Defining the True Sensitivity of Culture for the Diagnosis of Melioidosis Using Bayesian Latent Class Models

Background

Culture remains the diagnostic gold standard for many bacterial infections, and the method against which other tests are often evaluated. Specificity of culture is 100% if the pathogenic organism is not found in healthy subjects, but the sensitivity of culture is more difficult to determine and may be low. Here, we apply Bayesian latent class models (LCMs) to data from patients with a single Gram-negative bacterial infection and define the true sensitivity of culture together with the impact of misclassification by culture on the reported accuracy of alternative diagnostic tests.

Methods/Principal Findings

Data from published studies describing the application of five diagnostic tests (culture and four serological tests) to a patient cohort with suspected melioidosis were re-analysed using several Bayesian LCMs. Sensitivities, specificities, and positive and negative predictive values (PPVs and NPVs) were calculated. Of 320 patients with suspected melioidosis, 119 (37%) had culture confirmed melioidosis. Using the final model (Bayesian LCM with conditional dependence between serological tests), the sensitivity of culture was estimated to be 60.2%. Prediction accuracy of the final model was assessed using a classification tool to grade patients according to the likelihood of melioidosis, which indicated that an estimated disease prevalence of 61.6% was credible. Estimates of sensitivities, specificities, PPVs and NPVs of four serological tests were significantly different from previously published values in which culture was used as the gold standard.

Conclusions/Significance

Culture has low sensitivity and low NPV for the diagnosis of melioidosis and is an imperfect gold standard against which to evaluate alternative tests. Models should be used to support the evaluation of diagnostic tests with an imperfect gold standard. It is likely that the poor sensitivity/specificity of culture is not specific for melioidosis, but rather a generic problem for many bacterial and fungal infections.     

Biological relevance of colony morphology and phenotypic switching by Burkholderia pseudomallei

Melioidosis is a notoriously protracted illness and is difficult to cure. We hypothesize that the causative organism, Burkholderia pseudomallei, undergoes a process of adaptation involving altered expression of surface determinants which facilitates persistence in vivo and that this is reflected by changes in colony morphology. A colony morphotyping scheme and typing algorithm were developed using clinical B. pseudomallei isolates. Morphotypes were divided into seven types (denoted I to VII). Type I gave rise to other morphotypes (most commonly type II or III) by a process of switching in response to environmental stress, including starvation, iron limitation, and growth at 42 degrees C. Switching was associated with complex shifts in phenotype, one of which (type I to type II) was associated with a marked increase in production of factors putatively associated with in vivo concealment. Isogenic types II and III, derived from type I, were examined using several experimental models. Switching between isogenic morphotypes occurred in a mouse model, where type II appeared to become adapted for persistence in a low-virulence state. Isogenic type II demonstrated a significant increase in intracellular replication fitness compared with parental type I after uptake by epithelial cells in vitro. Isogenic type III demonstrated a higher replication fitness following uptake by macrophages in vitro, which was associated with a switch to type II. Mixed B. pseudomallei morphologies were common in individual clinical specimens and were significantly more frequent in samples of blood, pus, and respiratory secretions than in urine and surface swabs. These findings have major implications for therapeutics and vaccine development.     

Evaluating Burkholderia pseudomallei Bip proteins as vaccines and Bip antibodies as detection agents

Burkholderia pseudomallei is a biothreat agent and an important natural pathogen, causing melioidosis in humans and animals. A type III secretion system (TTSS-3) has been shown to be critical for virulence. Because TTSS components from other pathogens have been used successfully as diagnostic agents and as experimental vaccines, it was investigated whether this was the case for BipB, BipC and BipD, components of B. pseudomallei's TTSS-3. The sequences of BipB, BipC and BipD were found to be highly conserved among B. pseudomallei and B. mallei isolates. A collection of monoclonal antibodies (mAbs) specific for each Bip protein was obtained. Most recognized both native and denatured Bip protein. Burkholderia pseudomallei or B. mallei did not express detectable BipB or BipD under the growth conditions used. However, anti-BipD mAbs did recognize the TTSS needle structures of a Shigella strain engineered to express BipD. The authors did not find that BipB, BipC or BipD are protective antigens because vaccination of mice with any single protein did not result in protection against experimental melioidosis. Enzyme-linked immunosorbent assay (ELISA) studies showed that human melioidosis patients had antibodies to BipB and BipD. However, these ELISAs had low diagnostic accuracy in endemic regions, possibly due to previous patient exposure to B. pseudomallei.     

Copy Number Variation in Thai Population

Copy number variation (CNV) is a major genetic polymorphism contributing to genetic diversity and human evolution. Clinical application of CNVs for diagnostic purposes largely depends on sufficient population CNV data for accurate interpretation. CNVs from general population in currently available databases help classify CNVs of uncertain clinical significance, and benign CNVs. Earlier studies of CNV distribution in several populations worldwide showed that a significant fraction of CNVs are population specific. In this study, we characterized and analyzed CNVs in 3,017 unrelated Thai individuals genotyped with the Illumina Human610, Illumina HumanOmniexpress, or Illumina HapMap550v3 platform. We employed hidden Markov model and circular binary segmentation methods to identify CNVs, extracted 23,458 CNVs consistently identified by both algorithms, and cataloged these high confident CNVs into our publicly available Thai CNV database. Analysis of CNVs in the Thai population identified a median of eight autosomal CNVs per individual. Most CNVs (96.73%) did not overlap with any known chromosomal imbalance syndromes documented in the DECIPHER database. When compared with CNVs in the 11 HapMap3 populations, CNVs found in the Thai population shared several characteristics with CNVs characterized in HapMap3. Common CNVs in Thais had similar frequencies to those in the HapMap3 populations, and all high frequency CNVs (>20%) found in Thai individuals could also be identified in HapMap3. The majorities of CNVs discovered in the Thai population, however, were of low frequency, or uniquely identified in Thais. When performing hierarchical clustering using CNV frequencies, the CNV data were clustered into Africans, Europeans, and Asians, in line with the clustering performed with single nucleotide polymorphism (SNP) data. As CNV data are specific to origin of population, our population-specific reference database will serve as a valuable addition to the existing resources for the investigation of clinical significance of CNVs in Thais and related ethnicities.     

A simple scoring system to differentiate between relapse and re-infection in patients with recurrent melioidosis

Background

Melioidosis is an important cause of morbidity and mortality in East Asia. Recurrent melioidosis occurs in around 10% of patients following treatment either because of relapse with the same strain or re-infection with a new strain of Burkholderia pseudomallei. Distinguishing between the two is important but requires bacterial genotyping. The aim of this study was to develop a simple scoring system to distinguish re-infection from relapse.

Methods

In a prospective study of 2,804 consecutive adult patients with melioidosis presenting to Sappasithiprasong Hospital, NE Thailand, between1986 and 2005, there were 141 patients with recurrent melioidosis with paired strains available for genotyping. Of these, 92 patients had relapse and 49 patients had re-infection. Variables associated with relapse or re-infection were identified by multivariable logistic regression and used to develop a predictive model. Performance of the scoring system was quantified with respect to discrimination (area under receiver operating characteristic curves, AUC) and categorization (graphically). Bootstrap resampling was used to internally validate the predictors and adjust for over-optimism.

Findings

Duration of oral antimicrobial treatment, interval between the primary episode and recurrence, season, and renal function at recurrence were independent predictors of relapse or re-infection. A score of <5 correctly identified relapse in 76 of 89 patients (85%), whereas a score ≥5 correctly identified re-infection in 36 of 52 patients (69%). The scoring index had good discriminative power, with a bootstrap bias-corrected AUC of 0.80 (95%CI: 0.73–0.87).

Conclusions

A simple scoring index to predict the cause of recurrent melioidosis has been developed to provide important bedside information where rapid bacterial genotyping is unavailable.     

Insights into saquinavir resistance in the G48V HIV-1 protease: quantum calculations and molecular dynamic simulations

The spread of acquired immune deficiency syndrome has increasingly become a great concern owing largely to the failure of chemotherapies. The G48V is considered the key signature residue mutation of HIV-1 protease developing with saquinavir therapy. Molecular dynamics simulations of the wild-type and the G48V HIV-1 protease complexed with saquinavir were carried out to explore structure and interactions of the drug resistance. The molecular dynamics results combined with the quantum-based and molecular mechanics Poisson-Boltzmann surface area calculations indicated a monoprotonation took place on D25, one of the triad active site residues. The inhibitor binding of the triad residues and its interaction energy in the mutant were similar to those in the wild-type. The overall structure of both complexes is almost identical. However, the steric conflict of the substituted valine results in the conformational change of the P2 subsite and the disruption of hydrogen bonding between the -NH of the P2 subsite and the backbone -CO of the mutated residue. The magnitude of interaction energy changes was comparable to the experimental K(i) data. The designing for a new drug should consider a reduction of steric repulsion on P2 to enhance the activity toward this mutant strain.     

The genetic and molecular basis of O-antigenic diversity in Burkholderia pseudomallei lipopolysaccharide

Lipopolysaccharide (LPS) is one of the most important virulence and antigenic components of Burkholderia pseudomallei, the causative agent of melioidosis. LPS diversity in B. pseudomallei has been described as typical, atypical or rough, based upon banding patterns on SDS-PAGE. Here, we studied the genetic and molecular basis of these phenotypic differences. Bioinformatics was used to determine the diversity of genes known or predicted to be involved in biosynthesis of the O-antigenic moiety of LPS in B. pseudomallei and its near-relative species. Multiplex-PCR assays were developed to target diversity of the O-antigen biosynthesis gene patterns or LPS genotypes in B. pseudomallei populations. We found that the typical LPS genotype (LPS genotype A) was highly prevalent in strains from Thailand and other countries in Southeast Asia, whereas the atypical LPS genotype (LPS genotype B) was most often detected in Australian strains (~13.8%). In addition, we report a novel LPS ladder pattern, a derivative of the atypical LPS phenotype, associated with an uncommon O-antigen biosynthesis gene cluster that is found in only a small B. pseudomallei sub-population. This new LPS group was designated as genotype B2. We also report natural mutations in the O-antigen biosynthesis genes that potentially cause the rough LPS phenotype. We postulate that the diversity of LPS may correlate with differential immunopathogenicity and virulence among B. pseudomallei strains.