Taguchi optimisation of a multiplex pneumococcal serotyping PCR and description of 11 novel serotyping primers.

Recently, a PCR-derived method for serotyping Streptococcus pneumoniae has been devised to substitute the conventional antiserum phenotypic method. The method initially used a multiplex PCR reaction, dividing the isolates into 6 different groups based on the detected PCR gel pattern. In order to optimise and refine this crucial step, the Taguchi technique was employed, which can evaluate the individual effect of six parameters (in this case: primers, MgCl2, nucleotide mix, polymerase and buffer), with only 18 experiments; varying the parameter levels in an orthogonal matrix which suppresses the interactions between them. With this method, clear and sharp bands were observed in 5 experiments out of the 18, while the PCR did not work reliably in the remaining cases. In addition, the PCR-based technique could be rendered more economic by the 10-fold lowering of the quantities of two primers. The modified reaction yielded identical results to those obtained with the original method. Furthermore, we have designed serotype-specific primers for 11 new serotypes. The most important ones are those that can distinguish the very closely related, but equally important serotypes 6A and 6B.

Endotoxins do not influence transplacental transmission of lymphotropic human herpesviruses and human papillomaviruses into amniotic fluid taken from healthy mothers before parturition in Hungary.

Pregnant women were examined following healthy pregnancies at term. Amniotic fluids were sampled before arteficial rupture of membranes using closed vacutainer system. Blood samples were also taken from the pregnants simultaneously. Endotoxin concentrations of amniotic fluids were tested by the semiquantitative Limulus amebocyte lysate. Both amniotic fluids and blood samples were tested for the presence of DNA of lymphotropic human herpesviruses. The DNA of human papillomaviruses were tested only in the amniotic fluid samples. One-third of the amniotic fluids tested were found to contain measurable amounts of endotoxin. Lymphotropic herpesvirus DNA was deteced in every fourth amniotic fluid sample and in every 8th blood sample. The prevalence of papillomaviruses was 7 of 96 samples. No significant correlation was found between the presence of endotoxin and viruses in the amniotic fluids. Epstein-Barr virus, human cytomegalovirus and human herpesvirus type 7 were found more frequently in the amniotic fluids than in blood samples (7 to 1). The prevalence of human herpesvirus 6 and 8 was higher in the blood samples than that in the amniotic fluids. The mean weight of the neonates were not impaired significantly by the presence of either viruses or endotoxin. Possible post partum consequences, i.e. partial immunotolerance to viruses is discussed.

Planning of empirical antibiotic therapy for women with pelvic inflammatory diseases: a geographical area-specific study.

OBJECTIVE: Elaboration of an empiric antibiotic regimen for women with pelvic inflammatory disease (PID) for a geographical area in eastern Hungary. STUDY DESIGN: Pathogens were identified by culturing or polymerase chain reaction (PCR) from 2215 patients with suspected PID between 1 January 1999 and 31 December 2001. Empiric guidelines for PID treatment were based on susceptibility testing of the recovered bacteria, patient acceptance and cost-effectiveness of drugs and recommendations of earlier studies. RESULTS: Chlamydia trachomatis was detected in 11%, Neisseria gonorrhoeae in 2%, Streptococcus spp. in 17%, Enterococcus spp. 9%, genital mycoplasmas in 25%, all obligate anaerobic pathogens in 30% of the patients. All antibiotics chosen for our regimen were effective in vitro against one or more recovered pathogens at least in 80%; this regimen produced 98% clinical cure rate in mild cases of PID. CONCLUSION: Early detection and prompt empirical antimicrobial therapy adapted to the local microflora and its resistance pattern can lead to good clinical results.

Pelvic inflammatory disease is a risk factor for cervical cancer.

The acquisition of human papillomavirus (HPV), the most important etiological agent of cervical cancer, does not cause clinical complaints. Although HPV spreads together with agents causing pelvic inflammatory disease (PID) with complaints forcing the patient to seek medical advice, PID has not yet been evaluated as a predictor of cervical cancer. The present study aimed to determine the relationship between PID and HPV in order to evaluate the possible risk factor role of PID for cervical cancer. Two groups of patients were studied: (i) 2,215 women with PID; (ii) 4,217 women participating in a cervical cancer screening programme who were found to have cytological atypia, mucopurulent cervicitis or other colposcopically detected disorders but were free of symptoms of PID. The presence of HPV and other STD agents in cervical smears was detected with polymerase-chain reaction. HPV prevalence was 33.74% in patients with PID and 26.40% in the group of women without PID (p < 0.001). This suggests that patients suffering from PID apparently have a higher risk of cervical cancer.

The endosomal epsilon-coatomer protein is involved in human adenovirus type 5 internalisation.

The effects of bafilomycin A1 and of the reduced level of endosomal epsilon-COP (coatomer protein) on the infectivity of human adenovirus type 5 were investigated in Coxsackie adenovirus receptor- (CAR-) transfected Chinese hamster ovary (CHO) cells. The endosomal proton pump inhibitor bafilomycin A1 was able to cause only partial inhibition. Using Id1F cells (an epsilon-COP thermosensitive mutant CHO cell line) the reduction of epsilon-COP level also had partial inhibitory effect. Based on these results and comparing them to existing models of the adenovirus entry, we propose a refined model in which there are two pathways of adenoviral entry: the first one involves the epsilon-COP as the downstream effector of the acidification and can be blocked by bafilomycin A1 and the second one is a pH-independent pathway.