Validation of assembled nucleic acid-based tests in diagnostic microbiology laboratories

Medical microbiology and virology laboratories use nucleic acid tests (NAT) to detect genomic material of infectious organisms in clinical samples. Laboratories choose to perform assembled (or in-house) NAT if commercial assays are not available or if assembled NAT are more economical or accurate. One reason commercial assays are more expensive is because extensive validation is necessary before the kit is marketed, as manufacturers must accept liability for the performance of their assays, assuming their instructions are followed. On the other hand, it is a particular laboratory's responsibility to validate an assembled NAT prior to using it for testing and reporting results on human samples. There are few published guidelines for the validation of assembled NAT. One procedure that laboratories can use to establish a validation process for an assay is detailed in this document. Before validating a method, laboratories must optimise it and then document the protocol. All instruments must be calibrated and maintained throughout the testing process. The validation process involves a series of steps including: (i) testing of dilution series of positive samples to determine the limits of detection of the assay and their linearity over concentrations to be measured in quantitative NAT; (ii) establishing the day-to-day variation of the assay's performance; (iii) evaluating the sensitivity and specificity of the assay as far as practicable, along with the extent of cross-reactivity with other genomic material; and (iv) assuring the quality of assembled assays using quality control procedures that monitor the performance of reagent batches before introducing new lots of reagent for testing.

Humans, dogs and parasitic zoonoses - Unravelling the relationships in a remote endemic community in Northeast India using molecular tools

Canine parasitic zoonoses pose a continuing public health problem, especially in developing countries and communities that are socioeconomically disadvantaged. Our study combined the use of conventional and molecular epidemic, logical tools to determine the role of dogs in transmission of gastrointestinal (GI) parasites such as hookworms, Giardia and Ascaris in a parasite endemic teagrowing community in northeast India. A highly sensitive and specific molecular tool was developed to detect and differentiate the zoonotic species of canine hookworm eggs directly from faeces. This allowed epidemiological screening of canine hookworm species in this community to be conducted with ease and accuracy. The zoonotic potential of canine Giardia was also investigated by characterising Giardia duodenalis recovered from humans and dogs living in the same locality and households at three different loci. Phylogenetic and epidemiological analysis provided compelling evidence to support the zoonotic transmission of canine Giardia. Molecular tools were also used to identify the species of Ascaris egg present in over 30% of dog faecal samples. The results demonstrated the role of dogs as a significant disseminator and environmental contaminator of Ascaris lumbricoides in communities where promiscuous defecation practices exist. Our study demonstrated the usefulness of combining conventional and molecular parasitological and epidemiological tools to help solve unresolved relationships with regards to parasitic zoonoses.

Regulation and properties of KCNQ1 (K(v)LQT1) and impact of the cystic fibrosis transmembrane conductance regulator

The K+ channel KCNQ1 (K(V)LQT1) is a voltage-gated K+ channel, coexpressed with regulatory subunits such as KCNE1 (IsK, mink) or KCNE3, depending on the tissue examined. Here, we investigate regulation and properties of human and rat KCNQ1 and the impact of regulators such as KCNE1 and KCNE3. Because the cystic fibrosis transmembrane conductance regulator (CFTR) has also been suggested to regulate KCNQ1 channels we studied the effects of CFTR on KCNQ1 in Xenopus oocytes, Expression of both human and rat KCNQ1 induced time dependent K+ currents that were sensitive to Ba2+ and 293B. Coexpression with KCNE1 delayed voltage activation, while coexpression with KCNE3 accelerated current activation. KCNQ1 currents were activated by an increase in intracellular cAMP, independent of coexpression with KCNE1 or KCNE3. cAMP dependent activation was abolished in N-terminal truncated hKCNQ1 but was still detectable after deletion of a single PKA phosphorylation motif. In the presence but not in the absence of KCNE1 or KCNE3, K+ currents were activated by the Ca2+ ionophore ionomycin. Coexpression of CFTR with either human or rat KCNQ1 had no impact on regulation of KCNQ1 K+ currents by cAMP but slightly shifted the concentration response curve for 293B. Thus, KCNQ1 expressed in Xenopus oocytes is regulated by cAMP and Ca2+ but is not affected by CFTR.

Experimental susceptibility of some reef fish species to Benedenia lutjani (Monogenea : Capsalidae), a parasite of Lutjanus carponotatus (Pisces : Lutjanidae)

The susceptibility of species of lutjanid, lethrinid and serranid fish to infection by either larval or post-larval (juvenile and adult) specimens of the capsalid monogenean Benedenia lutjani Whittington and Kearn (1993) was examined experimentally. Four species of lutjanids became infected when exposed to larvae of B. lutjani, but three species of lethrinids and four species of serranids were not susceptible to larvae under the same conditions. Variability in the intensity of infection by larvae occurred within and between lutjanid species. Few post-larval specimens of B. lutjani transferred between individuals of the specific host Lutjanus carponotatus (Richardson 1842) in 60-l aquaria and none transferred between specimens of L. carponotatus in a 7,500-l concrete tank. These results indicate that transfer of post-larval B. lutjani between individuals of the specific host is unlikely to occur in the wild. Other lutjanid species did not become infected when exposed to specimens of L. carponotatus infected heavily by post-larval B. lutjani, but two lethrinid species were susceptible to infection under the same conditions. These data indicate that different factors may mediate host-specificity for larval and post-larval B. lutjani.

Prediction of many new exons and introns in Plasmodium falciparum chromosome 2

The current prediction or genes in the Plasmodium falciparum genome database relies upon a limited number of specially developed computer algorithms. We have re-annotated the sequence of chromosome 2 of P. falciparum by a computer-assisted manual analysis. which is described here. Of 161 newly predicted introns, we have experimentally confirmed 98. We regard 110 introns from the previously published analyses as probable, we delete 3, change 26 and add 135. We recognise 214 genes in chromosome 2. We have predicted introns in 121 genes. The increased complexity or gene structure on chromosome 2 is likely to be mirrored by the entire genome. (C) 2001 Elsevier Science B.V. All rights reserved.

Mutation rates in the dihydrofolate reductase gene of Plasmodium falciparum

A new method has been established to define the limits on a spontaneous mutation rate for a gene in Plasmodium falciparum. The method combines mathematical modelling and large-scale in vitro culturing and calculates the difference in mutant frequencies at 2 separate time-points. We measured the mutation rate at 2 positions in the dihydrofolate reductase (DHFR) gene of 3D7, a pyrimethamine-sensitive line of P. fulciparum. This line was re-cloned and an effectively large population was treated with a selective pyrimethamine concentration of 40 nM. We detected point mutations at codon-46 (TTA to TCA) and codon-108 (ACC to AAC), resulting in serine replacing leucine and asparagine replacing serine respectively in the corresponding gene product. The substitutions caused a decrease in pyrimethamine sensitivity. By mathematical modelling we determined that the mutation rate at a given position in DHFR was low and occurred at less than 2(.)5 x 10(-9) mutations/DHFR gene/replication. This result has important implications for Plasmodium genetic diversity and antimalarial drug therapy by demonstrating that even with lon mutation rates anti-malarial resistance will inevitably arise when mutant alleles are selected under drug pressure.

The sequence of a 200 kb portion of a Plasmodium vivax chromosome reveals a high degree of conservation with Plasmodium falciparum chromosome 3

Within a 199 866 base pair (bp) portion of a Plasmodium vivax chromosome we identified a conserved linkage group consisting of at least 41 genes homologous to Plasmodium falciparum genes located on chromosome 3. There were no P. vivax homologues of the P. falciparum cytoadherence-linked asexual genes clag 3.2, clag 3.1 and a var C pseudogene found on the P. vivax chromosome. Within the conserved linkage group, the gene order and structure are identical to those of P. falciparum chromosome 3. This conserved linkage group may extend to as many as 190 genes. The subtelomeric regions are different in size and the P. vivax segment contains genes for which no P. falciparum homologues have been identified to date. The size difference of at least 900 kb between the homologous P. vivax chromosome and P. falciparum chromosome 3 is presumably due to a translocation. There is substantial sequence divergence with a much higher guanine + cytosine (G + C) content in the DNA and a preference for amino acids using GC-rich codons in the deduced proteins of P. vivax. This structural conservation of homologous genes and their products combined with sequence divergence at the nucleotide level makes the P. vivax genome a powerful tool for comparative analyses of Plasmodium genomes. (C) 2001 Elsevier Science B.V. All rights reserved.

The application of statistical process control charts to the detection and monitoring of hospital-acquired infections

The monitoring of infection control indicators including hospital-acquired infections is an established part of quality maintenance programmes in many health-care facilities. However, surveillance data use can be frustrated by the infrequent nature of many infections. Traditional methods of analysis often provide delayed identification of increasing infection occurrence, placing patients at preventable risk. The application of Shewhart, Cumulative Sum (CUSUM) and Exponentially Weighted Moving Average (EWMA) statistical process control charts to the monitoring of indicator infections allows continuous real-time assessment. The Shewhart chart will detect large changes, while CUSUM and EWMA methods are more suited to recognition of small to moderate sustained change. When used together, Shewhart and EWMA methods are ideal for monitoring bacteraemia and multiresistant organism rates. Shewhart and CUSUM charts are suitable for surgical infection surveillance.

Time of treatment influences the appearance of drug-resistant parasites in Plasmodium falciparum infections

A deterministic mathematical model which predicts the probability of developing a new drug-resistant parasite population within the human host is reported, The model incorporates the host's specific antibody response to PfEMP1, and also investigates the influence of chemotherapy on the probability of developing a viable drug-resistant parasite population within the host. Results indicate that early, treatment, and a high antibody threshold coupled with a long lag time between antibody stimulation and activity, are risk factors which increase the likelihood of developing a viable drug-resistant parasite population. High parasite mutation rates and fast PfEMP1 var gene switching are also identified as risk factors. The model output allows the relative importance of the various risk factors as well as the relationships between them to be established, thereby increasing the understanding of the conditions which favour the development of a new drug-resistant parasite population.

The cloning and characterization of a second alpha-amylase of A-hydrophila JMP636

Aims: The aim of this study was to identify, clone and characterize the second amylase of Aeromonas hydrophila JMP636, AmyB, and to compare it to AmyA. Methods and Results: The amylase activity of A. hydrophila JMP636 is encoded by multiple genes. A second genetically distinct amylase gene, amyB, has been cloned and expressed from its own promoter in Escherichia coli. AmyB is a large alpha-amylase of 668 amino acids. Outside the conserved domains of alpha-amylases there is limited sequence relationship between the two alpha-amylases of A. hydrophila JMP636 AmyA and AmyB. Significant (80%) similarity exists between amyB and an alpha-amylase of A. hydrophila strain MCC-1. Differences in either the functional properties or activity under different environmental conditions as possible explanations for multiple copies of amylases in JMP636 is less likely after an examination of several physical properties, with each of the properties being very similar for both enzymes (optimal pH and temperature, heat instability). However the reaction end products and substrate specificity did vary enough to give a possible reason for the two enzymes being present. Both enzymes were confirmed to be alpha-type amylases. Conclusions: AmyB has been isolated, characterized and then compared to AmyA. Significance and Impact of Study: The amylase phenotype is rarely encoded by more than one enzyme within one strain, this study therefore allows the better understanding of the unusual amylase production by A. hydrophila.

Specificity of PCR and in situ hybridization assays designed for detection of Marteilia sydneyi and M-refringens

Primers and DNA probes designed for use in the specific detection of the paramyxean parasites Marteilia sydneyi and Marteilia refringens were tested for their potential to cross-react with closely related species in Polymerase Chain Reaction (PCR) and in situ hybridization. PCR primers and a DNA probe designed within the ITS1 rRNA of M. sydneyi were specific for M. sydneyi when compared with related species of Marteilia and Marteilioides. PCR primers designed within the 18S rRNA of M. refringens were specific in the detection of this species in PCR while a DNA probe (named Smart 2) designed on the same gene cross-reacted with M. sydneyi in tissue sections of Saccostrea glomerata as well as Marteilioides sp. infecting Striostrea mytiloides. Though not species specific, the Smart 2 probe provided a stronger signal in detection of all stages of M. sydneyi than the ITS1 probe. The ITS probe is proposed for use as a confirmatory diagnostic too] for M. sydneyi.

The effect of volatile fatty acids in acidified fermented piggery effluent on shiga-toxigenic and non-toxic resident strains of Escherichia coli

Aims: To examine the effects of acidified acidogenically fermented piggery effluent containing Volatile Fatty Acids (VFA) on shiga-toxigenic and resident strains of Escherichia coli (E. coli) as part of the development of a waste treatment process. Methods and Results: Four shiga-toxigenic E. coli strains (O157:H7, 091.H-, 0111.H-, and 0123.H-) and four non-toxic resident enzootic strains were all killed by 3 h treatment with fermented piggery effluent liquor (153 mmol l(-1) total VFA) at pH 4.3. The shiga-toxigenic strains showed greater sensitivity after 1 h of treatment. The fermented liquor at pH 6.8 was not inhibitory. Conclusions: The shiga-toxigenic strains were no more resistant to the toxic effects of VFA than the non-toxic strains tested. Significance and Impact of the Study: Shiga-toxigenic strains and resident enzootic non-toxigenic strains are equally susceptible to inactivation by this waste treatment process and by acidified VFA in general.

Identification and minisequencing-based discrimination of SHV beta-lactamases in nosocomial infection-associated Klebsiella pneumoniae in Brisbane, Australia

Extended-spectrum beta-lactamases (ESBLs) are active against oxyimino cephalosporins and monobactams. Twenty-one Klebsiella pneumoniae isolates obtained between 1991 and 1995 at the Princess Alexandra Hospital in Brisbane, Australia, were subject to amplification and sequencing of the SHV beta-lactamase-encoding genes. Thirteen strains were phenotypically ESBL positive. Of these, six strains carried the bla(SHV-2a) gene and seven strains carried the bla(SHV-12) gene. Eight strains were phenotypically ESBL negative. Of these, seven strains carried the non-ESBL bla(SHV-11) gene and one strain carried the non-ESBL bla(SHV-1) gene. There was complete correspondence between the ESBL phenotype and the presence or absence of an ESBL-encoding gene(s). In addition, it was determined that of the 13 ESBL-positive strains, at least 4 carried copies of a non-ESBL-encoding gene in addition to the bla(SHV-2a) or bla(SHV12) gene. A minisequencing-based assay was developed to discriminate the different SHV classes. This technique, termed first-nucleotide change, involves the identification of the base added to a primer in a single-nucleotide extension reaction. The assay targeted polymorphisms at the first bases of codons 238 and 240 and reliably discriminated ESBL-positive strains from ESBL-negative strains and also distinguished strains carrying bla(SHV-2a) from strains carrying bla(SHV-12). In addition, this method was used to demonstrated an association between the relative copy numbers of bla(SHV) genes in individual strains and the levels of antibiotic resistance.

Manipulation of colonic bacteria and volatile fatty acid production by dietary high amylose maize (amylomaize) starch granules

Aims : To study the effects of amylomaize starch and modified (carboxymethylated and acetylated) amylomaize starches on the composition of colonic bacteria and the production of volatile fatty acids, in mice. Methods and Results : Balb/c mice were fed with experimental diets containing various amount of amylomaize and modified amylomaize starches. Colonic bacterial populations and short-chain fatty acids were monitored. Results showed that the increases in indigenous bifidobacteria were detected in mice fed all starches tested; however, the highest numbers were observed in the group fed with 40% unmodified amylomaize starch. The starch type influenced the populations of indigenous Lactobacillus , Bacteroides and coliforms. High Lactobacillus numbers were achieved in the colon of mice fed with high concentration of amylomaize starch. Acetylated amylomaize starch significantly reduced the population of coliforms. In addition, orally dosed amylomaize utilizing bifidobacteria reached their highest levels when fed together with amylomaize or carboxymethylated amylomaize starch and in both cases butyrate levels were markedly increased. Conclusions: These results indicate that different amylomaize starches could generate desirable variation in gut microflora and that particular starches may be used to selectively modify gut function. Significance and Impact of Study: Amylomaize starch appeared to enhance the desirable composition of colonic bacteria in mice, and suggested it possessed the potential prebiotic properties.MTherefore, resistant starch and its chemical derivatives may exert beneficial impacts to the human colon.