Emergence of community-associated methicillin-resistant Staphylococcus aureus carriage in children in Cambodia

We previously described the first reported isolation of methicillin-resistant Staphylococcus aureus (MRSA) (a case series of pediatric community-associated MRSA infections) in Cambodia. We define the rate of pediatric MRSA carriage in the same population and characterize the associated bacterial genotypes by using pulsed-field gel electrophoresis and multilocus sequence typing. A prospective cohort study of MRSA carriage conducted over one month at the Angkor Hospital for Children, Siem Reap, Cambodia, identified MRSA carriage in 87 (3.5%) of 2,485 children who came to the outpatient department, and 6 (4.1%) of 145 inpatients, including at least two with cases of nosocomial acquisition. Genotyping of all 93 MRSA isolates resolved 5 genotypes. Most (91%) isolates were assigned to sequence type 834. Only 28 (32%) of 87 MRSA carriers identified in the outpatient department had no history of recent healthcare contact. The study findings have important implications for healthcare in a setting where diagnostic microbiology and access to antimicrobial drugs with efficacy against MRSA are limited.     

Accuracy of loop-mediated isothermal amplification for diagnosis of human leptospirosis in Thailand

There is a lack of diagnostic tests for leptospirosis in technology-restricted settings. We developed loop-mediated isothermal amplification (LAMP) specific for the 16S ribosomal RNA gene (rrs) of pathogenic and intermediate group Leptospira species. The lower limit of detection was 10 genomic equivalents/reaction, and analytical specificity was high; we observed positive reactions for pathogenic/intermediate groups and negative reactions for non-pathogenic Leptospira species and other bacterial species. We evaluated this assay in Thailand by using a case-control study of 133 patients with laboratory-proven leptospirosis and 133 patients with other febrile illnesses. Using admission blood, we found that the rrs LAMP showed positive results in 58 of 133 cases (diagnostic sensitivity = 43.6, 95% confidence interval [CI] = 35.0-52.5) and in 22 of 133 controls (diagnostic specificity = 83.5, 95% CI = 76.0-89.3). Sensitivity was high for 39 patients who were culture positive for Leptospira spp. (84.6, 95% CI = 69.5-94.1). The rrs LAMP can provide an admission diagnosis in approximately half of patients with leptospirosis, but its clinical utility is reduced by a lower specificity.     

Knowledge and attitudes towards vitiligo in the general population: A survey based on the simulation video of a real situation

Background/Objective

Vitiligo is a common pigmentary disorder. Significant psychological effects and social discrimination was noticed among vitiligo patients. The severity of the negative impact is related to the society's attitude and knowledge. Therefore, this study aims to investigate knowledge and attitudes among general population towards vitiligo.

Role of tonsillectomy in the search for a squamous cell carcinoma from an unknown primary in the head and neck

Cervical metastases of an unknown primary lesion accounts for 5%-10% of head and neck cancers. Tonsillar carcinoma is the third most common, with an incidence in the region of 18%-47% of unknown primaries, and the likelihood of synchronous tonsillar tumour of about 5%-10%. Current practice for investigating an unknown primary includes computed tomography (CT) with or without magnetic resonance imaging (MRI), and then, if necessary, 18-fluoro-deoxy-glucose positron emission tomography (FDG-PET). This is followed by panendoscopy with biopsy from the potential primary sites and tonsillectomy. A five-year retrospective review of our database of metastatic squamous cell carcinomas from unknown primary sites identified patients with synchronous tonsillar tumours (n=5). All had clinically normal-looking tonsils; MRI was within normal limit and a PET-CT identified the ipsilateral tonsillar primary in three cases only. Bilateral tonsillectomy rather than a tonsillar biopsy in the search for an unknown primary has been standard practice in our unit. Identification of a second primary dictates planning for the primary site and in the long term, influences patients' survival. We therefore recommend that bilateral tonsillectomy should be standard in the investigation of patients who present with cervical metastases from an unknown primary in the head and neck region.     

Relationship of conventional and fluorescent microscopic technique to assess in vitro semen quality status of Murrah buffalo males

In vitro fertility assessment using fluorescent technique is a better predictor of fertility status of bulls as compared to traditional semen quality assessment techniques, therefore, the study was planned to assess in vitro fertility status of bulls based on conventional and fluorescent techniques. Seventy-three ejaculates were collected from 12 Murrah buffalo bulls maintained at Artificial Breeding Research Centre, NDRI, Karnal, India for the experiment and subjected to statistical analysis using SYSTAT. The mean values of ejaculate volume (ml), mass activity, individual motility (%), sperm concentration (millions/ml), live sperm (%), total abnormalities (%), HOST (%) and acrosomal integrity (%) were 2.70 ± 0.28, 2.8 ± 0.14, 63.8 ± 2.16, 1749.7 ± 122.24, 77.3 ± 2.48, 6.2 ± 0.51, 75.1 ± 1.81 and 84.5 ± 2.26, respectively. The repeatability estimates were significant (P<0.05) for ejaculate volume (0.34 ± 0.137), acrosomal integrity (0.29 ± 0.134) and live percentage (0.28 ± 0.133), indicating sufficient bull to bull variation for the parameters. The mean values of seminal attributes of fluorescent based criteria of CMA3 (Chromomycin A3), SYBR-PI and FITC-PNA (fluorescent isothiocynate-conjugated peanut agglutinin) were 5.25 ± 0.41, 67.91 ± 1.24 and 82.00 ± 1.25 percent, respectively. Bulls were ranked on the basis of expected producing ability (EPA) for semen characteristics assessed by conventional and fluorescent criteria. Rank correlations were found to be significant for FITC with most of the parameters evaluated by conventional methods. In conclusion, among the conventional criteria, individual motility (%) revealed ranking of bulls almost similar to that of fluorescent criteria.Key Words: Conventional method, Fluorescent method, Murrah buffalo bull, Semen quality     

Leptospira Species in Floodwater during the 2011 Floods in the Bangkok Metropolitan Region,Thailand

Floodwater samples (N = 110) collected during the 2011 Bangkok floods were tested for Leptospira using culture and polymerase chain reaction (PCR); 65 samples were PCR-positive for putatively non-pathogenic Leptospira species, 1 sample contained a putatively pathogenic Leptospira, and 6 samples contained Leptospira clustering phylogenetically with the intermediate group. The low prevalence of pathogenic and intermediate Leptospira in floodwater was consistent with the low number of human leptospirosis cases reported to the Bureau of Epidemiology in Thailand. This study provides baseline information on environmental Leptospira in Bangkok together with a set of laboratory tests that could be readily deployed in the event of future flooding.Leptospirosis is an endemic infection throughout the tropics, where outbreaks are well-described, often in the context of heavy freshwater flooding.^1,2 Flooding of the Bangkok Metropolitan Region between late October of 2011 and January of 2012 posed a potential risk for a leptospirosis outbreak, and case reports were monitored by the Bureau of Epidemiology of Thailand. The purpose of this article is to describe two adjunctive methods (direct polymerase chain reaction [PCR] and culture) to determine whether pathogenic Leptospira spp. was present in the floodwater.A total of 110 floodwater samples was collected between November 14 and December 6 of 2011; 70 samples were taken within a radius of approximately 2 km around the Salaya Campus of Mahidol University in Nakhon Pathom province (sample code NP), and 40 samples were taken within a radius of approximately 8 km in Don Muang district, Bangkok province (sample code DM) (Figure 1). At each sampling point, 100 mL floodwater were collected at a depth of 20 cm into a sterile glass bottle. Samples were maintained at ambient temperature (25–32°C) during transportation to the laboratory; 50 mL sample were used for direct PCR assay, and the remaining 50 mL sample were used for culture.Open in a separate windowFigure 1.Map of the Bangkok Metropolitan Region showing the extent of flooding in 2011 and the two sampling zones. Flood data were obtained from the Geo-Informatics and Space Technology Development Agency in Thailand, and the map was generated using Google Earth (Google Inc.). The area flooded in 2011 is shown in blue, and the sampling zones are shown as red circles.A published PCR assay based on amplification and sequencing of a region of the 16S rRNA gene (rrs)³ was used to test all water samples. This testing was performed on a 50-mL sample that was first centrifuged at 3,000 × g for 30 minutes. The deposit was resuspended in 140 μL sterile water, and DNA was extracted from the suspension using the QIAamp Viral RNA Mini Kit (QIAGEN, Santa Clarita, CA) Five microliters DNA extract were used in the reaction, which was performed as described previously.³ The 433-bp amplicons were sequenced, and the species of Leptospira was inferred based on position in a maximum likelihood (ML) phylogenetic tree, which included 36 rrs sequences from GenBank for Leptospira species belonging to the pathogenic, intermediate (of intermediate pathogenicity), non-pathogenic, or unculturable groups Supplemental Table 1. The tree was constructed using the algorithm implemented in PhyML version 3.0.1,⁴ and the model of sequence evolution used was the generalized time-reversible (GTR) model with γ-distributed rate variation. An ML tree was constructed using the nearest neighbor interchange (NNI) method.⁴ The MEGA program⁵ was used to display and edit the tree. The rrs sequences generated during this study were submitted to GenBank, and they are provided in Supplemental Table 2.Fifty milliliters each water sample were passed through a sterile 0.2-μm filter (Sartorius AG, Gottingen, Lower Saxony, Germany); 0.5 mL filtrate were inoculated into a tube containing 3 mL Leptospira Vanaporn Wuthikanun (LVW) solid agar slant containing 1% Noble agar base and 10% rabbit serum.⁶ Slants were incubated at 30°C in 5% CO₂ for 2 days followed by 30°C in air for a total of 28 days.⁶ The surface fluid on each agar slant was examined two times weekly by dark-field microscopy. If spirochetes were observed, 100 μL surface fluid were spread-plated onto LVW agar⁶ supplemented with 2,6-dichlorophenolindophenol (10 mg/mL; Sigma-Aldrich) to enhance visibility of Leptospira colonies and incubated at 30°C in 5% CO₂ for 2 days followed by 30°C in air for up to 28 days. Plates were examined two times weekly for visible colonies. We hypothesized that water samples might contain more than one strain of Leptospira species, which may manifest as different colony morphologies on solid agar. In view of this possibility, a single colony of each morphology type on a given plate was picked for additional analysis. These colonies were inoculated into 3 mL Ellinghausen-McCullough-Johnson-Harris (EMJH) broth and incubated at 30°C in air for 5–7 days to achieve a Leptospira concentration of 10⁸ cfu/mL. DNA was then extracted using a boiling method and screened using a multiplex PCR assay developed during this study (using both rrs and lipL32 genes as targets) (Supplemental Text 1). Colonies were assigned to pathogenic, intermediate, or non-pathogenic groups (Supplemental Text 1). Samples that were positive for pathogenic and intermediate species were further evaluated using the rrs assay.³Direct rrs PCR of 110 floodwater samples yielded an amplicon that could be sequenced in 65 cases (59%), of which 45 cases were from Nakhon Pathom and 20 cases were from Bangkok. These 65 sequences were resolved into eight rrs alleles with two polymorphic sites. Analysis of eight alleles using the blastn algorithm implemented in BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) showed that the highest matches (nucleotide identities ranging from 98% to 99%) were rrs sequences cloned from unculturable bacteria in freshwater samples from several countries, including the United States, Korea, and China in 2003, 2008, and 2010, respectively (GenBank ID codes {"type":"entrez-nucleotide","attrs":{"text":"DQ065390","term_id":"67550984","term_text":"DQ065390"}}DQ065390, {"type":"entrez-nucleotide","attrs":{"text":"FJ164045","term_id":"205278401","term_text":"FJ164045"}}FJ164045, and {"type":"entrez-nucleotide","attrs":{"text":"FJ820401","term_id":"268308545","term_text":"FJ820401"}}FJ820401, respectively). On phylogenetic analysis, all eight alleles resided in the non-pathogenic Leptospira spp. cluster (Figure 2).Open in a separate windowFigure 2.Phylogenetic analysis of partial rrs sequences. An ML tree was based on a 443-nt region of rrs. Thirty-six reference sequences from GenBank are shown as color-coded circles that denote their species group: pathogenic (brown), intermediate pathogenicity (green), non-pathogenic (yellow), uncertain pathogenicity (white), or unculturable (grey). Black circles denote two members of the family Leptospiracae. Leptospira species sequences obtained from floodwater samples are shown as red triangles. Two samples are shown two times, because they each contained two different species.Culture yielded colonies from 87 of 110 samples (79.1%), which on dark-field microscopy, consisted of spirochetes (Supplemental Table 4). Picking one representative colony for each morphology type on a given plate yielded 140 single colonies for evaluation by the multiplex PCR assay. Results are presented using sample as the denominator, with additional information provided for samples containing more than one species. A single sample (sample code NP-29) was positive for pathogenic Leptospira species, and phylogenetic analysis of rrs placed this sample in the pathogenic clade on a discrete branch that did not contain any other isolates (Figure 2). Six samples were positive for intermediate Leptospira species. Using BLAST analysis, two samples were designated as L. licerasiae (NP-21 and NP-46), and two samples were designated as L. wolffii (NP-49 and NP-63). The remaining two samples (NP-30 and NP-64) were most closely related to L. licerasiae, with 99% and 97% nucleotide identity, respectively Supplemental Table 3, but they resided on their own branch in the intermediate Leptospira cluster (Figure 2). Samples NP-30 and NP-49 also contained non-pathogenic Leptospira along with an additional 74 samples. Six samples contained non-Leptospiraceae spirochetes. There was overlap between samples that were both culture-positive and positive on direct PCR for unculturable non-pathogenic Leptospira (N = 47).In summary, we found a low prevalence of pathogenic and intermediate Leptospira species. The culture and PCR assays used here require additional validation to determine their accuracy for environmental use, but our findings are consistent with the low number of human leptospirosis cases reported to the Bureau of Epidemiology of Thailand, with only 11 notified cases from the Bangkok Metropolitan Region between November of 2011 and January of 2012 (Annual Epidemiological Surveillance Report 2011; http://www.boe.moph.go.th/Annual/AESR2011/index.html). Although outbreaks of leptospirosis during periods of flooding are well-documented in Thailand and elsewhere in the world,^1,2 our evidence indicates that an outbreak was not the case during the 2011 Bangkok flood.     

Emergence of Pediatric Melioidosis in Siem Reap, Cambodia

We describe the first cases of pediatric melioidosis in Cambodia. Thirty-nine cases were diagnosed at the Angkor Hospital for Children, Siem Reap, between October 2005 and December 2008 after the introduction of microbiology capabilities. Median age was 7.8 years (range = 1.6–16.2 years), 15 cases were male (38%), and 4 cases had pre-existing conditions that may have pre-disposed the patient to melioidosis. Infection was localized in 27 cases (69%) and disseminated in 12 cases (31%). Eleven cases (28%) were treated as outpatients, and 28 (72%) cases were admitted. Eight children (21%) died a median of 2 days after admission; seven deaths were attributable to melioidosis, all of which occurred in children receiving suboptimal antimicrobial therapy and before bacteriological culture results were available. Our findings indicate the need for heightened awareness of melioidosis in Cambodia, and they have led us to review microbiology procedures and antimicrobial prescribing of suspected and confirmed cases.     

A comparison of the stability of single-piece and segmental Le Fort I maxillary advancements

This study retrospectively evaluated the stability of Le Fort I maxillary advancements and compared segmental and one-piece maxillary osteotomy procedures. A cephalometric analysis was performed on 26 cases of maxillary advancement. The sample comprised 11 cases of one-piece and 15 cases of segmental maxillary procedures. The tracings were superimposed and digitized by computer software, and the skeletal changes were analyzed before surgery, immediately after surgery, and at a minimum of 1 year of follow-up. Different values were compared by the paired and nonpaired t tests and were correlated by the Pearson correlation test. The significant value was set at a 95% confidence interval. The maxilla was advanced by a mean of 5.0 +/- 1.6 mm (P < 0.001), and the anterior maxilla was repositioned inferiorly by a mean of 1.5 +/- 3.3 mm (P < 0.05). The maxilla relapsed posteriorly by a mean of 0.6 +/- 1.2 mm (P < 0.05) and superiorly at the anterior maxilla by a mean of 0.8 +/- 1.1 mm (P < 0.001). Overjet and overbite did not significantly change (P > 0.05). It was concluded that maxillary advancement using rigid fixation and interpositional bone grafting in both groups was a stable procedure, particularly in the horizontal plane. In the one-piece group, there was a significantly higher relapse in the vertical plane than in the segmental group (P < 0.05), however. Minor skeletal relapse was compensated for by postoperative tooth movement, and segmental procedures are recommended when required to enhance occlusal results.