A point-of-care microfluidic biochip for quantification of CD64 expression from whole blood for sepsis stratification.

Sepsis, a potentially life-threatening complication of an infection, has the highest burden of death and medical expenses in hospitals worldwide. Leukocyte count and CD64 expression on neutrophils (nCD64) are known to correlate strongly with improved sensitivity and specificity of sepsis diagnosis at its onset. A major challenge is the lack of a rapid and accurate point-of-care (PoC) device that can perform these measurements from a minute blood sample. Here, we report a PoC microfluidic biochip to enumerate leukocytes and quantify nCD64 levels from 10 µl of whole blood without any manual processing. Biochip measurements have shown excellent correlation with the results from flow cytometer. In clinical studies, we have used PoC biochip to monitor leukocyte counts and nCD64 levels from patients' blood at different times of their stay in the hospital. Furthermore, we have shown the biochip's utility for improved sepsis diagnosis by combining these measurements with electronic medical record (EMR).

Assessment of anti-inflammatory and hepatoprotective potency of Polyalthia longifolia var. pendula leaf in Wistar albino rats.

The objective of the present study was to screen various solvent extracts of Polyalthia longifolia var. pendula (Annonaceae) leaf for anti-inflammatory activity and to evaluate the anti-inflammatory and hepatoprotective potency of the potent solvent extract. Successive extraction was performed with six different solvents, viz. petroleum ether, hexane, toluene, chloroform, acetone and methanol. Toluene, chloroform, acetone and methanol were used in acute inflammatory studies; the results revealed methanol as most potent extract. Hence three concentrations of methanolic extract (300, 600, 900 mg/kg) were used to evaluate its potential as an anti-inflammatory and hepatoprotective agent. Diclofenac sodium was used as the toxicant in hepatoprotective studies, in which various serum biochemical parameters and liver glycogen were assessed. The three concentrations of methanol showed anti-inflammatory activity comparable to that of the control (Diclofenac sodium). All the serum biochemical parameters studied revealed the hepatoprotective nature of the methanol extract, but a concentration effect was not observed. The results indicated that the methanolic extract of Polyalthia longifolia var. pendula leaf possesses significant anti-inflammatory and hepatoprotective activity. However, the appropriate effective concentration needs to be determined.

Invasive group A streptococcal infection in injecting drug users and non-drug users in a single UK city.

OBJECTIVES: Injecting drug users (IDU) represent an increasing proportion of patients with invasive group A streptococcal (GAS) disease. Our aims were to characterise the clinical presentation and strains causing GAS bacteremia in IDU from a single UK city (Brighton and Hove), and to compare this patient group with non-drug users (non-DU) with GAS bacteremia. METHODS: Consecutive GAS blood culture isolates from twenty-two IDU and twenty-two non-DU presenting to the city hospital were studied. Clinical features, strain emm typing and superantigen toxin genotyping were investigated. RESULTS: GAS invasive disease presented differently in IDU compared to non-DU with a predominance of injection site abscesses and lower mortality in IDU. GAS strains from IDU were predominantly emm82 and emm83 types, which are uncommon in the UK and emm82 strains appeared clonal. The non-DU GAS strains demonstrated a broader range of emm types including most frequently emm1 and emm89. There was no major difference in superantigen gene profile between the isolate groups. CONCLUSION: The distinct presentation of invasive GAS disease in IDU compared with non-DU was associated with distinct emm types, a predominance of abscesses, and low mortality, although the small numbers preclude definitive conclusions. Further study is required to establish if these findings reflect strain differences or epidemiological differences in colonisation patterns and injecting practice.

Does the use of a specialised paediatric retrieval service result in the loss of vital stabilisation skills among referring hospital staff?

AIMS: To compare the proportion of airway and vascular access procedures performed by referring hospital staff on critically ill children in two discrete time periods, before and after widespread use of a specialised paediatric retrieval service. METHODS: Transport data were obtained from retrieval logs of all children for whom a paediatric retrieval team was launched in each of two time periods (October 1993 to September 1994; and October 2000 to September 2001). RESULTS: The overall intubation rate was similar in the first and second time periods (83.9% v 79.1%). However, 31/51 (61%) retrieved children were intubated by referring hospital staff in 1993-94, compared to 227/269 (84%) in 2000-01. Referring hospital staff gained central venous access in 11% v 18% and arterial access in 22% v 19% of retrieved children in the first and second time periods respectively. This was in spite of a significant reduction in the proportion of children on whom these procedures were performed. CONCLUSION: Referring hospital staff are performing a greater proportion of initial airway and vascular access procedures undertaken in the stabilisation of sick children retrieved by a specialised paediatric retrieval team. The provision of this service has not resulted in the loss of vital skills at the local hospital.

Streptococcal toxic shock syndrome: a description of 14 cases from North Yorkshire, UK.

OBJECTIVE: To analyze the clinical and laboratory features of patients diagnosed with streptococcal toxic shock syndrome (TSS) in North Yorkshire from 1986 to 1999. METHODS: Records of patients with features satisfying the published criteria for streptococcal TSS were reviewed from laboratory and clinical records made at the time and from the hospital case notes. Isolates of streptococci were analyzed for serotype and genes encoding for the production of streptococcal pyrogenic exotoxins. RESULTS: Fourteen patients satisfied the entry criteria. In one district, where the data were complete, the annual incidence of detected streptococcal TSS rose from 1.1 to 9.5 cases per million population in the 1990s. TSS was associated with various M serotypes of group A streptococci and various exotoxin genotypes. Two cases (14% of the series) were associated with severe group G streptococcal infection. The fatality rate was 64%, and the mode of time to death was 4 days. Local tissue necrosis occurred in 71% of cases, including necrotizing fasciitis, intrathoracic and intra-abdominal forms. Non-steroidal anti-inflammatory drugs (NSAIDs) had been taken around the time of onset of disease by 92% of the patients with TSS. CONCLUSIONS: There has been a dramatic increase in the number of detected cases of streptococcal TSS over the 14 years since the first case was recognized here. There was a wide range of invasive forms of infection, a high fatality rate even in fit young adults, and a rapid course from onset to death. There was a high association of TSS with aggressive streptococcal infection producing local tissue necrosis.

Invasive M-type 3 Streptococcus pyogenes affecting a family and a residential home.

This paper describes the first recognised United Kingdom outbreak of M-type 3 streptococci for 12 years. Four epidemiologically-linked invasive infections occurred in a residential home in northern England over two weeks. The index patient was admitted from home with necrotising fasciitis of the leg. Infection was subsequently detected in her husband (fatal pneumonia) another resident (fatal pneumonia) and a member of the care staff (parapharyngeal abscess). Screening of staff and residents in the home did not reveal any further infection or carriage. There is a substantial risk of serious secondary infection amongst the contacts of a patient with invasive Streptococcus pyogenes infection. Guidance is lacking, but needed, on the advisability of chemoprophylaxis in these circumstances.

Unusual occurrence of M type 77, antibiotic-resistant group A streptococci in southern Sweden.

For many years group A streptococci of T type 28 (T28) have been common in southern Sweden; however, since 1995 resistance to both macrolide-lincosamide-streptogramin B (MLS) antibiotics and tetracycline was observed among T28 isolates, which prompted the present studies on clonal relatedness of antibiotic-resistant T28 strains. By extended T typing, 95 of 100 examined tetracycline-resistant strains showed the combination T9-T13-T28; of these, 94 belonged to M type 77 (M77) and one belonged to M73. Three strains were T28-M28 and two were T28-M nontypeable. The serological M77 was confirmed by PCR capture enzyme-linked immunosorbent assay, emm amplicon restriction profiling, and emm sequence typing. Fifty strains were examined for superantigen genes: speA was detected in three blood isolates only, whereas all isolates harbored speB, and only two of the strains were negative for speC. Eighty-nine of the 100 strains were also macrolide resistant, of which 59 were inducibly MLS resistant (IR) and 21 were constitutively MLS resistant (CR), 6 were noninducibly resistant (NI), and 3 had novel subphenotypes recently reported by our group. Resistance genes were determined by PCR and hybridization methods. Eighty-four of the 100 strains harbored tetM. ermB was detected in all CR and IR strains, and mefA was found in all NI strains; both ermB and mefA were identified in two strains with novel subphenotypes. Pulsed-field gel electrophoresis showed that these antibiotic-resistant M77 strains belonged to at least five different clones.

High rate of tetracycline resistance in Streptococcus pyogenes in Iran: an epidemiological study.

Streptococcus pyogenes, a major human pathogen, is still considered susceptible to beta-lactams, but for other relevant antibiotics, highly variable resistance rates have been reported. Since no data were available from Iran, we tested 1,335 throat isolates from two different regions of the country for their antibiotic susceptibilities and, for comparison, a collection of 80 strains isolated from 1989 to 1991. Erythromycin resistance was uncommon (0.6%), whereas an overall high rate of tetracycline resistance was found, increasing between 1989-1991 and 1995-1997 from 23 to 42%. The tetracycline-resistant strains belonged to more than 10 different T types, the majority being types 4, 11, and B3264. By conventional M typing of 406 tetracycline-resistant isolates, more than 20 different M types were found. Approximately 50% of the strains were nontypeable by T agglutination as well as serological M typing; however, by genotyping by a combined PCR-capture-enzyme-linked immunosorbent assay, many of these strains were successfully emm typed. We conclude that the high rate of tetracycline resistance among Iranian S. pyogenes isolates is due to multiclonal dissemination of resistance within the streptococcal population rather than epidemic spread of single clones.

Resistance to macrolides in Streptococcus pyogenes in France in pediatric patients.

A total of 1,500 recent throat isolates of Streptococcus pyogenes collected between 1996 and 1999 from children throughout France were tested for their susceptibility to erythromycin, azithromycin, josamycin, clindamycin, and streptogramin B. The erythromycin-resistant isolates were further studied for their genetic mechanism of resistance, by means of PCR. The clonality of these strains was also investigated by means of serotyping and ribotyping. In all, 6.2% of the strains were erythromycin resistant, and 3.4 and 2.8% expressed the constitutive MLS(B) and M resistance phenotypes and harbored the ermB and mefA genes, respectively; ermTR was recovered from one isolate which also harbored the ermB gene. Ten serotypes and 8 ribotypes were identified, but we identified 17 strains by combining serotyping with ribotyping. Among the eight ribotypes, the mefA gene was recovered from six clusters, one being predominant, while the ermB gene was recovered from four clusters, of which two were predominant.

An outbreak of skin sepsis in abattoir workers caused by an 'unusual' strain of Streptococcus pyogenes.

An outbreak of indolent skin infections due to an 'unusual' serological type of Streptococcus pyogenes that lasted for 3 months and affected eight workers in an abattoir is described. The group A streptococcal (GAS) isolates were serotyped as M-type 59; however, they possessed a T-protein pattern (T5/27/44) that is not commonly associated with M-type 59. Further genotypic characterisation studies revealed that all eight isolates were indistinguishable by pulsed-field gel electrophoresis (PFGE) and possessed the emm gene encoding for the M-type 59. Once identified, and after a combination of penicillin treatment, exclusion of workers with lesions and reinforcement of standard hygiene precautions, no further cases developed. Although common in the 1970s and 1980s, streptococcal infections in this situation are now reported infrequently. This report serves to highlight the issues surrounding working practices in abattoirs.

Fluorescent amplified-fragment length polymorphism analysis of an outbreak of group A streptococcal invasive disease.

Fluorescent amplified-fragment length polymorphism (FAFLP) analysis was carried out for an outbreak of group A streptococcal (GAS) invasive disease. Streptococcal genomic DNAs were digested with endonucleases EcoRI and MseI, site-specific adaptors were ligated, and PCR amplification was carried out with an EcoRI adaptor-specific primer labelled with fluorescent dye. Amplified fragments of up to 600 bp in size were separated on a polyacrylamide sequencing gel which contained internal size markers in each lane. These data were automatically scanned and analyzed, fragments were precisely sized (+/-1 bp), and electropherograms were generated for each genome with GeneScan 2.1 software. All isolates were compared in this way. Among 27 GAS isolates examined, we found 18 FAFLP profiles, compared with 12 macrorestriction profiles by pulsed-field gel electrophoresis. FAFLP readily distinguished genotypes for two clones of GAS serotype M77 which were responsible for outbreaks of invasive disease in a care-of-the-elderly system. It provided an automated analysis of the whole genome of bacterial isolates. It was reproducible, more discriminatory, and capable of higher throughput than other molecular typing methods. Given agreed conditions, FAFLP would be reproducible between laboratories for rapid characterization of outbreak strains.

Optical cross-sectional imaging of the macula with the retinal thickness analyzer in X-linked retinoschisis.

OBJECTIVE: To assess the morphologic characteristics of the foveal abnormality in juvenile X-linked retinoschisis using the scanning retinal thickness analyzer (RTA). This characteristic foveal abnormality is present in 83% to 100% of patients with X-linked retinoschisis and has not been demonstrated histopathologically. METHODS: The RTA is a noncontact imaging device. The RTA scans an obliquely oriented slit laser beam across the macula to obtain a series of optical cross sections, which are digitized. PARTICIPANTS: The RTA was used to examine 7 eyes of 5 patients with X-linked retinoschisis. RESULTS: The RTA demonstrated foveal schisis in all eyes examined. In 2 eyes of 2 patients, a single schisis cavity, with an inner leaf in a dome-shaped configuration, was present. In 4 eyes of 3 patients, a single schisis cavity containing fine strands was present. Some of these strands partially, and others completely, bridged the cavity. In 1 eye of 1 patient, 2 separate schisis cavities with bridging strands were present in the fovea. CONCLUSIONS: Scanning RTA is a noninvasive imaging modality capable of producing optical cross sections that demonstrate the extent and structural details of the foveal schisis in X-linked retinoschisis. Scanning RTA seems to be effective in the detection, characterization, and quantification of foveal schisis.

A community outbreak of invasive and non-invasive group A beta-haemolytic streptococcal disease in a town in South Wales.

An increase in the incidence of invasive and non-invasive infections caused by group A beta-haemolytic streptococci (GAS) was noted in and around the town of Glynneath (population approx. 4000) in West Glamorgan, South Wales between 1 January and 30 June 1995. A total of 133 cases was ascertained with 127 (96%) occurring between 1 March and 30 June 1995. Six patients had invasive disease (one died) and all presented at the peak of the outbreak. There were 127 non-invasive cases of whom 7 were hospitalized. The outbreak was investigated to determine its extent and whether it was caused by a single M-serotype of GAS. Serotyping showed that 13 different M-serotypes were involved with the M1 serotype predominating. The overall incidence of GAS invasive disease in West Glamorgan (population 365,000) increased sevenfold from a crude incidence of 0.5/10(5) per year in 1994 to 3.5/10(5) per year in 1995, but fell back to 0.75/10(5) per year in 1996. Eighty-two (80%) out of 102 individuals affected by GAS replied to a health questionnaire; sore throat was the commonest symptom reported (97%). Thirty-nine of these index cases identified at least one other member of their household who had experienced similar symptoms. The interval between the onset of illness in members of a single household was 0-83 days with a mean of 22 days. The mean duration of illness was 13.5 days and 61% of patients were treated with penicillin V for a mean duration of 9.3 days. Twenty-one per cent of GAS isolates were erythromycin-resistant and the M4 and M6 serotypes were especially resistant to erythromycin (87.5 and 100% resistance, respectively). Penicillin V failed to eradicate GAS from the throats of 25% of assessable patients. In this community, an outbreak of non-invasive disease caused by GAS was linked in time and place with an outbreak of serious invasive disease.

High-resolution genotyping elucidates the epidemiology of group A streptococcus outbreaks.

Streptococcus pyogenes strains were genotyped by a combination of molecular methods for high- resolution epidemiologic studies of disease outbreaks. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the emm gene is reported. Alone or in conjunction with other molecular techniques (16S ribotyping, pulsed-field gel electrophoresis, and detection of exotoxin genes), PCR-RFLP could differentiate outbreak-related strains from contemporaneous background strains of the same M serotype. Three outbreaks were studied: pharyngitis in a boarding school (serotype M5), cross-infection in a hospital burn unit (serotype M76), and severe invasive disease in two elderly care homes (serotype R28). It was possible, for example, to identify within serotype R28 a clone with particular potential for invasive disease. In all cases, the four molecular methods yielded complementary results that were hierarchically related. Strains could be assigned to the outbreak or the background in a precise, reproducible, and rapid manner.