Development of a target product profile for a point-of-care cardiometabolic device.

INTRODUCTION: Multi-parameter diagnostic devices can simplify cardiometabolic disease diagnosis. However, existing devices may not be suitable for use in low-resource settings, where the burden of non-communicable diseases is high. Here we describe the development of a target product profile (TPP) for a point-of-care multi-parameter device for detection of biomarkers for cardiovascular disease and metabolic disorders, including diabetes, in primary care settings in low- and middle-income countries (LMICs). METHODS: A draft TPP developed by an expert group was reviewed through an online survey and semi-structured expert interviews to identify device characteristics requiring refinement. The draft TPP included 41 characteristics with minimal and optimal requirements; characteristics with an agreement level for either requirement of ≤ 85% in either the survey or among interviewees were further discussed by the expert group and amended as appropriate. RESULTS: Twenty people responded to the online survey and 18 experts participated in the interviews. Twenty-two characteristics had an agreement level of ≤ 85% in either the online survey or interviews. The final TPP defines the device as intended to be used for basic diagnosis and management of cardiometabolic disorders (lipids, glucose, HbA1c, and creatinine) as minimal requirement, and offering an expanded test menu for wider cardiometabolic disease management as optimal requirement. To be suitable, the device should be intended for level 1 healthcare settings or lower, used by minimally trained healthcare workers and allow testing using self-contained cartridges or strips without the need for additional reagents. Throughput should be one sample at a time in a single or multi-analyte cartridge, or optimally enable testing of several samples and analytes in parallel with random access. CONCLUSION: This TPP will inform developers of cardiometabolic multi-parameter devices for LMIC settings, and will support decision makers in the evaluation of existing and future devices.

Diagnostics and monitoring tools for noncommunicable diseases: a missing component in the global response.

A key component of any health system is the capacity to accurately diagnose individuals. One of the six building blocks of a health system as defined by the World Health Organization (WHO) includes diagnostic tools. The WHO's Noncommunicable Disease Global Action Plan includes addressing the lack of diagnostics for noncommunicable diseases, through multi-stakeholder collaborations to develop new technologies that are affordable, safe, effective and quality controlled, and improving laboratory and diagnostic capacity and human resources. Many challenges exist beyond price and availability for the current tools included in the Package of Essential Noncommunicable Disease Interventions (PEN) for cardiovascular disease, diabetes and chronic respiratory diseases. These include temperature stability, adaptability to various settings (e.g. at high altitude), need for training in order to perform and interpret the test, the need for maintenance and calibration, and for Blood Glucose Meters non-compatible meters and test strips. To date the issues surrounding access to diagnostic and monitoring tools for noncommunicable diseases have not been addressed in much detail. The aim of this Commentary is to present the current landscape and challenges with regards to guidance from the WHO on diagnostic tools using the WHO REASSURED criteria, which define a set of key characteristics for diagnostic tests and tools. These criteria have been used for communicable diseases, but so far have not been used for noncommunicable diseases. Diagnostic tools have played an important role in addressing many communicable diseases, such as HIV, TB and neglected tropical diseases. Clearly more attention with regards to diagnostics for noncommunicable diseases as a key component of the health system is needed.

Informing Antibiotic Treatment Decisions: Evaluating Rapid Molecular Diagnostics To Identify Susceptibility and Resistance to Carbapenems against Acinetobacter spp. in PRIMERS III.

The widespread dissemination of carbapenem-resistant Acinetobacter spp. has created significant therapeutic challenges. At present, rapid molecular diagnostics (RMDs) that can identify this phenotype are not commercially available. Two RMD platforms, PCR combined with electrospray ionization mass spectrometry (PCR/ESI-MS) and molecular beacons (MB), for detecting genes conferring resistance/susceptibility to carbapenems in Acinetobacter spp. were evaluated. An archived collection of 200 clinical Acinetobacter sp. isolates was tested. Predictive values for susceptibility and resistance were estimated as a function of susceptibility prevalence and were based on the absence or presence of beta-lactamase (bla) NDM, VIM, IMP, KPC, and OXA carbapenemase genes (e.g., blaOXA-23, blaOXA-24/40, and blaOXA-58 found in this study) against the reference standard of MIC determinations. According to the interpretation of MICs, 49% (n = 98) of the isolates were carbapenem resistant (as defined by either resistance or intermediate resistance to imipenem). The susceptibility sensitivities (95% confidence interval [CI]) for imipenem were 82% (74%, 89%) and 92% (85%, 97%) for PCR/ESI-MS and MB, respectively. Resistance sensitivities (95% CI) for imipenem were 95% (88%, 98%) and 88% (80%, 94%) for PCR/ESI-MS and MB, respectively. PRIMERS III establishes that RMDs can discriminate between carbapenem resistance and susceptibility in Acinetobacter spp. In the context of a known prevalence of resistance, SPVs and RPVs can inform clinicians regarding the best choice for empiric antimicrobial therapy against this multidrug-resistant pathogen.

Rapid Molecular Diagnostics, Antibiotic Treatment Decisions, and Developing Approaches to Inform Empiric Therapy: PRIMERS I and II.

BACKGROUND: Rapid molecular diagnostic (RMD) platforms may lead to better antibiotic use. Our objective was to develop analytical strategies to enhance the interpretation of RMDs for clinicians. METHODS: We compared the performance characteristics of 4 RMD platforms for detecting resistance against ß-lactams in 72 highly resistant isolates of Escherichia coli and Klebsiella pneumoniae (PRIMERS I). Subsequently, 2 platforms were used in a blinded study in which a heterogeneous collection of 196 isolates of E. coli and K. pneumoniae (PRIMERS II) were examined. We evaluated the genotypic results as predictors of resistance or susceptibility against ß-lactam antibiotics. We designed analytical strategies and graphical representations of platform performance, including discrimination summary plots and susceptibility and resistance predictive values, that are readily interpretable by practitioners to inform decision-making. RESULTS: In PRIMERS I, the 4 RMD platforms detected ß-lactamase (bla) genes and identified susceptibility or resistance in >95% of cases. In PRIMERS II, the 2 platforms identified susceptibility against extended-spectrum cephalosporins and carbapenems in >90% of cases; however, against piperacillin/tazobactam, susceptibility was identified in <80% of cases. Applying the analytical strategies to a population with 15% prevalence of ceftazidime-resistance and 5% imipenem-resistance, RMD platforms predicted susceptibility in >95% of cases, while prediction of resistance was 69%-73% for ceftazidime and 41%-50% for imipenem. CONCLUSIONS: RMD platforms can help inform empiric ß-lactam therapy in cases where bla genes are not detected and the prevalence of resistance is known. Our analysis is a first step in bridging the gap between RMDs and empiric treatment decisions.

Rapid Diagnosis of Infection in the Critically Ill, a Multicenter Study of Molecular Detection in Bloodstream Infections, Pneumonia, and Sterile Site Infections.

OBJECTIVE: Early identification of causative microorganism(s) in patients with severe infection is crucial to optimize antimicrobial use and patient survival. However, current culture-based pathogen identification is slow and unreliable such that broad-spectrum antibiotics are often used to insure coverage of all potential organisms, carrying risks of overtreatment, toxicity, and selection of multidrug-resistant bacteria. We compared the results obtained using a novel, culture-independent polymerase chain reaction/electrospray ionization-mass spectrometry technology with those obtained by standard microbiological testing and evaluated the potential clinical implications of this technique. DESIGN: Observational study. SETTING: Nine ICUs in six European countries. PATIENTS: Patients admitted between October 2013 and June 2014 with suspected or proven bloodstream infection, pneumonia, or sterile fluid and tissue infection were considered for inclusion. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We tested 616 bloodstream infection, 185 pneumonia, and 110 sterile fluid and tissue specimens from 529 patients. From the 616 bloodstream infection samples, polymerase chain reaction/electrospray ionization-mass spectrometry identified a pathogen in 228 cases (37%) and culture in just 68 (11%). Culture was positive and polymerase chain reaction/electrospray ionization-mass spectrometry negative in 13 cases, and both were negative in 384 cases, giving polymerase chain reaction/electrospray ionization-mass spectrometry a sensitivity of 81%, specificity of 69%, and negative predictive value of 97% at 6 hours from sample acquisition. The distribution of organisms was similar with both techniques. Similar observations were made for pneumonia and sterile fluid and tissue specimens. Independent clinical analysis of results suggested that polymerase chain reaction/electrospray ionization-mass spectrometry technology could potentially have resulted in altered treatment in up to 57% of patients. CONCLUSIONS: Polymerase chain reaction/electrospray ionization-mass spectrometry provides rapid pathogen identification in critically ill patients. The ability to rule out infection within 6 hours has potential clinical and economic benefits.

Detection of prosthetic joint infection by use of PCR-electrospray ionization mass spectrometry applied to synovial fluid.

PCR coupled with electrospray ionization mass spectrometry applied to synovial fluid specimens had an 81% sensitivity and a 95% specificity for the diagnosis of prosthetic joint infection.

Diagnosis of prosthetic joint infection by use of PCR-electrospray ionization mass spectrometry.

We compared PCR-electrospray ionization mass spectrometry (PCR-ESI/MS) to culture using sonicate fluid from 431 subjects with explanted knee (n = 270) or hip (n = 161) prostheses. Of these, 152 and 279 subjects had prosthetic joint infection (PJI) and aseptic failure, respectively. The sensitivities for detecting PJI were 77.6% for PCR-ESI/MS and 69.7% for culture (P = 0.0105). The specificities were 93.5 and 99.3%, respectively (P = 0.0002).

Direct detection of indirect transmission of Streptobacillus moniliformis rat bite fever infection.

We describe the evaluation of culture-negative synovial fluid from a 3-year-old boy by PCR and electrospray ionization followed by mass spectrometry (PCR/ESI-MS). Our patient developed a diffuse rash and fever with systemic signs and symptoms of sepsis, but four sets of blood cultures obtained prior to initiation of antibiotics were negative. After 1 week of illness, he developed right-knee swelling. Analysis of synovial fluid was consistent with infection, but cultures of specimens obtained following initiation of antimicrobial treatment were negative for growth. PCR/ESI-MS detected Streptobacillus moniliformis in the synovial fluid sample. Our patient completed an appropriate course of antibiotic treatment and remained completely asymptomatic in follow-up evaluation. This unique case suggests that PCR/ESI-MS may be a useful diagnostic tool for direct detection of unusual or unexpected pathogens directly from clinical specimens, particularly when samples have been obtained from patients following initiation of antibiotic therapy.

Ureaplasma parvum prosthetic joint infection detected by PCR.

We describe the first reported case of Ureaplasma parvum prosthetic joint infection (PJI) detected by PCR. Ureaplasma species do not possess a cell wall and are usually associated with colonization and infection of mucosal surfaces (not prosthetic material). U. parvum is a relatively new species name for certain serovars of Ureaplasma urealyticum, and PCR is useful for species determination. Our patient presented with late infection of his right total knee arthroplasty. Intraoperative fluid and tissue cultures and pre- and postoperative synovial fluid cultures were all negative. To discern the pathogen, we employed PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS). Our patient's failure to respond to empirical antimicrobial treatment and our previous experience with PCR/ESI-MS in culture-negative cases of infection prompted us to use this approach over other diagnostic modalities. PCR/ESI-MS detected U. parvum in all samples. U. parvum-specific PCR testing was performed on all synovial fluid samples to confirm the U. parvum detection.

Identification of occult Fusobacterium nucleatum central nervous system infection by use of PCR-electrospray ionization mass spectrometry.

Anaerobic bacteria are often difficult to detect, especially after the initiation of antibiotics. We describe the application of PCR-electrospray ionization mass spectrometry (PCR/ESI-MS) using a sample of cerebrospinal fluid to identify an anaerobic Gram-negative bacillus, Fusobacterium nucleatum, in a patient with "culture-negative" meningitis and cerebral abscesses.

Improved sensitivity for molecular detection of bacterial and Candida infections in blood.

The rapid identification of bacteria and fungi directly from the blood of patients with suspected bloodstream infections aids in diagnosis and guides treatment decisions. The development of an automated, rapid, and sensitive molecular technology capable of detecting the diverse agents of such infections at low titers has been challenging, due in part to the high background of genomic DNA in blood. PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) allows for the rapid and accurate identification of microorganisms but with a sensitivity of about 50% compared to that of culture when using 1-ml whole-blood specimens. Here, we describe a new integrated specimen preparation technology that substantially improves the sensitivity of PCR/ESI-MS analysis. An efficient lysis method and automated DNA purification system were designed for processing 5 ml of whole blood. In addition, PCR amplification formulations were optimized to tolerate high levels of human DNA. An analysis of 331 specimens collected from patients with suspected bloodstream infections resulted in 35 PCR/ESI-MS-positive specimens (10.6%) compared to 18 positive by culture (5.4%). PCR/ESI-MS was 83% sensitive and 94% specific compared to culture. Replicate PCR/ESI-MS testing from a second aliquot of the PCR/ESI-MS-positive/culture-negative specimens corroborated the initial findings in most cases, resulting in increased sensitivity (91%) and specificity (99%) when confirmed detections were considered true positives. The integrated solution described here has the potential to provide rapid detection and identification of organisms responsible for bloodstream infections.

Rapid PCR/ESI-MS-based molecular genotyping of Staphylococcus aureus from nasal swabs of emergency department patients.

BACKGROUND: A limitation of both culture-based and molecular methods of screening for staphylococcal infection is that current tests determine only the presence or absence of colonization with no information on the colonizing strain type. A technique that couples polymerase chain reaction to mass spectrometry (PCR/ESI-MS) has recently been developed and an assay validated to identify and genotype S. aureus and coagulase-negative staphylococci (CoNS). METHODS: This study was conducted to determine the rates, risk factors, and molecular genotypes of colonizing Staphylococcus aureus in adult patients presenting to an inner-city academic emergency department. Participants completed a structured questionnaire to assess hospital and community risks for infection with methicillin-resistant S. aureus (MRSA). Nasal swabs were analyzed by PCR/ESI-MS to identify and genotype S. aureus and CoNS. RESULTS: Of 200 patients evaluated, 59 were colonized with S. aureus; 27 of these were methicillin-resistant strains. Twenty-four of the 59 S. aureus carriers were co-colonized with a CoNS and 140 of the 200 patients were colonized exclusively with CoNS. The molecular genotypes of the 59 S. aureus strains were diverse; 21 unique molecular genotypes belonging to seven major clonal complexes were identified. Eighty-five of 200 patients carried strains with high-level mupirocin resistance. Of these eighty-five participants, 4 were colonized exclusively with S. aureus, 16 were co-colonized with S. aureus and CoNS, and 65 were colonized exclusively with CoNS. CONCLUSION: The prevalence of S. aureus and methicillin-resistant S. aureus colonization in a random sample of patients seeking care in Emergency Department was 29.5% and 13.5%, respectively. A substantial fraction of the S. aureus-colonized patients were co-colonized with CoNS and high-level mupirocin-resistant CoNS. Determining the molecular genotype of S. aureus during intake screening may prove valuable in the future if certain molecular genotypes become associated with increased infection risk.

Culture negative empyema in a critically ill child: an opportunity for rapid molecular diagnostics.

BACKGROUND: Nucleic acid amplification technologies (NAAT) are advancing our ability to make rapid molecular diagnoses in patients with serious culture negative infections. This is the first report of PCR coupled to electrospray ionization mass spectrometry use in the evaluation of complicated community acquired pneumonia in a pediatric patient. CASE PRESENTATION: We present a case of culture negative empyema in a critically ill, Caucasian, 2-year-old girl who was treated with broad-spectrum empiric antibiotics, in which the length of stay was prolonged by adverse effects of the empiric antibiotic treatment. PCR coupled to electrospray ionization mass spectrometry was applied to culture negative fluid and tissue samples from the patient in order to determine the etiology of the empyema. CONCLUSIONS: Using this method, Streptococcus mitis/viridans was identified as the pathogen. A retrospective review of cases of empyema in children at our institution found that 87.5% of cases were negative for identification of a pathogen and antibiotics were administered to 100% of cases prior to collecting pleural fluid for culture. Understanding the role of Streptococcus mitis/viridans group in the etiology of empyema using an advanced NAAT coupled with mass spectrometry can enlighten clinicians as to the impact of this pathogen in community acquired pneumonia and help assist with antibiotic stewardship.

Rapid detection and differentiation of human noroviruses using RT-PCR coupled to electrospray ionization mass spectrometry.

The goal of this study was to develop an assay for the detection and differentiation of noroviruses using RT-PCR followed by electrospray ionization mass spectrometry (ESI-MS). Detection of hepatitis A virus was also considered. Thirteen primer pairs were designed for use in this assay and a reference database was created using GenBank sequences and reference norovirus samples. The assay was tested for inclusivity and exclusivity using 160 clinical norovirus samples, 3 samples of hepatitis A virus and 3 other closely related viral strains. Results showed that the assay was able to detect norovirus with a sensitivity of 92% and a specificity of 100%. Norovirus identification at the genogroup level was correct for 98% of samples detected by the assay and for 75% of a subset of samples (n = 32) compared at the genotype level. Identification of norovirus genotypes is expected to improve as more reference samples are added to the database. The assay was also capable of detecting and genotyping hepatitis A virus in all 3 samples tested. Overall, the assay developed here allows for detection and differentiation of noroviruses within one working day and may be used as a tool in surveillance efforts or outbreak investigations.

PCR and electrospray ionization mass spectrometry for detection of persistent enterococcus faecalis in cerebrospinal fluid following treatment of postoperative ventriculitis.

We describe the use of PCR and electrospray ionization followed by mass spectrometry (PCR/ESI-MS) to evaluate "culture-negative" cerebrospinal fluid (CSF) from a 67-year-old man who developed postoperative bacterial ventriculitis following a suboccipital craniotomy for resection of an ependymoma in the 4th ventricle. CSF samples were obtained on seven occasions, beginning in the operating room at the time of insertion of a right ventriculoperitoneal shunt (VPS) and continuing until his death, 6 weeks later. During the course of the illness, two initial CSF specimens taken before the initiation of antimicrobial treatment were notable for growth of Enterococcus faecalis. Once antimicrobial treatment was initiated, all CSF cultures were negative. PCR/ESI-MS detected genetic evidence of E. faecalis in all CSF samples, but the level of detection (LOD) decreased once antimicrobial treatment was initiated. When our patient returned with symptoms of meningitis 3 days after the completion of antibiotic treatment, CSF cultures remained negative, but PCR/ESI-MS again found genetic evidence for E. faecalis at levels comparable to the pretreatment levels seen initially. This unique case and these findings suggest that determination of CSF LOD by PCR/ESI-MS may be a very sensitive indicator of persistent infection in patients on antibiotic therapy for complex CNS infections and may have relevance for treatment duration and assessment of persistent or recurrent infection at the completion of therapy.

PCR-electrospray ionization mass spectrometry for direct detection of pathogens and antimicrobial resistance from heart valves in patients with infective endocarditis.

Microbiological diagnosis is pivotal to the appropriate management and treatment of infective endocarditis. We evaluated PCR-electrospray ionization mass spectrometry (PCR/ESI-MS) for bacterial and candidal detection using 83 formalin-fixed paraffin-embedded heart valves from subjects with endocarditis who had positive valve and/or blood cultures, 63 of whom had positive valvular Gram stains. PCR/ESI-MS yielded 55% positivity with concordant microbiology at the genus/species or organism group level (e.g., viridans group streptococci), 11% positivity with discordant microbiology, and 34% with no detection. PCR/ESI-MS detected all antimicrobial resistance encoded by mecA or vanA/B and identified a case of Tropheryma whipplei endocarditis not previously recognized.

Broad-spectrum biosensor capable of detecting and identifying diverse bacterial and Candida species in blood.

We describe an assay which uses broad-spectrum, conserved-site PCR paired with mass spectrometry analysis of amplicons (PCR/electrospray ionization-mass spectrometry [ESI-MS]) to detect and identify diverse bacterial and Candida species in uncultured specimens. The performance of the assay was characterized using whole-blood samples spiked with low titers of 64 bacterial species and 6 Candida species representing the breadth of coverage of the assay. The assay had an average limit of detection of 100 CFU of bacteria or Candida per milliliter of blood, and all species tested yielded limits of detection between 20 and 500 CFU per milliliter. Over 99% of all detections yielded correct identifications, whether they were obtained at concentrations well above the limit of detection or at the lowest detectable concentrations. This study demonstrates the ability of broad-spectrum PCR/ESI-MS assays to detect and identify diverse organisms in complex natural matrices that contain high levels of background DNA.

PCR followed by electrospray ionization mass spectrometry for broad-range identification of fungal pathogens.

Invasive fungal infections are a significant cause of morbidity and mortality among immunocompromised patients. Early and accurate identification of these pathogens is central to direct therapy and to improve overall outcome. PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) was evaluated as a novel means for identification of fungal pathogens. Using a database grounded by 60 ATCC reference strains, a total of 394 clinical fungal isolates (264 molds and 130 yeasts) were analyzed by PCR/ESI-MS; results were compared to phenotypic identification, and discrepant results were sequence confirmed. PCR/ESI-MS identified 81.4% of molds to either the genus or species level, with concordance rates of 89.7% and 87.4%, respectively, to phenotypic identification. Likewise, PCR/ESI-MS was able to identify 98.4% of yeasts to either the genus or species level, agreeing with 100% of phenotypic results at both the genus and species level. PCR/ESI-MS performed best with Aspergillus and Candida isolates, generating species-level identification in 94.4% and 99.2% of isolates, respectively. PCR/ESI-MS is a promising new technology for broad-range detection and identification of medically important fungal pathogens that cause invasive mycoses.

Enhanced diagnostic yields of bacteremia and candidemia in blood specimens by PCR-electrospray ionization mass spectrometry.

A prospective study was performed to determine the value of direct molecular testing of whole blood for detecting the presence of culturable and unculturable bacteria and yeasts in patients with suspected bloodstream infections. A total of 464 adult and pediatric patients with positive blood cultures matched with 442 patients with negative blood cultures collected during the same period were recruited during a 10-month study. PCR amplification coupled with electrospray ionization mass spectrometry (PCR-ESI-MS) plus blood culture reached an overall agreement of 78.6% in the detection and species-level identification of bacterial and candidal pathogens. Of 33 culture-negative/PCR-ESI-MS-positive specimens, 31 (93.9%) were judged to be truly bacteremic and/or candidemic based on a medical chart review and analytical metrics. Among the 15 culture-positive specimens in which PCR-ESI-MS detected additional bacterial or yeast species, 66.7% and 20.0% of the additional positive specimens by PCR-ESI-MS were judged to be truly or possibly bacteremic and/or candidemic, respectively. Direct analysis of blood samples by PCR-ESI-MS rapidly detects bacterial and yeast pathogens in patients with bloodstream infections. When used in conjunction with blood culture, PCR-ESI-MS enhances the diagnostics of septicemia by shortening test turnaround time and improving yields.

Survey of culture, goldengate assay, universal biosensor assay, and 16S rRNA Gene sequencing as alternative methods of bacterial pathogen detection.

Cultivation-based assays combined with PCR or enzyme-linked immunosorbent assay (ELISA)-based methods for finding virulence factors are standard methods for detecting bacterial pathogens in stools; however, with emerging molecular technologies, new methods have become available. The aim of this study was to compare four distinct detection technologies for the identification of pathogens in stools from children under 5 years of age in The Gambia, Mali, Kenya, and Bangladesh. The children were identified, using currently accepted clinical protocols, as either controls or cases with moderate to severe diarrhea. A total of 3,610 stool samples were tested by established clinical culture techniques: 3,179 DNA samples by the Universal Biosensor assay (Ibis Biosciences, Inc.), 1,466 DNA samples by the GoldenGate assay (Illumina), and 1,006 DNA samples by sequencing of 16S rRNA genes. Each method detected different proportions of samples testing positive for each of seven enteric pathogens, enteroaggregative Escherichia coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shigella spp., Campylobacter jejuni, Salmonella enterica, and Aeromonas spp. The comparisons among detection methods included the frequency of positive stool samples and kappa values for making pairwise comparisons. Overall, the standard culture methods detected Shigella spp., EPEC, ETEC, and EAEC in smaller proportions of the samples than either of the methods based on detection of the virulence genes from DNA in whole stools. The GoldenGate method revealed the greatest agreement with the other methods. The agreement among methods was higher in cases than in controls. The new molecular technologies have a high potential for highly sensitive identification of bacterial diarrheal pathogens.