Detection and genotyping of oocysts of Cryptosporidium parvum by real-time PCR and melting curve analysis

Several real-time PCR procedures for the detection and genotyping of oocysts of Cryptosporidium parvum were evaluated. A 40-cycle amplification of a 157-bp fragment from the C. parvum beta-tubulin gene detected individual oocysts which were introduced into the reaction mixture by micromanipulation. SYBR Green I melting curve analysis was used to confirm the specificity of the method when DNA extracted from fecal samples spiked with oocysts was analyzed. Because C. parvum isolates infecting humans comprise two distinct genotypes, designated type 1 and type 2, real-time PCR methods for discriminating C. parvum genotypes were developed. The first method used the same beta-tubulin amplification primers and two fluorescently labeled antisense oligonucleotide probes spanning a 49-bp polymorphic sequence diagnostic for C. parvum type 1 and type 2. The second genotyping method used SYBR Green I fluorescence and targeted a polymorphic coding region within the GP900/poly(T) gene. Both methods discriminated between type 1 and type 2 C. parvum on the basis of melting curve analysis. To our knowledge, this is the first report describing the application of melting curve analysis for genotyping of C. parvum oocysts.     

Green fluorescent protein as a marker in Plasmodium berghei transformation

We present a new marker that confers both resistance to pyrimethamine and green fluorescent protein-based fluorescence on the malarial parasite Plasmodium berghei. A single copy of the cassette integrated into the genome is sufficient to direct fluorescence in parasites throughout the life cycle, in both its mosquito and vertebrate hosts. Erythrocyte stages of the parasite that express the marker can be sorted from control parasites by flow cytometry. Pyrimethamine pressure is not necessary for maintaining the cassette in transformed parasites during their sporogonic cycle in mosquitoes, including when it is borne by a plasmid. This tool should thus prove useful in molecular studies of P. berghei, both for generating parasite variants and monitoring their behavior.     

Anti-idiotypes against autoantibodies and alloantibodies to VIII:C (anti-haemophilic factor) are present in therapeutic polyspecific normal immunoglobulins.

Therapeutic, polyspecific, normal immunoglobulins (IVIg) suppress anti-factor VIII (VIII:C) activity of anti-VIII:c autoantibodies in vivo and in vitro. In the present study anti-VIII:C activity was found to be inhibited by two different preparations of IVIg in the plasma of three of four patients with autoantibodies and two of three patients with alloantibodies. F(ab')2 fragments from IVIg inhibited anti-VIII:C activity in F(ab')2 fragments from the plasma of the patients. In patients in whom anti-VIII:C activity was inhibited by IVIg, anti-VIII:C F(ab')2 antibodies were specifically retained on an affinity column of Sepharose-bound F(ab')2 from IVIg. In patients in whom anti-VIII:C activity was not suppressed by IVIg in vitro, no binding of anti-VIII:C antibodies to Sepharose-bound IVIg was observed. In a patient in whom anti-VIII:C activity was only suppressed by one preparation of IVIg, specific binding of anti-VIII:C antibodies was only observed with that preparation but not with another. These results indicate that IVIg contain anti-idiotypes against autoantibodies and alloantibodies to VIII:C. The capacity of IVIg to inhibit anti-VIII:C activity in vitro is directly related to the presence of demonstrable anti-idiotypes against anti-VIII:C antibodies. The finding of anti-idiotypes against anti-VIII:C alloantibodies in IVIg suggests that, in addition to autoantibodies, some alloantibodies may be suppressed in vivo by IVIg.     

Collagen and steroid hormones

The authors analyse the effects of steroid hormones on collagen, from up to date datas previously published and personal works. Glucocorticoids have catabolic effects; their molecular effects are reviewed. Conversely, oestrogens and androgens have an anabolic effect.     

Stature estimation based on hand length and foot length

This study was carried out to estimate the relationship between hand length, foot length and stature using multiple linear regression analyses based on a sample of male and female adult Turks residing in Adana. Measurements of hand length, foot length and stature were taken from 155 adult Turks (80 male, 75 female) aged 17-23 years. The participants were students of the Medical Faculty of Cukurova University. A multiple linear regression model was fitted to the observed data. Stature was taken as the response or dependent variable, hand length and foot length were taken as explanatory variables or regressors. All possible (simple and multiple) linear regression models for each of males, females and both genders together were tested for the best model. The multiple linear regression model for both genders together was found to be the best model with the highest values for the coefficients of determination R2 = 0.861 and R2adjusted = 0.859, and multiple correlation coefficient R = 0.928.     

Coping and distress as predictors of glycemic control in diabetes

This study attempts to identify the relative contribution of coping styles (task-, emotion-oriented and avoidance) and anxiety and diabetes-related distress to glycemic control in IDDM (Insulin Dependent Diabetes Mellitus) patients. Recent research suggests that coping and distress may as well be determinants as consequences of certain characteristics of the illness and subject. This points to the need to control some of these variables in diabetic research. Ninety seven IDDM patients from an outpatient unit were included. Measures were self-report questionnaires (CISS, STAI-Y, PAID) and biological data (HbA1C i.e. metabolic control). When potential confounds were controlled for (current age, age at onset, gender, presence of complication), three main predictors of metabolic control could be isolated through hierarchical regression analysis: (1) presence of complications; (2) task-oriented coping style; and (3) diabetes-related distress. Three steps were included in predictors: illness and subject variables (step 1); coping styles (step 2); and negative emotions (step 3). Each step contributed to a significant increase in variance. Emotion-oriented coping style appeared as redundant with distress measures. Results suggest that the relations between psychological predictors and metabolic control would be closer in women. These results partly replicate other findings, and advocate for a better consideration of emotional factors in the prediction of blood glucose control in this disease.     

Comparison of mismatch amplification mutation assay with DNA sequencing for characterization of fluoroquinolone resistance in Neisseria gonorrhoeae

A mismatch amplification mutation assay (MAMA) was developed for identification of point mutations in quinolone resistance-determining region (QRDR) of gyrA at codons 91 and 95. MAMA PCR was used to detect mutations at codons 91 and 95 of gyrA in 117 Neisseria gonorrhoeae isolates (with ciprofloxacin MICs of 0.004 to >32 microg/ml) from Bangladesh during 1997 to 2001. The QRDR regions of the gyrA genes from 31 randomly selected isolates were sequenced, and the results were compared with those of MAMA PCR. Using mismatch PCR, a mutation at Ser91 could be detected in all 27 (resistant and intermediate) isolates, and an Asp95-to-Gly95 mutation could be detected in all 15 isolates, as detected by sequencing. MAMA PCR offers a simple, inexpensive, rapid, and easier alternative for detection of point mutations in fluoroquinolone resistance in N. gonorrhoeae.     

Regional anaesthesia for labour,operative vaginal delivery and caesarean delivery: a narrative review

This narrative review discusses recent evidence surrounding the use of regional anaesthesia in the obstetric setting, including intrapartum techniques for labour and operative vaginal delivery, and caesarean delivery. Pudendal nerve blockade, ideally administered by an obstetrician, should be considered for operative vaginal delivery if neuraxial analgesia is contraindicated. Regional techniques are increasingly utilised in clinical practice for caesarean delivery to minimise opioid consumption, reduce pain, improve postpartum recovery and facilitate earlier discharge as part of enhanced recovery protocols. The evidence surrounding transversus abdominis plane and quadratus lumborum blockade supports their use when: long-acting neuraxial opioids cannot be administered due to contraindications; if emergency delivery necessitates general anaesthesia; or as a postoperative rescue technique. Current data suggest quadratus lumborum blockade is no more effective than transversus abdominis plane blockade after caesarean delivery. Transversus abdominis plane blockade, wound catheter insertion and single shot wound infiltration are all effective techniques for reducing postoperative opioid consumption, with transversus abdominis plane blockade favoured, followed by wound catheters and then wound infiltration. Ilio-inguinal and iliohypogastric, erector spinae plane and rectus sheath blockade all require further studies to determine their efficacy for caesarean delivery in the presence or absence of long-acting neuraxial opioids. Future studies are needed to: compare approaches for individual techniques; determine which combinations of techniques and dosing regimens result in optimal analgesic and recovery outcomes following delivery; and elucidate the populations that benefit most from regional anaesthesia in the obstetric setting.