An important and serious complication of intestinal infection with Entamoeba histolytica is the involvement of the liver (hepatic amoebiasis). Hepatic amoebiasis is usually diagnosed by the clinical picture (pain in the right upper quadrant and fever), ultrasound examination and positive serology. However, none of these tests are definitive and the picture overlaps with pyogenic liver abscess caused by bacteria. It is for this reason that the feasibility of using polymerase chain reaction (PCR) for the detection of E. histolytica DNA in liver abscess pus was investigated. A comparative study was done to verify the sensitivity of ten pairs of primers specific for detecting E. histolytica in stools. Samples of liver abscess pus from 22 serology-positive patients were collected under ultrasound guidance; and these were used directly in PCR assays without any prior pre-treatment of the samples. Of the ten pairs of previously published primers tested, two pairs of primers (PI + P2 and P11 + P12) were found to give 100% sensitivity. Based on these results, we recommend that PCR assay can be successfully used to confirm the diagnosis of amoebic liver abscess with the primers identified.

A 0.5kb Spel-BclI fragment containing the pIJ101 korB ORF was cloned into pUC8 under the control of the lacZ promoter, creating pQR206. In vitro coupled transcription-translation of pQR206 identified a protein product of approximately 10kDa, which corresponds to the predicted molecular weight deduced from the korB sequence. pQR206 was used to express the 10kDa KorB protein in vivo in E. coli. Crude E. coli protein extracts containing KorB were shown to bind to a 0.8kb kilB fragment and a 0.5kb korB fragment in gel retardation assays. DNasel footprinting indicated that the DNA recognition sequence of the KorB protein lies within a 60bp protected region encompassing the kilB promoter and a 36bp region encompassing the korB promoter.

) ~~The indigenous plasmid pIJlOl is the parent of many cloning vectors used in Streptumyces. One early pIJlOl derivative, pIJ702, has been particularly widely used. PI 5702 lacks sti: cup/kurB and accumulates single-stranded DNA (ssDNA). The 1.2 kb BclI-BclI sti:cop/kuuB and 0.7 kb SpeI-BclI sti regions were isolated from pIJlOl and cloned into pIJ702 at the PstI site in both orientations. No ssDNA was detected in constructs containing sti present in its correct orientation with respect to the basic replicon, with or without cup/korB. Constructs which contained sti in the reverse orientation did accumulate ssDNA. Thus, sti is only active as the site for second-strand synthesis in its natural orientation. Furthermore, sti inserted in either orientation into the structurally unstable pIJ702-pUC8 shuttle vectors prevented them from rearranging in S. fividans. The sti function was defined to a 0.53 kb SpeI-Sac11 fragment and the probable site for second-strand initiation (ssi) was identified.

Seventeen pairs of published primer sets were compared for their relative sensitivity to detect malaria DNA extracted from blood samples, which were obtained from Pakistani patients suffering from malaria. The primer sets investigated consisted of: (i) 9 pairs of direct primers and 3 sets of nested primers for detecting Plasmodium falciparum, (ii) 2 pairs of direct primers and 2 sets of nested primers for detecting P. vivax, and (iii) 1 set of multiplex primers for detecting both P. falciparum and P. vivax, simultaneously. After a miniscreen of 9 DNA-extracted blood samples using the 17 primer sets stated above, 5 primer sets were short-listed (based on their superior sensitivity) and used for a maxi-screen of DNA extracted from 126 microscopy-positive blood samples from Pakistan, with the following results. (i) For the detection of P. falciparum, the direct primer pair 'PF1 + PF2' gave a sensitivity of 95% and the nested primer set 'RIT405 + RIT406/RIT371 + RIT372' gave a sensitivity of 97%. (ii) For the detection of P. vivax, the direct primer pair 'Forward + Reverse' and the nested primer set 'PLF + UNR/PLF + VIR' both gave a sensitivity of 94%. (iii) The nested multiplex primer set 'rPLU5 + rPLU6/rFAL1 + rFAL2 + rVIV1 + rVIV2' gave a sensitivity of 97% and 96% for P. falciparum and P. vivax, respectively. It was concluded that the nested multiplex primer set was the most optimal primer set to use for the detection of malaria DNA extracted from blood samples. Furthermore, the nested multiplex primer set has the advantage of simultaneously detecting and differentiating between P. vivax and P. falciparum.

Nosocomial isolates of Pseudomonas aeruginosa exhibit high rates of resistance to antibiotics, and are often multidrug resistant. P. aeruginosa clinical isolates (n = 56) were obtained from ICU patients in a hospital in Pakistan over a 3-y period. Antimicrobial susceptibility of the 56 P. aeruginosa clinical isolates was investigated using 7 antibiotics and the resistance rates were as follows: aztreonam (68% resistant), ceftazidime (67%), imipenem (66%), ofloxacin (59%), amikacin (56%), gentamicin (44%), and piperacillin-tazobactam (27%) (p < 0.01). In addition, 55% of the P. aeruginosa clinical isolates were resistant to 4 or more antibiotics. Imipenem-resistant strains were frequently associated with ceftazidime, ofloxacin, aztreonam, and more strikingly, amikacin resistance (p < 0.05). PCR (using P. aeruginosa-specific primers VIC1 + VIC2 and P1 + P2, respectively) was highly specific and sensitive, and was positive for all 56 P. aeruginosa isolates tested. Automated ribotyping was used to investigate the clonal diversity of the 56 P. aeruginosa isolates. Automated ribotyping indicated that the clinical isolates were clonally related and could be clustered into 4 major ribogroups based on their similarity index, with ribogroup II being the dominant one. The P. aeruginosa isolates in ribogroup II were correlated with their antibiotic resistance pattern and, interestingly, there seemed to be a gradual acquisition of multiple antibiotic resistance associated with the isolates within this group over time. The ribotyping data, together with the antibiotic resistance profile, provide valuable molecular epidemiology information for the control of hospital-acquired P. aeruginosa infections.