Evaluation of associations between single nucleotide polymorphisms in the FRMD3 and CARS genes and diabetic nephropathy in a Kuwaiti population.

Diabetic nephropathy is the leading cause of end-stage kidney disease in the world. Many single nucleotide polymorphisms (SNPs) have been associated with diabetic nephropathy. SNPs at the 4.1 protein ezrin, radixin, moesin domain 3 (FRMD3) and cysteinyl t-RNA synthetase (CARS) genes have a well-established relationship with diabetic nephropathy. However, this association has not been evaluated in a Kuwaiti population. DNA was extracted from blood samples obtained from patients with diabetic nephropathy (N = 38); the genes of interest were amplified, and the SNPs were genotypes. Diabetics without nephropathy (N = 64) were used as controls. The risk (G and C) and non-risk (C and T) allele frequencies of the SNPs at the rs1888747 and rs739401 loci of FRMD3 and CARS, respectively, did not differ significantly between the diabetics with (case) and without (control) nephropathy (P > 0.05). These findings suggest that the molecular mechanisms involved in diabetic nephropathy may be different in a Kuwaiti population, compared to other populations (such as Japanese and Caucasian Europeans). The discrepancies observed in our study could also be attributed to the smaller sample size analyzed in this study. Therefore, further analyses with larger samples are required to identify the susceptibility genes in a Middle-Eastern population.

Retrospective study of an outbreak in a Kuwaiti hospital of multidrug-resistant Klebsiella pneumoniae possessing the new SHV-112 extended-spectrum beta-lactamase.

Patients infected with bacteria producing extendedspectrum beta-lactamases (ESBL) are at higher risk of mortality and morbidity. Several mutations in genes encoding SHV, tem and CTX-M beta-lactamases have been associated with ESBL activity. This paper describes a new SHV mutation in ESBL-producing strains of Klebsiella pneumoniae isolated in Kuwait. The study included 13 K. penumoniae strains isolated from patients admitted to the Amiri hospital of Kuwait. The production of ESBL in all strains was confirmed by Vitek system and E-test. All the ESBL genes were amplified by PCR and examined by DNA sequencing. All these ESBL-positive isolates were resistant to ceftazidime and cefotaxime. DNA sequencing revealed an A815G point mutation in the bla (SHV )gene causing an asparagine (AAT) to aspartic acid (GAT) mutation at position 253 of the enzyme. This new mutation was assigned the unique number SHV-112, and the Genebank accession number EU477409. This study reports a new mutation in the SHV gene in K. pneumoniae with ESBL capability. There could be other mutations still to be found in ESBL genes of K. pneumoniae in Kuwait and probably in other middle eastern countries, and researchers in the region should make use of molecular techniques to look for more novel mutations in ESBL-producing strains of K. pneumoniae.

Detection of mutations in the gyrA gene in fluoroquinolone resistance Salmonella enterica serotypes typhi and paratyphi A isolated from the Infectious Diseases Hospital, Kuwait.

BACKGROUND: Enteric fever due to Salmonella enterica is a major health problem, and fluoroquinolones such as ciprofloxacin are mostly the antibiotic of choice for treatment. Resistance to ciprofloxacin has been noticed to increase due to the emergence of new mutations in the bacterial DNA. AIMS: To explore the fluoroquinolone resistance and molecular characterisation of reduced quinolone susceptibility in S typhi and S paratyphi A in Kuwait. METHODS: 136 clinical isolates of S typhi and 40 of S paratyphi A were collected over five years. The antimicrobial susceptibility was studied by various methods. DNA sequencing of gyrA, gyrB, parC and parE genes was performed in 31 isolates. RESULTS: There was a substantial difference in MIC range between the two serotypes, with the most common MIC for S typhi being 0.25 mg/l and for S paratyphi A being 1 mg/l. The proportion of nalidixic acid resistant strains increased gradually over the years. These strains had a significantly higher range of MIC of ciprofloxacin (0.023 mg/l to 1.0 mg/l) compared to the nalidixic acid sensitive strains (0.0016 mg/l to 0.125 mg/l). DNA sequencing of gyrA gene showed the presence of three different point mutations: Ser83-->Phe in 17 strains, Ser83-->Leu in 3 strains and Asp87-->Asn in 6 strains. No mutations in the other genes were found. CONCLUSIONS: It is very important to keep searching for new mutations and continuously monitor drug resistance in different parts of the world in order to efficiently manage cases with enteric fever.

Can we rely on one laboratory test in detection of extended-spectrum beta-lactamases among Enterobacteriaceae? An evaluation of the Vitek 2 system and comparison with four other detection methods in Kuwait.

BACKGROUND AND AIMS: Clinical hospitals need to correctly identify extended spectrum beta-lactamase (ESBL)-producing bacteria in infected patients to correctly treat the patient and avoid spreading antibiotic resistance. Kuwaiti hospitals use one laboratory test for detecting ESBL bacteria. This study evaluated whether that was sufficient to detect ESBL bacteria, and compared the Vitek system with other detection systems. METHODS: (Klebsiella pneumoniae, Escherichia coli, K oxytoca and Enterobacter cloacae) were collected from five different Kuwaiti main hospitals, all of which were flagged as ESBL-positive by the Vitek 2 system. The isolates were retested by the Vitek 2 system, and were also tested by double disc diffusion (DDD), the disc approximation test, the E-test and the MicroScan system for the detection of ESBLs. RESULTS: Retesting with the Vitek system revealed 100% compatibility with the results of the source hospitals. The MicroScan system, DDD, disc approximation test and E-test could detect ESBL in 199, 192, 178 and 205 isolates, respectively. CONCLUSIONS: Technically, the MicroScan and Vitek 2 systems were the least demanding method to detect ESBL as it is an integral part of the routine susceptibility test card. E-test strips were reliable but the most expensive of all techniques used. The DDD test and disc approximation, while relatively inexpensive, were technically subjective. The Vitek system may be very suitable in clinical laboratories, but would be better if accompanied with another test for detection of ESBL bacteria.

Salmonella enterica Serotype typhi in Kuwait and its reduced susceptibility to ciprofloxacin.

Salmonella enterica serotype typhi continues to be an important public health problem in Kuwait. Analysis of the isolates from 163 patients, collected between 1995 and 2003, showed that the majority were from patients from the Indian sub-continent, including 45 from Bangladesh, 38 from India and 30 from Pakistan. Fifty-four of the strains showed multiple antibiotic resistance (MDR). Twenty-five strains were from Kuwaitis, with 15 aged <18 years. Bacteriophage typing of 20 isolates from Kuwaitis revealed that they belonged to 8 different phage types, and that the 3 MDR strains were phage type E1. Random amplified polymorphic DNA typing showed genetic variability amongst isolates from Kuwaiti patients. This method conveniently demonstrated the identity of 4 isolates associated with a small outbreak. 48 isolates from 2002-3 were tested for reduced susceptibility to quinolones. 12 of 18 MDR strains and 7/30 susceptible strains showed reduced susceptibility to ciprofloxacin (minimum inhibitory concentration 0.125-0.5 mg/L). All 12 strains were tested for mutation in the quinolone resistance determining region (QRDR) of the gyr A gene. The mutation ser83 phe was detected in the 10 strains tested. Thus typhoid fever in Kuwait is predominantly associated with those who have traveled from endemic areas to work in Kuwait. The incidence of MDR strains remains at about 30%. Reduced susceptibility to ciprofloxacin in MDR S. typhi has increased from (11%) in 1995-1996 to (67%) in 2002-2003 and from (0%) to (23%) in susceptible strains. Mutation of the gyrA gene is the mechanism most often responsible.

Extended-spectrum beta-lactamase-producing Escherichia coli isolated in the Al-Amiri Hospital in 2003 and compared with isolates from the Farwania hospital outbreak in 1994-96 in Kuwait.

Extended-spectrum beta-lactamases (ESBLs) are a major problem in Kuwait and an accurate method for their detection is essential. This study was designed to evaluate the efficacy of the commercial system (Vitek 2) to identify ESBLs in clinical isolates of Escherichia coli and relate this to their identification by agar dilution methods for use in a diagnostic laboratory. The presence of the major ESBLs parental enzyme groups was confirmed by PCR and the similarity of the strains was determined by pulsed field gel electrophoresis (PFGE) on DNA, cleaved using XbaI endonuclease, to identify clonal spread.Seventy-one separate E. coli isolates from 65 patients were tested. Sixty-two isolates were from 56 patients from the Al-Amiri Hospital and nine isolates from neonates from Farwania Hospital. The isolates were screened for ESBL activity by the Vitek 2 system. Isolates showing positive results were further tested with Etest ESBL strips and by the disc approximation methods. All the isolates were flagged as ESBL-positive by the Vitek 2 advanced expert system (AES). Isolates from all the 65 patients were detected as ESBL positive by the Etest, only if both ESBL strips were used. The double disc approximation test using five different antibiotics could detect ESBL presence in isolates from only 46 patients. In this test, the synergy with cefepime was the most sensitive in ESBL detection, showing their presence in 41 isolates. PCR with primers for bla(TEM) and bla(SHV) demonstrated that one or both of these enzymes in all isolates. PFGE revealed that many different clones were present amongst the isolates. The epidemiology of ESBL E. coli in Kuwait is complex. Many distinct strains are already present in the population, as shown by the results of PFGE. Several testing methods may be required to detect all strains harboring ESBLs.

Characterization of extended-spectrum beta-lactamase (ESBL)-producing Kuwait and UK strains identified by the Vitek system, and subsequent comparison of the Vitek system with other commercial ESBL-testing systems using these strains.

Two hundred and fifty-one unique patient isolates of Klebsiella pneumoniae (123), Escherichia coli (114), Klebsiella oxytoca (7), Enterobacter cloacae (5) and Citrobacter freundii (2), flagged as extended-spectrum beta-lactamase (ESBL) positive by the Vitek system (GNS-526 card), were collected. These strains were isolated from a variety of clinical specimens submitted to the clinical bacteriology laboratories of the Royal Infirmary of Edinburgh (RIE), Edinburgh, UK (and associated GP practices), Hairmyers Hospital, Glasgow, UK, and the Amiri and Farwania Hospitals, Kuwait. Of the 101 RIE strains tested, 15 E. coli strains were found to be ESBL negative by Etest ESBL strips. On retesting the 15 E. coli strains with the Vitek GNS-532 card, 14 were found to be ESBL negative, despite being originally flagged as ESBL positive. The remaining 236 ESBL-producing strains were also subjected to the double disc-diffusion (DDD) technique for the detection of ESBLs. Of these, two were false negatives by Etest ESBL test strips (using both cefotaxime and ceftazidime strips), and 38 were false negatives by the DDD method. The Etest false-negative ESBL-producing strains of K. pneumoniae were positive by DDD. Technically, the Vitek method was the least demanding method to perform, as it was an integral part of the routine susceptibility test card. Etest strips were reliable, but were the most expensive of all the techniques used. The DDD test, while relatively inexpensive, was technically subjective, and in our hands, seven of the ESBL-positive strains that were confirmed by the other two techniques were not detected. Despite the false-positive ESBL-producing E. coli strains, the Vitek susceptibility card with its integral ESBL test offers the clinical laboratory a valuable and quick option to screen for ESBL-producing Klebsiella spp. and E. coli as part of the routine laboratory methodology.

Linkage of ciprofloxacin resistance with a single genotypic cluster of Klebsiella pneumoniae.

The objective of this study was to examine the epidemiology of ciprofloxacin-resistant, extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae strains. Sixty-nine unique patient isolates of K. pneumoniae isolated from a variety of clinical specimens submitted to the clinical bacteriology laboratories of The Royal Infirmary of Edinburgh and associated General Practices were identified and susceptibility testing was performed with the Vitek system. Strains flagged as ESBL-positive by the Vitek system were subjected to isoelectric focusing. The results suggested that all 69 isolates harboured at least one ESBL, which was later confirmed by polymerase chain reaction (PCR) with bla(TEM) and/or bla(SHV) primers. The purified PCR product was subjected to automated sequencing and the results were compared with the BLAST online search engine. Of the 69 isolates, 32 (46.4%) were found to be resistant to ciprofloxacin, 11 (15.9%) were intermediate and 26 (37.7%) were sensitive. To investigate the epidemiological relationship between the ciprofloxacin-resistant ESBL-positive strains, pulsed-field gel electrophoresis (PFGE) was performed. Rapidest software was used to calculate the genetic distance by the Nei distance method. PFGE analysis indicated that the clinical isolates belonged to four distinct genotype clusters (Groups A, B, C and D); each group or cluster was homogeneous or compact with respect to certain characteristics. Group A consisted of 25 isolates, group B of 3 isolates and Groups C and D of 2 isolates each. These results indicate that the spread of resistance is largely as a result of the dissemination of a single clonal strain. PCR was used to amplify the gyrA and parC genes from genomic DNA of the ciprofloxacin-resistant isolates. The amplified product was sent for analysis by automated DNA sequencing and the resulting DNA sequences were compared with the gyrA gene of K. pneumoniae. The sequencing results demonstrated that alteration of the GyrA subunit of DNA gyrase at amino acid 83 and/or amino acid 87 plays a central role in conferring high-level quinolone resistance in K. pneumoniae possessing ESBLs.

A comparative study on methods of measuring mesiodistal tooth diameters for interceptive orthodontic space analysis.

AIM: This study was designed to find the most reliable method of measurement of mesiodistal tooth diameter. METHODS: Measurements were made of all erupted permanent teeth of 14 orthodontic study casts. These measurements were made directly by using A) a digital calliper, B) measuring photocopies of casts with a calliper, C) a Magiscan Image Analysis using a photocopy of the casts. Measurements derived from the two methods were compared by statistical analysis. RESULTS: These showed that the electronic digital calliper was the most reliable method of measuring mesiodistal tooth diameter using dental study casts. The measurement of photocopies was unreliable and the image analysis method had a too high error factor.

HR2 haplotype in Arab population and patients with venous thrombosis in Kuwait.

BACKGROUND: Venous thromboembolism (VTE) occurs due to a number of hereditary and acquired disorders of hemostasis. A recently identified polymorphism in factor V gene (A4070G; named HR2) has been reported to be a possible risk factor for the development of VTE, with a high prevalence of 9.5%-15.2% in patients of different ethnic groups in different parts of the world. However, the prevalence of HR2 has not yet been tested in VTE patients of Arab ethnicity. OBJECTIVES: To study the prevalence and possible risk of HR2 haplotype in Arabs. PATIENTS/METHODS: Exactly 188 VTE patients and 100 healthy subjects, all being of Arab ethnicity, were examined for HR2 using Polymerase chain reaction, restriction fragment length polymorphism and agarose gel electrophoresis. RESULTS: Data showed that 31 patients and seven healthy subjects had HR2 haplotype, with a prevalence of 16.5% and 7%, respectively. Furthermore, 43 patients (22.9%) had more than one risk factor for VTE. CONCLUSIONS: The prevalence of HR2 in Arabs is quite high, with a 2.62-fold greater risk of developing VTE. Moreover, coexistence of two or more genetic/acquired defects of VTE is quite common in Arab patients.

Detection of genes encoding aminoglycoside-modifying enzymes in staphylococci by polymerase chain reaction and dot blot hybridization.

Dot blot hybridization and the polymerase chain reaction (PCR) were used to study aminoglycoside-modifying enzymes in aminoglycoside-resistant staphylococci isolated in hospitals in Kuwait. DNA encoding the acetyltransferase (AAC) (6')-phosphotransferase (APH) (2"), nucleotidyltransferase (ANT) (4') and APH (3') enzymes were detected in Staphylococcus aureus and coagulase negative staphylococci. ANT (4') was the most common enzyme detected. The majority of isolates contained genes for all three modifying enzymes, AAC (6')-APH (2"), ANT (4') and APH (3'); only few isolates carried genes for a single modifying enzyme. Genes encoding the AAC (6')-APH (2") were detected in all except two gentamicin-resistant isolates. In these isolates the genes for the AAC (6')-APH (2") enzyme could not be detected by PCR and dot blot hybridization. Whereas antibiotic resistance testing could be used to predict the presence of the AAC (6')-APH (2") enzyme it was not useful in predicting the presence of the ANT (4') or APH (3') enzymes in gentamicin-resistant isolates. Results obtained with dot blot hybridization were comparable to those obtained with PCR. However, PCR was fast and results were obtained within the same day. Therefore PCR would be preferred for the detection and confirmation of the presence of aminoglycoside-modifying enzymes in clinical microbiology laboratories.

Breast feeding, bottle feeding and dental caries in Kuwait, a country with low-fluoride levels in the water supply.

A study was carried out in Kuwait, a country with low levels of fluoride in the water supplies, to determine the prevalence and extent of caries in early childhood, and enquire into associated factors. Mothers of pre-schoolchildren were interviewed, and their children aged 18 to 48 months received a dental examination. Of the 227 children examined, 107 (47 per cent) were caries free, and 41 (18%) had five or more dmf teeth. 'Nursing caries', affecting at least two maxillary incisors, was seen in 19 per cent of the sample. Breast fed children were significantly more likely to be caries free than those who were bottle fed from birth, although 'nursing caries' was positively associated with the practice of breast feeding at night 'at will' after 6 months of age (P < 0.01). Bottle fed children were more likely to develop caries, including 'nursing caries', particularly when the practice was continued to an older age. It was concluded that 'nursing caries' constitutes a significant dental health problem in Kuwait.