Enhancing pharmaceutical crystallization in a flow crystallizer with ultrasound: Anti-solvent crystallization.

Continuous crystallization is a fast growing application domain in the pharmaceutical industry. Application of ultrasound has been proven to have positive effects like reduction in induction time and Metastable Zone Width (MSZW) in both batch and flow systems. Further understanding of flow-based sonocrystallization is required to achieve industrial level scale up. This work investigates the sonocrystallization of pharmaceutical compounds in a tubular flow crystallizer. Acetyl Salicylic Acid (ASA-Aspirin) is used as a model compound with ethanol and water as solvent and anti-solvent, respectively. Experiments were conducted in silent and sonicated conditions to study the MSZW. Ultrasound made it possible to achieve crystallization within the crystallizer which was not possible in silent conditions, under the tested conditions. Continuous crystallization was achieved at as low as 48 wt% of anti-solvent and crystallization was already seen at a supersaturation of 1.02. In some experiments, temperature rise with ultrasound caused the crystals to re-dissolve within the channels. Better crystallization - no re-dissolution - was achieved by using low ultrasonic power without any loss in the yield. Particle sizes of product crystals were in the range of 4-46 µm. In conclusion, ultrasound was highly effective in enabling anti-solvent crystallization of a pharmaceutical compound in a tubular flow crystallizer.

Influence of dissolved gases on sonochemistry and sonoluminescence in a flow reactor.

In the present work, the influence of gas addition is investigated on both sonoluminescence (SL) and radical formation at 47 and 248 kHz. The frequencies chosen in this study generate two distinct bubble types, allowing to generalize the conclusions for other ultrasonic reactors. In this case, 47 kHz provides transient bubbles, while stable ones dominate at 248 kHz. For both bubble types, the hydroxyl radical and SL yield under gas addition followed the sequence: Ar>Air>N2>>CO2. A comprehensive interpretation is given for these results, based on a combination of thermal gas properties, chemical reactions occurring within the cavitation bubble, and the amount of bubbles. Furthermore, in the cases where argon, air and nitrogen were bubbled, a reasonable correlation existed between the OH-radical yield and the SL signal, being most pronounced under stable cavitation at 248 kHz. Presuming that SL and OH originate from different bubble populations, the results indicate that both populations respond similarly to a change in acoustic power and dissolved gas. Consequently, in the presence of non-volatile pollutants that do not quench SL, sonoluminescence can be used as an online tool to qualitatively monitor radical formation.

Characterization of stable and transient cavitation bubbles in a milliflow reactor using a multibubble sonoluminescence quenching technique.

The bubble type, generated by an ultrasonic field, was studied in a batch and flow reactor using a multibubble sonoluminescence (MBSL) quenching technique with propanol and acetone. The influence of frequency and transducer configuration was evaluated using the same piezoelectric element in both setups. Results show that the bubble type not only depends on the frequency, but also on the input power or transducer configuration. Additionally, the effect of flow on sonoluminescence yield and bubble type was studied in the continuous setup at various frequencies. As the flow becomes turbulent, the sonoluminescence signal reaches a plateau for three out of four frequencies, and a transition from transient to stable cavitation occurs for frequencies below 200 kHz.

Impact of alginate overproduction on attachment and biofilm architecture of a supermucoid Pseudomonas aeruginosa strain.

The supermucoid Pseudomonas aeruginosa strain PDO300Deltaalg8(pBBR1MCS-5:alg8) showed strongly impaired attachment compared with the respective mucoid or nonmucoid strains and formed a thicker biofilm with large extended mushroom-like microcolonies. Alginate lyase treatment dissolved microcolonies. The data suggested that alginate overproduction impairs attachment but plays a structural role in microcolony formation.

A novel porA-based real-time PCR for detection of meningococcal carriage.

Real-time PCR based on the capsule transfer gene (ctrA) is a significant aid in the diagnosis of meningococcal infection but fails to detect a high proportion (60 %) of non-groupable strains associated with nasopharyngeal carriage. This study aimed to design a novel real-time (TaqMan) PCR that would detect more strains of meningococci and be suitable for large-scale carriage studies. Primer and probe sequences were based on the meningococcal porA gene and designed specifically to exclude the highly related porA pseudogene in Neisseria gonorrhoeae. The specificity of the assay was confirmed by testing strains of N. gonorrhoeae known to contain the porA pseudogene together with commensal strains of Neisseria lactamica and Neisseria sicca. None of these was detected in the assay. Neisseria meningitidis strains representing a wide range of serogroups together with non-groupable strains isolated from the nasopharynx were tested by ctrA assay and the novel porA-based TaqMan PCR. All carriage strains were detected by the porA-based assay including four that gave weak or no reaction with the ctrA assay. Comparison of ctrA and porA assays on 71 throat swabs obtained from university students showed that the porA assay detected meningococcal DNA in all samples that were ctrA positive plus three that were ctrA negative but culture positive. This novel porA-based TaqMan assay provides a highly specific method for detecting meningococcal DNA that is more sensitive than the ctrA assay for detecting meningococcal carriage and is particularly suitable for carriage studies where non-groupable strains and other Neisseria are present.

Development of immunity to serogroup B meningococci during carriage of Neisseria meningitidis in a cohort of university students.

Understanding the basis of protective immunity is a key requirement for the development of an effective vaccine against infection with Neisseria meningitidis of serogroup B. We have conducted a longitudinal study into the dynamics of meningococcal acquisition and carriage in first-year university students. The detection of carriage of serogroup B meningococci correlated with an increase in detection of serum bactericidal activity (SBA) against both colonizing and heterologous serogroup B strains. Once induced, SBA remained high throughout the study. Although students showed increases in antibodies reactive with capsular polysaccharide and lipopolysaccharide (LPS), these antibody responses were transitory, and their decline was not accompanied by a corresponding decline in SBA. In contrast, there was a significant correlation between the presence of antibodies to the PorA outer membrane protein and SBA against both homologous and heterologous strains. SBA induced by a PorA-negative mutant confirmed the contribution of PorA to heterologous activity. Increases in SBA against a range of serogroup B strains were also observed in students in whom no meningococcal carriage was detected. This heterologous protection could not be associated with the presence of antibodies reacting with capsule, LPS, PorA, PorB, Rmp, Opa, Opc, or pilin, demonstrating that other, as yet unidentified, antigens contribute to the development of immunity to serogroup B meningococci. Identification of such antigens with the ability to induce an effective cross-reactive bactericidal response to a range of strains would be a major step in the production of a universally effective vaccine against infections caused by serogroup B meningococci.

Detection of meningococcal carriage by culture and PCR of throat swabs and mouth gargles.

The standard method for detecting meningococcal carriage is culture of throat swabs on selective media, but the levels of carriage determined depend heavily on the skills of the individuals taking the swab and interpreting the cultures. This study aimed to determine the most sensitive detection method for meningococcal carriage. Throat swabs and saline mouth gargles, obtained from 89 university students, were processed in parallel by conventional culture and TaqMan ctrA PCR. Carriage of meningococci, as detected by the combined methods, was 20%. The sensitivities of throat swab culture, throat swab PCR, gargle culture, and gargle PCR were 72, 56, 56, and 50%, respectively, and the probabilities that these techniques would correctly identify the absence of carriage (negative predictive value [NPV]) were 93.4, 89.9, 89.9, and 88.8%. Culturing both throat swabs and gargles increased the NPV to 98.6%. The further addition of throat swab PCR increased this to 100%. Testing gargles by both culture and PCR was as sensitive as testing throat swabs by both methods, suggesting that gargles may be a suitable alternative for large-scale screening studies when throat swabs are difficult to obtain, although they required more lengthy laboratory processing. PCR was a useful adjunct to culture for detecting nasopharyngeal carriage, but it failed to detect some nongroupable strains. For maximum sensitivity, a combination of techniques was required. This study indicates the confidence with which health care professionals involved in meningococcal screening can regard laboratory results.

Amplification with molecular beacon primers and reverse line blotting for the detection and typing of human papillomaviruses.

A novel method for the detection and typing of human papillomavirus (HPV) was developed using molecular beacon primers. The method is based on the use of HPV-specific primers containing a hairpin loop structure in which fluorescent donor and quencher groups are held in close proximity such that fluorescence is quenched. Amplification of the target sequence results in the opening of the loop and the resulting fluorescence can be detected on a sequence detector system (SDS) 7700 (Applied Biosystems), as used for TaqMan assays. Fluorescent amplicons were identified on the SDS 7700 and then typed by a single hybridisation with specific probes immobilised in lines on a nylon membrane and detected on a fluorescent scanner. This novel beacon primer method compared well with conventional PCR for cervical scrape specimens. The combination of the beacon primer method and reverse line blotting should enable large-scale population studies of HPV infection.

Single-tube real-time nested polymerase chain reaction for detecting human papillomavirus DNA.

A single-tube real-time nested polymerase chain reaction (PCR) was developed to detect human Papillomavirus (HPV) DNA in a closed tube system. The oligonucleotide primers MY09/MY11 and GP5+/GP6+ were included in contiguous reactions, thus eliminating the need to transfer first round PCR product into a second tube. The sensitivity and specificity of the optimized single-tube nested PCR were comparable with that achieved by two separate reactions on a conventional thermal block system using serial dilutions derived from plasmids containing DNA of 20 HPV types. A minimum of 10 copies of HPV types 11 and 16 DNA could be detected by both systems. In clinical samples, HPV types 1A, 2, 3, 5, 6-8, 10, 11, 14, 16, 17, 18, 20, 31, 33, 35, 39, 45, 49, 50, 52-54, 57, 62, 66, 70, CP8304 and LVX82/MM7 could be detected by both PCR methods. A total of 145 samples collected from patients were tested for the presence of HPV DNA with the two PCR systems; 124 (86.1%) of 144 samples gave concordant results in both assays. The HPV DNA positive PCR amplicons were typed and concordant results were obtained in 47 of 67 positive samples tested in both amplicons. In samples containing multiple HPV types at least one type was common to both amplicons.

Detection and typing of human papillomavirus DNA in paired urine and cervical scrapes.

The prevalence of human papillomavirus (HPV) in paired cervical scrape and urine specimens from 144 women attending a clinic for genitourinary medicine was determined by polymerase chain reaction (PCR) and nested PCR, using degenerate and general primer pairs localized within the L1 region. HPV typing was by restriction fragment length polymorphism (RFLP), type-specific PCR (HPV 6, 11, 16, 18, 33), and partial DNA sequencing of PCR products. HPV DNA was detected in 114 (84%) women. HPV DNA was detected in the specimens of 58 patients after amplification with MY09/MY11 primers and in a further 54 patients after nested PCR with the GP5+/GP6+ primers. A total of 106/136 (78%) of women had HPV DNA positive cervical scrapes and 89 (65%) had HPV DNA positive urine specimens. Both the urine and cervical specimens of 81 women were positive. In 25 women HPV DNA was detected in the cervical specimen only, and in 8 women HPV DNA was detected in the urine specimens only. A total of 108 specimens from 75 patients were typed. For 33 patients HPV typing was achieved in both the cervical and the urine specimens and 19 women had identical types in paired specimens. Multiple HPV infections could be detected in 15 (20%) of 75 women where either the cervical and urine specimen or both of the specimens could be typed. More then one HPV type was found in 8 specimens and from multiple sites (cervix and urinary tract) in the same patients on 7 occasions. The results of this study indicate that the detection of HPVs in the urogenital tract can be maximised through the testing of both cervical scrapes and urine specimens in conjunction with the use of a nested PCR to increase the sensitivity of HPV DNA detection. Also, urine cannot be a direct substitute for a cervical scrape as different HPV types are often detected in the urine compared with those detected in the cervix.

Characterisation of non-capsulate Haemophilus influenzae by repetitive extragenic palindromic (REP)-PCR.

The use of repetitive extragenic palindromic (REP)-PCR to characterise non-capsulate Haemophilus influenzae (NCHI) for epidemiological studies was validated by application to four outbreak-associated and three epidemiologically unrelated NCHI strains of the same phenotype which had been well characterised by other methods. The REP-PCR patterns were reproducible and showed the unrelated isolates to be distinguishable from each other, whereas the outbreak-associated isolates were indistinguishable. The results were concordant with those from outer-membrane protein enriched profiles, ribotyping and randomly amplified polymorphic DNA analysis. When applied to six further isolates from two different suspected outbreaks, rapid results were obtained from boiled supernates prepared from one colony and indicated that the isolates in question were not related. REP-PCR provides a rapid method of strain characterisation suitable for NCHI, which is ideal for use in conjunction with other methods.

Genomic DNA digestion and ribotyping.

Bacteria may need to be characterized for a number of reasons. Newly discovered organisms are characterized to determine their taxonomic position; clinical isolates are characterized to provide an indication of pathogenic potential and likely antibiotic susceptibility. Such studies generally mvolve characterization to the genus and species level and provide an identification for the organism. Highly discrimmatory methods of intraspecies characterization are required for epidemiologic purposes in order to establish sources and routes of mfection to which control measures may be directed. For an mdlvidual patient, differentiation between relapsmg infection or reinfection may ard treatment choice and clinical management. The pattern of DNA fragments produced by digestion of genomic DNA with a restriction endonuclease, directly or after hybridization, can be highly discrimmatory and enable strain characterization, or typing, suitable for epidemiologic studies. The practice of obtaining these patterns (or profiles) for epidemiologic purposes is the subject of the present chapter, although the methods can also be applied to questions regarding identification at higher taxonomic levels.

Source of variation detected in ribotyping patterns of Haemophilus influenzae: comparison of traditional ribotyping, PCR-ribotyping and rDNA restriction analysis.

The pattern of EcoRI restriction fragments of chromosomal DNA that hybridize with a probe for genes encoding 16S and 23S rRNA is highly discriminatory for non-capsulate Haemophilus influenzae (NCHI). The source of variation detected by these probe-based ribotyping patterns was investigated by restriction analysis of rRNA operon (rrn) amplification products from nine representative strains. Digestion of rrn amplification products with EcoRI indicated one conserved EcoRI site within 16S rDNA and no EcoRI sites within the 16S-23S intergenic spacer region of the nine strains, and an EcoRI site at the 5' end of 23S rDNA from seven of the nine strains. Comparison of the EcoRI ribotyping patterns obtained with separate probes for 16S and 23S rDNA showed more variation with the 23S probe indicating variation in EcoRI sites downstream from the operon. Restriction analyses of 16S and 23S rDNA amplification products with AluI, HhaI, HaeIII and TaqI divided the nine 'traditional' ribotypes into a maximum of three and eight groups, respectively. Similar analyses of the 16S-23S intergenic regions (PCR-ribotyping) failed to distinguish any of the nine representative strains. Therefore, there is probably insufficient variation within the operon for it to form a good target for PCR-based typing methods. In contrast, 'traditional' ribotyping with cDNA from 16S plus 23S rRNA detects restriction site differences in the sequences flanking the operon, which show considerably more variation between strains. 'Traditional' ribotyping should therefore remain the standard for characterising NCHI in epidemiological investigations.

Pyrolysis mass spectrometry in epidemiological and population genetic studies of Haemophilus influenzae.

Haemophilus influenzae serotype b (Hib) vaccines have reduced the amount of invasive Hib disease in immunised infants. However, Hib disease remains in unvaccinated infants and adults and non-capsulate H. influenzae (NCHi) still causes infections, including outbreaks of respiratory disease. Characterisation of strains and the bacterial population as a whole is therefore necessary to detect outbreaks of infection with NCHi or changes in the population, for example, to vaccine-resistant clones of Hib. The rapid, simple and objective technique of pyrolysis mass spectrometry (PMS) was investigated as an alternative to current complex, subjective methods. PMS was compared with ribotyping and multilocus enzyme electrophoresis (MLEE) for population genetic analyses of Hib and with ribotyping and protein profiling for epidemiological analyses of NCHi. PMS clustered all the isolates of Hib together whereas MLEE and ribotyping distinguished certain clones - this is probably because the three methods examine different (and unrelated) characteristics of the organisms. The PMS results were essentially similar to those from ribotyping and protein profiling for the epidemiological analyses of outbreaks of NCHi disease. Therefore, PMS is probably unsuitable for comparisons of Hib populations but it is a useful addition to the arsenal of techniques for the characterisation of NCHi.

Epidemiological typing of coagulase-negative staphylococci from nosocomial infections.

Biotyping, antibiograms, bacteriophage typing, plasmid profile analysis and SDS-PAGE protein profiles were used to determine the relatedness of 44 Staphylococcus epidermidis and four S. haemolyticus isolates from 14 patients. A selection of these were further characterised by ribotyping. Biotyping classified the isolates into three major groups but was considered a poor strain marker. Although antibiograms classified the S. epidermidis isolates into 20 groups, some changes in the susceptibility patterns of related isolates from a single patient were demonstrated. Bacteriophage typing was the least discriminatory of the methods used. SDS-PAGE gave highly related patterns for the majority of S. epidermidis isolates. Plasmid profile analysis and ribotyping, with a minimum of two restriction endonucleases, were the most discriminatory methods for typing S. epidermidis. Nonetheless, some isolates from the same patient - probably representing a single strain - varied in plasmid profile indicating plasmid instability. One of six related isolates from a single patient lacked two bands from the ribotyping pattern of the other isolates. Although no single method proved entirely satisfactory on all occasions, the combination of typing methods was sufficient to provide evidence of the relatedness of S. epidermidis isolates from individual patients.

A molecular analysis of Greek and UK Haemophilus influenzae conjugative resistance plasmids.

Antibiotic resistance in Haemophilus influenzae has been associated with the presence of large, chromosomally integrated, conjugative plasmids. The plasmids of 10 beta-lactamase-positive, ampicillin-resistant strains, two from the UK and eight from Greece, were investigated. Plasmids were detected and isolated after transfer to a rec-deficient recipient. Purified whole plasmid was used as probe. In addition a 12 kb PstI fragment containing the putative point of recircularization in one plasmid, p1056, was cloned and used as a probe. All plasmids shared a high degree of sequence homology suggesting that plasmids of diverse geographical origin are highly related. All plasmids also shared sequence homology with the 12 kb PstI fragment containing the point of recircularization, suggesting that the sequences involved in excision and recircularization are conserved.

When should healthcare workers be screened for methicillin-resistant Staphylococcus aureus?

The role of screening of healthcare workers (HCWs) in the control of methicillin-resistant Staphylococcus aureus (MRSA) is controversial. It is recommended in guidelines by expert groups in both North America and the United Kingdom, although the role of MRSA carriage by HCWs in outbreaks is not clearly defined. The present report describes the spread of a distinct strain of MRSA to patients by a single HCW on three separate occasions over 27 months. The isolates from this HCW and patient contacts were shown to be indistinguishable by antibiogram and repetitive extragenic palindromic polymerase chain reaction (REP/PCR); none were typeable by lytic phage-typing. Throat carriage of the MRSA probably recurred in this HCW, despite attempts to eradicate it on three occasions. Over the same period, nine other small clusters were seen in the Oxford Hospital Group, involving 66 patients and 22 HCWs colonized, or occasionally infected, with a variety of MRSA strains. In none of these instances could HCWs be implicated in the initiation of an outbreak. The advantages of a screening policy include the determination of the full extent of MRSA-colonization and work exclusion; the disadvantages include detection of transient nasal carriage, disruption of staff routine and stigmatization. Screening of HCWs can be a valuable tool in the control of MRSA outbreaks but it should be used selectively. This strategy remains an important part of a control programme.

Analysis of the prevaccine population of noncapsulate Haemophilus influenzae and identification of a putative epidemic clone.

For six months prior to the introduction of Haemophilus influenzae serotype b vaccines, all noncapsulate Haemophilus influenzae received by our laboratory were characterised by biotyping, antibiogram, outer-membrane protein profiling, and ribotyping. Simpson's index of diversity (SID) showed the population was heterogeneous with multiple clones. The study identified a clone within noncapsulate Haemophilus influenzae biotype II that caused more disease than other strains. This clone was shown to have previously caused two outbreaks of respiratory disease and to possess a small extrachromosomal plasmid encoding ampicillin resistance. The study shows that describing the diversity within a bacterial population with SID may negate the need for retrospective subtyping comparisons.

The incidence and epidemiology of beta-lactam resistance in Haemophilus influenzae.

Consecutive isolates of Haemophilus influenzae were collected by the Public Health Laboratory, Bath between 1 June 1992 and 31 May 1993. Of 379 apparently distinct isolates, 216 originated from the respiratory tract, 102 from eyes and 61 from other sites. The minimum inhibitory concentrations of amoxycillin, amoxycillin/clavulanate, cefaclor, cefuroxime, cefotaxime and cefpodoxime were determined for each isolate. Forty strains (10.6%) were beta-lactamase producers. MIC50 and MIC90 values and the range of MICs were determined for all isolates. The overall resistance rates were: amoxycillin (MIC > 1.0 mg/L), 22.7%; amoxycillin/clavulanate (MIC > 1.0 mg/L), 14.8%; cefuroxime (MIC > 1.0 mg/L), 18.5%, (MIC > 4.0 mg/L), 5.5%; cefaclor (MIC > 8 mg/L), 15.6%; cefpodoxime (MIC > 1.0 mg/L), 0.3%; cefotaxime (MIC > 1.0 mg/L), 0%. Twenty non-beta-lactamase producing but beta-lactam resistant strains (cefuroxime MIC > 4.0 mg/L) were matched with 20 susceptible strains on the basis of patient age, sex, and specimen type. The strains were characterised by outer-membrane protein (OMP), random amplified polymorphic DNA (RAPD) and ribotyping patterns. Eleven of the 20 resistant strains were indistinguishable by the methods used, suggesting spread of a single beta-lactam resistant, non-beta-lactamase producing clone. The distribution of resistant strains within the local community was plotted geographically.