Raul V. Destura

Pyrosequencing for Rapid Detection of Mycobacterium tuberculosis Resistance to Rifampin, Isoniazid, and Fluoroquinolones

ABSTRACT After isoniazid and rifampin (rifampicin), the next pivotal drug class in Mycobacterium tuberculosis treatment is the fluoroquinolone class. Mutations in resistance-determining regions (RDR) of the rpoB, katG, and gyrA genes occur with frequencies of 97%, 50%, and 85% among M. tuberculosis isolates resistant to rifampin, isoniazid, and fluoroquinolones, respectively. Sequences are highly conserved, and certain mutations correlate well with phenotypic resistance. We developed a pyrosequencing assay to determine M. tuberculosis genotypic resistance to rifampin, isoniazid, and fluoroquinolones. We characterized 102 M. tuberculosis clinical isolates from the Philippines for susceptibility to rifampin, isoniazid, and ofloxacin by using the conventional submerged-disk proportion method and validated our pyrosequencing assay using these isolates. DNA was extracted and amplified by using PCR primers directed toward the RDR of the rpoB, katG, and gyrA genes, and pyrosequencing was performed on the extracts. The M. tuberculosis H37Rv strain (ATCC 25618) was used as the reference strain. The sensitivities and specificities of pyrosequencing were 96.7% and 97.3%, 63.8% and 100%, and 70.0% and 100% for the detection of resistance to rifampin, isoniazid, and ofloxacin, respectively. Pyrosequencing is thus a rapid and accurate method for detecting M. tuberculosis resistance to these three drugs.

Clostridium difficile Toxin A Induces Intestinal Epithelial Cell Apoptosis and Damage: Role of Gln and Ala-Gln in Toxin A Effects

The aim of this study was to investigate the effect of Clostridium difficile toxin A (TxA) on intestinal epithelial cell migration, apoptosis, and transepithelial resistance and to evaluate the effect of glutamine (Gln) and its stable derivative, alanyl-glutamine (Ala-Gln), on TxA-induced damage. Migration was measured in rat intestinal epithelial cells (IEC-6) 6 and 24 hr after a razor scrape of the cell monolayer. Cell proliferation was indirectly measured utilizing the tetrazolium salt WST-1. The cells were incubated with TxA (1–100 ng/ml) in medium without Gln or medium containing Gln or Ala-Gln (1–30 mM). Apoptosis was quantified in IEC-6 cells using annexin V assay. Transepithelial resistance was measured using an epithelial voltohmmeter across T84 cells seeded on a transwell filter. TxA-induced a dose-dependent reduction of migration and also caused dose and time-dependent apoptosis in IEC-6 cells. Gln and Aln-Gln significantly enhanced IEC-6 cell migration and proliferation. Gln and Ala-Gln also prevented the inhibition of migration, apoptosis, and the initial drop in transepithelial resistance induced by TxA. In conclusion, both peptides reduced toxin-induced epithelial damage and thus might play an adjunctive role in C. difficile-induced colitis therapy.

Laboratory diagnosis and susceptibility profile of Helicobacter pylori infection in the Philippines

BackgroundHelicobacter pylori diagnosis and susceptibility profile directs the applicability of recommended treatment regimens in our setting. To our knowledge, there is no published data on the culture and local susceptibility pattern of Helicobacter pylori in the Philippines.Methods52 dyspeptic adult patients undergoing endoscopy from the Outpatient Gastroenterology clinic of the University of the Philippines-Philippine General Hospital underwent multiple gastric biopsy and specimens were submitted for gram stain, culture, antimicrobial sensitivity testing, rapid urease test and histology. Antimicrobial susceptibility testing was done by Epsilometer testing (Etest) method against metronidazole, clarithromycin, amoxicillin, and tetracycline.ResultsSixty percent (60%) of the study population was positive for H. pylori infection (mean age of 44 years ± 13), 70% were males. H. pylori culture showed a sensitivity of 45% (95% CI [29.5–62.1]), specificity of 98% (95%CI [81.5–100%]), positive likelihood ratio of 19.93 (95% CI [1.254–317.04]) and a negative likelihood ratio of 0.56 (95% CI [0.406–0.772]). All H. pylori strains isolated were sensitive to metronidazole, clarithromycin, amoxicillin and tetracycline.ConclusionKnowledge of the antibiotic susceptibility patterns in our setting allows us to be more cautious in the choice of first-line agents. Information on antibiotic susceptibility profile plays an important role in empiric antibiotic treatment and management of refractive cases.

Clostridium difficile and Entamoeba histolytica infections in patients with colitis in the Philippines

Amoebiasis is a common cause of non-specific colitis in the Philippines. The prevalence of Clostridium difficile infection with colitis is unknown. Empiric use of metronidazole for colitis treatment is widely practiced. We investigated the association of C. difficile or Entamoeba histolytica infection with endoscopically/histopathologically proven colitis among adults in the Philippines. Two hundred and ten patients undergoing colonoscopy were enrolled. Demographic and clinical data were reviewed. Stool specimens were assayed for C. difficile and E. histolytica by ELISA. Microscopy was performed. The mean age of the patients was 53 y (range: 19-88 y) and 53% were male. Colitis was diagnosed in 39 of 205 patients. Clostridium difficile, E. histolytica and parasites were seen in 17 (43.6%), 10 (25.6%) and 11 (28.2%), respectively, of patients with colitis compared with 36 (21.7%; p=0.005), 13 (7.8%; p=0.001) and 56 (33.7%; p=0.51), respectively, of those without colitis. Diarrhoea and antibiotic intake history were significantly more common among patients with colitis than those without (43.6% and 20.5% vs 18.1% and 5.4%; p=0.001 and p=0.006, respectively). The mean duration of diarrhoea was 2.53 d shorter among patients with colitis. The most frequent antibiotics taken were fluoroquinolones and metronidazole (50% and 40% of antibiotic courses, respectively, in patients with colitis). This study suggests that C. difficile infection is common and might be overlooked in settings where amoebiasis and intestinal parasitism are endemic.

Antibiotic management of complicated intra-abdominal infections in adults: The Asian perspective.

Regional epidemiological data and resistance profiles are essential for selecting appropriate antibiotic therapy for intra-abdominal infections (IAIs). However, such information may not be readily available in many areas of Asia and current international guidelines on antibiotic therapy for IAIs are for Western countries, with the most recent guidance for the Asian region dating from 2007. Therefore, the Asian Consensus Taskforce on Complicated Intra-Abdominal Infections (ACT-cIAI) was convened to develop updated recommendations for antibiotic management of complicated IAIs (cIAIs) in Asia. This review article is based on a thorough literature review of Asian and international publications related to clinical management, epidemiology, microbiology, and bacterial resistance patterns in cIAIs, combined with the expert consensus of the Taskforce members. The microbiological profiles of IAIs in the Asian region are outlined and compared with Western data, and the latest available data on antimicrobial resistance in key pathogens causing IAIs in Asia is presented. From this information, antimicrobial therapies suitable for treating cIAIs in patients in Asian settings are proposed in the hope that guidance relevant to Asian practices will prove beneficial to local physicians managing IAIs.

Advancing Cryptosporidium Diagnostics from Bench to Bedside

Cryptosporidium is increasingly being recognized as an important cause of diarrhea worldwide. Although well known for its impact among HIV positive population, improved diagnostic tests have contributed to its emerging recognition one among the most prevalent causes of early childhood moderate to severe diarrhea, persistent diarrhea, and impaired neurocognitive development. The diagnosis of Cryptosporidiosis is generally carried out based on availability of skilled microscopist or advanced equipment for molecular- and immunologic-based assays. As an emerging enteric pathogen of medical importance, the need for point-of-care technology is deemed necessary for early identification of the pathogen and application of infection control measures for its potential risk of creating outbreaks. Current point-of-care technologies demonstrate varying sensitivities and specificities and may already address the present diagnostic need.

IAMP tackles a void in medical education: leadership

1 Legrand M, Mateo J, Aribaud A, et al. The use of dabigatran in elderly patients. Arch Intern Med 2011; 171: 1285–86. 2 Connolly SJ, Ezekowitz MD, Yusuf S, et al. Dabigatran versus warfarin in patients with atrial fi brillation. N Engl J Med 2009; 361: 1139–51. 3 Connolly SJ, Eikelboom J, Joyner C, et al. Apixaban in patients with atrial fi brillation. N Engl J Med 2011; 364: 806–17. 4 Patel MR, Mahaff ey KW, Garg J, et al. Rivaroxaban versus warfarin in nonvalvular atrial fi brillation. N Engl J Med 2011; 365: 883–91. 5 Eikelboom JW, Wallentin L, Connolly SJ, et al. Risk of bleeding with 2 doses of dabigatran compared with warfarin in older and younger patients with atrial fi brillation: an analysis of the randomized evaluation of long-term anticoagulant therapy (RE-LY) trial. Circulation 2011; 123: 2363–72. developed through peer and senior faculty interactions. Participants from developing countries acknowledged challenges of limited resources and infrastructure. For example, one participant had been asked to start a centre with US

Continued circulation of a single genotype of dengue virus serotype 2 in the Philippines

30 of funding. Common challenges in the developing and developed world were the diffi culty of breaking down silos, and that politics, as well as preference for seniority over talent, can often get in the way of success. Participants from all countries shared diffi culties in gaining credibility as young leaders and breaking into established hierarchies. Most important from this experience was the sense that the world is small; that the goals, aspirations, and challenges of our junior faculty remain similar despite vastly diff erent cultures and access to resources. Having pioneered this YPL programme for the young physician leaders, the next issue to address is the programme’s future. The IAMP executive board members will continue to work as personal mentors for each inaugural YPL member, providing their insight and guidance on future career decisions and linking them to other professional leaders in their fi elds. The inaugural class of IAMP’s YPL leadership programme felt that the workshop was a success and the IAMP asks for its member academies to continue supporting these young leaders as they return to their countries and support an annual programme and development of a growing network of young physician leaders. These eff orts will help address the dearth of leadership training programmes for young academicians and nurture them as they learn to shape global health policy for millions in need.

Detection of Epithelial-Cell Injury, and Quantification of Infection, in the HCT-8 Organoid Model of Cryptosporidiosis

OBJECTIVE To obtain descriptive information of behavioral pattern in Chinese school-aged children with cleft lip and palate. METHODS A total of 93 cleft lip and palate patients between the age of 6-11 year-old and treated at West China Stomatology Hospital were selected. And another 100 unaffected controls, matched for age and gender, were recruited randomly from a common primary school in Chengdu. Chart review of medical records was used to obtain psychosocial checklists. Scores were compared with published norms and controls to evaluate the risk of problems, separately for three diagnostic groups. RESULTS The patients group had lower scores of social and academic competencies, especially those with facial deformity or speech problem. No difference was found in the aspect of activity competency. All patients showed elevations in behavior problems. But the type of behavior problems varied in different genders. CONCLUSIONS Chinese school-aged children with cleft lip and palate are at raised risk for social and academic difficulties. Specific pattern of behavior problems displays differently depending on gender of the patient.