Determination of vWF, ADAMTS-13 and Thrombospondin-1 in Venous Thromboembolism and Relating Them to the Presence of Factor V Leiden Mutation.

Thrombophilia in venous thromboembolism (VTE) is multifactorial. Von Willebrand factor (vWF) plays a major role in primary hemostasis. While elevated vWF levels are well documented in VTE, findings related to its cleaving protease (ADAMTS-13) are contradicting. The aim of this study was to determine vWF, ADAMTS-13, and the multifactorial Thrombospondin-1 (TSP-1) protein levels in patients after 3-6 months following an unprovoked VTE episode. We also explored a possible association with factor V Leiden (FVL) mutation. vWF, ADAMTS-13 and TSP-1 were analyzed using ELISA kits in 60 VTE patients and 60 controls. Patients had higher levels of vWF antigen (P = .021), vWF collagen-binding activity (P = .008), and TSP-1 protein (P < .001) compared to controls. ADAMTS-13 antigen was lower in patients (P = .046) compared to controls but ADAMTS-13 activity was comparable between the two groups (P = .172). TSP-1 showed positive correlation with vWF antigen (rho = 0.303, P = .021) and negative correlation with ADAMTS-13 activity (rho = -0.244, P = .033) and ADAMTS-13 activity/vWF antigen ratio (rho = -0.348, P = .007). A significant association was found between the presence of FVL mutation and VTE (odds ratio (OR): 9.672 (95% confidence interval (CI) 2.074-45.091- P = .004), but no association was found between the mutation and the studied proteins (P > .05). There appears to be an imbalance between vWF and ADAMTS-13 in VTE patients even after 3-6 months following the onset of VTE. We report that the odds of developing VTE in carriers of FVL mutation are 9.672 times those without the mutation, but the presence of this mutation is not associated with the studied proteins.

The emergence of plasmid mediated quinolone resistance qnrA2 in extended spectrum ß-lactamase producing Klebsiella pneumoniae in the Middle East.

BACKGROUND: Klebsiella pneumoniae is one of the most important opportunistic pathogens causing serious complications in patients in hospitals and community. The clinical significance of K. pneumoniae is mainly due to its ability to acquire multiple antibiotic resistance genes. In this study we report the findings of a survey of plasmid mediated quinolone resistance in Extended-Spectrum ß-lactamase (ESBL)-producing K. pneumoniae in Kuwait. METHODS: Clinical samples were collected from the microbiology laboratories of three major hospitals. Isolates were confirmed as ESBL-producers by disc diffusion method and PCR for the presence of bla genes. Antimicrobial susceptibility testing and genetic analysis were performed to detect the presence of a number of genes conferring resistance to ß-lactam and fluoroquinolone antimicrobial agents including bla SHV, bla TEM, aac (6')-Ib-cr, qnrA, qnrB and qnrS. Pulsed-field gel electrophoresis (PFGE) was used for typing the isolates. RESULTS: In total 173 ESBL-producing K. pneumoniae were detected. qnr genes were identified in 27 (15.6 %) isolates and aac(6')-Ib Ib-cr gene in 26 (96 %). One (3.7 %) contained qnrA2, 21 harbored qnrB1 (78 %) and 5 (18.5 %) contained qnrS. Twenty one (78 %) isolates contained all three bla genes. PFGE showed diverse profiles. CONCLUSION: We identified for the first time the emergence of the mobile fluoroquinolone resistance qnrA2 in a clinical isolate in the middle east and also showed the dissemination of aac (6')-Ib-cr, qnrB, and qnrS genes among ESBL-producing K. pneumoniae in Kuwait. The abundance of plasmid mediated resistance to fluoroquinolones among ESBL-producing K. pneumoniae is alarming as it facilitates therapy failure. Preventing the spread of these isolates is crucial if we are to sustain the effectiveness of the limited choices we have left in antimicrobial therapy.

Klebsiella oxytoca with reduced sensitivity to chlorhexidine isolated from a diabetic foot ulcer.

In most hospitals, chlorhexidine is used as skin antiseptic prior to clinical procedures, in dressings and when bathing patients. We hereby report, for the first time, the isolation of a clinical Klebsiella oxytoca isolate with reduced sensitivity to chlorhexidine from a foot ulcer of a diabetic patient, which is a common and serious complication associated with diabetes. The Minimum Inhibitory Concentration of the K. oxytoca isolate to chlorhexidine was found to be 30 mg/L and the Minimum Bactericidal Concentration was 60 mg/L. An increased resistance to ethidium bromide (MIC 200 mg/ L) was also observed. Molecular tests revealed that the isolate contained blaCTXM15, blaT(EM-1) and bla(SHV). The other resistant genes detected were qnrB1 and aac(6')-Ib-cr. The resistant determinants were located on a class I integron integrase (intI1) containing qacE gene. DNA sequencing showed homology to K. oxytoca plasmid pACM1. Identification of K. oxytoca with reduced sensitivity to chlorhexidine raises concern regarding dilution standards in hospitals. Adherence to the hospitals' infection control policies should be strictly monitored to avoid continuous low level exposure of bacteria to biocides, specifically in developing countries.

The characterization and antibiotic resistance profiles of clinical Escherichia coli O25b-B2-ST131 isolates in Kuwait.

BACKGROUND: Escherichia coli O25b-B2-ST131 are considered virulent extra-intestinal pathogens causing serious clinical complications such as urinary tract infection and bacteraemia. Our main objectives in this study were to characterise the multi-drug resistant (MDR) isolates of this lineage in Kuwait, and to demonstrate whether reduced susceptibility is spread clonally. RESULTS: A subset of 83 (10%) non-duplicate and non-selective E. coli O25b-B2-ST131 out of 832 MDR E. coli was identified and collected. Minimum inhibitory concentrations of the isolates were determined and pulsed-field gel electrophoresis was used for typing.The majority (95.2%) of the 83 E. coli O25b-B2-ST131 harboured at least one bla gene with blaCTX-M-15 being the most prevalent. blaCTX-M-2 was present in one isolate. Also one isolate harboured blaCTX-M-56, qnrB1 and blaCMY-2 genes and carried IncF1 plasmids of about 97 kb and160 kb. qnrB and qnrS were found in 8 other blaCTX-M-15 containing isolates. The blaNDM, blaIMP, blaVIM and qnrA were not detected, however, the blaOXA-48 was present in two (2.4%). CONCLUSIONS: The majority of isolates harbouring qnr genes demonstrated relatedness (≥85%) by PFGE. However, the diversity in PFGE profiles for the other MDR isolates reflected the changes in population genetics of E. coli O25b-B2-ST131. We identified for the first time the appearance of blaCTX-M-2 in the Middle East and blaCTX-M-56 outside the Latin American countries. The isolate harbouring blaCTX-M-56 also contained qnrB1 and blaCMY-2 genes and carried IncF1 plasmids. The appearance of a highly virulent E. coli O25b-ST131 that is resistant to penicillins, most cephalosproins, ß-lactamase inhibitors as well as fluoroquinolones is a cause for concern.

Smoking, von Willebrand factor and ADAMTS-13 in healthy males.

While von Willebrand factor (vWF) has been reported to be elevated in smokers, there are no reports on the effects of smoking on its cleaving protease ADAMTS-13, particularly in subjects of Arab ethnicity. This study was conducted to determine the effects of smoking on vWF and ADAMTS-13 antigen and activity levels in Arab males. Venous blood samples from 80 smoking (at rest) and 80 non-smoking healthy males were collected after asking subjects to fast and refrain from smoking for 8 hours. Similar sampling was done for 40 smokers (acute smokers), who were asked to smoke one cigarette immediately before blood collection. Plasma was used to measure ADAMTS-13 antigen and activity levels, as well as vWF antigen and collagen binding activity levels using commercial ELISA kits. Compared to non-smokers, ADAMTS-13 and vWF activities were significantly lower in smokers at rest (p < 0.05). Acute smokers had significantly higher levels of vWF activity and ADAMTS-13 antigen and activity levels (p < 0.01), compared to smokers at rest. Our results suggest that high vWF activity is accompanied by an increase in ADAMTS-13 activity as a natural physiological mechanism to degrade the elevated vWF molecules. If not followed by a subsequent smoke, the activities of both proteins subside. It is possible that the repeated increase in vWF and constant degradation by ADAMTS-13 results in lower overall levels of both proteins in smokers (at rest) compared to nonsmokers who do not experience a similar (repeated) injury to the endothelium.

Thrombosis risk in carriers of the factor V Leiden mutation: is it associated with a defined skin color?

Factor V Leiden (FVL; G1691A) is an autosomal dominant mutation with a high risk for thrombosis. Speculation that founders of FVL lived in the Middle East is supported by a prevalence of FVL that is higher in Arabs residing in Israel, Jordan, Lebanon, and Syria (12-14%) than in other white populations like Europeans (4-5%, up to 15% in the South of Sweden). We sought to verify the appropriate use of skin color as a clinical sign by which Arab individuals in Kuwait are included or excluded from testing for FVL. After institutional approval, 200 healthy Arabs residing in Kuwait consented to participate. Skin type was distinguished for the participants by Fitzpatrick natural skin color classification: 76 (38%) skin type II (white), 96 (48%) Mediterranean skin type IV (brown), and 28 (14%) skin type VI (black). FVL was tested by real-time PCR, and the percentage of carriers was calculated in each group. FVL was positive in 17 (8.5%) of the total subjects: 8 (10.5%) skin type II, 7 (7.3%) skin type IV, and 2 (7.1%) skin type VI. Therefore, FVL shows an even distribution in Arabs, and all Arabs residing in Kuwait should be tested for FVL irrespective of skin color.

Race differences in the prevalence of the factor V Leiden mutation in Kuwaiti nationals.

Factor V Leiden mutation (FVL; G1691A) is an established risk factor for venous thromboembolic disorders. FVL was reported with high prevalence in Caucasians (1-15%) but was absent in non-Caucasians like Africans and Asians. Studies reported FVL in 5-27% of Arabs and non-Arabs living in the Middle Eastern countries northern to the Arabian Peninsula, but was almost absent in Arabs in the Arabian Peninsula itself. Kuwait is an Arabic country present on the northern border of the Arabian Peninsula, and Kuwaitis are originally from Saudi Arabia (Southern to Kuwait and within the Arabian Peninsula) or from Iran and Iraq (northern to Kuwait and the Arabian Peninsula). This study was conducted to study FVL in Kuwaitis in relation to their origin. Real-time PCR was performed on DNA samples of 285 apparently healthy Kuwaitis using specially designed primers and probes for FVL. There were 109 Kuwaitis of Iranian origin, 71 of Iraqi origin and 105 of Saudi origin. FVL was present in 7 and 5 Kuwaitis of Iranian and Iraqi origin, respectively. None of the Kuwaitis of Saudi origin had the mutation. Prevalence of FVL in Kuwaitis of Iranian (6.42%) and Iraqi (7.04%) origin were statistically different from prevalence in Kuwaitis of Saudi (0%) origin (P-value<0.05). No difference was found between females and males (P-value>0.6). In conclusion, FVL is present in Kuwaitis of Iranian or Iraqi origin only. Therefore, testing and providing genetic consultation for FVL may be needed in those Kuwaitis only which should save time, cost and efforts. However, this assumption should be confirmed by other studies and on larger number of cases.

Epidemiology of Prothrombin G20210A Mutation in the Mediterranean Region.

There are many genetic and acquired risk factors that are known to cause venous thromboembolic disorders (VTE). One of these is the Prothrombin G20210A mutation, which has been identified in 1996. Prothrombin G20210A mutation causes higher levels of the clotting factor prothrombin in the blood of carriers, which creates a higher tendency towards blood clotting (hypercoagulability), and therefore the carriers become at higher risk of developing VTE. High prevalence of Prothrombin G20210A mutation was reported in Caucasian populations, but the prevalence was almost absent in non-Caucasians. That was most obvious in countries of South Europe and the Mediterranean region. This review article discusses Prothrombin G20210A mutation, how it causes VTE, the origin of the mutation, and its distribution worldwide with special concentration on the Mediterranean area.

Epidemiology of activated protein C resistance and factor v leiden mutation in the mediterranean region.

Venous thromboembolic disorders (VTE) are serious disorders with high morbidity and mortality rates. Many genetic and acquired risk factors were identified to cause VTE. The most common genetic risk factor is Factor V Leiden mutation (FVL). FVL was found in high percentage of populations of Caucasian origin but was almost absent in non-Caucasians. It was also reported in populations living in North Africa and the Middle East. This review article briefly explains FVL and how it causes VTE, the distribution of FVL worldwide, and then it elaborates on the epidemiology of FVL in the Mediterranean Region and how this brought speculations that FVL might have originated in the Eastern Mediterranean area.

High prevalence of activated protein C resistance and factor V Leiden mutation in an Arab population and patients with venous thrombosis in Kuwait.

INTRODUCTION: Activated protein C resistance (APC-R) because of clotting factor V Leiden mutation (FVL; Arg506Gln; G1691A) is a risk factor for the development of venous thromboembolic disorders (VTE). APC-R/FVL was reported to be very high in White patients with VTE (15% to 65%) and healthy populations (1% to 15%), and to be very low or absent in non-White patients. Studies on Arab patients and populations were very inconsistent. This study reports APC-R and FVL in Arabs living in Kuwait. MATERIALS AND METHODS: Whole venous blood samples were collected from 400 patients with VTE and 200 healthy controls, all of whom were of Arab ethnicity living in Kuwait. The samples were used to separate plasma for an APC-R test, and DNA extraction for polymerase chain reaction and restricted fragment length polymorphism were performed. APC-R was on an automated hemostasis analyzer, and values less than 2.0 were reported as APC-R. Polymerase chain reaction and restricted fragment length polymorphism tests were performed using standard methods, and the results were reported as normal wild-type homozygous GG, FVL homozygous AA, or FVL heterozygous GA. RESULTS: Sixty-three out of 400 patients (15.75%) and 4 out of 200 healthy controls (2%) had APC-R and at least one copy of FVL. Fifty-one patients and 4 controls were heterozygous whereas only 12 patients were homozygous. CONCLUSION: The prevalence of APC-R and FVL is quite high in Arabs living in Kuwait, being comparable with the prevalence reported in Whites, although being toward the lowest values reported there.

Factor V Leiden mutation in Arabs in Kuwait by real-time PCR: different values for different Arabs.

Factor V Leiden (FVL) mutation (G1691A) is a risk factor for development of venous thromboembolic disorders. FVL was found mostly in Caucasians (1-15%) but was almost absent in non-Caucasians. Studies on Arab patients and populations revealed very inconsistent results. This study reports FVL in Arabs living in Kuwait with a focus on the nationality of the Arab subjects studied. Whole-blood samples were collected from 400 healthy Arabs who were 268 Kuwaitis (67%), 50 Syrians (12.5%), 34 Jordanians (8.5%), 8 Palestinians (2%) and 40 Egyptians (10%). DNA extraction was carried out for these blood samples and real-time PCR was performed to detect the presence of FVL. Generally, 36 cases (9%) had the mutation (33 were heterozygous and 3 were homozygous), with an allelic frequency of 0.049. The prevalence of FVL differed in different Arabic cases: Kuwaitis 4.5%, Egyptians 15%, Syrians 16%, Jordanians 23.5% and Palestinians 25%. The allelic frequency was 0.022 in the Kuwaitis and 0.088-0.132 in non-Kuwaitis. The three homozygous cases were from Syria, Jordan and Egypt. In conclusion, the prevalence of FVL in Arabs living in Kuwait is as high as in Caucasians. There is a difference in prevalence among Arabs themselves, being relatively lower in Kuwaitis than in non-Kuwaitis.

Transmission of a Klebsiella pneumoniae clone harbouring genes for CTX-M-15-like and SHV-112 enzymes in a neonatal intensive care unit of a Kuwaiti hospital.

The spread of antibiotic-resistant bacteria has become a large problem in most countries including Kuwait. This antibiotic resistance is usually due to the production of extended-spectrum beta-lactamase (ESBL) enzymes such as SHV, TEM and CTX-M. This study reports the emergence and spread of an ESBL-producing Klebsiella pneumoniae clone in a neonatal intensive care unit (NICU) in a Kuwaiti hospital. Eight ESBL-producing K. pneumoniae isolates were from blood cultures of seven neonates, and two were from the fingers of two healthcare workers in a NICU in Al Jahra Hospital, Kuwait. All isolates were obtained in February-March 2006, except for one, which was obtained in August 2005. Identification of the bacteria was based on traditional bacteriological and biochemical tests using the Vitek system. Antibiotic susceptibility was tested by the disc diffusion method using 16 different antibiotics. ESBLs were detected using disc approximation and double-disc synergy methods and confirmed as ESBLs using Etest. PCR and DNA sequencing were performed to determine the genotypes and mutations in the beta-lactamase genes (blaTEM, blaSHV and blaCTX-M). Genetic relatedness was determined by PFGE. All isolates were confirmed to have ESBLs by the Vitek system, disc approximation test, double-disc diffusion test and Etest, being resistant to cefotaxime, ceftazidime, cefepime, gentamicin, tobramycin and ciprofloxacin but susceptible to tetracycline and trimethoprim-sulfamethoxazole. Molecular studies showed the isolates to have TEM-1 beta-lactamase, a CTX-M-15-like ESBL and the newly discovered SHV-112 ESBL. PFGE showed that all isolates had identical banding patterns. The results indicate that a single clone of ESBL-producing K. pneumoniae caused bloodstream infections among babies in a NICU of a Kuwaiti hospital, and may have emerged at least 5 years ago. This clone was also present on the hands of healthcare workers, suggesting that they may have been involved in its transmission. Further studies are recommended to determine whether this clone is also spreading in other Kuwaiti hospitals.

Whole-blood polymerase chain reaction and restriction fragment length polymorphism: a simplified method by microwave irradiation.

OBJECTIVES: The aim of the present study was to develop a simple, quick and cheap method to process whole-blood samples for the molecular techniques polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) without the use of expensive reagents or sophisticated machines. MATERIALS AND METHODS: Venous whole-blood samples were collected from 40 individuals. The samples were frozen at -80 degrees C, and then rapidly thawed at 37 degrees C. Each sample was incubated with distilled water, then boiled in a microwave and centrifuged. The supernatant was taken directly for PCR and RFLP. For comparison, PCR and RFLP were performed on DNA purified from the same samples using the phenol-chloroform method and two commercial DNA extraction kits. RESULTS: PCR/RFLP results using the presented method were qualitatively similar to those obtained by DNA extracted using the other three methods. CONCLUSION: The presented method proved to be a simpler and cheaper way of processing whole-blood samples for PCR and RFLP analyses.

Ciprofloxacin-resistant Salmonella enterica serovar typhi from Kuwait with novel mutations in gyrA and parC genes.

Blood isolates of Salmonella enterica serovar Typhi from two recently returned Bangladeshi patients in Kuwait were ciprofloxacin resistant, with ciprofloxacin MICs of 12 mg/liter for both isolates. Both isolates had three novel gyrA mutations (55-Leu-->Trp, 87-Asp-->Ala, and 106-Gln-->Arg) and three novel parC mutations (84-Glu-->Lys, 106-Trp-->Gly, and 128-Tyr-->Asp).

Factor V Kuwait: a novel mutation in the coagulation factor V gene discovered in Kuwait.

OBJECTIVE: It was the aim of this study to report a new point mutation in the clotting factor V gene in the general Arab population. SUBJECTS AND METHODS: The HR2 haplotype was tested in 288 Arabs living in Kuwait--188 patients with venous thromboembolic disorders (VTE) and 100 healthy subjects--using polymerase chain reaction and restriction fragment length polymorphism techniques. The presence of the new mutation was verified by DNA sequencing. RESULTS: Two (1.06%) VTE patients had guanine instead of the wild-type adenine at nucleotide number 3935 (A3935G) of the factor V gene. This mutation caused a histidine to arginine change in amino acid number 1254 of the factor V molecule. The new mutation is termed 'factor V Kuwait' (His1254Arg) and was absent in the 100 healthy subjects. CONCLUSION: It appears that factor V Kuwait could be a risk factor for developing VTE in Arabs. A larger study is needed to confirm this observation.