Biochanin A - A G6PD inhibitor: In silico and in vitro studies in non-small cell lung cancer cells (A549).

Secondary metabolites from medicinal plants have a well-established therapeutic potential, with many of these chemicals having specialized medical uses. Isoflavonoids, a type of secondary metabolite, have little cytotoxicity against healthy human cells, making them interesting candidates for cancer treatment. Extensive research has been conducted to investigate the chemo-preventive benefits of flavonoids in treating various cancers. Biochanin A (BA), an isoflavonoid abundant in plants such as red clover, soy, peanuts, and chickpeas, was the subject of our present study. This study aimed to determine how BA affected glucose-6-phosphate dehydrogenase (G6PD) in human lung cancer cells. The study provides meaningful insight and a significant impact of BA on the association between metastasis, inflammation, and G6PD inhibition in A549 cells. Comprehensive in vitro tests revealed that BA has anti-inflammatory effects. Molecular docking experiments shed light on BA's high binding affinity for the G6PD receptor. BA substantially decreased the expression of G6PD and other inflammatory and metastasis-related markers. In conclusion, our findings highlight the potential of BA as a therapeutic agent in cancer treatment, specifically by targeting G6PD and related pathways. BA's varied effects, which range from anti-inflammatory capabilities to metastasis reduction, make it an appealing option for future investigation in the development of new cancer therapeutics.

Comparative transcriptome analysis elucidates positive physiological effects of foliar application of pyraclostrobin on tomato (Solanum lycopersicum L.).

Strobilurins, including pyraclostrobin have frequently been reported showing positive physiological effects in various agricultural crops apart from fungicidal activity. Present study elucidates comparative transcriptome analysis of control and pyraclostrobin treated tomato leaf and identifies metabolic pathways and key genes responsible for positive effects of pyraclostrobin on tomato. Pair-end raw reads, generated by Illumina Hi-seq platform were pre-processed and good quality reads were mapped onto tomato reference genome using HISAT2 alignment programme. Transcript assembly and quantification were performed using StringTie assembler. Differential Gene Expression analysis by DESeq2 identified 1,952 upregulated genes including genes encoding pathogenesis related proteins and 835 downregulated genes. RT-PCR study showed increase in expression of RBCs (2.5-fold), GA20o (3-fold), and NR (1.4-fold) genes, which are the key genes of photosynthesis, gibberellic acid synthesis, and nitrogen assimilation pathways respectively identified in KEGG pathway analysis. Pyraclostrobin treated plants showed 1.6-folds increase in plant height, 3.3-folds increase in number of leaves, and 2.8-folds increase in number of flowers. Total protein content increased 1.7, 1.4, 1.2, 1.2, and 1.4 folds at 1 day after application (DAA), 4DAA, 7DAA, 10DAA, and 13DAA respectively in treated plants. Moreover, content of phenol also increased 1.14, 1.5, 2.4, and 1.5 folds in 4DAA, 7DAA, 10DAA, and 13DAA respectively. Nitrate reductase activity increased 2-fold, 1.8-fold, 1.5-fold and 1.15-fold in 1DAA, 7DAA, 10DAA and 13DAA respectively. Carbohydrate decreased in treated plants up to 7DAA. The present study is the first report of transcriptome analysis elucidating positive physiological effects of strobilurin on plant. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01191-7.

Oral Toxicity Study for Salmonella Killing Lytic Bacteriophage NINP13076 in BALB/c Mice and Its Effect on Probiotic Microbiota.

Viruses that infect bacteria are emerging as attractive biocontrol agents and biopreservatives for foods. Since these bacteriophages kill the target pathogens by lysis and are also consumed along with food, it is essential to evaluate their collateral toxicity on the probiotic gut microbiota. In this study, we examined the acute oral toxicity of a Salmonella phage isolated from sewage in mice. Acute oral administration of the Salmonella phage for five consecutive days did not show any significant pathological changes in the vital organs like lung, kidneys, heart, liver, and intestine. In addition, growth of typical probiotic microbiota remained unaffected even after incubation up to 24 h with the Salmonella phage. The results of this study clearly showed that oral administration of the lytic Salmonella phage did not have any significant adverse effects on the animals, may not harm the probiotic gut microbiota, and are likely to be safe for use in food preservation.

Molecular cloning, heterologous expression, and functional characterization of a cellulolytic enzyme (Cel PRII) from buffalo rumen metagenome.

A cellulase encoding gene, Cel PRII, was identified from Mehsani buffalo rumen metagenome, and cloned and expressed in Escherichia coli BL21(DE3)pLysS. The 1170 bp full length gene encodes a 389 residue polypeptide (Cel PRII) containing a catalytic domain belonging to glycosyl hydrolase (GH) 5 family. The fusion protein consisting of the Cel PRII, thioredoxin tag and 6x Histidine tag with predicted molecular weight of 63 kDa when recovered from inclusion bodies under denaturing conditions, exhibited cellulolytic activity against carboxymethyl cellulose (CMC). Recombinant Cel PRII was stable in the pH range 4.0-10.0 with pH optima 6.0. The optimal reaction temperature of Cel PRII was 30 °C with more than 50% of its activity retained at the temperatures ranging from 0 to 50 °C. Cel PRII exhibited enhanced enzymatic activity in the presence of Mn2+ ions and was inhibited in the presence of chelating agent EDTA. The K m and V max values for CMC were found to be 166 mg/mL and 1292 IU/mg, respectively. Cel PRII identified in the present study may act as an excellent candidate for industrial applications, and may aid in lignocellulosic biomass conversion because of its potential cellulolytic activity, thermostability, and excellent pH stability.

Phytol induces ROS mediated apoptosis by induction of caspase 9 and 3 through activation of TRAIL, FAS and TNF receptors and inhibits tumor progression factor Glucose 6 phosphate dehydrogenase in lung carcinoma cell line (A549).

A number of drugs as well as lead molecules are isolated from natural sources. Phytol is one of such lead molecule belongs to terpenes group distributed widely in medicinal plants. In the present work, we investigated the cytotoxic behavior of phytol on human lung carcinoma cells (A549). Phytol was found to cause characteristic apoptotic morphological changes and generation of ROS in A549 cells. The mechanism of phytol involved the activation of TRAIL, FAS and TNF-α receptors along with caspase 9 and 3. In silico molecular docking studies revealed that phytol has a good binding affinity with glucose-6-phosphate dehydrogenase (G6PD), which is known to promote tumor proliferation. The ability of phytol to become potential drug candidate has been revealed from the pharmacokinetic study performed in the present study.

Maslinic Acid Inhibits Proliferation of Renal Cell Carcinoma Cell Lines and Suppresses Angiogenesis of Endothelial Cells.

Despite the introduction of many novel therapeutics in clinical practice, metastatic renal cell carcinoma (RCC) remains a treatment-resistant cancer. As red and processed meat are considered risk factors for RCC, and a vegetable-rich diet is thought to reduce this risk, research into plant-based therapeutics may provide valuable complementary or alternative therapeutics for the management of RCC. Herein, we present the antiproliferative and antiangiogenic effects of maslinic acid, which occurs naturally in edible plants, particularly in olive fruits, and also in a variety of medicinal plants. Human RCC cell lines (ACHN, Caki-1, and SN12K1), endothelial cells (human umbilical vein endothelial cell line [HUVEC]), and primary cultures of kidney proximal tubular epithelial cells (PTEC) were treated with maslinic acid. Maslinic acid was relatively less toxic to PTEC when compared with RCC under similar experimental conditions. In RCC cell lines, maslinic acid induced a significant reduction in proliferation, proliferating cell nuclear antigen, and colony formation. In HUVEC, maslinic acid induced a significant reduction in capillary tube formation in vitro and vascular endothelial growth factor. This study provides a rationale for incorporating a maslinic acid-rich diet either to reduce the risk of developing kidney cancer or as an adjunct to existing antiangiogenic therapy to improve efficacy.

Mutant prevention concentration, pharmacokinetic-pharmacodynamic integration, and modeling of enrofloxacin data established in diseased buffalo calves.

The pharmacokinetic-pharmacodynamic (PK/PD) modeling of enrofloxacin data using mutant prevention concentration (MPC) of enrofloxacin was conducted in febrile buffalo calves to optimize dosage regimen and to prevent the emergence of antimicrobial resistance. The serum peak concentration (Cmax ), terminal half-life (t1/2 K10) , apparent volume of distribution (Vd(area) /F), and mean residence time (MRT) of enrofloxacin were 1.40 ± 0.27 µg/mL, 7.96 ± 0.86 h, 7.74 ± 1.26 L/kg, and 11.57 ± 1.01 h, respectively, following drug administration at dosage 12 mg/kg by intramuscular route. The minimum inhibitory concentration (MIC), minimum bactericidal concentration, and MPC of enrofloxacin against Pasteurella multocida were 0.055, 0.060, and 1.45 µg/mL, respectively. Modeling of ex vivo growth inhibition data to the sigmoid Emax equation provided AUC24 h /MIC values to produce effects of bacteriostatic (33 h), bactericidal (39 h), and bacterial eradication (41 h). The estimated daily dosage of enrofloxacin in febrile buffalo calves was 3.5 and 8.4 mg/kg against P. multocida/pathogens having MIC90 ≤0.125 and 0.30 µg/mL, respectively, based on the determined AUC24 h /MIC values by modeling PK/PD data. The lipopolysaccharide-induced fever had no direct effect on the antibacterial activity of the enrofloxacin and alterations in PK of the drug, and its metabolite will be beneficial for its use to treat infectious diseases caused by sensitive pathogens in buffalo species. In addition, in vitro MPC data in conjunction with in vivo PK data indicated that clinically it would be easier to eradicate less susceptible strains of P. multocida in diseased calves.

Antimicrobial behavior of biosynthesized silica-silver nanocomposite for water disinfection: a mechanistic perspective.

The biosynthesis of nano-silica silver nanocomposite (NSAgNC) and it is as antibacterial effect on gram-negative bacteria viz.Escherichia coli and Pseudomonas aeruginosa has been investigated for disinfection of water. The as-synthesized NSAgNC exhibited antibacterial activity in a dose dependent manner and ∼ 99.9% of E. coli and P. aeruginosa were killed at a concentration of 1.5 mg/mL of NSAgNC (5.1 wt% Ag) within 5h. The NSAgNC showed similar antibacterial activities both in oxic and anoxic conditions. The results further demonstrated that NSAgNC exhibited reactive oxygen species (ROS) independent "particle specific" antibacterial activity through multiple steps in absence of leached out Ag(+) ions. The initial binding of NSAgNC on the cell wall caused loss of cell membrane integrity and leakage of cytoplasmic materials. Inhibition of respiratory chain dehydrogenase by NSAgNC caused metabolic inactivation of the cells and affecting the cell viability. Genomic and proteomic studies further demonstrated the fragmentations of both plasmid and genomic DNA and down regulation of protein expression in NSAgNC treated cells, which leading to the cell death. Thus the biosynthesized NSAgNC has great potential as disinfectant for water purification while minimizing the toxic effects.

Crystal structure of 2-(2,4-di-chloro-phen-yl)-4-hydroxy-9-phenyl-sulfonyl-9H-carbazole-3-carbaldehyde.

In the title compound, C25H15Cl2NO4S, the di-chloro-phenyl ring is twisted by 68.69 (11)° from the mean plane of the carbazole ring system [r.m.s. deviation = 0.084 (2)°]. The hy-droxy group is involved in an intra-molecular O-H⋯O hydrogen bond, which generates an S(6) graph-set motif. In the crystal, pairs of C-H⋯Cl hydrogen bonds link mol-ecules into inversion dimers with an R (2) 2(26) motif. Weak C-H⋯O inter-actions further link these dimers into ribbons propagating in [100].

Panax ginseng has anti-infective activity against opportunistic pathogen Pseudomonas aeruginosa by inhibiting quorum sensing, a bacterial communication process critical for establishing infection.

Virulent factors produced by pathogens play an important role in the infectious process, which is regulated by a cell-to-cell communication mechanism called quorum sensing (QS). Pseudomonas aeruginosa is an important opportunistic human pathogen, which causes infections in patients with compromised immune systems and cystic fibrosis. The QS systems of P. aeruginosa use N-acylated homoserine lactone (AHL) as signal molecules. Previously we have demonstrated that Panax ginseng treatment allowed the animals with P. aeruginosa pneumonia to effectively clear the bacterial infection. We postulated that the ability to impact the outcome of infections is partly due to ginseng having direct effect on the production of P. aeruginosa virulence factors. The study explores the effect of ginseng on alginate, protease and AHL production. The effect of ginseng extracts on growth and expression of QS-controlled virulence factors on the prototypic P. aeruginosa PAO1 and its isogenic mucoid variant (PAOmucA22) was determined. Ginseng did not inhibit the growth of the bacteria, enhanced the extracellular protein production and stimulated the production of alginate. However, ginseng suppressed the production of LasA and LasB and down-regulated the synthesis of the AHL molecules. Ginseng has a negative effect on the QS system of P. aeruginosa, may explain the ginseng-dependent bacterial clearance from the animal lungs in vivo in our previous animal study. It is possible that enhancing and repressing activities of ginseng are mutually exclusive as it is a complex mixture, as shown with the HPLC analysis of the hot water extract. Though ginseng is a promising natural synergetic remedy, it is important to isolate and evaluate the ginseng compounds associated with the anti-QS activity.

Macrophage-specific Mycobacterium tuberculosis genes: identification by green fluorescent protein and kanamycin resistance selection.

Mycobacterium tuberculosis survives and multiplies inside macrophages of its host by modulating the expression of several genes essential for in vivo survival. An in vivo expression system has been developed, based on green fluorescent protein and kanamycin resistance, to identify M. tuberculosis genes which appear to be up-regulated in infected macrophages. A promoter-trap shuttle vector, pLL192, was constructed, containing a streptomycin resistance gene as selection marker and an artificial bicistronic operon composed of the promoterless green fluorescent protein (gfp) gene, followed by the kanamycin resistance gene. A unique BamHI site upstream of the gfp gene allowed for insertion of promoter libraries. The vector was validated by the use of known regulated or constitutive M. tuberculosis promoters. In addition, an M. tuberculosis genomic DNA library was inserted into pLL192 and then introduced into Mycobacterium bovis BCG. The recombinant BCG cells were then used to infect the J774A.1 murine macrophage-like cell line in the presence of kanamycin. Several recombinant BCG cells were thereby selected that were resistant to kanamycin within infected macrophages, but were sensitive to kanamycin when grown in vitro. The kanamycin resistance phenotype was paralleled by the fluorescence phenotype. After nucleotide sequencing, the corresponding genes were identified as mce1A, PE_PGRS63(RV3097c), Rv2232, Rv1026, Rv1635c, viuB, Rv2231(cobC) and Rv0997. Real-time PCR analysis using RNA isolated at various time points from M. tuberculosis and M. bovis BCG grown in vitro and within macrophages, confirmed the up-regulation of these genes. The level of up-regulation varied from 2- to 40-fold in macrophages compared to growth in vitro.

Improved diagnosis of pulmonary tuberculosis by detection of free and immune complex-bound anti-30 kDa antibodies.

The 30 kDa secreted antigen of Mycobacterium tuberculosis was purified to homogeneity by serial chromatography, and enzyme linked immunosorbent assay (ELISA) was used to evaluate its diagnostic value in patients with pulmonary tuberculosis. The immunoglobulin (Ig) antibodies G, A, and M were estimated in the two groups: patients who were smear- and culture-positive (S+C+) for pulmonary tuberculosis and normal healthy subjects (NHS). Sensitivity of 67.4%, 14.8%, and 14.3%, with the specificity of 99%, 96.7%, and 92% were obtained for the 3 isotypes respectively. Combination of the results of IgG and IgA increased the sensitivity to 71%, with 97% specificity. Polyethylene glycol precipitation of the circulating immune complexes (CIC) in sera was carried out. The CIC bound antibodies offered a sensitivity of 92.5%, 85.4%, and 68.7%, respectively for the S+C+, S-C+, and S-C- patients, while the specificity was 96.6%. Thus CIC-bound antibodies promise to be a better diagnostic tool in the detection of tuberculosis.

Cloning, expression, and purification of the 27 kDa (MPT51, Rv3803c) protein of Mycobacterium tuberculosis.

A limited number of proteins of Mycobacterium tuberculosis have been characterized so far for their use as potential candidates for diagnosis and vaccine studies. This study was aimed at cloning, expression, and purification of a 27 kDa protein (otherwise known as the MPT51 or Rv3803c protein) of M. tuberculosis. The Rv3803c gene was PCR amplified using primers that contain specific restriction sites. The amplified product was inserted initially into pTOPO and then sub-cloned into pET15b and pET24d vectors, such that the recombinant protein is predicted to contain an N-terminal or a C-terminal histidine tag, respectively. The recombinant plasmids were introduced into Escherichia coli BL21 (DE3) and the recombinant proteins were purified from the cytosolic fractions of the E. coli sonicates by nickel-NTA chromatography. The purity, molecular mass, and the conformation of the proteins were determined by high performance liquid chromatography (HPLC), matrix assisted laser desorption-ionization-time-of-flight (MALDI-TOF), and circular dichroism (CD) studies, respectively. The purified proteins were found to be immunogenic and useful for immunodiagnostic studies of tuberculosis by enzyme linked immunosorbent assay (ELISA), with a sensitivity of 71% and specificity of 95%.

Isotype-specific anti-38 and 27 kDa (mpt 51) response in pulmonary tuberculosis with human immunodeficiency virus coinfection.

There is a need for rapid diagnostic methods to identify tuberculosis among human immunodeficiency virus-positive cases (HIV-TB). This study evaluated the serodiagnostic potential of the native 38 kDa and recombinant 27 kDa (mpt 51) antigens of Mycobacterium tuberculosis purified in the laboratory, when applied to HIV-TB patients. The antibody response was studied using enzyme-linked immunosorbent assays (ELISA). In the HIV-TB group, anti-38 kDa antibody of the immunoglobulin G (IgG), IgA and IgM isotypes was found in 38%, 43% and 7%, of patients, respectively. Antibodies to the 27 kDa antigen occurred in 50%, 31%, and 1%, for IgG, IgA and IgM, respectively. The sensitivity increased upon combination of the results of IgG and IgA isotypes for each of the antigens, without compromising specificity. When the results were analysed based on the smear positivity, 71-78% and 54-69% were positive among smear-positive and smear-negative HIV-TB cases, respectively. A higher sensitivity (71% and 69%) was obtained using the 27 kDa antigen. The use of both antigens offered a sensitivity of 82% in smear-positive and 69% in smear-negative cases. There was no difference in antibody response among the HIV-TB cases, related to CD4 counts. Thus, the combination of the 38 and 27 kDa (mpt 51) antigens proved to be of diagnostic utility in HIV-TB, irrespective of the severity of immunosuppression, in smear-positive and smear-negative TB.

Antibody response to Mycobacterium tuberculosis 30 and 16kDa antigens in pulmonary tuberculosis with human immunodeficiency virus coinfection.

There is urgent need for rapid diagnostic methods to identify tuberculosis among the HIV positive cases, since the mortality is high. We have isolated and evaluated the serodiagnostic potential of the 30kDa secreted antigen and 16kDa cytosolic antigen of M. tuberculosis, among the HIV-TB patients. Antibody response was studied using Enzyme linked immunosorbent assay. In the HIV-TB group, antibody was found to be 65%, 69% and 6% positive for IgG, A and M isotypes, respectively, against 30kDa. The sensitivity increased to 84%, upon combination of the results of 3 isotypes. Anti-16kDa was detected in 15% (G), 50% (A) and 3% (M) of cases. Combination of results improved the positivity to 57%. There was no difference in antibody response among the HIV-TB cases, related to CD4 counts. Thus the 30kDa antigen proved to be of diagnostic utility in HIV-TB.

Immunoglobulin G, A, and M responses in serum and circulating immune complexes elicited by the 16-kilodalton antigen of Mycobacterium tuberculosis.

The 16-kDa cytosolic antigen of M. tuberculosis was purified to homogeneity by molecular sieving chromatography, and the diagnostic potential of the antigen was evaluated in various categories of patients by enzyme-linked immunosorbent assay (ELISA). The immunoglobulin G (IgG), IgA, and IgM antibody levels to 16-kDa antigen were estimated in the two polar groups, namely, smear- and culture-positive pulmonary tuberculosis (S(+)C(+)) patients and healthy subjects (HS). Sensitivities of 62, 52 and 11% with specificities of 100, 97, and 95% were obtained for the three isotypes, respectively. The total number of positives by a combination of the three isotypes was analyzed in the polar groups, and the sensitivity improved to 83% with a specificity of 93%. Even when a combination of IgG and IgA alone was considered, the sensitivity was 82% with a specificity of 97%. Polyethylene glycol precipitation of the circulating immune complex (CIC) in sera was carried out. The CIC-bound antibodies to 16-kDa antigen were assessed by ELISA in the S(+)C(+), S(-)C(+), and S(-)C(-) categories of patients. Measuring the IgG-IgA-IgM combination positivities of the CIC-bound antibodies gave sensitivities of 97.5, 100, and 45.3%, respectively. The specificity of the assay with these combinations was maintained at 95.4%.

Purification and characterization of three immunodominant proteins (38, 30, and 16 kDa) of Mycobacterium tuberculosis.

Specific mycobacterial antigens are an important prerequisite in the serodiagnosis of tuberculosis. Many studies have reported the use of both native and recombinant proteins. Even though recombinant proteins can form standardized reagents with unlimited supply, their diagnostic test characteristics were not satisfactory in some cases. In this study we have purified the 38-, 30- (antigen 85B), and 16-kDa native antigens of Mycobacterium tuberculosis by procedures with limited number of steps. Starting with the secreted antigens of M. tuberculosis H37Rv, the 38-kDa form was purified by preparative isoelectric focusing, followed by preparative electrophoresis. Separation of antigen 85 components was achieved by anion-exchange chromatography, followed by hydrophobic interaction chromatography. Gel-permeation chromatography was employed for the isolation of the 16-kDa form, from the cytosol fraction of M. tuberculosis H37Rv. By using a minimal number of steps, considerable yields of these proteins were obtained without loss of immunological activity. The native proteins purified were characterized by analytical two-dimensional electrophoresis, HPLC, and circular dichroism studies. Conformation of the native 38-kDa form purified in our laboratory was different from that of the recombinant 38-kDa form from the WHO Bank. The identities of these native antigens were established by immunoblotting with known monoclonal antibodies from the WHO Bank.

Qualitative and quantitative analysis of antibody response in childhood tuberculosis against antigens of Mycobacterium tuberculosis.

PURPOSE: Serodiagnosis of tuberculosis in children, using available crude antigens, has been difficult. The tests lack sufficient sensitivity and/or specificity. In this study, western blot analysis of M. tuberculosis H37Rv culture filtrate antigen (CFA) was carried out, to identify diagnostically useful antigens. In addition, the CFA was also used in enzyme linked immunosorbent assay (ELISA), to measure antibodies of multiple isotypes. METHODS: Specific IgG, IgA and IgM antibodies were estimated in the sera from 26 clinically/bacteriologically diagnosed cases of childhood tuberculosis (CTB) and 61 normal children (CNHS), using culture filtrate antigen. Western blot analysis with culture filtrate antigen was carried out to qualitatively compare the antibody profile among the CTB, with childhood normal controls and adult TB. RESULTS: IgG positivity was only 7.6% with culture filtrate antigen in the CTB group, while 3.2% among the controls were also positive. However, the results of IgA and IgM isotypes were better. By combination of all the three isotypes an increased sensitivity of 57.7% with a specificity of 93.5%, was obtained. Immunoblot analysis revealed marked difference among antibodies in the region of 16, 19, 38 and 45 kDa between CTB and CNHS. CONCLUSIONS: Our findings point to a limited sensitivity of 57.7% in ELISA with culture filtrate antigen. However, antibodies around 16, 19, 38 and 45 kDa region may be useful in differentiating the CTB patients from CNHS by immunoblot assay.

Specific and early detection of IgG, IgA and IgM antibodies to Mycobacterium tuberculosis 38kDa antigen in pulmonary tuberculosis.

The objective was to apply the purified 38kDa protein antigen of Mycobacterium tuberculosis in ELISA to estimate the IgG, IgA and IgM antibody levels in sera and circulating immune complexes of tuberculosis patients. Sera from smear and culture positive tuberculosis patients were positive for anti 38kDa IgG, IgA and IgM antibodies, with a sensitivity of 61%, 30% and 10%, respectively, and with a specificity of 100% for IgG. The sensitivity of the test improved to a level of 68% for IgG+IgA and of 71.4% for IgG+IgA+IgM without significantly compromising the specificity (IgG of 100%, IgG+IgA of 96%, IgG+IgA+IgM of 90%). Among the smear, culture-negative but X-ray-positive cases, 60% were serum positive for IgG antibody, while in smear-negative but culture-positive cases, 54% were positive for IgG antibody. Measurement of 38kDa antibodies showed a greater than 95% sensitivity in smear and culture-positive, and smear-negative and culture-positive patients, through a combination of assays for serum IgG and circulating immune complex antibodies, while the specificity was 100%.