Insights to the diphtheria toxin encoding prophages amongst clinical isolates of Corynebacterium diphtheriae from India

Diphtheria is a dreadful disease caused by Corynebacterium diphtheriae. Lysogenised bacteriophages carrying toxin gene in C. diphtheriae can make the strain toxigenic. However, such phage disseminates the toxin genes to other strains when it undergoes lytic phase. As little is known about the phage diversity in C. diphtheriae in India, the present study was undertaken to investigate the prophages integrated into the genome of 29 clinical isolates of C. diphtheriae using whole-genome shotgun sequencing. Amongst these isolates, 27 were toxigenic, while 2 were non-toxigenic strains. Of the 27 toxigenic strains, all harbored known phages carrying toxin gene and two other phages with unknown function. However, the two non-toxin strains did not harbour any of the phages in the genome. It is imperative to devise prevention strategies that hinder the dissemination of toxin by prophages, as it may increase the complications of diphtheria post-immunisation.

Surface disinfection by exposure to germicidal UV light.

The present study was aimed to design a simple model to check efficacy of germicidal UV tube, to standardise the position, distance and time for UV light and also to find out its efficacy against medically important bacteria, the bacterial spores and fungi. The microbial cultures tested included gram positive and gram negative bacteria, bacterial spores and fungal spores. The microbes streaked on solid media were exposed to UV light. The inactivation of the order of four logs was observed for bacteria. UV light can have efficient inactivation of bacteria up to a distance of eight feet on either side and exposure time of 30 minutes is adequate.

Elisa kit evaluation for IGG and IGM antibodies to A-60 tubercular protein antigen.

AIMS: The purpose of this study is to evaluate the A-60 antigen-based enzyme-linked immuno sorbent assay (ELISA) test for its sensitivity, specificity, and other related statistical parameters. SETTINGS AND DESIGN: Sera from 114 healthy volunteers, 105 bacteriologically confirmed cases of pulmonary tuberculosis (PTB), 59 sera from family contacts of PTB, and 40 sera from cases of lung infections other than tuberculosis collected from September to December 2003 were used for the kit evaluation. METHODS AND MATERIALS: Enzyme-linked immuno sorbent assay test using tuberculosis A-60 antigen-based kit manufactured by Anda Biologicals, France was used for the evaluation. STATISTICAL ANALYSIS: Differences in the optical density (OD) values for immunoglobulins G (IgG), and immunoglobulins M (IgM) antibodies in various groups were studied using t-test. RESULTS: On the basis of the findings the threshold value was setup as 400 U for IgG and mean OD for sera from healthy volunteers +2SD as the threshold for IgM. The sensitivity was 80% and specificity 95.8% for the IgG antibody test. The efficiency and predictive values were also high. The sensitivity for IgM was low (28.5%) but the specificity was high (95.7%). None of the 40 nontubercular lung infection cases were positive for the IgG and IgM antibody test for A-60, whereas five and three cases of 59 family contacts of PTB were positive for IgG and IgM antibody test. The test reproducibility was good for both IgG and IgM. CONCLUSION: IgG antibody test using A-60 antigen has good sensitivity and specificity, whereas IgM antibody test had high specificity but low sensitivity. Multicentric trials suggested evaluation of the diagnostic utility of the test for the extra-PTB.

Drug resistance among tubercle bacilli from pulmonary tuberculosis cases in central India.

An estimate of drug resistance is extremely important in the epidemiology and control of tuberculosis. Data on drug resistance among mycobacterial isolates from sputum samples analysed at Microbiology dept. of Choithram Hospital and Research Centre, Indore, M.P. is presented here. Drug sensitivity testing was carried out on 1426 Mycobacterial isolates by the method of proportion using critical concentration in Lowenstein Jensen medium. Resistance for Isoniazid, streptomycin, and pyrazinamide was found to be high (54.2%, 41.5% and 50% respectively) and was followed by resistance to rifampin (25%) and ethambutol (22%). Resistance for kanamycin, p-aminosalicylic acid, thiacetazone and ciprofloxacin was much lower (18%, 13%, 6.5% and 3.6% respectively). Only 12% of the isolates were sensitive to all the anti-TB drugs while resistance to two, three, and four or more drugs was in the range of 20-25%. Pattern wise, simultaneous resistance to INF and Rifampin with or without resistance to other drugs was observed in 8.1% while resistance for Isoniazid + pyrazinamide and Isoniazid + streptomycin was 11.9 and 11.5% respectively. Resistance for Isoniazid + ethambutol was the lowest (5.1%). Growing multiple drug resistance among tubercle bacilli warrant urgent attention in tuberculosis control programme.

Smears from rat liver suspension as modified substrate for antinuclear antibody test.

Immunofluorescence still remains a standard method for documenting antinuclear antibodies (ANA). Cryostat cut sections of rodent liver or Hep2 cell nuclei have been used as substrate in the test but are often difficult to arrange in laboratories in developing countries. Hence, a modification was developed using smears from rat liver suspensions. The smears were compared with the cryostat cut sections over 338 sera samples of suspected cases of collagen diseases, rheumatoid arthritis, thyroid disorders, hepatitis B, enteric fever, tuberculosis and normal subjects. The sera from suspected collagen diseases cases were also compared with ANA test using Hep2 cells. The modified smear technique was well comparable and the clarity of the immunofluorescence was even better than for cryostat cut sections. Using the modified smear technique 272 sera out of 2,851 sera gave positive test for ANA. The homogenous, speckled and peripheral patterns were seen for 203, 66 and 3 samples respectively. To conclude: The smears prepared from homogenised rat liver suspension and fixed like bacterial smears offer a very convenient and reliable tissue substrate for ANA test.

Studies on blood factors responsible for rise in ESR.

Rise in ESR following inflammation processes is well known. However, the exact factors in blood responsible for the rise remain largely unknown. The present experimental work on mixing of plasma from pulmonary tuberculosis cases with RBCs from healthy volunteers indicates that ESR rising properties reside in plasma and not in RBCs. Levels of C-reactive proteins (CRP) are also known to rise following inflammation and hence CRP could contribute to rise in ESR. However, absorption of C-reactive protein from the plasma does not reduce the ESR and suggests that C-reactive proteins are not directly involved in raising ESR. Further experimental work was carried out to see whether cytokines released by mononuclear cells are responsible. However, cell culture supernatants added to whole blood samples did not cause rise in ESR. The ESR rising substances in plasma were non-dialysable and thus were of larger molecular weight.

Acridine orange stained blood wet mounts for fluorescent detection of malaria.

The age old Romanowsky stained thick blood smear examination for malarial parasites may fail to reveal the low parasitaemia. The commercial 'QBC' like acridine orange stained capillary tube preparation has a limitation of precise species identification and the detection of extra-erythrocytic parasites. Hence, the present study was aimed to improve malarial parasite detection by using acridine orange to stain large blood drops in the form of wet coverglass mounts. The acridine orange stained blood wet mounts over 2420 suspected malaria cases from Indore city were examined under fluorescent microscope and the results compared with the Leishman's stained thick blood smears in a blind study. The positivity of malarial parasites reported by the modified acridine orange staining was 248 against 109 by Leishman's stained thick blood smears. The modified acridine orange stained method is simple, instant and more efficient, requires less scanning time and skill, allows scanning of larger blood volume (75 ul) at lower magnification and the morphological details at higher magnification helps to make the precise species identification.