comparison of histopathological findings and polymerase chain reaction in lesions with primary clinical diagnosis of cutaneous leishmaniasis with negative smear

Cutaneous leishmaniasis [CL] is a common skin disease in Middle East region which involves all ranges of ages and can affects both sexes. With regard to this fact that on time diagnosis and treatment would lead to reduction in its permanent complications and because it resembles other skin diseases, various techniques have been introduced to diagnose its agent in affected tissue. The reliability of pathological examination and PCR technique for diagnosis of CL was investigated in present study. In total 30 patients with clinically CL lesions having negative direct smear, were investigated. Tissue biopsies were taken from the patients, histopatholgic sections were prepared and the sections were examined to confirm or rule out the disease. DNA was extracted from biopsy samples by using standard DNA extraction kit and PCR assay was performed according to standard protocol using Leishmania tropica species-specific primers. Among 30 biopsies from patients with clinical diagnosis of CL under investigation, five were positive on PCR [16.7%] and 4 were positive on histopathologic examination [13.3%], There was not any meaningful statistical difference between PCR assay and pathological examination [P= 0.87] for diagnosis of CL cases with a negative smear. The present study indicated that PCR is a reliable technique for detection of some cases of suspected patients to CL with negative direct smear, but did not find any priority of PCR compared to histopathology in respect to similarity and economics

Application of restriction enzyme analysis technique based on 65KDA heat shock protein gene for fingerprinting and differentiation of mycobacterium tuberculosis clinical strains isolated from tuberculosis patients in Ahwaz, Iran

Application of identification methodology of restriction enzyme analysis [REA] for fingerprinting of the expanded population of Mycobacterium tuberculosis [MTB] isolates. A total of 150 clinical isolates from patients referred to TB reference laboratory, Public Health Centre, Ahwaz, Iran, were identified as MTB by using conventional culture and biochemical tests from January to December 2004. The PCR-REA method uses a PCR step based on amplification of a 439 bp fragment of hsp65 gene involving genus specific primers and restriction enzyme analysis by digestion of products with HaeIII and BstEII enzymes were employed. The identical restriction patterns similar to MTB reference strains equal to 160 / 145 / 72bp fragments for Hae III and 250 /120/82bp fragments for Bst EII digests were seen in 145 isolates [96.6%]. The diverse patterns were observed for five isolates in Hae III digest as 180 / 100 / 80 bp, 194/ 72 bp and 160/ 145 bp representing the possible intra-species variation within studied MTB strains, while their Bst EII digestion patterns showed no variation. The PCR-REA technique revealed three different new patterns for Hae III digest. However to verify that they are indeed MTB isolates, a sequence-based analysis of the exceptional isolates should be performed

Bacteriologic study of diabetic foot ulcer

To study the relative frequency of bacterial isolates cultured from diabetic foot infections and assess their in vitro susceptibility to the commonly used antibacterial agents. In total 32 hospitalized diabetic patients with foot infections were investigated. Deep tissue biopsies were inoculated into freshly prepared Thioglycollate broth medium. Bacterial agents were identified by conventional bacteriologic methods. Sensitivity tests were performed according to standard disc diffusion method of Kirby and Bauer. Clinical grading and bacteriological study of 32 patients with diabetic foot lesions revealed polymicrobial etiology in 16 [50%] and single etiology in 10 [31.2%] and six negative cultures. Aerobic Gram-positive bacteria accounted for 42.9%. Staphylococcus aureus was the most frequent microorganism yielded [26.2%], and Staphylococcus epidermidis was regularly associated with the lesions [14.3%]. Gram-negative rods accounted for 54.8%. Escherichia coli was the most predominant gram negative organism [23.8%]. No anaerobes were isolated from the ulcers. All the microorganisms isolated showed high resistance to used antibiotics, amongst them, Staphylococcus aureus and Pseudomonas aeruginosa were the most resistant bacteria in present study. Staphylococcus aureus, Escherichia coli, Staphylococcus epidermidis and Proteus vulgaris were the most common causes of diabetic foot infections in present study. And the rate of antibiotic resistance was 65% among the isolates. Due to polymicrobial infection and antibiotic resistance, surgical intervention must be concerned

Detection of isoniazid and rifampin resistant mycobacterium tuberculosis isolated from tuberculosis patients using conventional method and PCR

To investigate the drug susceptibility patterns among TB isolates from patients in Ahvaz, Iran. Descriptive study. TB reference laboratory, PHLS, Ahvaz, Iran from May 2001 to December 2001. A total of 100 sputum samples from patients suspected of having tuberculosis were collected for detection of M. tuberculosis [MTB]. For identification of the isolates acid fast stain, cultural and biochemical techniques were used. The isolates were examined for INH and RIF resistance using conventional MIC method and PCR technique in the next step. MIC were done based on proportion method and PCR was performed according to manufacturer's instruction and by using specific INH [Kat G] and RIF [rpo B] resistant primers. Eighty samples were identified as MTB. Using susceptibility testing, 7 isolates were resistant to both INH and RIF by MIC method. In PCR technique, 5 and 6 out of 7 above mentioned strains showed resistant to INH and RIF respectively. The prevalence of resistance to INH and RIF is high in the region of study. The conventional MIC method despite being time consuming is more sensitive for evaluation of drug resistance among TB isolates. However, PCR as a rapid and sensitive technique is recommended additionally to conventional method for having quicker results to start treatment and disease control management

Application of polymerase chain reaction technique for laboratory diagnosis of cutaneous tuberculosis

Evaluation of the role of polymerase chain reaction [PCR] for the detection of Mycobacterium tuberculosis [MTB] DNA as a diagnostic aid in cutaneous tuberculosis. Descriptive study. TB reference laboratory, PHLS, Ahvaz, Iran from May 2001 to December 2001. Thirty formalin-fixed, paraffin-embedded samples belonging to 28 patients were analyzed. Tissue sections were treated by lysis buffer containing proteinase K and DNA was extracted by using standard extraction kit. PCR amplification was performed using assay based on a repetitive sequence IS 6110 of MTB according to standard procedure. PCR was positive in six samples. Amongst them, two of the samples [7.1%] belonged to patients with chronic granulomatosis, which was previously confirmed histopathologically, were positive in entire applied tests, i.e. direct smear, culture and PCR. Using PCR technique, six out of the total specimens tested [21.4%], were positive for the presence of M. tuberculosis DNA. Statistically the difference between applied methods was significant [P<0.0016]. Accounting histopathology as gold standard, the sensitivity of PCR in this study was determined as 75%. Our study showed that from 8 cases of skin tuberculosis diagnosed by histopathology, 6 were positive by PCR technique, which shows the superiority of previous method to molecular technique. However, PCR assay has priority to conventional bacteriologic methods for detection of M. tuberculosis from cutaneous tuberculosis cases, and can be only used when the staining for acid fast bacilli is negative and there is a lack of growth on culture or when fresh material has not been collected for culture

Isolation of brucella melitensis and brucella abortus from brucellosis patients by conventional culture method and polymerase chain reaction technique

Isolation of B. melitensis and B. abortus from brucellosis patients by application of conventional culture and PCR methods and comparison of sensitivity of both techniques in diagnosis of human brucellosis. Infectious diseases Ward, Ahwaz University's hospitals over a period of 9 months [September 2003 to May 2004]. A total of 30 peripheral blood samples were collected from patients with brucellosis referred to infectious Diseases Unit of Ahwaz University's hospitals. The samples were inoculated into Castaneda medium and after 7-30 days, the isolated colonies were identified by inoculation into brucella agar containing thionine and fuschin and by performance of biochemical tests. DNA was extracted from colonies and serum samples and examined by PCR involving specific primers for B. melitensis and B. abortus based on IS 711 in the brucella chromosome. We identified 8 isolates of B. melitensis using culture and biochemical methods. When PCR technique was applied to serum samples, 28 cases were positive for B. melitensis and we were able to isolate more cases compared to culture method and the difference was significant [P<0.05]. The sensitivities of culture and PCR in this study were determined as 26.6% and 93.3% respectively. Neither of examined cultures or sera subjected to PCR were positive for B. abortus. The results of present study showed that PCR assay is a rapid and sensitive technique for diagnosis of brucellosis compared to culture method. However it is more valuable when coupled with conventional methods

Evaluation of the antibacterial activity of the seed hull of quercus brantii on some gram negative bacteria

Due to rapid increase of antibiotic resistance especially among gram negative bacteria that cause diarrheal diseases, the role of methanol extract of oak seed hull [Quercus brantii] was evaluated on few gram negative entric bacilli and compared with some in-use antibiotics. Nine months from October 2001 to July 2002 at the schools of Medicine and pharmacy, Ahwaz Jondi Shapour University of Medical Sciences, Ahwaz, Iran. Cold maceration with 70% methanol was used for extraction of seed hulls and different concentrations were prepared from the concentrated extract. The antimicrobial activity of the extract was examined using the standard MIC and disc diffusion method on E. coli, Salmonella typhimurium, Shigelia flexneri and Proteus mirabilis and the activity was compared with those of gentamycin, nalidixic acid and co-trimoxazole in the next step. The antibacterial effect of the methanol extract on P mirabilis and E. coli was significant and was directly concentration- related but had no significant effect on S. flexneri. Some concentrations had a similar or even better effect compared with nalidixic acid or co-trimoxazole, while the effect of 80% extract was not significant in general, except for S. typhimurium where its effect was equivalent to that of 25micro g co-trimoxazole. Although oak seed hull has some antibacterial activity apparently its anti-diarrheal effect is due to water absorption and protein precipitation in the body