Diagnostic role of stool culture & toxin detection in antibiotic associated diarrhoea due to Clostridium difficile in children.

BACKGROUND & OBJECTIVE: Mere diagnosis of Clostridium difficile by culture does not help in the diagnosis of antibiotic associated diarrhoeae (AAD) due to C. difficile. Detection of toxins A and B form the mainstay in the diagnosis of AAD due to C. difficile. This study was undertaken to find out the role of stool culture and toxin detection in the diagnosis of AAD due to C. difficile. As there are very few documented reports from India about AAD due to C. difficile in children in the age group of 5-12 yr, this age group was selected. METHODS: Faecal samples were collected from 250 hospitalized children in the age group of 5-12 yr who developed diarrhoea on receiving antibiotics for different medical problems for more than five days duration. Also faecal samples of 250 age and sex matched controls were collected. Culture for C. difficile was done on cycloserine cefoxitin fructose egg yolk agar (CCFA) and colonies were identified by standard laboratory techniques. ELISA for toxins A and B detection and tissue culture on HeLa cells for toxin B detection were also done. RESULTS: Overall positivity was 18 per cent in this study group compared to the controls (P<0.001). Maximum positive cases were in 5-8 yr age group (84.4%). Severe diarrhoea, liquid stool with mucus and blood, faecal leucocytes >5/high power field, altered flora and presence of Gram-positive bacilli with oval subterminal spores on Gram stain were sensitive predictors for diagnosis of AAD due to C. difficile. Amongst positive cases, 68.9 per cent responded to discontinuation of antibiotics and 31.1 per cent to metronidazole therapy. INTERPRETATION & CONCLUSION: C. difficile was an important pathogen responsible for antibiotic associated diarrhoea (AAD) in children of 5-12 yr age group. Conservative use of antibiotics would be beneficial to decrease the incidence of AAD.

Phaeohyphomycotic ulcer caused by Phialophora verrucosa: successful treatment with itraconazole.

We report the first well documented case of subcutaneous phaeohyphomycotic infection caused by Phialophora verrucosa in India. Examination of the biopsied tissue from an ulcerating lesion on the shin of the left leg of a 45-year-old woman from Bombay, India, showed numerous dematiaceous, septate, branching hyphal elements and thick-walled cells characteristic of phaeohyphomycosis. Cultures of the scrapings from the lesion and of the biopsied tissue yielded a pigmented fungus that was identified as P. verrucosa. Initial treatment with fluconazole followed by oral itraconazole for 30 days and local application of a copper sulphate solution resulted in complete resolution of the lesion. Treatment with itraconazole was continued for an additional 3 months after complete healing. No new lesions developed and the patient did not show jaundice, hepatosplenomegaly or any other signs of toxicity.

Assessment of enzyme linked immunosorbent assay based diagnostic kits (Toxokit-G and Toxokit-M) for the detection of IgG and IgM antibodies to Toxoplasma gondii in human serum.

An enzyme linked immunosorbent assay (ELISA) using penicillinase was developed in the form of diagnostic kits (Toxokit-G and Toxokit-M) for the detection of IgG and IgM antibodies to Toxoplasma gondii. The performance of both the kits was compared with commercially available diagnostic kits, i.e. Enzygnost-Toxoplasmosis/IgG (Behring Co., Germany), TOXOTEK-G (Flow Lab., U.K.) and Toxoplasma IgM Microassay (Diamedix Corp., U.S.A.) by testing toxoplasma-suspected human serum samples. The results indicate a good reliability between these diagnostic kits. Toxokit-G has 86.66 and 96.05% sensitivity and specificity respectively. The main advantage of Toxokit-G is that the end result can be assessed visually without using sophisticated instruments. Toxokit-M has 100% sensitivity and specificity and test results were not affected by the presence of antitoxoplasma IgG antibodies, rheumatoid factor or antinuclear antibodies.

Disease prevalence in women attending the STD clinic in Mumbai (formerly Bombay), India.

Our objectives were to determine the prevalence of Neisseria gonorrhoeae and its association with other STD causing organisms. Three hundred and thirty-six consecutive women (female sex workers (FSWs) and married contacts), attending a sexually transmitted disease (STD) clinic in Mumbai, were screened for N. gonorrhoeae, Chlamydia trachomatis and Trichomonas vaginalis. Per speculum examination was performed and clinical signs were recorded. Symptoms perceived by the women were also recorded. The mean age for married contacts, FSWs and gonorrhoea-positive women was 27.9, 29.7 and 27.5, respectively. 9.7% of the women were positive for N. gonorrhoeae, 23.2% were chlamydia-positive and 5.9% had trichomoniasis. N. gonorrhoeae was isolated more frequently from FSWs as compared to the married contacts. The prevalence of HIV was significantly higher among women with multiple sex partners (FSWs) (P<0.001). Gonococcal infection is significantly associated with the presence of HIV. A significant association between sexual habits and prevalence of gonorrhoea, trichomoniasis and HIV was observed. The prevalence of gonorrhoea over 1988 to 1996 remained approximately the same.

PIP: This clinical trial was conducted to determine the prevalence of Neisseria gonorrhea and its correlation with other sexually transmitted disease (STD) causing organisms. A total of 336 consecutive women (female sex workers (FSWs) and married contacts) attending an STD clinic in Mumbai, India, were screened for N. gonorrhea, Chlamydia trachomatis, and Trichomonas vaginalis. Speculum examinations were performed, the vulva and external genitalia were assessed for ulcers and warts, and clinical signs were recorded. The symptoms reported by the women were also documented. The mean age of married contacts was 27.9 years, while that of FSWs was 29.7 years. The mean age of gonorrhea-, chlamydia-, and trichomonas-positive women was 27.3, 28.0, and 28.8 years, respectively. This study showed that 9.7% of the women were gonorrhea-positive, 23.3% were chlamydia-positive, and 5.9% were trichomonas-positive. N. gonorrhea was more prevalent among FSWs than married contacts. Moreover, HIV was more prevalent among FSWs (P 0.001). Gonococcal infection is significantly associated with HIV. An important correlation between sex behavior and the prevalence of gonorrhea, trichomoniasis, and HIV was found. Between 1988 and 1996 the prevalence of gonorrhea remained approximately the same.

Microbiological study of urethritis in men attending a STD clinic.

A microbiological study of 275 male patients suffering from urethritis and 100 healthy male controls showed that Neisseria gonorrhoeae (130), Ureaplasma urealyticum (81), Staphylococcus aureus (38), and alpha and beta streptococci (34) were the common isolates. Specificity and sensitivity of the direct fluorescent antibody technique in the detection of N. gonorrhoeae in 130 urethral samples, were found to be 100 per cent. Penicillin (10 units/disc) resistance was found in 36.93 per cent of N. gonorrhoeae. Minimum inhibitory concentration of penicillin for 75 isolates of N. gonorrhoeae (including 5 beta lactamase producers) varied from 0.01-5 micrograms/ml with a 95 per cent confidence limit range of 0.26-0.61 microgram/ml. Most of the N. gonorrhoeae isolates tested were sensitive to norfloxacin and spectinomycin. Inclusion bodies of Chlamydia trachomatis were observed in 25 patients.

Enzyme linked immunosorbent assay for immunoglobulins G to Mycoplasma hominis.

Sera (187) from women patients attending the STD, Obstetrics and Gynaecology, and Family Planning Clinics were screened for the presence of the IgG class of immunoglobulins against M. hominis, using the ELISA technique. Sonicates of locally isolated M. hominis serotype (CS1) and standard PG 21 strain were used as antigens. The test was standardized using penicillinase as an enzyme. The ELISA showed 90.48 per cent sensitivity and 84.8 per cent specificity, and was also rapid (as compared to culture) and reproducible.

Murine pathogenicity of Trichomonas vaginalis & its susceptibility to metronidazole.

Eight isolates of T. vaginalis obtained from clinical specimens were tested for their pathogenicity in the murine model using the subcutaneous route of inoculation. In general, all isolates produced localised purulent abscesses in the mice. On comparison with a reference strain, all but two isolates were found to be significantly more pathogenic for mice than the reference strain, while two were comparable in their pathogenicity. All eight isolates were sensitive in vivo to metronidazole. In six of the eight isolates, metronidazole (30 mg/kg body wt) administered for four days to subcutaneously infected mice effected a 100 per cent cure. The ED50 values ranged from 3.4 to 23.94 mg/kg body weight. None of the isolates could be termed as resistant.

Mycoplasma hominis infections in female genital tract & use of immunofluorescence for antibody detection.

A total of 325 women with genital tract infection and 108 healthy controls were screened for the presence of mycoplasmas. Of these, 325 patients, mycoplasmas were recovered in 186. Thirty five isolates were M. hominis and 151 isolates of Ureaplasma urealyticum. The fluorescent-antibody technique (FAT) has been employed for the rapid identification of mycoplasma colonies growing on agar plates. The method was found to be rapid for detection of M. hominis growing on primary isolation plates. The antibody titre of patient's serum was also detected by FAT. Patients' sera were diluted four fold from 1:4 to 1:256 dilutions. In 7 patients fluorescence was seen with 1:16 serum dilution, in 18 with 1:64 dilution and in 3 with 1:256. The immunofluorescence test gave a sensitivity of 96.66 per cent, specificity of 95.0 per cent, with percentage of false negativity and false positivity being 3.45 and 5.0 per cent respectively.

Microbiological evaluation of female patients in STD clinics.

A total of 215 women patients attending the STD clinic were evaluated in an attempt to isolate the different microorganisms in sexually transmitted diseases (STD). Mycoplasmas (30.22%), Candida species (20.00%), Trichomonas vaginalis (wet mount study; 15.81%), beta haemolytic streptococci (13.48%), Neisseria gonorrhoeae (9.30%), Staphylococcus aureus (13.95%), inclusion bodies of Chlamydia trachomatis (11.60%) and Gram negative organisms (9.30%) were isolated from these patients. Sera of all patients screened for HBsAg by ELISA showed a carrier rate of 12.5 per cent; 29.8 per cent sera were reactive in the VDRL test at the dilutions varying from 1:4 to 1:64.

PIP: The incidence of 10 sexually transmitted diseases (STDs) in 215 prostitutes attending a STD clinic was determined by culture and other standard techniques. A control group of 100 women attending an outpatient family planning clinic in L.T.M. Medical College, Bombay, matched for age and socioeconomic level was the reference. The organisms assayed, the percent of study group found positive, and the percent of controls found positive were: M. hominis, 11.2 and 0.0%; U. urealyticum, 19.1 and 12.0%; Candida to species level, 20.0 and 70.%; beta-hemolytic streptococci, 13.5 and 3.0%; group B streptococci, 6.7 and 0.0%; T. vaginalis, 15.8 and 3.0%; C. trachomatis (by inclusion bodies), 11.6 and 1.0%; coagulase-positive S. aureus, 13.95 and 5.0%; N. gonorrhoeae, 9.3 and 0.0%; and Gram negative bacteria, 9.3 and 1.0%. The VDRL (Venereal Disease Reference Laboratory) seropositivity was 29.5% in STD patients. HBsAG (hepatitis B surface antigen) were present in 12.5%. Most of the women had vaginitis, cervicitis, vulvitis or warts with secondary infections. These incidence rates were discussed relative to several other Indian surveys.

Microbiological evaluation of women with bad obstetrics history.

In the present study a total of 300 pregnant women were evaluated, 200 women with bad obstetrics history (BOH) and 100 clinically normal women. Cervical culture studies, as well as serological evaluation was carried out in each woman. It was noted that among the various microbial agents detected, the presence of genital mycoplasmas, chlamydia, Toxoplasma gondii and cytomegalovirus was significant. Foetal outcome could be noted in some of the BOH patients. Toxoplasmosis was associated with complete abortion (38%), stillbirths (6%), premature delivery (16%) and congenital anomalies (6%). Cytomegalovirus infection was associated with complete abortion (41.66%), preterm delivery (33.33%) and congenital anomalies (8.33%). Ureaplasma infection in BOH patients resulted in preterm delivery with premature rupture of membranes in 45 per cent of women and complete abortion in 35 per cent.

Detection of Chlamydia trachomatis antigen & Toxoplasma gondii (IgM) & Mycoplasma hominis (IgG) antibodies by ELISA in women with bad obstetric history.

A total of 365 non pregnant women with bad obstetrics history (BOH) were studied with a control group of 100 women who had delivered full term clinically normal infants. The presence of C. trachomatis antigen and T. gondii (IgM) and M. hominis (IgG) antibodies was assessed by ELISA test. C. trachomatis antigen was detected in 28.2 per cent of women with BOH whereas T. gondii and M. hominis specific antibodies were found in 43.83 and 27.1 per cent respectively, these were highly significant (P < 0.001) in comparison with the control group. In case, facilities for culture are not available then detection of antigen and IgM class of antibodies by ELISA can pinpoint current infection.

Standardization of a dot blot immunoassay for antigen detection in cases of pulmonary tuberculosis & its evaluation with respect to the conventional techniques.

A simple dot (blot) ELISA test for detecting tubercular antigen in sputum samples of patients of pulmonary tuberculosis has been standardized using nitrocellulose paper. The sensitivity of the assay is 20 ng/ml. The cut-off value was 80 ng/ml. Of the 1042 patients in the study group, the percentage positivity by smear and culture was 54.51 and 57.93 per cent respectively; 68.7 per cent of the ELISA positives were confirmed by smear. The dot blot ELISA could be used as a rapid and specific test as it not only picked up 88.88 per cent of the smear positive, culture positive cases but also 81.89 per cent of the smear negative, culture positive cases. If the results of smear and dot blot ELISA are combined, 91.08 per cent of the culture positive cases were picked up as positive. If such a noninvasive test is commercialized and used in conjunction with smear, the pick up rate of tuberculosis cases will improve considerably.

Brucella spondylitis: an important treatable cause of low backache.

OBJECTIVES: This study was undertaken to see if brucella spondylitis existed as a cause of backache in Mumbai and to identify the clinical setting in patients of backache where brucella serology is indicated. METHODS: In 18 months (June 1996-Dec. 1997) we performed tube agglutination test (TAT) for Brucella melitensis and abortus on 72 patients of low backache from Orthopaedics Department of a teritary health centre. All 72 patients satisfied the inclusion and exclusion criteria designed to exclude radiologically detectable congenital or degenerative cause of backache. RESULTS: Six out of 72 patients were seropositive for brucellosis. All six patients had either history of animal contact or ingestion of raw milk or milk product (cheese or paneer). The lumbosacral backache was severe, radiating to the legs and straight leg raising test was significantly positive, they had marked tenderness on spinous process of lower lumbar vertebrae. Changes of brucella spondylitis were present on plain radiogram of lumbosacral spine in three patients. Four patients had abnormalities on bone scintigraphy. CONCLUSION: Low backache of brucella spondylitis closely simulates pain of prolapsed intervertebral disc. Serologic testing for brucellosis is an important step in management of such patients, especially when history of animal contact or raw milk or milk product ingestion is present, as the disease can be eminantly treated with antibiotics.

Prevalence of gram negative anaerobic bacilli in routine clinical specimens.

A total of 206 gram negative anaerobic bacilli were isolated from 2591 routine clinical specimens. Majority of them (78.2%) were part of a mixed flora. A high incidence of gram negative anaerobic bacilli was found in post-operative wound infections and diabetic foot infections (30.1%), followed by necrotizing fasciitis and crush injuries (18%), pus of empyema and lung abscesses (14.5%) and aspirates from other deep abscesses (10.7%). Bacteroides fragilis was the most frequently recognized species (42.2%), followed by Fusobacterium sp. (17.4%), Prevotella sp. (15.1%) and Porphyromonas sp. (11.2%). Neomycin blood agar plate yielded the maximum number (72.8%) of strains in pure culture. Gram negative anaerobic bacilli in infective processes are significantly on the rise and so suspected cultures should be processed for both aerobic and anaerobic organisms.

Early detection of tuberculous meningitis using one step competitive ELISA.

A one step competitive Enzyme-Linked Immunosorbent assay (ELISA) method was developed to detect mycobacterial antigen in cerebrospinal fluid (CSF) for the diagnosis of tuberculous meningitis and compared with a standard competitive ELISA method. Indigenously prepared soluble extract of Mycobacterium tuberculosis H37 Rv was used as antigen. The study was conducted using CSF of 230 clinically diagnosed cases of tuberculous meningit is and 208 control subjects. A cutoff value of 0.57 ng/ml by the one step ELISA and 0.5 ng/ml by the standard ELISA method were determined. The specificity of both methods were 100% and positivity was 68.26% and 70.43% respectively. A follow up study was conducted in 63 cases at various interval of time after starting anti-tubercular therapy i.e. at 3 weeks (63 cases), 6 weeks (27 cases) and > or = 4-12 months (13 cases). It was observed that antigen levels decreased gradually, but were much above the cutoff range. Indigenously prepared antigen was compared with antigen prepared in other laboratories and standard molecular weight markers using SDS PAGE (Sodium Do-decyl Sulphate Polycrylamide Gel Electrophoresis).

C-reactive protein and buffy coat smear in early diagnosis of childhood septicemia.

Out of 200 cases of septicemia in children (age group 0-14 years), 111 had positive C-Reactive Protein (CRP > 12 mg/l) and 84 were buffy coat smear positive. Blood culture was positive in 98 cases, with predominant organism being Klebsiella pneumoniae, followed by Staphylococcus aureus. CRP test showed 100% sensitivity and 87.3% specificity, while buffy coat smear showed 76.5% sensitivity and 91.2% specificity. As blood culture reports are not available before 48-72 hours, combination of CRP test and buffy coat smear examination will be very helpful in early diagnosis of childhood septicemia.

Enzyme-linked immunosorbent-assay for antigen detection in tuberculous meningitis.

Two systems of Competitive Enzyme linked immunosorbent assay (ELISA) were developed to detect Mycobacterial antigen in cerebrospinal fluid (CSF) for the diagnosis of tuberculous meningitis (TBM)--one by using indigenously prepared Anti-M.tuberculosis H37Rv-Penicillinase conjugate (Method I) and another by using commercially available Anti M. bovis BCG-Horse Radish Peroxidase (HRP) conjugate--(Method II). The tests were used to analyse CSF of 148 patients clinically confirmed as having TBM and 278 control subjects. By using > or = 10 ng/ml as the cut-off value for Method I and > or = 1 ng/ml as that for Method II, the specificity for both were 100% and positivity was 79.73% and 67.57% respectively. A follow up study in 26 TBM cases after 2 weeks (16 cases), 4 weeks (13 cases) and 4-12 months (10 cases) of antituberculous treatment revealed that mycobacterial antigen persisted in the majority of cases even after 4 weeks of the treatment.

Drug resistance to the first line of antitubercular regimen (a preliminary report).

Sputum samples from 100 patients of pulmonary tuberculosis were processed. These patients were admitted in group of Tuberculosis Hospital at Sewri, Mumbai, which is a referral tuberculosis hospital. Isolates were identified as M. Tuberculosis by biochemical tests. Antitubercular sensitivity testing for Isoniazid, Rifampicin, Ethambutol and Streptomycin was performed by resistance ratio method. Total resistance was 36% in our study. Resistance to Isoniazid was 61%: to Rifampicin was 50%, to Ethambutol was 8% and to Streptomycin was 41%. Primary drug resistance to Isoniazid was 45% to Rifampicin was 27%, to Ethambutol was 9%, and to Streptomycin was 54%. Secondary drug resistance to Isoniazid was 68% to Rifampicin was 60%, to Ethambutol was 8% and to Streptomycin was 36%. Secondary drug resistance to Isoniazid and Rifampicin is rising as compared to primary drug resistance to Isoniazid and Rifampicin. This is statistically significant (p < 0.001). 11 out of 36 cases (30%) showed multi drug resistance to Isoniazid and Rifampicin.