Molecular Characterization of Carbapenem-Resistant Acinetobacter baumannii Isolated from Intensive Care Unit Patients in Jordanian Hospitals.

Acinetobacter baumannii is a common cause of healthcare-associated infections (HAI) worldwide, mostly occurring in intensive care units (ICUs). Extended-spectrum beta lactamases (ESBL)-positive A. baumannii strains have emerged as highly resistant to most currently used antimicrobial agents, including carbapenems. The most common mechanism for carbapenem resistance in this species is ß-lactamase-mediated resistance. Carbapenem-hydrolyzing class D oxacillinases are widespread among multidrug-resistant (MDR) A. baumannii strains. The present study was conducted to determine the presence and distribution of blaOXA genes among multidrug-resistant A. baumannii isolated from ICU patients and genes encoding insertion sequence (IS-1) in these isolates. Additionally, the plasmid DNA profiles of these isolates were determined. A total of 120 clinical isolates of A. baumannii from various ICU clinical specimens of four main Jordanian hospitals were collected. Bacterial isolate identification was confirmed by biochemical testing and antibiotic sensitivity was then assessed. PCR amplification and automated sequencing were carried out to detect the presence of blaOXA-51, blaOXA-23, blaOXA-24, and blaOXA-58 genes, and ISAba1 insertion sequence. Out of the 120 A. baumannii isolates, 95% of the isolates were resistant to three or more classes of the antibiotics tested and were identified as MDR. The most frequent resistance of the isolates was against piperacillin (96.7%), cephalosporins (97.5%), and ß-lactam/ß-lactamase inhibitor combinations antibiotics (95.8%). There were 24 (20%) ESBL-producing isolates. A co-existence of blaOXA-51 gene and ISAba1 in all the 24 ESBL-producing isolates was determined. In addition, in the 24 ESBL-producing isolates, 21 (87.5%) carried blaOXA-51 and blaOXA-23 genes, 1 (4.2%) carried blaOXA-51 and blaOXA-24, but all were negative for the blaOXA-58 gene. Plasmid DNA profile A and profile B were the most common (29%) in ESBL-positive MDR A. baumannii isolates while plasmid DNA profile A was the most common in the ESBL-negative isolates. In conclusion, there was an increase in prevalence of MDR-A. baumannii in ICU wards in Jordanian hospitals, especially those having an ESBL phenotype. Thus, identification of ESBL genes is necessary for the surveillance of their transmission in hospitals.

Vitamin D status of female UAE college students and associated risk factors.

Objective: Vitamin D deficiency is now recognized as a pandemic with implications for bone health and chronic diseases. The study investigated the vitamin D status and risk factors of subnormal serum vitamin D levels in female college students. Design: Cross-sectional study. Setting: American University of Sharjah, United Arab Emirate. Serum 25-hydroxyvitamin D (25(OH)D) levels were measured for the participating female undergraduate college students using a radioimmunoassay kit. All participants answered a questionnaire that included 30 questions, which covered among others the demographic information, dietary intake, sun exposure and autoimmune diseases. Subjects: Undergraduate college female students (n, 480), aged 18-26 years. Results: Overall, 47.92% had suboptimal serum vitamin D levels. Results indicated that vitamin D deficiency and other health problems are prevalent among female university students. Risk factors included: wearing hijab by 37.5% of the students that might have interfered with the penetration of UVB radiation into the skin, short time sun exposure, use of sunscreens and limited intake of foods rich in vitamin D. Conclusions: Vitamin D deficiency is a problem in female college students due to lifestyle, and avoidance of sun exposure. Poor vitamin D status has been associated with increased risk for development of several autoimmune diseases, and other health conditions. This problem needs to be addressed, where prevention of future health consequences in this young group is still possible.

Protozoan parasites in irritable bowel syndrome: A case-control study.

AIM: To investigate the putative role of protozoan parasites in the development of irritable bowel syndrome (IBS). METHODS: The study included 109 IBS consecutive adult patients fulfilling the Rome III criteria and 100 healthy control subjects. All study subjects filled a structured questionnaire, which covered demographic information and clinical data. Fresh stool samples were collected from patients and control subjects and processed within less than 2 h of collection. Iodine wet mounts and Trichrome stained smears prepared from fresh stool and sediment concentrate were microscopically examined for parasites. Blastocystis DNA was detected by polymerase chain reaction, and Cryptosporidium antigens were detected by ELISA. RESULTS: A total of 109 IBS patients (31 males, 78 females) with a mean age ± SD of 27.25 ± 11.58 years (range: 16 -60 years) were enrolled in the study. The main IBS subtype based on the symptoms of these patients was constipation-predominant (88.7% of patients). A hundred healthy subjects (30 males, 70 females) with a mean ± SD age of 25.0 ± 9.13 years (range 18-66 years) were recruited as controls. In the IBS patients, Blastocystis DNA was detected in 25.7%, Cryptosporidium oocysts were observed in 9.2%, and Giardia cysts were observed in 11%. In the control subjects, Blastocystis, Cryptosporidium and Giardia were detected in 9%, 0%, and 1%, respectively. The difference in the presence of Blastocystis (P = 0.0034), Cryptosporidium (P = 0.0003), and Giardia (P = 0.0029) between IBS patients and controls was statistically significant by all methods used in this study. CONCLUSION: Prevalence of Blastocystis, Cryptosporidium and Giardia is higher in IBS patients than in controls. These parasites are likely to have a role in the pathogenesis of IBS.

Community-acquired urinary tract infections caused by Burkholderiacepacia complex in patients with no underlying risk factor.

Introduction. Urinary tract infections (UTIs) remain common infections diagnosed in outpatients as well as hospitalized patients. Community-acquired UTIs are generally caused by Escherichia coli and other members of the family Enterobacteriaceae.Burkholderiacepacia is an opportunistic pathogen mainly affecting immunocompromised and hospitalized patients, particularly those who have received prior broad-spectrum antibacterial therapy. Case presentation. Urine samples were collected from 157 outpatients clinically diagnosed with UTI and from 100 healthy control subjects. Samples were cultured on differential media and non-motile lactose-non-fermentors were identified via the Remel RapID ONE system. The isolates were tested by the disc diffusion method against 17 antimicrobial agents. Burkholderia was isolated as a single organism from four patients having uncomplicated infections, and one from recurrent infection. None of these patients had an underlying risk factor for this pathogen. Identification of these isolates by the Remel-RapID ONE system was confirmed by recA gene amplification. The four isolates were resistant to lincomycin, nalidixic acid, oxacillin and penicillin G. These cases received monotherapy of oral co-trimoxazole. Conclusions. Our findings alert urologists and diagnostic laboratories to the potential of B.cepacia complex infections in similar cases, and that this bacterium should not be ruled out.

Foodborne bacterial pathogens recovered from contaminated shawarma meat in northern Jordan.

INTRODUCTION: The purpose of the study was to isolate, identify, and determine the antimicrobial resistance of the bacterial pathogens recovered from shawarma (donair) sandwiches served to the public in Jordan. METHODOLOGY: Bacterial contamination of 100 shawarma sandwiches with pathogenic bacteria was studied by culture on selective media, serology, PCR assay, and antimicrobial susceptibility testing. RESULTS: One hundred and forty-five bacterial isolates were identified. The predominant species was Escherichia coli (28.3%), with six isolates of serotype O157:H7, followed by Salmonella spp. (25.5%). Higher contamination rates were found in chicken sandwiches. The majority of these bacteria expressed high resistance to several antimicrobials, especially tetracycline and streptomycin. Citrobacter freundii was isolated from 15.9% and Staphylococcus aureus was isolated from 8.3% of the sandwiches. The presence of these pathogens is of primary concern because some strains are capable of producing a heat-stable enterotoxin that causes food poisoning in humans, and should therefore be taken into account in risk assessment. CONCLUSIONS: Results signify the importance of sustained surveillance of foodborne pathogens in shawarma sandwiches to minimize the risk of contamination. Availability of data on the isolated pathogens and modes of transmission in food from different countries would provide a common ground for reaching international agreement on food safety regulations.

Unusual case presentation of intestinal Sarcocystis hominis infection in a healthy adult.

INTRODUCTION: Sarcocystosis is mainly a veterinary problem; however, humans can serve as the definitive host for at least two species (Sarcocystis hominis and Sarcocystis suihominis). Intestinal infections occur in the definitive host after ingesting the intramuscular cysts (sarcocysts) in the intermediate host, which initiate sexual stages in the intestine that terminate in oocysts excreted in the faeces. CASE PRESENTATION: A 19-year-old male presented with diffuse abdominal pain, watery non-bloody diarrhoea, nausea, vomiting and intermittent low-grade fever that lasted for more than 3 weeks. Multiple stool cultures on enriched and selective media gave negative results. Microscopic examination of wet mounts of stool prepared from formalin/ethyl acetate concentrates, together with permanent staining helped in making a definitive diagnosis and ruling out other coccidian parasites. Diagnosis of the parasite as S. hominis was made based on the size and morphology of the individual sporocysts that were observed in the wet-mount preparations. This severe case of intestinal sarcocystosis in a healthy adult after eating undercooked beef shawarma meat is described. CONCLUSION: The unusual presentation of intestinal sarcocystis described in this case is very rare. The clinical signs and size and morphology of both oocysts and sarcocysts observed in concentrated wet mounts of stool helped in the definitive diagnosis. The food ingested prior to the appearance of symptoms was important in making the definitive diagnosis of the parasite as S. hominis, as well as the incubation period and treatment.

Escherichia albertii, a newly emerging enteric pathogen with poorly defined properties.

Escherichia albertii is a newly emerging enteric pathogen that has been associated with sporadic infections among humans and birds. Selected coliform isolates were screened for allelic variation in 2 housekeeping genes (lysP and mdh) specific for E. albertii. The 48 strains that were identified as E. albertii were tested for 15 virulence markers and biochemical and serogical properties. All E. albertii strains were non-motile, fermented D-glucose (with gas), D-mannitol, and D-mannose, but failed to ferment lactose and other sugars. Variable positive reactions were noted for other tests. Most strains were rough or failed to agglutinate with Shigella boydii 13 antisera and E. coli antisera with few exceptions. All strains were positive for the eaeA gene, and variable numbers were positive for the cdtB, phoE, ehxA, and stx2f genes. Results illustrate the variability extent within this lineage and highlight the importance of accurately distinguishing it within the genus Escherichia and including information within commercial databases to improve their identification.

Detection of mutations associated with multidrug-resistant Mycobacterium tuberculosis clinical isolates.

Antimicrobial resistance was studied in 100 Mycobacterium tuberculosis strains selected randomly from sputum cultures of newly diagnosed tuberculosis patients. Resistance of the isolates to rifampicin, isoniazid, and ethambutol was tested by both drug susceptibility testing (DST) and allele-specific PCR (AS-PCR). A total of 19 (19%) isolates were found resistant to at least one of the antituberculosis drugs investigated by PCR compared with 14 (14%) resistant isolates detected by DST. Eleven mutations were detected by AS-PCR in the rpoB gene (codons 516, 526, and 531), associated with rifampicin resistance, a marker of multidrug-resistant tuberculosis (MDR-TB), 14 mutations in the katG gene codon 315 that confers resistance to isoniazid, and nine mutations in the embB gene codon 306 that confers resistance to ethambutol. Mutations in the six multidrug-resistant isolates were confirmed by DNA sequencing. Results were compared with phenotypic DST data. Nineteen different mutation types to at least one of the drugs were found; six isolates (6%) were classified as MDR-TB, defined as resistance to at least rifampicin and isoniazid. The rates of concordance of the PCR with the phenotypic susceptibility test were 71.4, 54.5, and 44.4 for isoniazid, rifampicin, and ethambutol, respectively. These results highlight the importance of molecular epidemiology studies of tuberculosis in understudied regions with a tuberculosis burden to uncover the true prevalence of the MDR-TB.

Oh my aching gut: irritable bowel syndrome, Blastocystis, and asymptomatic infection.

Blastocystis is a prevalent enteric protozoan that infects a variety of vertebrates. Infection with Blastocystis in humans has been associated with abdominal pain, diarrhea, constipation, fatigue, skin rash, and other symptoms. Researchers using different methods and examining different patient groups have reported asymptomatic infection, acute symptomatic infection, and chronic symptomatic infection. The variation in accounts has lead to disagreements concerning the role of Blastocystis in human disease, and the importance of treating it. A better understanding of the number of species of Blastocystis that can infect humans, along with realization of the limitations of the existing clinical laboratory diagnostic techniques may account for much of the disagreement. The possibility that disagreement was caused by the emergence of particular pathogenic variants of Blastocystis is discussed, along with the potential role of Blastocystis infection in irritable bowel syndrome (IBS). Findings are discussed concerning the role of protease-activated receptor-2 in enteric disease which may account for the presence of abdominal pain and diffuse symptoms in Blastocystis infection, even in the absence of fever and endoscopic findings. The availability of better diagnostic techniques and treatments for Blastocystis infection may be of value in understanding chronic gastrointestinal illness of unknown etiology.

Helicobacter pylori genotypes identified in gastric biopsy specimens from Jordanian patients.

BACKGROUND: The genetic diversity of Helicobacter pylori can be analyzed at two different levels: the genomic variation between strains originating from different individuals, and the variation in bacterial populations within an individual host. We reported for the first time the H. pylori genotypes in Jordanian patients with gastrointestinal diseases. METHODS: Upper endoscopy was performed on 250 patients with symptoms of gastrointestinal diseases. Multiple gastric biopsy specimens were taken from the antrum. All the biopsies were tested by PCR for the H. pylori virulence genes vacA, cagA, and iceA, and 151 were tested by histology. RESULTS: The biopsies positive for H. pylori by PCR were 110/250 (44%), and by histology 117/151 (77.5%), and these results were highly associated (P < 0.02). Analyses of virulence genes revealed that iceA2 (73.6%) was the predominant genotype, the vacAs2 allele was more frequently identified than the vacAs1 allele, while the cagA genotype was low (26.4%). The presence of certain genotypes might be associated with each other, but the presence of certain genotypes was not significantly associated with the age, or gender of the patient. CONCLUSION: The results illustrate the geographic nature of the genetic diversity of H. pylori, as the identified genotypes are similar to those reported in neighboring countries. This study provides a baseline data of H. pylori genotypes identified in gastric biopsy specimens from Jordan, serving as a powerful epidemiological tool for prospective investigations to better understand the genetic diversity of this pathogen.

Polymicrobial infections in children with diarrhoea in a rural area of Jordan.

Polymicrobial infections associated with diarrhoea are common in developing countries. Stool specimens were collected from 220 patient children and 100 controls. Potential pathogenic agents isolated from 143 (65%) children were identified by molecular and standard microbiological methods. Co-infections with two or more agents were detected in 50 (35%) cases. Escherichia coli, Shigella dysenteriae, Giardia and Entamoeba histolytica were found to be predominant. The etiologic agents could not be determined in 77 (35%) cases. The most significant risk factors were the age, the education level of the mother and the use of non-chlorinated water. The high infection rate of diarrhoeal diseases is a strong indication that these pathogens circulate easily through the population.

Detection of Toxoplasma gondii DNA and specific antibodies in high-risk pregnant women.

Primary maternal infection with toxoplasmosis during pregnancy is frequently associated with transplacental transmission to the fetus. This study was conducted to test the utility of a polymerase chain reaction (PCR) assay to detect recent infections with Toxoplasma in pregnant women. One hundred forty-eight women with high-risk pregnancies who had abnormal pregnancy outcomes (cases) and 100 with normal pregnancies (controls) were tested for the presence of Toxoplasma DNA in their blood by a nested PCR and specific antibodies to Toxoplasma by an enzyme-linked immunosorbent assay. The IgG results of the cases differed significantly from those of the controls (54% and 12%, respectively; P < 0.02). Four (2.7%) of the cases were IgM positive, but none of the controls were positive. Detection of Toxoplasma DNA in 20 (8.1%) of the IgG-positive cases suggests a recent infection. The risk factors associated with the infection were eating raw meat and contact with soil. The diagnostic serology of recent infection in early pregnancy could be confirmed by a positive Toxoplasma-specific PCR result in blood samples collected in the first half of pregnancy, even in the presence of serologic results difficult to interpret due to the lack of sequential follow-up during pregnancy.

Cyclospora cayetanensis and other intestinal parasites associated with diarrhea in a rural area of Jordan.

Cryptosporidium spp. and Cyclospora cayetanensis have emerged as important causes of epidemic and endemic diarrhea in immunocompetent and immunocompromised hosts. The exact modes of transmission in certain rural areas are still unclear. Reports of water-borne and food-borne outbreaks suggest that fecally contaminated water or food acts as a vehicle of transmission. Two hundred stool samples of patients with gastroenteritis from four health centers in a rural area of Jordan were examined using formalin-ethyl acetate concentration, wet preparation, and modified acid-fast staining methods. Oocysts of C. cayetanensis and Cryptosporidium spp. were found in 6% and 8% of the samples respectively, mainly those of children. Parasites such as Entamoeba histolytica, Giardia lamblia, and other enteropathogens were also observed. The results reflect the seasonality of natural cyclosporiasis and cryptosporidiosis, being higher in the spring. The risk factors that were found by the Fisher test to be significant and might be associated with illness are the source of drinking water, contact with animals, and eating unwashed vegetables ( p<0.028, p<0.0005, p<0.00005 respectively).

Diagnosis of Mycobacterium tuberculosis in cases of pulmonary infections from Jordanian hospitals.

The aim of this study is to use the diagnostic utility of smear technique for the detection of Mycobacterium tuberculosis in clinical specimens obtained from patients suspected of having pulmonary tuberculosis. A total of 305 respiratory specimens (broncoalveolar lavage, sputum and pleural fluid) were collected from 298 patients having pulmonary infections as bronchitis, tuberculosis, pneumonia and other respiratory diseases. Results revealed that 25 specimens collected from 22 patients were positive (8.2%) by acid fast (AF) smear. Data indicated that the specificity and positive predictive value of the conventional smear assay were 100%, however, the sensitivity of the smear examination was 73.5%. Conventional smear technique actually detected M. tuberculosis in respiratory specimens and it could be applied for early and specific diagnosis of tuberculosis in such patients.

Diagnosis of recent and relapsed cases of human brucellosis by PCR assay.

BACKGROUND: Brucellosis affects human populations in many developing countries including the Middle East, and Latin America where it is still endemic. It has been prevalent in Jordan for years, where 7842 cases of human brucellosis were registered at the Ministry of Health during 10 year-period. This study was initiated by the recent increase in the number of human cases diagnosed in a rural area in the Northern Jordan to help assess the status of the disease in that area. For this purpose blood specimens from brucellosis suspected cases were tested by serology, culture and PCR. METHODS: Peripheral blood specimens from 50 healthy control subjects and 165 seropositive patients having compatible signs and symptoms that were clinically diagnosed to have brucellosis were tested by blood culture, and by PCR. The PCR assay used genus-specific primers from the conserved region of the 16S rRNA sequence, which showed high specificity for the Brucella spp. RESULTS: Diagnosis of Brucella was established by PCR in 120 cases (72.7%). All of them were seropositive and 20 were positive by culture. Forty-eight of 58 (82.8%) of the relapsed cases two months after completing the treatment with an increase in the previous serological titers were positive by PCR. The assay has 85.7% positive predicative value, 100% sensitivity and specificity since it correctly identified all cases that were positive by blood cultures, 95.8% by serology and none of the control group was positive. CONCLUSIONS: Results showed that PCR assay can be applied with serology for the diagnosis of brucellosis suspected cases and relapses regardless of the duration or type of the disease without relying on the blood cultures, especially in chronic cases.

Leishmania species and zymodemes isolated from endemic areas of cutaneous leishmaniasis in Jordan.

BACKGROUND: Cutaneous leishmaniasis (CL) is endemic in the Middle Eastern countries. New cases are emerging in areas previously free of the disease. In Jordan, the diagnosis of cases during the 1960s and 1970s was mainly reported in military hospitals in Amman. Endemicity of the disease was ascertained after reporting a total of 524 cases during 1973-1978. RESULTS: Leishmania major and Leishmania tropica were isolated from seventy-six autochthonous and imported cases of CL, during eight-year period. The highest infection rates recorded were in the central part of Jordan (60.5%), in males (72.4%) and in the age group 21-30 years (30.5%). Lesions were on the exposed sites of the body, mainly on the face (40%). Both Leishmania spp. were isolated from all parts of the country, although L. major was the predominant species (75% of cases) in all areas except in the north part of Jordan. Isoenzyme characterization of the isolates identified four previously undescribed zymodemes (Z). Four Leishmania major zymodemes were found, one of which was a new zymodeme (ZMON-103 variant in GLUD220); L. major ZMON-103 was the most common zymodeme. Four Leishmania tropica zymodemes were identified, of which three were previously unreported. Of these, ZMON-54 var PGD96-97 was isolated from autochthonous cases, whereas ZMON-59 var MDH100 and ZMON-75 var FH110 were obtained from both autochthonous and imported cases, or from an imported CL case, respectively. CONCLUSION: The findings of this study indicate the emergence of the CL disease in new areas. New foci are reported, where the sporadic nature of the cases indicates recent spread of the disease to these areas and the urge for the implementation of control measures.

Genetic diversity of Pneumocystis carinii f. sp. hominis based on variations in nucleotide sequences of internal transcribed spacers of rRNA genes.

A variety of genes have been used to type Pneumocystis carinii. In the present study, nucleotide sequence variations in the ITS1 and ITS2 internal transcribed spacer (ITS) regions of the rRNA genes were used to type Pneumocystis carinii f. sp. hominis DNA obtained from the lungs of 60 human immunodeficiency virus-infected individuals. These regions were amplified by PCR, cloned, and sequenced. Multibase polymorphisms were identified among samples. Several new genotypes are reported on the basis of the nucleotide sequence variations at previously unreported positions of both the ITS1 and the ITS2 regions. Twelve new ITS1 sequences were observed, in addition to the nine sequence types reported previously. The most common was type E, which was observed in 60.5% of the samples. The sequence variations in the ITS1 region were mainly located at positions 5, 12, 23, 24, 45, 53, and 54. Sixteen new ITS2 types were also identified, in addition to the 13 types reported previously. The most common was type g (26.6%). The sequences of the ITS2 regions in most specimens were different from the previously published sequence at bases 120 and 166 through 183. The most common variations observed were deletions at positions 177 through 183. The presence of more than one sequence type in some patients (60%) suggested the occurrence of coinfection with multiple P. carinii strains. The genetic polymorphism observed demonstrates the degree of diversity of Pneumocystis strains that infect humans. Furthermore, the high degree of polymorphism suggests that these genes are evolving faster than other genes. Consequently, the sequence information derived is useful for purposes such as examination of the potential of person-to-person transmission and recurrent infections but perhaps not for other genotyping applications that rely on more stable genetic loci.