Dynamics of MDR Enterobacter cloacae outbreaks in a neonatal unit in Nepal: insights using wider sampling frames and next-generation sequencing.

OBJECTIVES: There are limited data on Enterobacter cloacae outbreaks and fewer describing these in association with NDM-1. With whole-genome sequencing, we tested the hypothesis that a cluster of 16 E. cloacae bacteraemia cases in a Nepali neonatal unit represented a single clonal outbreak, using a wider set of epidemiologically unrelated clinical E. cloacae isolates for comparison. METHODS: Forty-three isolates were analysed, including 23 E. cloacae and 3 Citrobacter sp. isolates obtained from blood cultures from 16 neonates over a 3 month period. These were compared with two contemporaneous community-associated drug-resistant isolates from adults, a unit soap dispenser isolate and a set of historical invasive isolates (n=14) from the same geographical locality. RESULTS: There were two clear neonatal outbreaks and one isolated case in the unit. One outbreak was associated with an NDM-1 plasmid also identified in a historical community-associated strain. The smaller, second outbreak was likely associated with a contaminated soap dispenser. The two community-acquired adult cases and three sets of historical hospital-associated neonatal isolates represented four additional genetic clusters. CONCLUSIONS: E. cloacae infections in this context represent several different transmission networks, operating at the community/hospital and host strain/plasmid levels. Wide sampling frames and high-resolution typing methods are needed to describe the complex molecular epidemiology of E. cloacae outbreaks, which is not appropriately reflected by routine susceptibility phenotypes. Soap dispensers may represent a reservoir for E. cloacae and bacterial strains and plasmids may persist in hospitals and in the community for long periods, sporadically being involved in outbreaks of disease.

Genome sequencing of an extended series of NDM-producing Klebsiella pneumoniae isolates from neonatal infections in a Nepali hospital characterizes the extent of community- versus hospital-associated transmission in an endemic setting.

NDM-producing Klebsiella pneumoniae strains represent major clinical and infection control challenges, particularly in resource-limited settings with high rates of antimicrobial resistance. Determining whether transmission occurs at a gene, plasmid, or bacterial strain level and within hospital and/or the community has implications for monitoring and controlling spread. Whole-genome sequencing (WGS) is the highest-resolution typing method available for transmission epidemiology. We sequenced carbapenem-resistant K. pneumoniae isolates from 26 individuals involved in several infection case clusters in a Nepali neonatal unit and 68 other clinical Gram-negative isolates from a similar time frame, using Illumina and PacBio technologies. Within-outbreak chromosomal and closed-plasmid structures were generated and used as data set-specific references. Three temporally separated case clusters were caused by a single NDM K. pneumoniae strain with a conserved set of four plasmids, one being a 304,526-bp plasmid carrying bla(NDM-1). The plasmids contained a large number of antimicrobial/heavy metal resistance and plasmid maintenance genes, which may have explained their persistence. No obvious environmental/human reservoir was found. There was no evidence of transmission of outbreak plasmids to other Gram-negative clinical isolates, although bla(NDM) variants were present in other isolates in different genetic contexts. WGS can effectively define complex antimicrobial resistance epidemiology. Wider sampling frames are required to contextualize outbreaks. Infection control may be effective in terminating outbreaks caused by particular strains, even in areas with widespread resistance, although this study could not demonstrate evidence supporting specific interventions. Larger, detailed studies are needed to characterize resistance genes, vectors, and host strains involved in disease, to enable effective intervention.

Haemophilus influenzae type b carriage and novel bacterial population structure among children in urban Kathmandu, Nepal.

Haemophilus influenzae type b (Hib) is a major cause of invasive bacterial infection in children that can be prevented by a vaccine, but there is still uncertainty about its relative importance in Asia. This study investigated the age-specific prevalence of Hib carriage and its molecular epidemiology in carriage and disease in Nepal. Oropharyngeal swabs were collected from children in Kathmandu, Nepal, from 3 different settings: a hospital outpatient department (OPD), schools, and children's homes. Hib was isolated using Hib antiserum agar plates, and serotyping was performed with latex agglutination. Hib isolates from children with invasive disease were obtained during active microbiological surveillance at Patan Hospital, Kathmandu, Nepal. Genotyping of disease and carriage isolates was undertaken using multilocus sequence typing (MLST). Swabs were taken from 2,195 children, including 1,311 children at an OPD, 647 children attending schools, and 237 children in homes. Overall, Hib was identified in 5.0% (110/2,195; 95% confidence interval [95% CI], 3.9% to 6.4%). MLST was performed on 108 Hib isolates from children carrying Hib isolates and 15 isolates from children with invasive disease. Thirty-one sequence types (STs) were identified, and 20 of these were novel STs. The most common ST isolates were sequence type 6 (ST6) and the novel ST722. There was marked heterogeneity among the STs from children with disease and children carrying Hib. STs identified from invasive infections were those commonly identified in carriage. This study provides evidence of Hib carriage among children in urban Nepal with genetically diverse strains prior to introduction of universal vaccination. The Hib carriage rate in Nepal was similar to the rates observed in other populations with documented high disease rates prior to vaccination, supporting implementation of Hib vaccine in Nepal in 2009.

Clinical profile of invasive pneumococcal diseases in Patan Hospital, Nepal.

BACKGROUND: Pneumococcal infection is one of the leading causes of pneumonia, meningitis and septicemia in developing countries. It accounts for one million deaths each year in children. OBJECTIVES: The objective of this study is to see the clinical profile of invasive pneumococcal disease, antibiotics sensitivity pattern and prevalent serotypes in children admitted at Patan Hospital. METHODS: This is a retrospective analytical study conducted in the department of Paediatrics, Patan hospital. The lab data of those children who grew pneumococci in their blood, cerebrospinal fluid or body fluids over a period of 3 years (January 2007 to Dec 2009) were collected and the case files were then studied. RESULTS: Out of 42 cases of invasive pneumococcal diseases studied admitted diagnoses included pneumonia, febrile seizure, bacteremia or septicemia, meningitis, acute gastroenteritis and glomerulonephritis. Twenty seven of them were children under five. The male to female ratio was 1.7:1. On investigation 64%, 52% and 5% of the patients had leucocytosis, anaemia, and leucopenia respectively. Twenty six of them had radiological changes suggestive of pneumonia. Streptococcus pneumoniae grew in 38 blood samples, 5 cerebrospinal fluid and 3 pleural fluids. Almost all of these isolates were sensitive to penicillin, cefotaxime, amoxycillin, choloramphenicol, erythromycin and ofloxacin and resistant to cotrimoxazole and gentamicin. Pneumococcal serotypes found in our study were 1, 14, 5, 23B, 6B, 8, 9A, 9V, 10A, 15 and 23F (11 serotypes). CONCLUSIONS: Penicillin is still the most effective antibiotic for streptococcal infection in our study. Of the pneumococcal serotypes identified; 36% were covered by the 7-valent pneumococcal conjugate vaccine, 54% each by PCV-10 and PCV-13, and 72% by the e 23 valent vaccines.

Macbeth in the resource room: students with learning disabilities study Shakespeare.

A teacher describes a secondary English unit on Shakespeare implemented in a manner designed to take advantage of the learning differences of resource room students. After developing and investigating questions relating to Macbeth, students then read and enjoyed the drama. As a result of acquiring an understanding of Shakespeare exceeding that of students in general classes, self-esteem and academic motivation increased. Finally, connections to practice and behavioral, constructivist, feminist, and critical theories are discussed. Appendices describe specific schedules and routines.

Trends in the teaching of neurology to family practice residents, 1981-1990.

A decade ago, less than one-half of the family practice (FP) residency programs surveyed provided a defined rotation in neurology. To learn how neurology is currently being taught in FP residencies, we sent questionnaires to 384 approved FP residencies in the United States, receiving responses from 241 (63%). Seventy-eight percent provide a defined rotation in neurology, usually in the form of a preceptorship with a community neurologist, and 57% systematically present the core curriculum recommended by the American Academy of Neurology and the American Academy of Family Physicians in 1987. Asked about the level of cooperation they encounter in recruiting neurologists to teach, 54% of the respondents reported "too little" or "no" cooperation from academic neurologists, while just 20% reported these responses in regard to community neurologists. Most (87%) reported that involvement in the residency program leads to increased patient referrals for the participating neurologist. Most (81%) respondents would welcome more participation by neurologists.

Early treatment of Campylobacter jejuni enteritis.

The bacteriologic and clinical effects of early antibiotic treatment of Campylobacter jejuni enteritis were studied. Erythromycin rapidly eliminated C. jejuni from stools, whereas trimethoprim-sulfamethoxazole did not. Despite its bacteriologic effectiveness, erythromycin did not reduce the duration or severity of diarrhea, abdominal pain, or other symptoms.

Evaluation and diagnosis of acute infectious diarrhea.

The appropriate approach to the diagnosis and management of acute infectious diarrhea is determined by the frequency and setting of the illness, the recognizable causes or syndromes, the cost and yield of available diagnostic tests, and the treatability of the disease. Acute diarrhea affects everyone throughout the world from one to more than six times each year, depending on age, location, and living conditions. The range of identifiable viral, bacterial, and parasitic etiologies is great, and the cost of indiscriminate use of etiologic studies for diagnosis is prohibitive. Because of its insensitivity for many organisms and poor selection of cases for testing, routine stool culture has been one of the most costly and ineffective microbiologic tests; the cost per positive result has traditionally exceeded $900 to $1,000. The appropriate treatment for the vast majority of cases (independent of their cause) is simple and effective: oral glucose- and electrolyte-containing rehydration solution. On the basis of an appropriate history and understanding of pathogenesis, fecal specimens can be selectively obtained and promptly examined for leukocytes and parasites, and the common noninflammatory diarrheas can be separated from the inflammatory infections in order to focus further studies on the latter group. The bacteria for which specific antimicrobial therapy should be considered usually cause inflammatory diarrhea in the United States. Therefore, only when the history or fecal leukocyte findings indicates an inflammatory process is it appropriate to culture for the routine invasive bacterial pathogens. In sporadic inflammatory diarrhea, culture methods should include those for Campylobacter jejuni as well as Salmonella and Shigella. Several special circumstances may prompt a consideration of parasites (including Giardia, Entamoeba, Strongyloides, Cryptosporidium), Vibrio, Yersinia, Clostridium difficile, enterotoxigenic Escherichia coli, food-borne agents, or sexually transmitted pathogens. The practical value of specific identification of rotaviruses (by enzyme-linked immunosorbent assay, Rotazyme, or electron microscopy) is primarily epidemiologic, particularly in hospitalized infants or young children. Using such a selective approach to fecal culture will greatly increase its yield and can reduce the cost per positive result from $1,000 to less than $150.

Needle aspiration biopsy in cystic papillary carcinoma of the thyroid.

The records of 11 patients with cystic papillary carcinoma of the thyroid who had undergone preoperative sonography and fine-needle aspiration biopsy were retrospectively reviewed. The lesions varied from 1.5 to 5 cm in diameter. In only six (55%) of the 11 patients was the correct diagnosis made preoperatively. In the other 45%, the lesion was initially misdiagnosed as a benign or hemorrhagic cyst. These data indicate that needle aspiration often yields false-negative results in patients with cystic papillary carcinoma. All patients diagnosed on sonography and fine-needle aspiration as having benign cysts should have continued clinical follow-up. If lesions do not disappear either clinically or by sonography, a more aggressive approach should be taken.

Variability of arterial blood gas values in stable patients in the ICU.

To establish guidelines for the interpretation of changes in arterial blood gas (ABG) values, we studied 29 clinically stable ICU patients for spontaneous variability in PaO2, PaCO2 and pH. ABGs were sampled six times over a 50-minute period, during which all patients received a fixed FIO2 of 0.5 via endotracheal tube and underwent no therapeutic interventions. Each sample was analyzed in duplicate with careful attention to method of collection and measurement. The range separating the lowest and highest PaO2 varied from 1 to 45 mm Hg (16.2 +/- 10.9 mm Hg [mean +/- SD] ). For PaCO2 this range was from 1 to 8 mm Hg (3.0 +/- 1.9 mm Hg). Coefficient of variation for PaO2 and PaCO2 averaged 5.1 +/- 3.2 percent (mean +/- SD) and 3.0 +/- 1.5 percent respectively. pH varied within 0.03 +/- 0.02 units. Percentage change in PaO2 between sequential intrapatient samples averaged 5.3 +/- 2.8 percent (mean +/- SD) and 7.1 +/- 7.9 percent over ten- and 50-minute intervals, respectively. Various clinical features were analyzed by multiple regression analysis for their relation to PaO2 variation. Only leukocyte count and mean arterial oxygen content were statistically significant associations (p less than 0.05), but together explained less than 35 percent of the variation observed. Because considerable spontaneous variation occurs, even in stable patients, clinicians should base therapeutic decisions on trends in PaO2 values rather than on isolated changes interpreted without appropriate clinical correlation.

Experimental salmon poisoning disease in juvenile coyotes (Canis latrans).

Salmon poisoning disease (SPD) was experimentally induced in juvenile coyotes (Canis latrans). The disease was lethal in 11 of 12 coyotes within 15 days after inoculation with 1,000 or 4,000 metacercariae of Nanophyetus salmincola. Clinical manifestations of the disease included lymph node enlargement, anorexia, pyrexia, diarrhea and death. Coccoid bodies indistinguishable from rickettsiae were observed in macrophages of spleen, liver, lymph nodes, and duodenum. Percentage recovery of adult trematodes from metacercariae administered was 23% from 12 inoculated coyotes, compared to 13% in one inoculated dog. Juvenile coyotes appear to be highly susceptible to experimental SPD.

The paradox of inhibition and enhancement of the formation of urinary stones.

Nucleation (Bo) and linear crystal growth (G) rates, average particle size (L1,0) and total mass (MT) of calcium oxalate dihydrate crystals were measured in artificial urine with and without polylysine, polyglutamic acid or heparin. The purpose of the study was to see if any of these polymers had effects on crystallisation similar to those created by addition of 5% natural urine to artificial urine, wherein Bo had increased but G, L1,0 and MT decreased. Polylysine addition had insignificant effects. Heparin increased Bo and decreased G, L1,0 and MT significantly, and polyglutamate had similar but more marked effects than did heparin. It is concluded that properly structured organic polymers can significantly inhibit calcium oxalate dihydrate crystallisation by paradoxical enhancement of nucleation. It is possible that such polymers may act as nucleation substrates.

Effects of urinary organic macromolecules on crystallization of calcium oxalate: enhancement of nucleation.

The urine of normal persons, as well as that of stone formers, contains inhibitors to crystallization of calcium oxalate. To ascertain differences, if any, between these 2 groups we measured nucleation rate, growth rate and total mass produced in an analytic system with 5 per cent urine added to 95 per cent synthetic urine that did not contain large organic molecules. We also observed the effects of addition of uromucoid to the synthetic solution. Normal urine and stone-forming urine contained significant inhibitors to total mass of calcium oxalate dihydrate crystals precipitated but total crystal mass was not significantly different between the 2 groups. Growth rate was significantly less in experiments with the urine of stone formers. The other major difference found in stone-forming urine was significant enhancement of nucleation rate when compared to normal urine. Since addition of uromucoid produced these same growth inhibition and nucleation enhancement effects but did not affect total crystal mass, we hypothesize that uromucoid has an important part in producing the differences noted between and stone-forming urine.

Effects of urinary organic macromolecules on crystallization of calcium oxalate: enhancement of nucleation.

The urine of normal persons, as well as that of stone formers, contains inhibitors to crystallization of calcium oxalate. To ascertain differences, if any, between these 2 groups we measured nucleation rate, growth rate and total mass produced in an analytic system with 5% urine added to 95% synthetic urine that did not contain large organic molecules. We also observed the effects of addition of uromucoid to the synthetic solution. Normal urine and stone-forming urine contained significant inhibitors to total mass of calcium oxalate dihydrate crystals precipitated but total crystal mass was not significantly different between the 2 groups. Growth rate was significantly less in experiments with the urine of stone formers. The other major difference found in stone-forming urine was significant enhancement of nucleation rate when compared to normal urine. Since addition of uromucoid produced these same growth inhibition and nucleation enhancement effects but did not affect total crystal mass, we hypothesize that uromucoid has an important part in producing the differences noted between normal and stone-forming urine.

Evaluation of a new in-vitro blood test for determining thyroid status: the effective thyroxine ratio.

Estimation of serum thyroxine concentration in test serum was performed in the presence of test serum itself and expressed as a fraction (Effective Thyroxine Ratio, E.T.R.) of serum thyroxine concentration similarly determined in normal serum. From studies of serum obtained from euthyroid, hyperthyroid, and hypothyroid patients and from euthyroid pregnant women and women taking contraceptive (oestrogen) medication, the E.T.R. estimation has been shown to have a diagnostic accuracy (99%) in defining thyroid status comparable to that shown by the free thyroxine index (98%). Because of the ease and rapidity with which the procedure can be completed, estimation of E.T.R. shows promise as a single, accurate measure of thyroid status.