Eiman Mokaddas

Recent advances in the diagnosis and treatment of multidrug-resistant tuberculosis

Tuberculosis (TB) is a major infectious disease killing nearly two million people, mostly in developing countries, every year. The increasing incidence of resistance of Mycobacterium tuberculosis strains to the most-effective (first-line) anti-TB drugs is a major factor contributing to the current TB epidemic. Drug-resistant strains have evolved mainly due to incomplete or improper treatment of TB patients. Resistance of M. tuberculosis to anti-TB drugs is caused by chromosomal mutations in genes encoding drug targets. Multidrug-resistant (resistant at least to rifampin and isoniazid) strains of M. tuberculosis (MDR-TB) evolve due to sequential accumulation of mutations in target genes. Emergence and spreading of MDR-TB strains is hampering efforts for the control and management of TB. The MDR-TB is also threatening World Health Organizations target of tuberculosis elimination by 2050. Proper management of MDR-TB relies on early recognition of such patients. Several diagnostic methods, both phenotypic and molecular, have been developed recently for rapid identification of MDR-TB strains from suspected patients and some are also suitable for resource-poor countries. Once identified, successful treatment of MDR-TB requires therapy with several effective drugs some of which are highly toxic, less efficacious and expensive. Minimum treatment duration of 18-24 months is also long, making it difficult for health care providers to ensure adherence to treatment. Successful treatment has been achieved by supervised therapy with appropriate drugs at institutions equipped with facilities for culture, drug susceptibility testing of MDR-TB strains to second-line drugs and regular monitoring of patients for adverse drug reactions and bacteriological and clinical improvement.

Tobacco Agar, a New Medium for Differentiating Candida dubliniensis from Candida albicans

ABSTRACT Isolates of Candida dubliniensis may be misidentified as Candida albicans in microbiological laboratories if only the germ tube and/or the chlamydospore test is used for identification to the species level. In this study, we have evaluated the efficacy of tobacco agar for the differentiation of C. dubliniensis from C. albicans. On this medium at 28°C, all 30 C. dubliniensis isolates produced yellowish-brown colonies with hyphal fringes and abundant chlamydospores, whereas 54 C. albicans isolates formed smooth, white-to-cream-colored colonies with no chlamydospore production. This medium provides a simple tool for presumptive differentiation of C. dubliniensis from C. albicans.

Characterization of rpoB mutations in rifampin-resistant clinical Mycobacterium tuberculosis isolates from Kuwait and Dubai

Mutations conferring resistance to rifampin in rifampin-resistant clinical Mycobacterium tuberculosis isolates occur mostly in the 81 bp rifampin-resistance-determining region (RRDR) of the rpoB gene. In this study, 29 rifampin-resistant and 12 -susceptible clinical M. tuberculosis isolates were tested for characterization of mutations in the rpoB gene by line probe (INNO-LiPA Rif. TB) assay and the results were confirmed and extended by DNA sequencing of the PCR amplified target DNA. The line probe assay identified all 12 susceptible strains as rifampin-sensitive and the DNA sequence of RRDR in the amplified rpoB gene from two isolates matched perfectly with the wild-type sequence. The line probe assay identified 28 resistant isolates as rifampin-resistant with specific detection of mutation in 22 isolates including one isolate that exhibited hetro-resistance containing both the wild-type pattern as well as a specific mutation within RRDR while one of the rifampin-resistant strain was identified as rifampin-susceptible. DNA sequencing confirmed these results and, in addition, led to the specific detection of mutations in 5 rifampin-resistant isolates in which specific base changes within RRDR could not be determined by the line probe assay. These analyses identified 8 different mutations within RRDR of the rpoB gene including one novel mutation (S522W) that has not been reported so far. The genotyping performed on the isolates carrying similar mutations showed that majority of these isolates were unique as they exhibited varying DNA banding patterns. Correlating the ethnic origin of the infected TB patients with the occurrence of specific mutations at three main codon positions (516, 526 and 531) in the rpoB gene showed that most patients (11 of 15) from South Asian region contained mutations at codon 526 while majority of isolates from patients (6 of 11) of Middle Eastern origin contained mutations at codon 531.

Variations in the Occurrence of the S315T Mutation Within the katG Gene in Isoniazid-Resistant Clinical Mycobacterium tuberculosis Isolates from Kuwait

The worldwide threat of drug-resistant tuberculosis (TB) to human health has led to the development of molecular methods for rapidly determining the resistance of clinical Mycobacterium tuberculosis isolates to the two front-line antituberculous drugs, isoniazid and rifampin. The prevalence of the S315T mutation within the katG gene, which confers clinically significant resistance to isoniazid, was determined in isoniazid-resistant clinical M. tuberculosis isolates recovered from TB patients in Kuwait. A total of 67 isoniazid-resistant and 18 susceptible clinical M. tuberculosis isolates were tested. The mutation S315T was found in 46 (69%) of the 67 resistant strains, whereas none of the susceptible strains contained this mutation. The prevalence of this mutation was highest (32 of 40, 80%) in isolates recovered from patients of South Asian origin and lowest in isolates from patients of Middle Eastern origin (8 of 18, 44%). The genotyping performed on isolates carrying the S315T mutation showed that the isolates belong to several different types as they exhibited unique DNA banding patterns. The results point to a varying prevalence of the S315T mutation within the katG gene in clinical M. tuberculosis isolates recovered from patients of different ethnic groupings within the same country. The results also suggest that detection of the S315T mutation in the katG gene may be used as a rapid screening method for identifying isoniazid-resistant clinical M. tuberculosis isolates recovered from majority of patients in some ethnic groupings.

In vitro activity of 15 antimicrobial agents against clinical isolates of Clostridium difficile in Kuwait

A total of 73 clinical isolates of Clostridium difficile isolated from stool/rectal swabs of patients admitted to the intensive care units at Mubarak Hospital, Ibn Sina Hospital Burn unit and Haematology wards at the Kuwait Cancer Control Centre, were investigated for their susceptibility to 15 antibiotics using the Etest. Amoxycillin-clavulanic acid, ampicillin, meropenem, metronidazole, penicillin, piperacillin, piperacillin/tazobactam, teicoplanin and vancomycin had excellent activities with MIC(90)s of 0.38, 0.5, 1, 0.19, 1.5, 2, 3, 0.25 and 0.75 mg/l, respectively. Of the 73 C. difficile isolates, 86% were resistant to imipenem (MIC(90) >32 mg/l) and almost 97% were resistant to trovafloxacin (MIC(90)>256 mg/l). Forty eight percent of the isolates were resistant to clindamycin. A total of 18 isolates were highly clindamycin-resistant with an MIC of >256 mg/l; 10 of these were toxin producers. Multiple antibiotic resistance (two or more antibiotics) was noted in 63 isolates. These were more common among the toxigenic strains than the non-toxigenic strains by a ratio of 2.5:1.

Levels of (1→3)-β-D-glucan, Candida mannan and Candida DNA in serum samples of pediatric cancer patients colonized with Candida species

BackgroundSurveillance cultures may be helpful in identifying patients at increased risk of developing invasive candidiasis. However, only scant information exists on the effect of Candida colonization on serum levels of diagnostic biomarkers. This prospective surveillance study determined the extent of Candida colonization among pediatric cancer patients and its possible impact on serum levels of (1-3)-β-D-glucan (BDG), Candida mannan and Candida DNA.MethodsA total of 1075 swabs originating from oropharynx (n = 294), nostrils (n = 600), rectum (n = 28), groin (n = 50), ear (n = 54), and axilla (n = 49) of 63 pediatric cancer patients were cultured for the isolation of Candida spp. Patients yielding Candida spp. from any sites were considered as colonized. Serum samples were collected from patients at the time of first surveillance culture for detection of BDG by Fungitell kit and Candida mannan by Platelia Candida Ag. Candida DNA was detected by using panfungal primers and identification was carried out by using species-specific primers and DNA sequencing.ResultsSeventy-five (7.6%) swab cultures from 35 (55.5%) patients yielded Candida spp. These isolates included C. albicans (n = 62), C. dubliniensis (n = 8), C. glabrata and C. tropicalis (n = 2 each) and C. krusei (n = 1). Eleven patients were colonized at three or more sites. Eight of 36 serum samples from 6 colonized patients yielded BDG values higher than the currently recommended cut-off value of ≥80 pg/ml. However, none of the serum samples yielded Candida mannan levels ≥0.5 ng/ml and PCR test for Candida DNA was also negative in all the serum samples of colonized patients. During the study period, only two colonized patients subsequently developed candidemia due to C. tropicalis. Besides positive blood cultures, C. tropicalis DNA, BDG and Candida mannan were also detected in serum samples of both the patients.ConclusionsThe present study demonstrates that while mucosal colonization with Candida species in pediatric cancer patients is common, it does not give rise to diagnostically significant levels of Candida mannan or Candida DNA in serum specimens. However, BDG values may be higher than the cut-off value in some pediatric patients without clinical evidence of invasive Candida infection. The study suggests the utility of Candida mannan or Candida DNA in the diagnosis of invasive candidiasis, however, the BDG levels in pediatric cancer subjects should be interpreted with caution.

Antibacterial resistance and their genetic location in MRSA isolated in Kuwait hospitals, 1994-2004

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) continues to be a major cause of serious infections in hospitals and in the community worldwide. In this study, MRSA isolated from patients in Kuwait hospitals were analyzed for resistance trends and the genetic location of their resistance determinants.MethodsBetween April 1994 and December 2004, 5644 MRSA isolates obtained from different clinical samples were studied for resistance to antibacterial agents according to guidelines from the National Committee for Clinical Laboratory Standards and the British Society for Antimicrobial Chemotherapy. The genetic location of their resistance determinants was determined by curing and transfer experiments.ResultsThey were resistant to aminoglycosides, erythromycin, tetracycline, trimethoprim, fusidic acid, ciprofloxacin, chloramphenicol, rifampicin, mupirocin, cadmium acetate, mercuric chloride, propamidine isethionate and ethidium bromide but susceptible to vancomycin, teicoplanin and linezolid. The proportion of the isolates resistant to erythromycin, ciprofloxacin and fusidic acid increased during the study period. In contrast, the proportion of isolates resistant to gentamicin, tetracycline, chloramphenicol and trimethoprim declined. High-level mupirocin resistance increased rapidly from 1996 to 1999 and then declined. They contained plasmids of 1.9, 2.8, 3.0, 4.4, 27 and 38 kilobases. Genetic studies revealed that they carried plasmid-borne resistance to high-level mupirocin resistance (38 kb), chloramphenicol (2.8 – 4.4 kb), erythromycin (2.8–3.0 kb) and cadmium acetate, mercuric chloride, propamidine isethionate and ethidium bromide (27 kb) and chromosomal location for methicillin, the aminoglycosides, tetracycline, fusidic acid, ciprofloxacin and trimethoprim resistance. Thus, the 27 kb plasmids had resistance phenotypes similar to plasmids reported in MRSA isolates in South East Asia.ConclusionThe prevalence of resistance to erythromycin, ciprofloxacin, high-level mupirocin and fusidic acid increased whereas the proportion of isolates resistant to gentamicin, tetracycline, chloramphenicol and trimethoprim declined during the study period. They contained 27-kb plasmids encoding resistance to cadmium acetate, mercuric chloride, propamidine isethionate and ethidium bromide similar to plasmids isolated in MRSA from South East Asia. Molecular typing of these isolates will clarify their relationship to MRSA from South East Asia.

Current status and future trends in the diagnosis and treatment of drug-susceptible and multidrug-resistant tuberculosis

The global burden of tuberculosis (TB) is still large. The increasing incidence of drug-resistant, multidrug-resistant (MDR) (resistant to at least rifampicin and isoniazid), and extensively drug-resistant (XDR) (additionally resistant to a fluoroquinolone and kanamycin/amikacin/capreomycin) strains of Mycobacterium tuberculosis and the association of active disease with human immunodeficiency virus coinfection pose a major threat to TB control efforts. The rapid detection of M. tuberculosis strains and drug susceptibility testing (DST) for anti-TB drugs ensure the provision of effective treatment. Rapid molecular diagnostic and DST methods have been developed recently. Treatment of drug-susceptible TB is effective in ≥95% of disease cases; however, supervised therapy for ≥6 months is challenging. Non-adherence to treatment often results in the evolution of drug-resistant strains of M. tuberculosis due to mutations in the genes encoding drug targets. Sequential accumulation of mutations results in the evolution of MDR and XDR strains of M. tuberculosis. Effective treatment of MDR-TB involves therapy with 5-7 less effective, expensive, and toxic second-line and third-line drugs for ≥24 months and is difficult in most developing countries. XDR-TB is generally an untreatable disease in developing countries. Some currently existing drugs and several new drugs with novel modes of action are in various stages of development to shorten the treatment duration of drug-susceptible TB and to improve the outcome of MDR-TB and XDR-TB.

Surveillance of antibacterial resistance in Staphylococcus aureus isolated in Kuwaiti hospitals.

Objective: To investigate the prevalence of antibiotic resistance among Staphylococcus aureus isolated in Kuwaiti hospitals. Materials and Methods:S. aureus were isolated and identified following standard microbiological methods. Antibacterial susceptibility test was performed by disk diffusion and the measurement of minimum inhibitory concentration with E-test strips. Results: A total of 1,846 S. aureus isolates were analyzed from 13 hospitals between 1 March and 30 October 2005. They were isolated from 1,765 (95.6%) inpatients and 81 (4.4%) outpatients. Methicillin resistance was detected in 588 (32.0%) of the isolates. The methicillin-resistant S. aureus (MRSA) consisted of 461 (78%) multiresistant and 127 (22%) nonmultiresistant isolates. The nonmultiresistant MRSA consisted of epidemic MRSA-15 and community-associated MRSA. The community-associated MRSA was detected in all hospitals with MRSA, indicating its establishment in Kuwaiti hospitals. The proportion of isolates resistant to gentamicin, kanamycin, erythromycin, tetracycline, ciprofloxacin, fusidic acid and trimethoprim was higher among MRSA than methicillin-susceptible S. aureus (MSSA) isolates. Twenty-four and 22% of MRSA and MSSA isolates, respectively, expressed reduced susceptibility to vancomycin (minimum inhibitory concentration = 3–4 mg/l). Conclusion: The study revealed the presence of methicillin resistance in 32% of S. aureus isolated in Kuwaiti hospitals and revealed an increase in the number of MRSA and MSSA with reduced susceptibility to vancomycin.

Hospital-Acquired Clostridium difficile Infection amongst ICU and Burn Patients in Kuwait

Objectives: To prospectively study the prevalence of nosocomially acquired Clostridium difficile, a major cause of diarrhoea in hospitalized patients, in the intensive care units (ICUs) and burn unit (BUs) of three teaching hospitals in Kuwait. Methods: During a 1-year prospective study, stool/rectal swabs were obtained from 344 patients admitted into the ICUs of Mubarak Hospital (ICU-1), the Kuwait Cancer Control Centre (ICU-2), and the BU of Ibn Sina Hospital. The presence of C. difficile and/or its toxin was detected by serially culturing the specimens on differential, selective and enriched media and the use of TOX-A/B test, on admission and at subsequent 1-weekly interval until discharge. Results: Out of the 344 patients admitted into these units, over a study period of 1 year, only 263 (77%) were evaluable. All of them had negative stool culture/toxin on admission. Overall, 25 (9.5%) of these 263 patients acquired C. difficile during their hospitalization. Thirteen (7%) of 187 patients acquired C. difficile in ICU-1, 9 (36%) of 25 on ICU-2 and 3 (5.9%) of 51 patients in BU. Eight (32%) developed diarrhoea attributable only to C. difficile and/or toxin, and the remaining 17 (68%) were asymptomatic: none had pseudomembranous colitis. The diarrhoea in these patients was associated with antibiotic use, the main trigger-antibiotics being the third-generation cephalosporins. Acquisition occurred within 4–53 days of admission, with the majority occurring in the first 15 days. Conclusion: Overall, the prevalence of hospital-acquired C. difficile infection/colonization was less than 10%. The use of third-generation cephalosporins was high and was related to the development of diarrhoea. Once acquired, diarrhoea developed in about one third of C. difficile-positive cases, an indication that C. difficile infection/colonization endemic in the hospital ICUs studied is usually transmitted among the hospitalized patients.