Patent Application: US-90385504-A

Abstract:
a method and kit for the detection of an infectious disease , particularly tuberculosis , wherein lymphocytes of a subject are incubated with a disease - specific antigen , and the level of antibody production is measured , the production of antibodies above a baseline level being indicative of infection .

Description:
adult patients with suspected pulmonary tuberculosis from the national institute of diseases of the chest and hospital ( nidch ) in dhaka , bangladesh were prospectively studied . the diagnosis of tuberculosis was established by the clinical presentation , chest x - ray examination and sputum smear positivity . clinical evaluation included lung opacity , pyrexia , weight loss , high erythrocyte sedimentation rate ( esr ) and positive sputum smear . sputum was collected twice on consecutive days for mycobacterial culture from each patient after enrollment . diagnosis was further confirmed when sputum culture was found to be positive . all patients received standard therapy that included rifampicin , isoniazid , pyrazinamide and ethambutol . most of these patients had been ill for 3 - 5 months prior to inclusion in the study . tuberculin skin test was not performed in these patients since in bangladesh bcg is given to about 80 % of people , exposure to environmental mycobacteria is considered to be widespread and incidence of tuberculosis infection is high rendering the skin test less specific [ 5 , 34 , 35 ). the history and inspection for typical scar included detecting a scar resulting from previous vaccination with m . bovis bcg . although it is also possible that some patients may not develop a scar after vaccination and may be falsely grouped as non - vaccinated , most of these patients were however able to tell whether they were vaccinated or not with the exception of two who were unsure of their vaccination status and did not have the scars . they were grouped as non - vaccinated . patients attending the hospital with symptoms suggestive of tuberculosis with lung opacity , high esr but sputum smear negative and culture negative were enrolled as non - tuberculosis patients . healthy laboratory personnel ( with no known exposure to m . tuberculosis ) were also selected as healthy controls . tuberculin skin test was performed on these healthy controls only . blood was collected from each patient at enrollment with & lt ; 4 weeks of antimycobacterial drug treatment . in addition , blood was also collected from the non - tuberculosis patients and healthy subjects . the study was approved by the ethical review committee of the international centre for diarrhoeal disease research , bangladesh ( icddr , b ): international center for health and population research in dhaka . signed informed consent was obtained from each study subject according to the guidelines of the ethical review committee . sputum from patients were collected at the nidch and were cultured for m . tuberculosis at the icddr , b on lowenstein jensen medium using standard culture techniques . peripheral blood mononuclear cells ( pbmc ) were separated from blood upon ficoll - paque by differential centrifugation , and were suspended in 24 - well tissue culture plates ( costar , cambridge , mass .) in rpmi 1640 culture medium ( gibco brl , grand island , n . y .) containing 10 % fetal bovine serum ( gibco ), 2 mm l - glutamine and 1 % amphotericin b - penicillin - streptomycin - mix ( sigma chemicals co ., st ; louis , mo .). different dilutions of pbmc ( 1 × 10 6 , 2 × 10 6 , 5 × 10 6 and 1 × 10 7 cells / ml ) were incubated at 37 ° c . with 5 % co 2 . culture supernatants were collected at 24 , 48 , 72 and 96 hours post incubation . a cocktail of protease inhibitors ( 4 - aminoethyl benzenesulfonyl flouride , 0 . 2 μg / ml ; aprotinin , 1 μg / ml ; leupeptin 10 μm ; sodium azide 1 mg / ml in pbs ) were added to the supernatants and were stored at − 70 ° c . until used for the assay . antigens tested for the method were bcg ( freeze - dried , glutamate - bcg vaccine for intradermal use , lot # 1861 , japan bcg laboratories , japan ; no preservatives added , saline used as diluent ), and purified protein derivative ( ppd , sigma chemical co , st louis , mo .). antibody ( igg ) titers were measured in supernatants by the enzyme - linked immunosorbant assay ( elisa ). polystyrene microtitre plates ( nunc - maxisorp ) were first coated with bcg vaccine ( 1 μg / well ) or ppd ( 1 μg / well ) in carbonate buffer ( 0 . 1 m sodium bicarbonate and 5 mm magnesium chloride , ph - 9 . 8 ) and incubated overnight at 4 ° c . after washing , the plates were blocked with 10 % fbs in phosphate buffered saline ( pbs , ph 7 . 2 ) and incubated at 37 ° c . for 60 minutes . lymphocyte supernatants were thawed and brought to room temperature . following washing with pbs - tween , lymphocyte supernatants of appropriate dilutions ( diluted in 10 % fbs in pbs ) were added ( 100 μl / well ) and incubated for 2 hours at 37 ° c . plates were washed and rabbit anti human igg hrp conjugate ( 1 : 100 ) in pbs containing 10 % fbs was added and incubated for 2 hours at room temperature . after washing , freshly prepared substrate ( o - phenylenediamine ( opd ; 1 mg / ml in 0 . 1m sodium citrate ( ph - 4 . 5 ) buffer and h 2 o 2 ) was added and the plates were developed . the enzyme reaction was stopped with 1 . 0 m h 2 so 4 and optical density ( od ) was measured after 20 min at 492 nm . pooled sera from m . tuberculosis culture positive patients were used as positive control ( od & gt ; 1 . 0 ). antigen - specific responses were expressed as relative titers , which were defined as the optical density multiplied by the dilution factor of the specimen [ 36 ]. statistical analyses were carried out using the sigmastat software ( jandel scientific , san rafael , calif .). comparisons between the groups were made using the one way analysis of variance ( anova ) or anova on ranks as appropriate . p - values were considered significant when ≦ 0 . 05 . receiver - operator characteristic ( roc ) curves were constructed to describe the relation between the sensitivity and specificity at varying cutoff levels of bcg - or ppd - specific igg titers in lymphocyte secretions ( als ). forty - nine patients with suspected pulmonary tb were recruited from institute of diseases of the chest and hospital ( idch ). only those patients who had two consecutive - sputum specimens positive for acid - fast bacilli ( afb ) were included in the study . out of 49 patients with smear positive pulmonary tb , 45 patients were culture positive for m . tuberculosis ( 92 %) and 2 had contaminated culture and 2 were culture negative . all patients received the standard treatment and for therapy - resistant cases , the treatment was modified . median age of the patients was 30 years with a range of 18 to 57 years . thirty - six of forty - nine tb patients were males and thirteen were females . among them , thirty - five were bcg vaccinated ( having a bcg scar ). patients with non - tuberculosis illness ( n = 35 ) included patients with bronchiectasis ( n = 22 ), lung cancer ( n = 7 ), lung abscess ( n = 4 ) and aspergillosis ( n = 2 ). the diagnosis was confirmed by histology or cytology . thirty - five healthy individuals ( laboratory personnel ) were included in the study as healthy controls all of whom except one were bcg vaccinated . culture supernatants from different concentrations of cell suspensions and different incubation time points were used to determine antigen - specific igg titers . with higher concentration of pbmc , higher bcg - specific igg titers were obtained ( fig1 ). bcg - specific igg titers were significantly higher in supernatants of 2 , 5 and 10 million cells compared to that in 1 million cells ( p & lt ; 0 . 001 ). for 2 to 10 × 10 6 pbmc / ml , the supernatants need to be diluted 2 - 4 times . however with the cell concentration of 1 × 10 6 pbmc / ml , undiluted supernatants had to be used . since the pbmc counts are usually low in moderate to severely sick tb patients , we opted for one million cell / ml suspensions . a gradual increase in relative titers of bcg - specific antibody was found from 48 to 72 hours with a slight decline in the titers at 96 hours ( fig2 ). the titers at 24 h were low and only studied in a few subjects . the optimum time point was found to be 72 hours . pulmonary tb patients had significantly higher bcg - specific igg antibody titers than healthy subjects ( p & lt ; 0 . 001 ), and non - tb patients ( p & lt ; 0 . 001 ) at all time points ( fig3 a ). response to ppd ( fig3 b ) was similar to that seen with bcg - vaccine . there was no significant difference in the bcg - specific antibody titers between patients with ( 35 vaccinated ; geometric mean ( gm ) of relative titer - 0 . 67 ) or without bcg vaccination ( 14 non - vaccinated ; gm = 0 . 75 ) ( p = 0 . 5 ). roc curves were constructed from the als responses to bcg or ppd comparing tb patients with healthy controls . the selection of the best cutoff point value was based on the level at which the accuracy was maximum . the best cutoff point was found to be 0 . 42 with a sensitivity of 92 . 5 % and a specificity of 80 % for the bcg - als assay ( fig4 a ). for ppd - specific response , the best cut - off value was 0 . 32 with a sensitivity of 73 % and a specificity of 80 % for the als assay ( fig4 b ). the sensitivity and specificity for bcg - als assay were higher than those of ppd - specific als assay . the positive predictive value of the assay was 97 %. a rapid diagnostic assay that can detect patients with active tuberculosis is urgently needed to control and prevent the spread of pulmonary tuberculosis . we report a novel technique to rapidly identify such patients by culturing peripheral blood lymphocytes and detection of tuberculosis - specific antibodies in lymphocyte secretions . comparison between bacteriologically confirmed tb patients and non - tuberculosis patients ( having illness in which tb was part of the differential diagnosis ) or healthy controls showed a significant difference in the bcg antigen - specific - igg antibody responses in the secretions . the sensitivity and specificity of the test were about 93 % and 80 % respectively indicating that the combination of the als and elisa assays using bcg vaccine as an antigen would enable rapid detection of m tuberculosis infection ( within 4 - 5 days ) in patients with active tuberculosis . prior bcg vaccination did not hamper the test for identification of tb and could successfully differentiate between bcg - vaccinated and m . tuberculosis infected patients . the positive predictive value of the test was 97 %. detection of antigen specific antibody secreting cells ( asc ) have been used for monitoring therapeutic responses in tb patients [ 37 ]. we evaluated the als technique because we hypothesized that active tuberculosis would provide continuous antigen stimulation resulting in antibody producing cells in circulation . by contrast inactive tuberculosis might result in high antibody titers in serum but would be less likely to stimulate antibody producing cells in circulation . in addition , it is easier to perform als assay and the supernatant can also be stored for future use to detect antigen - specific antibodies , novel antigens , cytokines and other mediators . bcg - vaccine and ppd were chosen as antigens for the easy availability and assessment of a broad spectrum of tb - specific antibodies , since they cover a vast array of protein and lipid antigens . als response to both bcg and ppd were similar however , the sensitivity and specificity of the bcg - specific als response were higher . our ongoing follow - up study of family contacts indicates that increased als responses to bcg or ppd are associated with increased risk of developing active tb . various purified protein antigens have been tested for diagnostic applications ; for example , esat - 6 , which is a small molecular weight peptide expressed by m . tuberculosis , m . bovis and m . africanum and is absent from all strains of m . bovis and most of the environmental mycobacteria . recent studies have found esat - 6 to be a highly promising antigen for immunodiagnosis of active m . tuberculosis infection in nonendemic regions [ 38 - 41 ]. however , in regions endemic for tuberculosis such as the gambia , india and bangladesh , contacts of tb patients had significantly higher esat - 6 specific response than tb patients [ 29 , 42 ] thereby limiting the use of the method to nonendemic countries . there is a long term persistence of esat - 6 specific antibodies in patients in remission from pulmonary tb in endemic areas making it difficult to discriminate between latent tb or remission from tb [ 43 ]. in conclusion , the use of the als specimens with the standard elisa technique holds potential as a future tb - specific diagnostic test . with the extensive availability of elisa technology in developing country settings , this method should be applicable both in developing countries endemic for tb as well as industrialized countries for screening of suspected patients . since this method does not require specimen from the site of disease , it should also be useful in diagnosis of paucibacillary childhood tb . 1 . who report 1999 . global tuberculosis control : communicable diseases , who . geneva : world health organization , 1999 . 2 . director general ( dg ) health services : report on the national prevalence survey on tuberculosis in bangladesh , 1987 - 88 . dhaka : dhaka : ministry of health and family welfare . 1989 . 3 . kumaresan j a r m , murray c j l . tuberculosis . in : murray c l j l a , ed . global health statistics — global burden of disease and injury series . vol . 2 . boston , mass . : harvard university press , 1996 : 142 - 7 . 4 . global tuberculosis control : surveillance , planning , financing . in : organization wh , ed . who report 2002 : geneva , switzerland , 2002 . 5 . surveillance for multidrug resistant mycobacterium tuberculosis . 2001 - 2002 . icddr , b : health and science bulletin . vol . 1 , 2002 : 6 - 10 . 6 . ameglio f , giannarelli d , cordiali - fei p , et al . use of discriminant analysis to assess disease activity in pulmonary tuberculosis with a panel of specific and nonspecific serum markers . am j clin pathol 1994 ; 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