Use of CHROMagar Candida for the presumptive identification of Candida species directly from clinical specimens in resource-limited settings

Introduction: Identification of yeast isolated from clinical specimens to the species level has become increasingly important. Ever-increasing numbers of immuno-suppressed patients, a widening range of recognized pathogens, and the discovery of resistance to antifungal drugs are contributing factors to this necessity. Material and methods: A total of 487 yeast strains were studied for the primary isolation and presumptive identification, directly from clinical specimen. Efficacy of CHROMagar Candida has been evaluated with conventional methods including morphology on Corn meal-tween 80 agar and biochemical methods by using API 20 C AUX. Results: The result of this study shows that CHROMagar Candida can easily identify three species of Candida on the basis of colonial color and morphology, and accurately differentiate between them i.e. Candida albicans, Candida tropicalis, and Candida krusei. The specificity and sensitivity of CHROMagar Candida for C. albicans calculated as 99%, for C. tropicalis calculated as 98%, and C. krusei it is 100%. Conclusion: The data presented supports the use of CHROMagar Candida for the rapid identification of Candida species directly from clinical specimens in resource-limited settings, which could be very helpful in developing appropriate therapeutic strategy and management of patients.Keywords: CHROMagar Candida; resource-limited settings; presumptive identificatio

Seroprevalence of Hepatitis B and C Genotypes Among Young Apparently Healthy Females of Karachi-Pakistan

INTRODUCTION: Although the prevalence of hepatitis virus infections in Pakistan is still unknown, limited data indicate that the exposure rate to HBV is 35-38% with 4% being carriers and 32% having anti-HBV surface antibodies through natural conversion [1,2,3]. Studies in Pakistan have shown that the prevalence rate of HCV is 4.8-14% for, and that it is continuously increasing. Hence there is an urgent need to create awareness about the prevalence of both hepatitis B and C, and to develop preventive measures aimed at minimizing the prevalence of these diseases in the country.Study Design: Prospective, descriptive study. The study took place from March 2002 till October 2006 at two university campuses in Karachi.MATERIALS AND METHODS: A total of 4000 healthy female students were screened for HBs Ag, anti-HBs antibodies and anti-HCV antibodies by rapid immunochromatography, ELISA and PCR.RESULTS: A total of 3820 volunteers (95.5%) were negative by all three methods, 181 (4.5%) tested positive for HB surface antigen and 20 (0.5%) were positive for anti HB surface antibodies; 208 volunteers (5.2%) were positive for HCV. Double infection with HBV and HCV was found in only one patient (0.025%). Out of 180 HBs antigen positive samples 151 (83.89%) were genotype D, 28 (15.56%) showed mixed infection with genotypes B and D, and one patient (0.56%) showed mixed infection with genotypes C and D. Out of 208 samples positive for HCV antibodies, 107 (51.44%) were genotype 3a, 50 (24.04%) were mix of genotype 3a and 3b, 33 (15.87%) were genotype 3b, 10 (4.81%) were genotype 1b while, 8 (3.84%) samples could not be typed.CONCLUSION: Although the presence of these pathogenic viruses was not very high in our young healthy female population, it is still a matter of concern to control the unregulated spread of these deadly infections by promoting increased awareness and regular immunization programs in the community. Local manufacturing of vaccines and related products may reduce these infections

Acquisition of the Sda1-encoding bacteriophage does not enhance virulence of the serotype M1 Streptococcus pyogenes strain SF370

The resurgence of invasive disease caused by Streptococcus pyogenes (group A Streptococcus [GAS]) in the past 30 years has paralleled the emergence and global dissemination of the highly virulent M1T1 clone. The GAS M1T1 clone has diverged from the ancestral M1 serotype by horizontal acquisition of two unique bacteriophages, encoding the potent DNase Sda1/SdaD2 and the superantigen SpeA, respectively. The phage-encoded DNase promotes escape from neutrophil extracellular traps and is linked to enhanced virulence of the M1T1 clone. In this study, we successfully used in vitro lysogenic conversion to transfer the Sda1-encoding phage from the M1T1 clonal strain 5448 to the nonclonal M1 isolate SF370 and determined the impact of this horizontal gene transfer event on virulence. Although Sda1 was expressed in SF370 lysogens, no capacity of the phage-converted strain to survive human neutrophil killing, switch to a hyperinvasive covRS mutant form, or cause invasive lethal infection in a humanized plasminogen mouse model was observed. This work suggests that the hypervirulence of the M1T1 clone is due to the unique synergic effect of the M1T1 clone bacteriophage-specific virulence factor Sda1 acting in concert with the M1T1 clone-specific genetic scaffold

A Naturally Occurring Mutation in ropB Suppresses SpeB Expression and Reduces M1T1 Group A Streptococcal Systemic Virulence

Epidemiological studies of group A streptococcus (GAS) have noted an inverse relationship between SpeB expression and invasive disease. However, the role of SpeB in the course of infection is still unclear. In this study we utilize a SpeB-negative M1T1 clinical isolate, 5628, with a naturally occurring mutation in the gene encoding the regulator RopB, to elucidate the role of RopB and SpeB in systemic virulence. Allelic exchange mutagenesis was used to replace the mutated ropB allele in 5628 with the intact allele from the well characterized isolate 5448. The inverse allelic exchange was also performed to replace the intact ropB in 5448 with the mutated allele from 5628. An intact ropB was found to be essential for SpeB expression. While the ropB mutation was shown to have no effect on hemolysis of RBC's, extracellular DNase activity or survival in the presence of neutrophils, strains with the mutated ropB allele were less virulent in murine systemic models of infection. An isogenic SpeB knockout strain containing an intact RopB showed similarly reduced virulence. Microarray analysis found genes of the SpeB operon to be the primary target of RopB regulation. These data show that an intact RopB and efficient SpeB production are necessary for systemic infection with GAS

Inactivation of DltA Modulates Virulence Factor Expression in Streptococcus pyogenes

D-alanylated lipoteichoic acid is a virtually ubiquitous component of gram-positive cell walls. Mutations in the dltABCD operon of numerous species exhibit pleiotropic effects, including reduced virulence, which has been attributed to increased binding of cationic antimicrobial peptides to the more negatively charged cell surface. In this study, we have further investigated the effects that mutating dltA has on virulence factor expression in Streptococcus pyogenes.Isogenic Delta dltA mutants had previously been created in two distinct M1T1 isolates of S. pyogenes. Immunoblots, flow cytometry, and immunofluorescence were used to quantitate M protein levels in these strains, as well as to assess their ability to bind complement. Bacteria were tested for their ability to interact with human PMN and to grow in whole human blood. Message levels for emm, sic, and various regulatory elements were assessed by quantitative RT-PCR. Cell walls of Delta dltA mutants contained much less M protein than cell walls of parent strains and this correlated with reduced levels of emm transcripts, increased deposition of complement, increased association of bacteria with polymorphonuclear leukocytes, and reduced bacterial growth in whole human blood. Transcription of at least one other gene of the mga regulon, sic, which encodes a protein that inactivates antimicrobial peptides, was also dramatically reduced in Delta dltA mutants. Concomitantly, ccpA and rofA were unaffected, while rgg and arcA were up-regulated.This study has identified a novel mechanism for the reduced virulence of dltA mutants of Streptococcus pyogenes in which gene regulatory networks somehow sense and respond to the loss of DltA and lack of D-alanine esterification of lipoteichoic acid. The mechanism remains to be determined, but the data indicate that the status of D-alanine-lipoteichoic acid can significantly influence the expression of at least some streptococcal virulence factors and provide further impetus to targeting the dlt operon of gram-positive pathogens in the search for novel antimicrobial compounds

Development Of An In-House Enzyme-Linked Immunosorbent Assay Based On Helicobacter Pylori Sonicate Whole Cell Antigen For Diagnosis Of Gastroduodenal Ulcer Disease In Karachi, Pakistan

Helicobacter pylori (H. pylori) is a causative agent of gastritis, gastroduodenal ulcers and gastric adenocarcinoma. Improper diagnostic facilities are responsible for the increasing incidence of H. pylori infections in Pakistan. ELISA is a noninvasive, less expensive assay for diagnosis of H. pylori. ELISA performance is mainly based on the H. pylori strain and nature of antigen. In this study, a total of 214 gastritis and ulcer patient’s serum samples were screened for anti- H. pylori IgG antibody. A 96-well plate coated with 10 µg/ml sonicate antigen and hundred-fold diluted patient’s serum was allowed to react. After extensive washing with buffer, 1:2,000 diluted conjugated secondary antibody was added. Later substrate was added to observe positivity by measuring the intensity of color. Statistical analyses were performed, and p value of <0.01 was taken as significant; 84 % male patients and 96 % female patients, respectively, tested positive for H. pylori, while agewise distribution was 35–45 years males (40 %) and 35–45 years females (73 %) were found highest number of H. pylori infected patients. In-house ELISA based on sonicate whole cell antigen (sELISA) showed a sensitivity of 98 %, specificity of 100 %, accuracy 98 % and κ value 0.906 with significant correlation R—0.956; p < 0.0001. We conclude that ELISA for H. pylori sero-diagnostic infection should be based on the local strain for better sensitivity and specificity. sELISA is better and reliable diagnostic assay for the diagnosis of H. pylori infection in gastric patients of Karachi, Pakistan

Development Of An In-House Enzyme-Linked Immunosorbent Assay Based On Helicobacter Pylori Sonicate Whole Cell Antigen For Diagnosis Of Gastroduodenal Ulcer Disease In Karachi, Pakistan

Helicobacter pylori (H. pylori) is a causative agent of gastritis, gastroduodenal ulcers and gastric adenocarcinoma. Improper diagnostic facilities are responsible for the increasing incidence of H. pylori infections in Pakistan. ELISA is a noninvasive, less expensive assay for diagnosis of H. pylori. ELISA performance is mainly based on the H. pylori strain and nature of antigen. In this study, a total of 214 gastritis and ulcer patient’s serum samples were screened for anti- H. pylori IgG antibody. A 96-well plate coated with 10 µg/ml sonicate antigen and hundred-fold diluted patient’s serum was allowed to react. After extensive washing with buffer, 1:2,000 diluted conjugated secondary antibody was added. Later substrate was added to observe positivity by measuring the intensity of color. Statistical analyses were performed, and p value of <0.01 was taken as significant; 84 % male patients and 96 % female patients, respectively, tested positive for H. pylori, while agewise distribution was 35–45 years males (40 %) and 35–45 years females (73 %) were found highest number of H. pylori infected patients. In-house ELISA based on sonicate whole cell antigen (sELISA) showed a sensitivity of 98 %, specificity of 100 %, accuracy 98 % and κ value 0.906 with significant correlation R—0.956; p < 0.0001. We conclude that ELISA for H. pylori sero-diagnostic infection should be based on the local strain for better sensitivity and specificity. sELISA is better and reliable diagnostic assay for the diagnosis of H. pylori infection in gastric patients of Karachi, Pakistan