen ( 33 ) .
 background : disseminated bacillus calmette guerin ( bcg ) infection is a rare but life threatening complication of bcg vaccination . 
 it has been mainly seen in severe immune deficiency . 
 a precise and rapid diagnosis is crucial for prompt initiation of an aggressive anti - mycobacterial treatment . 
 polymerase chain reaction ( pcr ) is directly applicable to smear - positive clinical specimens , proven to be a rapid and specific diagnostic test.objectives:the aim of this study was to investigate disseminated bcg infection among 34 children in southern iran , mainly confirmed by pcr.patients and methods : we included all the patients hospitalized with disseminated bcg infection at a referral teaching hospital in southern iran between years 1990 and 2007 . 
 the clinical and laboratory data including the immunological workups were obtained through a review of the medical files . 
 we recalled all pathology samples from pathology specimen banks and used an in - house pcr specific for mycobacterium 
 bovis bcg substrain to confirm the diagnosis.results:from the total of 34 children hospitalized with disseminated bcg infection , 21 were categorized as definite and 13 probable . 
 thirty - one patients ( 91% ) were under two years of age and 41% were male . 
 the most common clinical findings were fever in 31 ( 91.2% ) , axillary s lymphadenopathy in 26 ( 76.5% ) , hepatosplenomegaly in 25 ( 73.5% ) , stunted growth in 21 ( 61.8% ) , and distant lymphadenopathy in 16 ( 47.1% ) . 
 polymerase chain reaction positivity rate was 100% ( 9 of 9 ) in bone marrow smear slides and 84.2% ( 16 of 19 ) for formalin - fixed and paraffin - embedded tissue specimens . 
 immunodeficiency state was detected in 50% and the overall mortality rate was 58.8% ( 20 of 34).conclusions : disseminated bcg infection should be considered in the differential diagnosis of infants and young children with fever , hepatosplenomegaly , lymphadenopathy , and history of bcg vaccination . 
 the pcr method has a high positivity rate and can serve as a useful tool for the rapid and specific identification of m. bovis bcg substrain infection . 
1. Background
2. Objectives
3. Patients and Methods
4. Results
5. Discussion