suhail ahmad - Academia.edu (original) (raw)

Papers by suhail ahmad

Journal of Clinical Microbiology, 2002

The rapid detection and identification of Candida species in clinical laboratories are extremely ... more The rapid detection and identification of Candida species in clinical laboratories are extremely important for the management of patients with hematogenous candidiasis. The presently available culture and biochemical methods for detection and species identification of Candida are time-consuming and lack the required sensitivity and specificity. In this study, we have established a seminested PCR (snPCR) using universal and species-specific primers for detection of Candida species in serum specimens. The universal outer primers amplified the 3 end of 5.8S ribosomal DNA (rDNA) and the 5 end of 28S rDNA, including the internally transcribed spacer 2 (ITS2), generating 350-to 410-bp fragments from the four commonly encountered Candida species, viz., C. albicans, C. tropicalis, C. glabrata, and C. parapsilosis. The species-specific primers, complementary to unique sequences within the ITS2 of each test species, amplified species-specific DNA in the reamplification step of the snPCR. The sensitivity of Candida detection by snPCR in spiked serum specimens was close to 1 organism/ml. Evaluation of snPCR for specific identification of Candida species with 76 clinical Candida isolates showed 99% concordant results with the Vitek and/or ID32C yeast identification system. Further evaluation of snPCR for detection of Candida species in sera from culture-proven (n ‫؍‬ 12), suspected (n ‫؍‬ 16), and superficially colonized (n ‫؍‬ 10) patients and healthy subjects (n ‫؍‬ 12) showed that snPCR results were consistently negative with sera from healthy individuals and colonized patients. In culture-proven candidemia patients, the snPCR results were in full agreement with blood culture results with respect to both positivity and species identity. In addition, snPCR detected candidemia due to two Candida species in five patients, compared to three by blood culture. In the category of suspected candidemia with negative blood cultures for Candida, nine patients (56%) were positive by snPCR; two of them had dual infection with C. albicans and either C. tropicalis or C. glabrata. In conclusion, the snPCR developed in this study is specific and more sensitive than culture for the detection of Candida species in serum specimens. Moreover, the improved detection of cases of candidemia caused by more than one Candida species is an additional advantage.

Diagnostic Microbiology and Infectious Disease, 2002

Mutations in an 81-bp region of the rpoB gene associated with rifampin resistance were studied in... more Mutations in an 81-bp region of the rpoB gene associated with rifampin resistance were studied in 41 rifampin-resistant clinical strains of Mycobacterium tuberculosis isolated in Turkey. Fourteen different rpoB alleles, three of which had not been reported before, were found. A reverse hybridization-based line probe assay (the Inno-LiPA Rif.TB test) for rapid detection of the mutations was evaluated with these isolates. Rifampin resistance was correctly identified in 23 of 41 isolates (56.1%) with the kit's R probes specific for these mutations. Seventeen of 41 isolates (41.5%) yielded hybridization patterns, with at least one negative signal obtained with the S probes for the wild type. One isolate was identified as rifampin sensitive by the line probe assay. The rate of concordance of the results of the line probe assay with the results of the in vitro susceptibility test was high (97.6%). These results demonstrate that the line probe assay kit may be useful for the rapid diagnosis of rifampin-resistant tuberculosis.

International Journal of Antimicrobial Agents, 2004

The presence of ACC and other mutations at codon 315 in the katG gene was detected by PCR amplifi... more The presence of ACC and other mutations at codon 315 in the katG gene was detected by PCR amplification followed by restriction fragment length polymorphism (PCR-RFLP) generated with restriction enzymes Msp I and MspA1 I in 37 isoniazid-resistant and 22-susceptible Mycobacterium tuberculosis isolates from Kuwait obtained in 2001. The mutation AGC to ACC was detected in 22 (60%) isolates while any mutation at codon 315 of the katG gene was present in 24 (65%) of 37 isoniazid-resistant isolates. The typing studies showed that majority of the isolates carrying mutations at codon 315 exhibited unique DNA banding patterns. The results were extended by additional analysis of 67, 28 and 17 isoniazid-resistant and 18, seven and six-susceptible M. tuberculosis isolates from Kuwait, Dubai and Beirut, respectively, that were analyzed previously for ACC mutation alone. These studies showed that one of 21, one of 10 and two of 11 isolates (all recovered from patients of Middle Eastern origin) with no AGC to ACC mutation from Kuwait, Dubai and Beirut, respectively, contained other mutations at codon 315 of the katG gene. None of the susceptible strains contained any mutation at codon 315. The PCR-RFLP with MspA1 I that detects all mutations at codon 315, compared with Msp I that detects only ACC mutation, identified more isoniazid-resistant strains with mutations at codon 315 in the katG gene. The data also showed that mutations other than AGC to ACC at codon 315 in the katG gene occur frequently in M. tuberculosis isolates recovered from Middle Eastern patients and should be incorporated in a rapid screen for the detection of mutations for isoniazid-resistance in the katG gene from this ethnic group.

Journal of Medical Microbiology, 2004

Candida dubliniensis is an emerging pathogen capable of causing oropharyngeal, vaginal and bloods... more Candida dubliniensis is an emerging pathogen capable of causing oropharyngeal, vaginal and bloodstream infections. Although C. dubliniensis is similar to Candida albicans in several phenotypic characteristics, it differs from it with respect to epidemiology, certain virulence factors and the ability to develop resistance to fluconazole rapidly. In this study, the first seven isolations of C. dubliniensis from Kuwait are described, all originating from non-human immunodeficiency virus (HIV)-infected patients. The isolates were initially identified by the Vitek 2 yeast identification system, positive germ tube test, production of rough colonies and chlamydospores on Staib agar and by their inability to assimilate xylose, trehalose or methyl AE-D-glucoside. The species identity of the isolates was subsequently confirmed by specific amplification of rDNA targeting the internally transcribed spacer 2 (ITS2), restriction endonuclease digestion of the amplified DNA and direct DNA sequencing of the ITS2. Using the E-test method, the MICs of C. dubliniensis test isolates were in the range 0 . 125-0 . 75 ìg ml À1 for fluconazole, 0 . 002-0 . 75 ìg ml À1 for itraconazole, 0 . 006-0 . 125 ìg ml À1 for ketoconazole, 0 . 002-0 . 5 ìg ml À1 for amphotericin B and 0 . 002-0 . 016 ìg ml À1 for voriconazole. Two of the isolates were resistant to 5-flucytosine (.32 ìg ml À1 ), but none against fluconazole. The study reinforces the current view that C. dubliniensis has a much wider geographical and epidemiological distribution.

Diagnostic Microbiology and Infectious Disease, 2000

International Journal of Antimicrobial Agents, 2005

Mutations associated with rifampicin (RIF) resistance in two regions of the rpoB gene were studie... more Mutations associated with rifampicin (RIF) resistance in two regions of the rpoB gene were studied by line probe (INNO-LiPA Rif. TB) assay (LiPA) and/or DNA sequencing in 32 RIF-resistant and 21 RIF-susceptible Mycobacterium tuberculosis strains isolated from 53 tuberculosis patients in Kuwait. The LiPA identified all susceptible strains as RIF sensitive, and the DNA sequences of the 81 bp rifampicin resistance-determining region (RRDR) and the N-terminal region of the rpoB gene from five isolates were identical to the wild-type sequences from M. tuberculosis H 37 Rv. The LiPA identified 24 of 32 (75%) phenotypically documented RIF-resistant M. tuberculosis isolates as RIF resistant, with specific detection of mutation in 19 isolates, whilst 8 strains were identified as RIF susceptible. DNA sequencing of the RRDR confirmed the results for the 19 RIF-resistant isolates and identified precise mutations in five isolates for which specific base changes could not be determined by LiPA, as well as identifying a mutation (insertion 514TTC) in three isolates and no mutation in five of the eight isolates that were detected as RIF susceptible by LiPA. Two of the latter five isolates contained a V146F mutation in the N-terminal region of the rpoB gene and were recovered from patients of Middle Eastern origin. These analyses identified 11 different mutations in two regions of the rpoB gene. The majority of the isolates carrying identical or no mutations in the rpoB gene exhibited unique DNA banding patterns in fingerprinting studies. The occurrence of rare mutations in the rpoB gene, namely insertion 514TTC in 3 (9%) and V146F in 2 (6%) of 32 RIF-resistant M. tuberculosis isolates from Kuwait, is the highest reported so far.

Clinical Microbiology and Infection, 2004

This study evaluated sunflower (Helianthus annuus) seed agar (SSA) for differentiation of Candida... more This study evaluated sunflower (Helianthus annuus) seed agar (SSA) for differentiation of Candida dubliniensis from Candida albicans on the basis of colony characteristics and chlamydospore production. Simplified SSA without creatinine and KH 2 PO 4 was also used . On both media, C. dubliniensis isolates (n = 25) developed rough colonies and formed abundant chlamydospores after incubation for 24-48 h at 28°C, while C. albicans isolates (n = 53) showed smooth colonies with no evidence of chlamydospore formation. Cryptococcus neoformans isolates (n = 10) formed brown colonies on both media. Simplified SSA offers a simple and inexpensive tool for presumptive differentiation of C. dubliniensis from C. albicans in clinical microbiology laboratories.

International Journal of Medical Microbiology, 2004

We have developed a semi-nested PCR-enzyme immunoassay (snPCR-EIA) for the detection of Candida s... more We have developed a semi-nested PCR-enzyme immunoassay (snPCR-EIA) for the detection of Candida species in serum specimens, and the sensitivity of amplicon detection was compared with the detection of amplified product by agarose gel electrophoresis (AGE). The universal outer primers amplified the 3 0 end of 5.8S and the 5 0 end of 28S rDNA including the internally transcribed spacer 2 (ITS2) in PCR with genomic DNA as template from all the tested Candida species. The biotin-labeled species-specific primers derived from ITS2 from the four commonly encountered Candida species, viz. C. albicans, C. tropicalis, C. parapsilosis and C. glabrata, together with digoxigenin-labeled reverse primer amplified species-specific DNA in the reamplification step of the snPCR. The snPCR-EIA was positive for genomic DNA recovered from 0.06 Candida cells in culture and one organism/ml in spiked serum specimens. Evaluation of snPCR-EIA and snPCR-AGE for specific identification of Candida species with 26 clinical Candida isolates showed 100% concordant results with Vitek and ID32C yeast identification systems. Further evaluation of snPCR-EIA and snPCR-AGE for detection of Candida species in serum samples from culture proven (n ¼ 6) and suspected (n ¼ 10) patients showed concordance with the corresponding species isolated in culture. The serum samples from none of the healthy volunteers (n ¼ 10) were positive for the presence of Candida DNA by snPCR-EIA or snPCR-AGE. Our results show that the snPCR-EIA has the same sensitivity as snPCR-AGE, however, it offers additional advantages of simultaneous testing of a large number of serum samples and avoids the use of ethidium bromide, a potent mutagen. The snPCR-EIA could, therefore, be a method of choice for the diagnosis of candidemia.

Several clinical observations suggest the superiority of icodextrin compared with 4.25% dextrose ... more Several clinical observations suggest the superiority of icodextrin compared with 4.25% dextrose in optimizing peritoneal ultrafiltration (UF), but no rigorous controlled evaluation has hitherto been performed. For comparing icodextrin and 4.25% dextrose during the long dwell of automated peritoneal dialysis, a multicenter, randomized, double-blind trial was conducted in 92 patients (control, 45; icodextrin, 47) with 4-h dialysate to plasma ratio creatinine >0.70 and D/D 0 glucose <0.34. Long-dwell net UF and the UF efficiency ratio (net UF volume per gram of dialysate carbohydrate absorbed) were determined at baseline, week 1, and week 2. The control and treatment groups were comparable at baseline (all patients using 4.25% dextrose for the long dwell) with regard to mean (؎SEM) net UF (201.7 ؎ 103.1 versus 141.6 ؎ 75.4 ml, respectively; P ‫؍‬ 0.637) and the percentage of patients with negative net UF (control, 37.8%; treatment, 42.6%; P ‫؍‬ 0.641). During the study period, net UF was unchanged from baseline in the control group but increased significantly (P < 0.001) in the icodextrin group from 141.6 ؎ 75.4 to 505.8 ؎ 46.8 ml at week 1 and 540.2 ؎ 46.8 ml at week 2. In the icodextrin group, the incidence of negative net UF was significantly lower (P < 0.0001) than in the control group. Findings were similar for UF efficiency ratio. Rash was reported significantly more often in the icodextrin group. This study showed that in high-average and high transporters, icodextrin is superior to 4.25% dextrose for long-dwell fluid and solute removal.