Molecular Screening of Carbapenem-Resistant K. pneumoniae (CRKP) Clinical Isolates for Concomitant Occurrence of Beta-Lactam Genes (CTX-M, TEM, and SHV) in the Kingdom of Bahrain.

The emergence of extended-spectrum ß-lactamase-producing Klebsiella pneumoniae, including CRKP infections, has resulted in significant morbidity and mortality worldwide. We aimed to explore the presence of bla genes (CTX-M, TEM, and SHV) in CRKP isolates. A total of 24 CRKP isolates were randomly selected from the Salmaniya Medical Complex Microbiology Laboratory. These isolates, which were positive for carbapenemases, were further explored for CTX-M, TEM, and SHV genes using PCR. All the CTX-M PCR amplicons were sent for sequencing. To determine genetic relatedness, molecular typing by ERIC-PCR was performed. The bla gene testing demonstrated that a significant proportion of these isolates harbored SHV, CTX-M, and TEM genes (100%, 91.6%, and 45.8%), respectively. Bioinformatic analyses confirmed CTX-M-15 in these isolates. ERIC-PCR analysis showed three clusters demonstrating genetic relatedness. The study findings reveal the concomitant carriage of the SHV and CTX-M-15 and a comparatively lower carriage of TEM genes in CRKP isolates. Our findings highlight the significance of routinely reporting the presence of antibiotic resistance genes along with regular antibiotic sensitivity reports, as this will aid clinicians in prescribing appropriate antibiotics.

Clinical carbapenem-resistant Klebsiella pneumoniae isolates simultaneously harboring bla NDM-1, bla OXA types and qnrS genes from the Kingdom of Bahrain: Resistance profile and genetic environment.

The prevalence of Carbapenem-resistant Klebsiella pneumoniae (CRKP) is currently increasing worldwide, prompting WHO to classify it as an urgent public health threat. CRKP is considered a difficult to treat organism owing to limited therapeutic options. In this study, a total of 24 CRKP clinical isolates were randomly collected from Salmaniya Medical Complex, Bahrain. Bacterial identification and antibiotic susceptibility testing were performed, on MALDI-TOF and VITEK-2 compact, respectively. The isolates were screened for carbapenem resistance markers (bla NDM, bla OXA-23, bla OXA-48 and bla OXA-51) and plasmid-mediated quinolone resistance genes (qnrA, qnrB, and qnrS) by monoplex PCR. On the other hand, only colistin-resistant isolates (n=12) were screened for MCR-1, MCR-2 and MCR-3 genes by monoplex PCR. Moreover, the Genetic environment of bla NDM, integrons analysis, and molecular characterization of plasmids was also performed. Antibiotic susceptibility revealed that all the isolates (100%) were resistant to ceftolozane/tazobactam, piperacillin/tazobactam, 96% resistant to ceftazidime, trimethoprim/sulfamethoxazole, 92% resistant to meropenem, gentamicin and cefepime, 88% resistant to ciprofloxacin, imipenem, and 37% resistant to amikacin. Ceftazidime/avibactam showed the least resistance (12%). 75% (n=12/16) were resistant to colistin and 44% (n=7/16) showed intermediate susceptibility to tigecycline. The detection of resistant determinants showed that the majority (95.8%) of CRKP harbored bla NDM-1, followed by bla OXA-48 (91.6%) bla OXA-51 (45.8%), and bla OXA-23 (41.6%). Sequencing of the bla NDM amplicons revealed the presence of bla NDM-1. Alarmingly, 100% of isolates showed the presence of qnrS. These predominant genes were distributed in various combinations wherein the majority were bla NDM-1 + bla OXA-51+ qnrS + bla OXA-48 (n =10, 41.7%), bla NDM-1 + bla OXA-23+ qnrS + bla OXA-48 (n=8, 33.3%), among others. In conclusion, the resistance rate to most antibiotics is very high in our region, including colistin and tigecycline, and the genetic environment of CRKP is complex with the carriage of multiple resistance markers. Resistance to ceftazidime/avibactam is uncommon and hence can be used as a valuable option for empirical therapy. Molecular data on resistance markers and the genetic environment of CRKP is lacking from this geographical region; this would be the first report addressing the subject matter. Surveillance and strict infection control strategies should be reinforced in clinical settings to curb the emergence and spread of such isolates.

Perception of online and face to face microbiology laboratory sessions among medical students and faculty at Arabian Gulf University: a mixed method study.

BACKGROUND: The COVID-19 pandemic has impacted all spheres of society including medical education and healthcare systems. In response to the pandemic, there has been a transition in medical education practice from traditional forms of teaching to online instruction delivery and virtual learning. Effective clinical microbiology education involves a combination of 'hands-on' practical learning and instructional delivery of scientific knowledge. Microbiology practical laboratories are critical learning environments offering 'hands-on' learning experiences that cannot be replicated through online learning. We conducted a mixed-methods study to understand the perception of online and face-to-face microbiology laboratory sessions among the medical students and microbiology faculty at Arabian Gulf University (AGU). METHODS: The study participants were third and fourth-year undergraduate medical students and faculty involved in delivering microbiology labs at AGU. The questionnaire consisted of questions ranging from perceived learning style to attitude towards online delivery of microbiology curriculum. After the questionnaire administration (google form), focus group discussion (FGD) was conducted for students and microbiology faculty separately. RESULTS: Among 168 students, 50.6% preferred face-to-face lab sessions as compared to 30.4% who preferred online labs, and 51.8% considered online labs to be an essential addition to face-to-face labs. Among the faculty, 85.7% preferred the face-to-face mode of teaching. All the faculty (100%) disagreed that all the microbiology labs teaching should be online. 57.2% considered online labs to be an essential addition to traditional face-to-face labs. Both faculty and students hold that a blended mode of instructional delivery is vital and indispensable for the transfer of skills and knowledge for microbiology students. CONCLUSION: The blended mode of delivering microbiology laboratory sessions in medical school is successful and well-received by both students and faculty. Students take the responsibility for furthering their own learning and understanding of concepts. Instructors have also noticed that blending learning strategies also successfully enhances the development of cognitive skills and problem-solving abilities in students. A review of the microbiology lab curriculum is necessary to identify content areas that can be delivered effectively through online, face-to-face lab sessions, or both, supported with appropriate tools and infrastructure.

Variability of ophthalmology residents' perception toward different major training programs in Saudi Arabia.

PURPOSE: The constant demand for ophthalmologists has nationally resulted in creating more programs in different regions of the kingdom. We have previously reported the overall residents' satisfaction with the current local ophthalmology curriculum and the competency of the ophthalmic training (clinical and surgical) in our local programs in Saudi Arabia compared to international standards. In this study, we aim at comparing the major local training programs and analyze the differences among them aiming at improving our training. METHODS: This is a cross-sectional study using a questionnaire that was completed by residents and graduates of the local ophthalmology programs in Riyadh, Eastern and Western regions. A closed-ended questionnaire was validated and circulated online and a hard copy was distributed to residents and recently graduated ophthalmologists (2009-2015). Data were categorized by demographic variables, and basic statistics were done. The study has been approved by the Institutional Review Board (IRB) and Human Ethics Committee (HEC) of King Saud University. RESULTS: Of the 200 individuals surveyed, 175 (87.5%) completed their responses. The average age was 29.6 years (range: 24-39) with 67.5% males and 32.5% females. The overall satisfaction among senior residents and graduates showed a statistically significant higher result among graduates in Riyadh area (P=<0.001). Satisfaction of senior residents with the program director's support was higher in the Eastern region (P=<0.001). Clinic-based training was generally satisfactory. Refractive surgery was reported to be significantly insufficient in 70.6% (P=0.003). Most of the graduates in Riyadh area achieved the surgical requirements for training compared to other regions. CONCLUSION: Saudi postgraduate ophthalmology training programs show a variable level of satisfaction among senior residents and graduates. Better surgical exposure has been observed in Riyadh region, however reassessment of the current curriculum and the parameters for training are needed to fulfill the requirements with special attention to the surgical training in all programs.

Student Perception of Microbiology Laboratory Skills Learning Through a Problem-Based Learning Curriculum: Arabian Gulf University Experience.

INTRODUCTION: The curriculum at medical school at Arabian Gulf University is centered on small group learning and real-life problems provided to students and guiding students to learn actively. In microbiology, laboratory skills are taught in an innovative manner using mini cases and different lab sessions and are integrated with other basic sciences. This article describes the format and pattern of laboratory skills sessions conducted using PBL methods at Arabian Gulf University and discusses the perception of students towards PBL in laboratory skill learning and way forward for the same. METHODS: The study sample size was 110. The students' perception of the laboratory skills teaching methods was assessed through an exit survey at the end of each session. A semi-structured self-administered survey instrument was prepared, and the questions were arranged in two sessions and focused on identifying the relevance, timing, strengths, and weaknesses of the teaching method and recommendations to improve the same. RESULTS: We observed that more than 50% of the participants agreed or strongly agreed that the time given for PBL was adequate, topics discussed were relevant, presentations were clear, pre-session briefing and Case-Based Studies (team-based learning (TBL)) helped in their learning. The participants identified the demonstration of experiments and hands-on experience provided in the laboratory were most helpful. When enquired about the difficulty, among 48% of the participants, 80% observed that the slides used in the learning/teaching were lengthy. CONCLUSION: The use of PBL in a lab setting promotes active learning. In the heart of PBL, TBL is a powerful tool in the educational process offering the students deep comprehension and allowing them to gain practical and intellectual skills.

Is there a role for HLA-G in the induction of regulatory T cells during the maintenance of a healthy pregnancy?

PROBLEM: Pregnancy remains an immune challenge for the uterus that has to adapt to a semi-allogeneic fetus using various regulatory mechanisms. Both HLA-G and regulatory T cells (CD4+  CD25+  FOXP3+  Tregs ) are upregulated in successful pregnancy, but not in abortion. It is unclear if HLA-G plays a role in the upregulation of regulatory cells. METHOD OF STUDY: We measured the level of both sHLA-G and Treg  cells in the blood of healthy pregnant multigravida, unexplained recurrent spontaneous abortions (URSA) and healthy non-pregnant and nulliparous females. We cultured peripheral blood lymphocytes of healthy non-pregnant multigravida females who never had an abortion with lymphocytes of their partners at ratio of 1:1, with and without sHLA-G to detect changes in number of Treg  cells, or relevant cytokines. RESULTS: Soluble HLA-G concentrations and Treg  cells percentage were significantly lower in women with URSA as compared to healthy pregnant multigravida women and were comparable to healthy non-pregnant nulliparous women. Percentage of Tregs  increased between zero time and mixed lymphocyte cultures (MLC) in both cultures with and without recombinant sHLA-G but no significant difference between the two cultures. When stimulated with sHLA-G the mean extracellular IL-10 concentration was unchanged, while the mean INF-γ concentration was slightly higher with no significant difference. Intracellular TGF-ß was higher in CD4+  cells after incubation with sHLA-G. CONCLUSION: The results of this study are consistent with previous studies on the role of sHLA-G and Treg  cells in inducing immune-tolerance in pregnancy. The results also suggest a possible role for HLA-G in the enrichment of Treg  cells.

Evaluation of Curricular Adaptations Using Digital Transformation in a Medical School in Arabian Gulf during the COVID-19 Pandemic.

BACKGROUND: Several institutions adopted innovative approaches to ensure continued learning for their students during the COVID-19 pandemic. All curricular innovations should undergo curriculum evaluation; hence, the objective of this paper was to share the salient features of evaluation using faculty and student's feedback on curricular adaptations implemented through digital transformation in a Medical School in Arabian Gulf during the COVID-19 pandemic, using a structured questionnaire. METHODOLOGY: After getting informed consent, feedback about acceptability and limitations regarding various aspects of curricular adaptations was obtained from students and faculty, using a structured and validated questionnaire. The response rate from faculty and students was 90% and 60%, respectively. The qualitative responses were analyzed using thematic analysis. RESULTS: About 97% agreed that Modular Object-Oriented Dynamic Learning Environment, ZOOM, and Examsoft platforms were effective for curriculum delivery and assessment. 85% agreed that they were able to maintain online interactivity and 92% conveyed their willingness to continue to use these digital innovations even after the end of pandemic. "Lack of interactivity," "missed clinical training," "live sessions were more engaging than recorded ones" were the prominent themes emerged out of thematic analysis. All faculty and students expressed concern over the lack of clinical training involving real patients. All of them expressed appreciation to the university and faculty for their enormous efforts. CONCLUSION: Innovative ways should be considered to start clinical teaching with real patients, during pandemic. The learning outcomes of digital learning should be validated across all institutions. New indicators related to "digital learning" should be considered for accreditation of medical schools.

Evaluating the Reliability and Validity of the Maastricht Clinical Teaching Questionnaire in Bahrain.

OBJECTIVES: The Maastricht Clinical Teaching Questionnaire (MCTQ) was developed to evaluate clinical teachers' supervisory skills during undergraduate clinical rotations. Evidence exists supporting the reliability and validity of this questionnaire. Our study sought to examine the reliability and validity of the MCTQ in a Middle Eastern context. METHODS: Between 2016 and 2017, we evaluated a total of 549 medical students in their final year who were undergoing clinical rotations using the MCTQ. The construct validity was assessed using exploratory and confirmatory factor analysis. Internal consistency reliability was measured using Cronbach's alpha. Factor scores were compared with other outcomes to explore the relationship with other relevant variables. RESULTS: A four-factor model demonstrated an adequate fit with the data. The findings showed good internal consistency reliability. The following results were obtained for the four-factor model: chi-square divided by degrees of freedom was 5.026, and the comparative index, goodness of fit index, normalized fit index, and non-normalized fit index were all above 0.800 (0.955, 0.858, 0.950, and 0.952, respectively). The standardized root mean square residual was 0.016, and the root mean square error of approximation score was 0.086. Acceptable reliability was achieved with 10 evaluations per teacher. We observed a strong correlation between factors and overall judgment. CONCLUSIONS: Our study suggests that the MCTQ is a valid and reliable instrument to evaluate teachers' performance during clinical rotations in Bahrain.

T-regulatory cells in chronic rejection versus stable grafts.

OBJECTIVES: Studying regulatory T cells in kidney allograft acceptance versus chronic rejection may help in the understanding of more mechanisms of immune tolerance and, in the future, may enable clinicians to induce immune tolerance and decrease the use of immunosuppressive drugs. The aim of the current study was to evaluate regulatory T cells in kidney transplant patients with stable graft versus transplant with biopsy-proven chronic rejection. MATERIALS AND METHODS: The 3 groups that were studied included: kidney transplanted patients with no rejection episodes (n = 43); transplanted patients with biopsy-proven renal rejection (n = 27); and healthy age-matched nontransplanted individuals as controls (n = 42).The percentage of regulatory T cells (CD4+CD25+Foxp3+) in blood was determined by flow cytometry. RESULTS: The regulatory T cell percentage was significantly lower in chronic rejection patients than control or stable graft groups. No significant difference was observed in regulatory T cell percentage between the stable graft and control groups. In the stable graft group, patients on rapamycin had a significantly higher regulatory T cell percentage than patients on cyclosporine. No effect of donor type, infection, or duration after transplant was observed on regulatory T cell percentage. CONCLUSIONS: The results of the current study are consistent with previous studies addressing the function of regulatory T cells in inducing immunotolerance after kidney transplant. Considering the established role of regulatory T cells in graft maintenance and our observation of high regulatory T cell percentage in patients receiving rapamycin than cyclosporine, we recommend including rapamycin when possible in immunosuppressive protocols. The findings from the current study on the chronic rejection group support ongoing research of having treatment with regulatory T cells, which may constitute a novel, efficient antirejection therapy in the future.

Human leukocyte antigen G and renal allograft transplant.

OBJECTIVES: Studying immune tolerance induced by HLA-G in kidney allograft acceptance may help understanding of its mechanisms, hoping in the future to boaster it and decrease the immunosuppressive drugs given that are well known to have serious adverse effects. MATERIALS AND METHODS: The current study sought to evaluate soluble HLA-G in 3 groups: kidney transplanted patients with no rejection episodes, transplanted patients with biopsy-proven renal rejection, and healthy age-matched non transplanted individuals. Three groups were studied: kidney transplanted patients with no rejection episodes (n = 43); transplanted patients with biopsy-proven renal rejection (n = 27); healthy, age-matched, nontransplanted individuals as controls (n = 42). Soluble HLA-G level was measured in the serum by a quantitative sandwich enzyme linked immunosorbent assay. RESULTS: sHLAG level was significantly higher in the transplanted patients compared with the control. Prograf and not cyclosporine or Rapamune had positive effects on sHLAG levels. Patients with chronic rejection had a significant lower level of sHLAG compared with a graft stable group. No effect of donor type, infection or duration posttransplant, on sHLAG levels was found. CONCLUSIONS: The results of the current study are consistent with previous studies addressing the role of sHLAG in inducing immunotolerance postkidney transplant. The findings from the current study on the chronic rejection group, supports the on-going research of having a treatment with HLA-G/or derivate, which may constitute in the future a novel efficient antigraft rejection therapy.

Characterization of cephalosporin-resistant clinical Enterobacteriaceae for CTX-M ESBLs in Bahrain.

OBJECTIVE: To detect the presence of specific CTX-M class of extended spectyum ß-lactamases (ESBLs) in a collection of cephalosporin-resistant Enterobacteriaceae isolates from Bahrain. METHODS: A subset of 80 cephalosporin-resistant Enterobacteriaceae collected from Salmaniya Medical Complex, Bahrain, were characterized further for the presence of specific genogroups of CTX-M ß-lactamases by multiplex- and monoplex- PCRs. The primers used for the multiplex and monoplex PCRs were of genogroups- 1, 2, 8, 9 and 25. Sequencing of the representative isolates was performed to find the circulating CTX-M-types. RESULTS: A total of 93.8% (75/80) isolates showed the amplicons corresponding to any of the genogroups (1, 2, 8, 9, 25) and the remaining 6.2% isolates turned out negative in multiplex PCR. Some of the isolates demonstrated multiple bands corresponding to the sizes of different genogroups. Further confirmation with respective monoplex PCR on these 75 isolates demonstrated that 93.3% (70/75) harbored CTX-M genogroup-1 and 6.7% (5/75) harbored genogroup-9. We did not find the presence of genogroups 2, 8, and 25 in these isolates by monoplex PCR. Sequencing results of genogroup-1 isolates demonstrated the presence of CTX-M-15-like ESBL, however, discrepant results were noticed in genogroup-9 isolates, sequencing showed them as CTX-M-55-like ESBL. CONCLUSIONS: This is the first report from Bahrain characterizing the CTX-M genogroups of ESBLs and reporting the emergence of blaCTX-M-55-like gene in this region.

Campylobacter jejuni induces diverse kinetics and profiles of cytokine genes in INT-407 cells.

OBJECTIVE: To examine the kinetic ability of embryonic human epithelial INT-407 cells to express messenger ribonucleic acid mRNA for various cytokines and chemokines in response to Campylobacter jejuni C. jejuni stimulation. METHODS: In an experimental single-blind study, cultured embryonic human epithelial INT-407 cells were treated with different concentrations of viable C. jejuni, its sonicated, and filtered supernatant. A modified non-radioactive in situ hybridization using probe cocktails was used to measure mRNA levels for the pro-inflammatory cytokines interleukin IL-1beta, IL-6, interferon-gamma IFN-gamma, tumour necrosis factor TNF-alpha, transforming growth factor TGF-beta1, and IL-8, and the anti-inflammatory cytokines, IL-4 and IL-10. The study was carried out from September 2005 to March 2007 at the Department of Microbiology, Immunology, and Infectious Diseases, College of Medicine, Arabian Gulf University, Bahrain. RESULTS: Viable C. jejuni, sonicated bacteria and filtered supernatant induced high mRNA expression for the pro-inflammatory cytokines IL-1beta, IL-6, IFN-gamma, TNF-alpha, TGF-beta1, and IL-8, which peaked at the 12 hours post stimulation. Anti-inflammatory cytokines IL-4 and IL-10 mRNA expression were induced maximally at 3 hours post stimulation mainly by sonicated bacteria and filtrated supernatant, however, not with living bacteria. Untreated embryonic human epithelial INT-407 cells expressed low amount of mRNA for the various cytokines and chemokines at all time points. For each cytokine, 4 samples were used per time hour. CONCLUSION: This study demonstrated that embryonic human epithelial INT-407 cells in response to viable C. jejuni or its cytotoxins can alter cytokine and chemokine mRNA expression patterns and kinetics suggesting a potential role for theses mediators in the immunopathogenesis of the infection caused by this pathogen, which might be relevant for future immunotherapeutic interventions during severe bacterial infections.

Progress towards a Leishmania vaccine.

Leishmaniasis is a vector-born protozoan disease. Approximately 12 million individuals are affected worldwide with an estimated annual incidence of 1.5-2 million. Two clinical manifestations are recognized, cutaneous, and visceral, both of which are common in the Middle East. In both forms, infection is chronic, with potential deformities, persistence following cure, and lifelong risk of reactivation. Attempts to develop an effective human Leishmania vaccine have not yet succeeded. Leishmanization, a crude form of live vaccination historically originated in this part of the world. Experimental vaccination has been extensively studied in model animals in the past 2 decades. In this review, major human killed vaccine trials are surveyed, and modern trends in Leishmania vaccine development, including subunit vaccines, naked DNA vaccines, and transmission blocking vaccines are explored. Recent findings of a link between persistence of live parasites, and maintenance of long-term immunity suggest live vaccination with attenuated strains, as a future vaccination strategy.

Clinical relevance of virulence genes in Campylobacter jejuni isolates in Bahrain.

There are no data describing the genetic make-up of Campylobacter strains (an important aetiological agent of diarrhoea) circulating in the Arabian Gulf region. Here, the molecular characterization of two virulence genes in Campylobacter jejuni from Bahrain and the relationship with clinical infection are reported. Molecular screening for cytolethal distending toxin (cdtB) and invasion-associated marker (iam) genes was carried out on C. jejuni stool isolates collected from January 2002 to January 2004 in Bahrain. The molecular characterization was correlated with the patients' socio-demographic and clinical parameters. Of the 96 C. jejuni strains tested, 50 (52 %) were cdtB+/iam+, 30 (31 %) were cdtB+/iam- and 16 (17 %) were cdtB-/iam-. Sixty-nine per cent (66/96) of patients were less than 3 years old, with significantly higher detection of cdtB+/iam+ and cdtB+/iam- strains (P < 0.001 and P < 0.01, respectively) in this age group. Seventy patients (73 %) were symptomatic. In the group that were less than 3 years old, 62 and 85 % of those with cdtB+/iam+ and cdtB+/iam- strains, respectively, were symptomatic compared with 100 % for those over 3 years of age. However, the presence of cdtB-/iam- strains still resulted in clinical infection in the children under 3 years but not in the older patients. This is the first report describing the molecular characterization of virulence genes in Campylobacter isolates from this region. The findings indicate that strains of different virulence genetic make-up are circulating in the population, with children under the age of 3 years being most vulnerable. Further work on the molecular characterization, gene expression and determination of the invasive phenotypes of C. jejuni strains circulating in different regions is needed.

Intraocular pressure measurement after hyperopic and myopic LASIK.

PURPOSE: To assess the effects of hyperopic and myopic laser in situ keratomileusis (LASIK) on the intraocular pressure (IOP) measurement by Goldmann applanation tonometry. METHODS: Intraocular pressure was measured by Goldmann applanation tonometry pre- and postoperatively in 48 hyperopic eyes (26 patients) and 56 myopic eyes (28 patients). RESULTS: The mean preoperative spherical equivalent refraction was +3.97 +/- 2.48 diopters (D) in the hyperopic group and -6.73 +/- 4.79 D in the myopic group. Attempted correction was equivalent to preoperative refraction in each group. Mean preoperative IOP was 14.22 +/- 2.56 mmHg and 13.70 +/- 2.09 mmHg in the hyperopic and myopic groups, respectively. Following LASIK, the mean IOP was 11.85 +/- 2.52 mmHg and 11.38 +/- 3.08 mmHg in the hyperopic and myopic groups, respectively. CONCLUSIONS: The IOP measurement was significantly reduced (P < .001) by 2.37-2.25 mmHg and 2.32 +/- 2.89 mmHg following hyperopic and myopic LASIK, respectively.

Serodiagnosis of toxoplasmosis in Bahrain.

OBJECTIVE: We report here an analysis of results of anti-Toxoplasma immunoglobulin M (IgM) and immunoglobulin G (IgG) measurements reported from the Central Laboratory of Bahrain over a period of 3 years and 9 months. METHODS: This study included all blood samples received at the Salmaniya Medical Complex, Manama, Bahrain serology laboratory for the determination of Toxoplasma-specific antibodies during the period of January 2000 to September 2003. A total of 4,739 specimens were assayed for IgG and 1,947 for IgM. RESULTS: An overall seropositivity of 21.8% for IgG and 10.3% for IgM antibodies were found with no gender differences for either IgG or IgM. In addition, no statistically significant positivity rate differences (p=0.723) were observed between Bahrainis and non-Bahraini residents. The prevalence of Toxoplasma gondii (T. gondii)-specific IgG amongst post partum women was 15.8% and 6.3% for IgM, while for women of child-bearing age IgG was higher at 22.3% and 11.6% for IgM. The IgG seropositivity in neonates (<1 month old) was 16.5%, decreasing to 9.3% in preschool children, while for IgM, it was 3.7% at birth increasing to 7.3% in the preschool group. The IgG seroprevalence increased within the first 15 years of life, and leveled thereafter, for IgM however, it was low at birth and increased within the first 12 months of life then leveled-off at the age of 20-40 to approximately 11-14%, with a further increase after 40 years to 17%. CONCLUSION: The seropositivity rates of T. gondii in the samples examined during the present study fall within the range of other Gulf Cooperative Council countries. True prevalence in the general population may actually be lower.

Antigen requirements for efficient priming of CD8+ T cells by Leishmania major-infected dendritic cells.

CD4(+) and CD8(+) T-cell responses have been shown to be critical for the development and maintenance of acquired resistance to infections with the protozoan parasite Leishmania major. Monitoring the development of immunodominant or clonally restricted T-cell subsets in response to infection has been difficult, however, due to the paucity of known epitopes. We have analyzed the potential of L. major transgenic parasites, expressing the model antigen ovalbumin (OVA), to be presented by antigen-presenting cells to OVA-specific OT-II CD4(+) or OT-I CD8(+) T cells. Truncated OVA was expressed in L. major as part of a secreted or nonsecreted chimeric protein with L. donovani 3' nucleotidase (NT-OVA). Dendritic cells (DC) but not macrophages infected with L. major that secreted NT-OVA could prime OT-I T cells to proliferate and release gamma interferon. A diminished T-cell response was observed when DC were infected with parasites expressing nonsecreted NT-OVA or with heat-killed parasites. Inoculation of mice with transgenic parasites elicited the proliferation of adoptively transferred OT-I T cells and their recruitment to the site of infection in the skin. Together, these results demonstrate the possibility of targeting heterologous antigens to specific cellular compartments in L. major and suggest that proteins secreted or released by L. major in infected DC are a major source of peptides for the generation of parasite-specific CD8(+) T cells. The ability of L. major transgenic parasites to activate OT-I CD8(+) T cells in vivo will permit the analysis of parasite-driven T-cell expansion, differentiation, and recruitment at the clonal level.

Conditions influencing the efficacy of vaccination with live organisms against Leishmania major infection.

Numerous experimental vaccines have been developed with the goal of generating long-term cell-mediated immunity to the obligate intracellular parasite Leishmania major, yet inoculation with live, wild-type L. major remains the only successful vaccine in humans. We examined the expression of immunity at the site of secondary, low-dose challenge in the ear dermis to determine the kinetics of parasite clearance and the early events associated with the protection conferred by vaccination with live L. major organisms in C57BL/6 mice. Particular attention was given to the route of vaccination. We observed that the rapidity, strength, and durability of the memory response following subcutaneous vaccination with live parasites in the footpad are even greater than previously appreciated. Antigen-specific gamma interferon (IFN-gamma)-producing T cells infiltrate the secondary site by 1.5 weeks, and viable parasites are cleared as early as 2.5 weeks following rechallenge, followed by a rapid drop in IFN-gamma(+) CD4(+) cell numbers in the site. In comparison, intradermal vaccination with live parasites in the ear generates immunity that is delayed in effector cell recruitment to the rechallenge site and in the clearance of parasites from the site. This compromised immunity was associated with a rapid recruitment of interleukin-10 (IL-10)-producing CD4(+) T cells to the rechallenge site. Treatment with anti-IL-10-receptor or anti-CD25 antibody enhanced early parasite clearance in ear-vaccinated mice, indicating that chronic infection in the skin generates a population of regulatory cells capable of influencing the level of resistance to reinfection. A delicate balance of effector and regulatory T cells may be required to optimize the potency and durability of vaccines against Leishmaniasis and other intracellular pathogens.

Coinfection with Listeria monocytogenes potentlxtes the response of BALB/c mice against Leishmania major.

Protection against L. major is dependent on the stimulation of an anti-leishmanial T helper1 (Th1) response and the production of Interferon (IFN)-y. BALB/c mice develop a Th2 response and fatal infection with Leishmania major. Strategies that boost IL-12 production have shown to be protective. The innate response to Listeria monocytogenes is associated with IL-12 production. The co-infection of BALB/c mice with L. monocytogenes attenuates the course of L. major infection. Lesion sizes were smaller, and co-infected mice out-survived controls injected with L. major alone. The parasite load was reduced at site of injection, in draining lymph nodes (LN) and spleen. During the first week of infection, in-vitro Leishmania-restimulated LN cells from co-infected mice produced higher levels of IFN-7 and undetectable levels of IL-4 compared to controls. Significant IL-4 mRNA expression was detected in LN cells of control but not in co-infected mice.

Coinjection with CpG-containing immunostimulatory oligodeoxynucleotides reduces the pathogenicity of a live vaccine against cutaneous Leishmaniasis but maintains its potency and durability.

The inoculation of live, nonattenuated Leishmania major to produce a lesion in a selected site that heals, referred to as leishmanization, is to date the only vaccine against leishmaniasis that has proven to be effective in humans. Its use has been restricted or abandoned entirely, however, due to safety concerns. In an attempt to develop a leishmanization protocol that minimizes pathology while maintaining long-term protection, live parasites were coinjected with CpG-containing immunostimulatory oligodeoxynucleotides (CpG ODNs) alone or in combination with whole-cell lysates of heat-killed L. major promastigotes bound to alum (ALM). C57BL/6 mice infected intradermally by using L. major plus CpG ODN with or without ALM developed few or no dermal lesions and showed an early containment of parasite growth, while mice infected with L. major with or without ALM developed sizable dermal lesions that required up to 10 weeks to heal. The CpG ODNs provoked a transient inflammation that included an early recruitment and accumulation of gamma interferon-producing CD4(+) lymphocytes in the site. Attenuation of the live vaccine did not compromise its ability to confer long-term immunity, as mice receiving L. major and CpG ODN plus ALM were totally protected against reinfection with L. major for up to 6 months. By comparison, the immunity elicited by two efficient nonlive vaccines began to wane by 6 months. Our results suggest that immune modulation using CpG ODNs might be a practical approach to improving the safety of a highly effective live vaccine that has already been widely applied.