Molecular characterisation of Panton-Valentine leucocidin-producing methicillin-resistant Staphylococcus aureus clones isolated from the main hospitals in Taif, KSA.

INTRODUCTION: Panton-Valentine leucocidin (PVL) is a bicomponent pore-forming cytolytic toxin encoded by the lukF-PV and lukS-PV genes. Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) may carry the pvl genes which may be related to increased disease severity. This study aimed to characterise the PVL-producing MRSA recovered from different Taif Hospitals, Saudi Arabia. METHODS: The study included 45 hospital-acquired-MRSA (HA-MRSA) and 26 CA-MRSA strains which were identified from 445 S. aureus strains isolated from different clinical samples. MRSA strains were identified by standard oxacillin salt agar screening procedure and by the detection of the mecA gene by the polymerase chain reaction (PCR). Detection of the S. aureus-specific femA, mecA and pvl genes was performed by multiplex PCR. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis was done for coagulase (coa) gene. RESULTS: The staphylococcal cassette chromosome mec types of the 45 HA-MRSA strains were Type I (n = 24), Type II (n = 7) and Type III (n = 14) whereas the 26 CA-MRSA strains were Type IV (n = 14), Type V (n = 11) and one isolate was non-typeable. All the HA-MRSA and six CA-MRSA strains were PVL-negative PCR-RFLP analysis of coa gene showed that PVL-positive MRSA (n = 20) isolates showed six different patterns, and five patterns were shared by PVL-positive methicillin-susceptible S. aureus (MSSA). The eighth pattern was the most frequent in both MRSA and MSSA. CONCLUSION: PVL is more frequent among CA-MRSA than MSSA. All the HA-MRSA and 25% of CA-MRSA strains were negative for PVL. The pvl gene was related to the severity of infection but not related to coa gene RFLP pattern.

An Improved PCR-RFLP Assay for Detection and Genotyping of Asymptomatic Giardia lamblia Infection in a Resource-Poor Setting.

Laboratory workers, in resource-poor countries, still consider PCR detection of Giardia lamblia more costly and more time-consuming than the classical parasitological techniques. Based on 2 published primers, an in-house one-round touchdown PCR-RFLP assay was developed. The assay was validated with an internal amplification control included in reactions. Performance of the assay was assessed with DNA samples of various purities, 91 control fecal samples with various parasite load, and 472 samples of unknown results. Two cysts per reaction were enough for PCR detection by the assay with exhibited specificity (Sp) and sensitivity (Se) of 100% and 93%, respectively. Taking a published small subunit rRNA reference PCR test results (6%; 29/472) as a nominated gold standard, G. lamblia was identified in 5.9% (28/472), 5.2%, (25/472), and 3.6% (17/472) by PCR assay, RIDA(®) Quick Giardia antigen detection test (R-Biopharm, Darmstadt, Germany), and iodine-stained smear microscopy, respectively. The percent agreements (kappa values) of 99.7% (0.745), 98.9% (0.900), and 97.7% (0.981) were exhibited between the assay results and that of the reference PCR, immunoassay, and microscopy, respectively. Restriction digestion of the 28 Giardia-positive samples revealed genotype A pattern in 12 and genotype B profile in 16 samples. The PCR assay with the described format and exhibited performance has a great potential to be adopted in basic clinical laboratories as a detection tool for G. lamblia especially in asymptomatic infections. This potential is increased more in particular situations where identification of the parasite genotype represents a major requirement as in epidemiological studies and infection outbreaks.

Identification of Giardia lamblia and the human infectious-species of Cryptosporidium in drinking water resources in Western Saudi Arabia by nested-PCR assays.

The presence of Cryptosporidium and/or Giardia in drinking water represents a major public health problem. This study was the first report concerned with the occurrence of these protozoa in drinking water in Saudi Arabia. The study was undertaken in Al-Taif, a high altitude region, Western Saudi Arabia. Eight underground wells water, six desalinated water and five domestic brands of bottled water samples, 10 liter each, were monthly collected between May 2013 and April 2014. All samples (n = 228), were processed using an automated wash/elution station (IDEXX Laboratories, Inc.). Genomic DNA was directly isolated and purified from samples concentrates with QIAamp® Stool Mini Kit (Qiagen). The target protozoan DNA sequences were amplified using two previously published nested-PCR protocols. Of all the analyzed water, 31 samples (≈14%) were found contaminated with the target protozoa. Giardia lamblia was detected in ≈10% (7/72) of desalinated water and in ≈9% (9/96) of wells water. On the other hand, Cryptosporidium was identified in ≈8% (8/72) of desalinated water and in ≈7% (7/96) of wells water. All bottled water samples (n = 60) were (oo)cysts-free. Protozoan (oo)cysts were more frequently identified in water samples collected in the spring than in other seasons. The methodology established in our study proved sensitive, cost-effective and is amenable for future automation or semi-automation. For better understanding of the current situation that represent an important health threat to the local inhabitants, further studies concerned with (oo)cyst viability, infectivity, concentration and genotype identification are recommended.

Internal Amplification Control for a Cryptosporidium Diagnostic PCR: Construction and Clinical Evaluation.

Various constituents in clinical specimens, particularly feces, can inhibit the PCR assay and lead to false-negative results. To ensure that negative results of a diagnostic PCR assay are true, it should be properly monitored by an inhibition control. In this study, a cloning vector harboring a modified target DNA sequence (≈375 bp) was constructed to be used as a competitive internal amplification control (IAC) for a conventional PCR assay that detects ≈550 bp of the Cryptosporidium oocyst wall protein (COWP) gene sequence in human feces. Modification of the native PCR target was carried out using a new approach comprising inverse PCR and restriction digestion techniques. IAC was included in the assay, with the estimated optimum concentration of 1 fg per reaction, as duplex PCR. When applied on fecal samples spiked with variable oocysts counts, ≈2 oocysts were theoretically enough for detection. When applied on 25 Cryptosporidium-positive fecal samples of various infection intensities, both targets were clearly detected with minimal competition noticed in 2-3 samples. Importantly, both the analytical and the diagnostic sensitivities of the PCR assay were not altered with integration of IAC into the reactions. When tried on 180 randomly collected fecal samples, 159 were Cryptosporidium-negatives. Although the native target DNA was absent, the IAC amplicon was obviously detected on gel of all the Cryptosporidium-negative samples. These results imply that running of the diagnostic PCR, inspired with the previously developed DNA extraction protocol and the constructed IAC, represents a useful tool for Cryptosporidium detection in human feces.

Association of serum cytokines levels, interleukin 10 -1082G/A and interferon-γ +874T/A polymorphisms with atopic asthma children from Saudi Arabia.

The aim of this study was to clarify the role of IL-4, IL-10, IL-13 and interferon (IFN) -γ levels in atopic asthma patients by studying the relation between their serum levels and severity of the disease. The effect of IL-10 -1082G/A and IFN-γ +874T/A SNPs was also studied. The study included 200 atopic children with asthma and 50 age- and gender matched healthy children as controls. The levels of both IL-4 and IL-13 were significantly (p<0.001) higher, while IFN-γ was significantly (p<0.001) lower in patients compared to that of the controls. There was a significant effect of gene polymorphisms of IL-10 (p<0.05) and IFN-γ (p<0.001) in occurrence of atopic asthma and increased IgE level. Polymorphism of IFN-γ gene had an effect on the serum level of IFN-γ. In conclusion, IFN-γ gene polymorphism at position +874 and IL-10 gene polymorphism at position -1082A/G are genetic determinants which contribute to susceptibility to atopic asthma in children from Saudi Arabia.

Levels of serum steroid hormones in intrauterine contraceptive device users.

Serum progesterone, estradiol, testosterone and cortisol were assayed in the mid-follicular and mid-luteal phases of the menstrual cycles in 30 Lippe's loop and 30 Cu T-200 IUCD users, compared to 24 controls. Mean serum progesterone and estradiol levels were significantly higher in the mid-luteal, compared to the mid-follicular phase in each group (p less than 0.01). There were no significant differences between the mean levels of progesterone, testosterone and cortisol in IUCD users and controls in both the mid-follicular and mid-luteal phases. IUCD users had significantly higher levels of estradiol (p less than 0.01) in the mid-luteal phase but not in the mid-follicular phase, compared to controls. There was hormonal evidence of corpus luteum insufficiency in 8.3%, 14.3% and 20% in the controls, Lippe's loop and Cu T-200 IUCD users, respectively.

PIP: Physicians divided 84 gynecologic patients aged 18-35 years who had not used any hormonal contraception or any drug that affects hormone homeostasis for at least 1 year at the outpatient clinic of Benha University Hospital in Egypt into 3 comparable groups to document the impact of IUDs on the human hypothalamic-pituitary-ovarian axis. Lippes loop users experienced a significantly shorter cycle length than either the copper T-200 IUD (Cu T-200) group or the control group (p.05). The Cu T-200 group had a significantly longer education duration of flow than did the other 2 groups (p.05). Neither the Lippes loop nor the Cu T-200 affected ovulation, except in 1 case of a Lippes loop user. Nevertheless, corpus luteum insufficiency did occur in 14.3% of Lippes loop cases, 205 of Cu T-200 cases, and in 8.3% of control group cases. The mean serum estradiol level in the midluteal phase stood significantly higher in the Lippes loop users than in both those using the Cu T-200 and no IUD (p.01). No significant differences existed, however, in mean serum progesterone, testosterone, and cortisol levels between IUD users and the control group during the midfollicular and midluteal phases. For all groups, the midluteal phase exhibited higher mean serum levels of estradiol and progesterone than the midfollicular phase (p.01), but no significant differences existed for testosterone and cortisol.