ADAMTS-13 antigen and activity levels in thrombocytopenic disorders including thrombotic thrombocytopenic purpura in Kuwait.

BACKGROUND/AIMS: While deficiency of a disintegrin and metalloprotease with thrombospondin-1-like domains (ADAMTS-13) was reported as the basis for the pathogenesis of thrombotic thrombocytopenic purpura (TTP), low levels have also been found in other thrombocytopenic disorders. This study was conducted to characterize the activity and antigen levels of ADAMTS-13 and von Willebrand factor (vWF) in patients with different thrombocytopenic disorders in Kuwait. METHODS: Forty healthy subjects and 41 patients with different thrombocytopenic disorders were recruited for this study. ELISA tests were used to measure ADAMTS-13 and vWF activity and antigen levels in patients and controls. RESULTS: All TTP patients had severely deficient ADAMTS-13 activity (<5%), which was significantly lower than that of controls (p < 0.001). Severe deficiency of ADAMTS-13 was also found in some, but not all, patients with idiopathic thrombocytopenic purpura, acute leukemia and sepsis. CONCLUSIONS: This study is the first to report ADAMTS-13 levels in this part of the world. ADAMTS-13 was found to be severely deficient in TTP patients. We present evidence that significantly lower levels of ADAMTS-13 were not specific for TTP and can be found in other thrombocytopenic disorders. We also hypothesize that clinical manifestation of TTP may not be solely due to ADAMTS-13 deficiency, and there might be other contributing factors, since the deficiency was also found in some healthy controls.

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.

Megaloblastosis: from morphos to molecules.

OBJECTIVE: Megaloblastosis (i.e., megaloblastic transformation of erythroid precursor cells in the bone marrow) is the cytomorphological hallmark of megaloblastic anemia resulting from vitamin B12 and folate deficiency. It is characterized by a finely stippled lacy pattern of nuclear chromatin, which is believed to be an expression of deranged cellular DNA synthesis. However, the molecular basis of these cytomorphological aberrations still remains obscure. The current presentation describes the results of our studies on some molecular events associated with the development of megaloblastosis. METHODS: Transmission electron microscopy was used to study megaloblasts as well as DNA fibers extracted from megaloblastic and normoblastic bone marrows with and without treatment with proteinase K during the extraction procedure; cellular DNA synthesis in bone marrow cultures was studied by incorporation of 3H-thymidine and deoxyuridine suppression test, while histone biosynthesis in bone marrow cells was studied by in vitro incorporation of 3H-tryptophan, 3H-lysine and 3H-arginine into histones. RESULTS: Derangement of DNA synthesis occurred due to an impaired de novo pathway of thymidylate synthesis in both vitamin-B12- and folate-deficient human megaloblastic bone marrows as well as in the bone marrows of rhesus monkeys and rats with experimentally induced folate deficiency. Interestingly, folate-deficient monkeys developed frank megaloblastic bone marrows, but folate-deficient rats did not. On the other hand, megaloblastic changes in the bone marrow of human patients with myelodysplastic syndrome and erythroleukemia were not associated with this DNA synthetic abnormality. Biosynthesis of predominantly arginine-rich histones in megaloblastic bone marrows was markedly reduced as compared to normoblastic bone marrows, which was consistently associated with elongation and despiralization of chromosomes and finely stippled nuclear chromatin in megaloblasts. CONCLUSION: The impaired biosynthesis of predominantly arginine-rich nuclear histones appeared to be a common molecular event (a denominator) underlying the development of megaloblastosis with or without abnormal DNA synthesis.

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.

Zinc and liver cirrhosis: biochemical and histopathologic assessment.

Experimental liver cirrhosis was produced by administration of thioacetamide. Cirrhotic animals were divided into two groups: one group was given zinc sulphate and the second kept as cirrhotic control. Zinc-treated animals showed a restoration of normal hepatic and plasma zinc and copper levels. Similarly, plasma levels of aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl aminotransferase, and total bilirubin decreased significantly. Light microscopic studies showed that most of the hepatocytes appeared normal in zinc-treated as compared with untreated cirrhotic animals. The amount of fibrin, reticulin, and collagen, which was high in the cirrhotic livers, decreased following zinc treatment. Staining with periodic acid Schiff's reagent showed the ability of hepatocytes to store glycogen after zinc treatment. These results revealed that zinc may have some beneficial effect in the treatment of liver cirrhosis.