Role of 30 kDa antigen of enteric bacterial pathogens as a possible arthritogenic factor in post-dysenteric reactive arthritis.

Background: Reactive arthritis (ReA)/Reiter’s syndrome (RS) may be caused as a sequel of infections caused by enteric bacterial pathogens, although the mechanisms through, which different pathogens cause similar disease are not clear. Aim: This study was done to look for the presence and role of any common bacterial antigen among the pathogens isolated from such patients. Materials and Methods: A total of 51 patients of ReA and 75 controls (three groups of 25 subjects each: Group 1: Patients who did not develop arthritic complications within 3 months after bacillary dysentery/diarrhea; Group 2: Patients with other arthritic diseases and Group 3: Normal healthy subjects) were included. The isolated enteric pathogens were tested to detect the immunodominant antigens. Results and Conclusions: A common 30 kDa antigen was found to be specifi cally present among seven arthritogenic enteric bacterial strains belonging to three genera, Salmonella, Shigella and Hafnia. Post-dysenteric ReA patients’ sera show higher levels of immunoglobulin G, immunoglobulin M and immunoglobulin A antibodies against this antigen as compared to the controls. Lymphocytes of ReA patients recognize this antigen, proliferate and produce interleukin-2 in response to this antigen more than the lymphocytes of controls. 30 kDa antigen may be a common arthritogenic factor associated with postdysenteric ReA/RS. The association of Hafnia alvei with post-dysenteric ReA is described for the fi rst time. Four cases of mycobacterial ReA had an association with this antigen, suggesting that the arthritogenic antigen of mycobacteria and enteric bacteria may be of a similar nature.

Mycoplasma genitalium: An emerging sexually transmitted pathogen.

Mycoplasma genitalium is a member of genital mycoplasmas, which is emerging as an important causative agent of sexually transmitted infections both in males and females. The advent of polymerase chain reaction and other molecular methods have made studies on M. genitalium more feasible, which is otherwise a difficult organism to isolate. Besides Chlamydia trachomatis, M. genitalium is now an important and established cause of non gonococcal urethritis (NGU) in men, more so in persistent and recurrent NGU. Multiple studies have also shown a positive association of M. genitalium with mucopurulent cervicitis and vaginal discharge in females as well. The evidences for M. genitalium pelvic inflammatory diseases and infertility are quite convincing and indicate that this organism has potential to cause ascending infection. Lack of clear association with M. genitalium has been reported for bacterial vaginosis and adverse pregnancy outcomes. Diagnosis of M. genitalium infections is performed exclusively using nucleic acid amplification tests (NAATs), owing to poor or slow growth of bacterium in culture. Although there are no guidelines available regarding treatment, macrolide group of antimicrobials appear to be more effective than tetracyclines. The present review provides an overview of the epidemiology, pathogenesis, clinical presentation and management of sexually transmitted infections due to M. genitalium.

Characterization of low molecular weight antimicrobial peptide from human female reproductive tract.

Background & objectives: The mechanisms that protect female upper genital tract from ascending infection by microbes present in vagina are only partially understood. It is expected that epithelial cells in mucosal surfaces and their secretions directly interfere with microbial colonization and invasion. This study was aimed to demonstrate the expression of 2 kDa antimicrobial peptide which was identified and purified from female genital tract tissues using chromatographic techniques. Methods: Low molecular weight proteins were isolated from human female reproductive tract tissues obtained from premenopausal women. Antimicrobial activity of these LMW proteins was assessed against different reproductive tract pathogens viz., Neisseria gonorrhoeae, Group B streptococcus, Gardnerella vaginalis, Escherechia coli and Candida albicans. The expression of these peptides were also documented in reproductive tract tissues with the help of hyperimmune sera raised against the rabbits. The purified peptide was characterized by N-terminal sequencing. Results: Immunohistochemical and immunofluorescence studies demonstrated that 2 kDa peptide was expressed in the stratified squamous epithelial cells of the ectocervix while it was absent in columnar epithelial cells of upper genital tract. Upregulation of the expression of this peptide was observed in patients of chronic non-specific cervicitis and acute on chronic cervicitis. This purified antimicrobial peptide also showed broad spectrum antimicrobial activity against different reproductive tract pathogens. Interpretation & conclusions: Considering the emerging bacterial resistance against conventional antibiotics, isolation and understanding of the expression of antimicrobial peptides from female reproductive tissue extracts may provide some leads towards the development of strategies for the treatment of reproductive tract infections.

Efficacy of an in-house polymerase chain reaction assay for rapid diagnosis of Mycobacterium tuberculosis in patients with tubercular lymphadenitis: Comparison with fine needle aspiration cytology and conventional techniques.

Introduction: Tubercular lymphadenitis (TB-L) is the most common manifestation of extrapulmonary tuberculosis. Excisional biopsy with histopathological examination, Ziehl-Neelsen staining (ZNS) and culture and fine needle aspiration (FNA) cytology, although useful in the diagnosis of TB-L, cannot diagnose a substantial proportion of cases. We investigated the role of an in-house polymerase chain reaction (PCR) assay targeting the IS6110 gene from the FNA material in the diagnosis of the disease. Materials and Methods: The clinical profile of 150 patients with lymphadenopathy was noted and the fine needle aspirate was collected. After cytological processing, ZNS and culture on Lowenstein-Jensen media, mycobacterial DNA was isolated from the residual aspirate material and IS6110 gene PCR was performed. Results of cytology, ZNS, culture and IS6110 gene PCR were compared. Results: There were 49 confirmed patients of TB-L based on laboratory parameters (either culture isolation of Mycobacterium tuberculosis or any two of cytology, ZNS, PCR positive) and clinical response to therapy. Sensitivity and specificity of FNA was 89.8% and 96%, of ZNS was 40.8% and 99%, of culture was 40.8% and 100% and of IS6110 gene PCR test was 100% and 92.1%. Conclusion: IS6110 PCR can be considered a valuable adjunct to cytology, ZNS and culture techniques in the diagnosis of TB-L.

Association of genital mycoplasmas including Mycoplasma genitalium in HIV infected men with nongonococcal urethritis attending STD & HIV clinics.

Background & objectives: Acute nongonococcal urethritis (NGU) is one of the commonest sexually transmitted infections affecting men. The role of genital mycoplasmas including Mycoplasma genitalium in HIV infected men with NGU is still not known. The aim of this study was to determine the isolation pattern/detection of genital mycoplasma including M. genitalium in HIV infected men with NGU and to compare it with non HIV infected individuals. Methods: One hundred male patients with NGU (70 HIV positive, 30 HIV negative) were included in the study. Urethral swabs and urine samples obtained from patients were subjected to semi-quantitative culture for Mycoplasma hominis and Ureaplasama urealyticum, whereas M. genitalium was detected by PCR from urine. The primers MgPa1 and MgPa3 were selected to identify 289 bp product specific for M. genitalium. Chalmydia trachomatis antigen detection was carried out by ELISA. Results: M. genitalium and M. hominis were detected/isolated in 6 per cent of the cases. M. genitalium was more common amongst HIV positive cases (7.1%) as compared to HIV negative cases (3.3%) but difference was not statistically significant. Co-infection of C. trachomatis and U. urealyticum was found in two HIV positive cases whereas, C. trachomatis and M. hominis were found to be coinfecting only one HIV positive individual. M. genitalium was found to be infecting the patients as the sole pathogen. Interpretation & conclusions: Patients with NGU had almost equal risk of being infected with M. genitalium, U. urealyticum or M. hominis irrespective of their HIV status. M.genitalium constitutes one of the important causes of NGU besides other genital mycoplasmas.

Seroprevalence of Chlamydia pneumoniae in asthmatic children from Northern India.

Serum from children with chronic persistent asthma was subjected to C. pneumoniae IgG antibody determination. C. pneumoniae IgG serology suggestive of persistent infection was significantly higher in chronic persistent asthma group than in the control group. Seropositivity was significantly more in moderate and severe persistent groups than in the control subjects. There was no evidence of acute C. pneumoniae infection (IgM serology done in duplicate) in acute exacerbations.

Emergence of ciprofloxacin resistant Neisseria gonorrhoeae in north India.

BACKGROUND AND OBJECTIVES: Though quinolones have been recommended as a single dose treatment for uncomplicated gonorrhoea, there have been reports of treatment failure with fluoroquinolones. In this study we determined the antimicrobial susceptibility levels of consecutive isolates of Neisseria gonorrhoeae to examine the emergence of ciprofloxacin resistance N. gonorrhoeae. METHODS: Minimum inhibitory concentration (MIC) of ciprofloxacin and other drugs (penicillin, tetracycline, ciprofloxacin and ceftriaxone) was determined by agar dilution method. MIC was interpreted according to the NCCLS guidelines. beta lactamase production was detected by iodometric method and chromogenic cephalosporin method using nitrocefin disc. RESULTS: A total of 45 consecutive isolates of N. gonorrhoeae were obtained from patients with suspected acute gonococcal uretheritis. Of the 45 isolates, 35 (77.7%) were resistant to ciprofloxacin, 16 (35.5%) showed MIC value greater than 8 microg/ml. All isolates were sensitive to ceftriaxone while 21 isolates (46.6%) were resistant to penicillin and 23 (51%) to tetracycline. Ten isolates (22%) were found to be beta-lactamase producers. INTERPRETATION AND CONCLUSION: Ciprofloxacin resistant N. gonorrhoeae is on the rise in and around Chandigarh (north India). Thus, periodic surveillance of susceptibility levels of N. gonorrhoeae is essential to prevent the dissemination of drug resistant strains in the community.

Neurosyphilis in a tertiary care hospital in north India.

BACKGROUND AND OBJECTIVES: The clinical diagnosis of neurosyphilis is very rarely encountered today in the developed world although syphilis remains a significant health problem in few areas of the industrialized countries and in most of the third world nations. This apparent decline may be due to increase in number of asymptomatic neurosyphilis and cases presenting as subtle, illdefined syndromes rather than classic presentation of tabes dorsalis and general paresis in the post penicillin era. This retrospective study was carried out to report the neurosyphilis cases diagnosed at a tertiary care hospital in North India, and to analyse the laboratory and clinical parameters of these cases. METHODS: Suspected cases of neurosyphilis presenting at Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh over a period of 13 yr (January 1990 to December 2002) were identified. Diagnosis of neurosyphilis was based on clinical presentation, prior history of syphilis, routine CSF biochemistry (protein and leukocytes) and serological evidence [serum and CSF venereal disease research laboratory (VDRL) and Treponema pallidum particle agglutination (TPPA) tests]. RESULTS: A total of 25 cases of neurosyphilis were identified, 18 (72%) with reactive CSF-VDRL, 22 (88%) with elevated CSF protein and 24 (96%) with CSF mononuclear leukocytosis. Serum VDRL was reactive in all 25 cases. Three patients were asymptomatic (2 primary syphilis; 1 early latent stage), 8 had secondary and 14 had tertiary syphilis. Two of the neurosyphilis cases were also seropositive for HIV. Radiology was abnormal in 7 (28%) patients. INTERPRETATION AND CONCLUSION: Neurosyphilis still remains a problem in a country like India and a high index of suspicion and clinical expertise are required for appropriate diagnosis and proper management especially in the era of AIDS pandemic.

Association of Leu125Val polymorphism of platelet endothelial cell adhesion molecule-1 (PECAM-1) gene & soluble level of PECAM-1 with coronary artery disease in Asian Indians.

BACKGROUND & OBJECTIVES: Platelet endothelial cell adhesion molecule-1 (PECAM-1) plays a key role in the transendothelial migration of circulating leukocytes (diapedesis) during vascular inflammation. We hypothesized that genetic variation and the level of soluble PECAM-1 could be associated with the development of atherosclerosis and conducted a study on gene polymorphisms of PECAM-1 and soluble PECAM-1 levels in Asian Indian patients with coronary artery disease (CAD) in Singapore. METHODS: Of the 137 angiographically confirmed patients (> or =70% stenosis) of CAD and 110 controls in Asian Indian population, two single nucleotide polymorphisms (SNPs) of PECAM-1 gene, C+373G (Leu125Val) at exon 3 and G+1688A (Ser563Asn) at exon 8 were analyzed by polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) strategy. In addition, plasma soluble PECAM-1, P-selection and lipid profile were measured. Chi- square test and student t test were adopted to compare categorical and continuous variables, respectively. RESULTS: A significant decrease in C allele frequency but increase in G allele frequency of the Leu125Val (C/G) polymorphism were observed in CAD patients as compared with controls (0.54/0.46 vs 0.663/0.337 respectively, P=0.008). Alteration in genotype distributions (CC, CG and GG) of the Leu125Val polymorphism between CAD patients and controls (P=0.009) was also significant. A similar trend was observed on the allele frequencies (G/A) and genotype distributions of Ser563Asn (G/A) polymorphism, though the difference did not reach significance. On the other hand, plasma level of soluble PECAM-1 (sPECAM-1) was markedly elevated in CAD patients (P=0.006), and associated with soluble P-selectin and lipid profiles. INTERPRETATION & CONCLUSION: Our study showed that Leu125Val polymorphism of PECAM-1 gene and elevated soluble PECAM-1 were related to severe coronary artery stenosis in CAD patients of Asian Indian origin in Singapore. Our data also suggest that PECAM-1 plays an important role in the development of atherosclerosis.

Drug susceptibility of Mycobacterium tuberculosis to primary antitubercular drugs by nitrate reductase assay.

BACKGROUND & OBJECTIVES: Rapid susceptibility testing of Mycobacterium tuberculosis strains is imperative for therapy selection but traditional drug susceptibility tests take weeks or are expensive. In this study we evaluated nitrate reductase assay which utilizes the detection of nitrate reduction as an indication of growth and therefore results can be obtained faster than by visual detection of colonies. METHODS: One hundred clinical isolates of M. tuberculosis were tested for four first line antitubercular drugs by nitrate reductase assay (NRA) and were compared with standard proportion method. The bacteria were inoculated on Lowenstein-Jensen (LJ) medium with primary antitubercular drugs and potassium nitrate was incorporated. After incubation for 7- 14 days, nitrate reduction indicating growth could be detected by colour change when reagents were added. RESULTS: Resistance of isolates as determined by both methods for isoniazid, rifampicin, streptomycin and ethambutol was 32, 35, 62 and 15 per cent respectively. Agreement between NRA and proportion method was 99 per cent for isoniazid and ethambutol. Complete agreement (100%) was found for rifampicin and streptomycin. Results were available in 7-14 days by NRA as compared to proportion method which takes 4-6 wk. INTERPRETATION & CONCLUSION: Nitrate reductase assay is a rapid and inexpensive method for susceptibility testing of M. tuberculosis for primary antitubercular drugs and could be an appropriate alternative to existing methods, particularly in resource-poor settings.

Leptospirosis in northern India: a clinical and serological study.

A total of 400 serum samples collected from patients, clinically suspected of leptospirosis, were evaluated for antibodies by LEPTO dipstick and microscopic agglutination test (MAT). Twenty of these patients (5%) had serological evidence of leptospirosis. Leptospira interrogans serovars Autumnalis and Icterohaemorrhagiae, Canicola and Javanica were serogroups recorded serologically. Fever and jaundice were the most common clinical presentations. Male preponderance was seen in the leptospirosis cases. Outdoor activities, agricultural activities, contact with animals were significantly associated with seropositivity for Leptospira. This study highlights that leptospirosis is a significant health problem in northern India, though grossly under reported due to the absence of routine laboratory diagnostic facilities for this disease.

Evidence of chlamydial infection in infertile women with fallopian tube obstruction.

The study was carried out to find the prevalence of Chlamydia trachomatis seropositivity among women with infertility of tubal origin. Forty women with tubal infertility (verified at hysterosalpingography and laproscopy), 20 women with infertility due to variety of other reasons and 20 healthy fertile women of reproductive age were enrolled in the study. It was found that the presence of Chlamydia specific IgG antibody was significantly higher (70%) in women with infertility of tubal origin as compared to 35% seropositivity in healthy fertile women and 55% seropositivity in infertile women with cause of infertility other than tubal factor. Seventy eight percent of women with frequency of coitus 3-4 times/week were seropositive as compared to 34.7% when frequency of coitus was 1-2 times/week. Study also showed the silent nature of this infection as history suggestive of past pelvic inflammatory disease (PID) was lacking in majority of the seropositive women (63.75%). In the study group, both the ends of the fallopian tubes (cornual and distal block) were involved with almost equal frequency. Eighty three percent of women with seropositivity had unilateral or bilateral hydrosalpinx and 75% of women had marked pelvic adhesions. These results support the fact that there is strong association between serum anti-Chlamydial antibodies and tubal factor as a cause of infertility in infertile women.

Enhanced detection of Mycobacteria stained with rhodamine auramine at 37 degrees C.

Sputum smear microscopy is the most efficient and rapid technique for detection of acid-fast bacilli (AFB). Fluorochrome method of staining is preferred for Mycobacteria in the overburdened laboratories as the fluorescing bacilli are more readily detected than the fuchsin stained bacilli in shorter period of time. A total of 300 sputum samples obtained from suspected cases of Tuberculosis were collected and were subjected to staining by rhodamine auramine at 37 degrees C and also at room temperature (conventional method). The smears were then blindly evaluated. Fifty-eight samples were positive by both methods and 5 were positive at 37 degrees C only. Staining at 37 degrees C increased the smear positivity by 8.6% over conventional staining at room temperature. No smears were positive only with staining at room temperature alone. Out of 58 smears positive by both methods, 25 had equal number of AFB in both smears, 22 had more AFB in smear stained at 37 degrees C and 11 had greater number of AFB in smears stained at room temperature. Our study, therefore, indicates that rhodamine auramine staining at 37 degrees C is superior to conventional auramine method at room temperature for detecting AFB in sputum smears.

Rapid detection of mutations in rpoB gene of rifampicin resistant Mycobacterium tuberculosis strains by line probe assay.

BACKGROUND & OBJECTIVES: Multidrug resistant (MDR) tuberculosis (TB) is a problem of increasing importance in the world due to limited treatment options. Resistance to rifampicin results from nucleotide changes in the gene encoding the beta subunit of the RNA polymerase (rpoB) of Mycobacterium tuberculosis. Rifampicin resistance is considered as a marker for MDR TB. The nature and frequency of mutations in the rpoB gene of rifampicin resistant clinical isolates vary considerably according to the geographical location and very little information is available on specific mutational patterns in India. This study was undertaken to detect and characterize the rpoB gene mutation associated with rifampicin resistance in M. tuberculosis by line probe assay. METHODS: A total of 36 strains of M. tuberculosis were analysed by INNO-LiPA Rif TB and compared with the results of conventional susceptibility testing method. After PCR amplification of the region of RNA polymerase involved in rifampicin resistance, the amplified product was hybridized with a set of 10 oligonucleotides immobilized onto a membrane strip. From the pattern obtained, the presence or absence of rifampicin resistance in the M. tuberculosis strains was assessed. RESULTS: It was found that the M. tuberculosis probe was 100 per cent specific; the most frequently observed mutation was His-526-Tyr in the rpoB gene; and correlation between the results of the LiPA and those obtained by the classical susceptibility testing was excellent (100%). INTERPRETATION & CONCLUSION: INNO LiPA was found to be a reliable, simple, rapid and informative tool for the early detection and characterization of rpoB mutation associated with rifampicin resistance in M. tuberculosis in the clinical laboratory setting and may constitute an important molecular method for the control of tuberculosis.