Will Antigen Testing Remain Relevant in the Point-of-Care Testing Environment?

Before the molecular age, cell culture was the gold standard for confirmatory diagnosis of viral and atypical infectious diseases. Typical cell culture methodologies are costly, require days (or weeks) for results, and require significant technical expertise. As a result, cell culture is impractical for timely diagnostic testing in most of the health care environments. Traditional bacterial culture methods, also have disadvantages due to the need for incubation, subsequent identification of pathogens, and significant technical expertise. This article discusses the general considerations of antigen and molecular assays and the merits and factors to consider when implementing diagnostic assays for several common pathogens.

Localized cutaneous infections in immunocompetent individuals due to rapidly growing mycobacteria.

Rapidly growing mycobacteria (RGM) cause skin infections that are refractory to standard antibiotic regimens. Although typically associated with disseminated cutaneous or other systemic infections in immunocompromised patients, RGM sometimes cause localized cutaneous infections in immunocompetent hosts. These infections are almost always associated with precedent skin trauma and inoculation, and therefore have been implicated in outbreaks involving contaminated tattoo ink and inadequately sterilized acupuncture needles. Histologic features often include suppurative granulomatous inflammation, and microorganisms are rarely visualized with stains for acid-fast bacilli. The differential diagnosis includes granulomatous fungal and non-RGM bacterial infections as well as noninfectious suppurative or sarcoidlike conditions. Because no pathognomonic histologic features exist for cutaneous RGM infections, clinical suspicion and appropriate workup are essential to reach an accurate and timely diagnosis. Most localized cutaneous RGM infections in immunocompetent individuals respond well to either clarithromycin or amikacin, in combination with surgical debridement.

Characterization of infections with extended-spectrum ß-lactamase producing Escherichia coli and Klebsiella species at a major military medical center.

This study represents a review of the incidence of extended-spectrum ß-lactamase (ESBL) producing Escherichia coli and Klebsiella species causing infections over a 7-year period and provides a comparison of patient demographics, comorbidities, and ESBL subtypes between community-associated (CA) versus health care-associated (HA) infections. All ESBL-producing bacterial isolates between 2003 and May 2011 at Madigan Army Medical Center were evaluated and reviewed. Polymerase chain reaction (PCR) for ESBL subtypes TEM, SHV, and CTX-M was performed. Demographics and comorbidities associated with infection, ESBL subtype, and antibiotic susceptibility were compared for HA and CA infection. A total of 122 isolates were included in the analysis. From 2005 to 2010, incidence of ESBLs in E. coli increased from 0.13% to 1.0%, and incidence in Klebsiella species rose from 1.0% to 2.55%. CA infections were more likely in females (p < 0.01), age <60 (p < 0.01), urinary source (p < 0.01), and recurrent urinary tract infections (p = 0.02). 42% of CA infections had no associated comorbidity. CTX-M was the predominant subtype in CA infections. Coresistance was high in both HA and CA infection. These data emphasize the need for ongoing monitoring of local microbial epidemiologic trends as changes in prescribing practices may become necessary if resistance continues to spread.

Future-generation sequencing and clinical microbiology.

Sequencing technologies are changing the way both laboratory medicine and clinical practice impact patient care. This article focuses on the clinical microbiology laboratory and the potential benefits and limitations of coming generations of sequencing technology. Nucleic acid sequencing technology is rapidly outpacing the infrastructure needed to accurately educate, analyze, and interpret complex massive data sets that are rapidly becoming integrated into clinical practice.

Serratia infections: from military experiments to current practice.

Serratia species, in particular Serratia marcescens, are significant human pathogens. S. marcescens has a long and interesting taxonomic, medical experimentation, military experimentation, and human clinical infection history. The organisms in this genus, particularly S. marcescens, were long thought to be nonpathogenic. Because S. marcescens was thought to be a nonpathogen and is usually red pigmented, the U.S. military conducted experiments that attempted to ascertain the spread of this organism released over large areas. In the process, members of both the public and the military were exposed to S. marcescens, and this was uncovered by the press in the 1970s, leading to U.S. congressional hearings. S. marcescens was found to be a certain human pathogen by the mid-1960s. S. marcescens and S. liquefaciens have been isolated as causative agents of numerous outbreaks and opportunistic infections, and the association of these organisms with point sources such as medical devices and various solutions given to hospitalized patients is striking. Serratia species appear to be common environmental organisms, and this helps to explain the large number of nosocomial infections due to these bacteria. Since many nosocomial infections are caused by multiply antibiotic-resistant strains of S. marcescens, this increases the danger to hospitalized patients, and hospital personnel should be vigilant in preventing nosocomial outbreaks due to this organism. S. marcescens, and probably other species in the genus, carries several antibiotic resistance determinants and is also capable of acquiring resistance genes. S. marcescens and S. liquefaciens are usually identified well in the clinical laboratory, but the other species are rare enough that laboratory technologists may not recognize them. 16S rRNA gene sequencing may enable better identification of some of the less common Serratia species.

Evaluation of a selection strategy before use of 16S rRNA gene sequencing for the identification of clinically significant gram-negative rods and coccobacilli.

Although 16S ribosomal RNA (rRNA) gene sequencing is well established for correctly identifying bacteria, its most efficient use in a routine clinical laboratory is not clear. We devised and evaluated a strategy to select gram-negative rods and coccobacilli (GNRCB) for which sequencing might be necessary before routine identification methods had been exhausted. The prospectively applied selection criteria were primarily based on the isolate's display of unusual or discordant phenotypic results and/or disease correlation. By using this strategy, we selected a total of 120 GNRCB (representing only ∼2% of all identified). The strategy was demonstrated to be efficient because the preliminary phenotypic identification for 79.2% of those isolates needed revision (18.2% were novel and about a third would have required further extensive testing). The knowledge that 1.6% (ie, 79% of 2%) of isolated GNRCB might benefit from sequence identification could provide guidelines for routine clinical laboratories toward efficient use of sequence analysis.

Achromobacter xylosoxidans infection presenting as a pulmonary nodule mimicking cancer.

Achromobacter xylosoxidans is typically isolated from pulmonary sources, presenting as pneumonia in immunosuppressed individuals. We describe a novel clinical presentation of A. xylosoxidans infection presenting as multiple spiculated, pulmonary nodules mimicking cancer for which the patient underwent a wedge resection of the lung for diagnosis and staging of presumptive cancer.

Severe human parechovirus sepsis beyond the neonatal period.

Here we describe a case of viral sepsis beyond the neonatal period caused by human parechovirus subtype 3 (HPeV-3), which manifested as cardio-respiratory failure, hepatitis, and necrotizing enterocolitis (NEC). HPeV-1 and 2 were originally classified as enteroviruses but the advent of sequence analysis led to them being recognized as a new genus in the picornavirus family. Subsequently, nine additional HPeV strains have been reported including HPeV-3 in 1999.(1) The spectrum of disease that these viruses may cause is still unknown, and they are rarely screened for in clinical practice.

Significantly larger numbers of methicillin-resistant Staphylococcus aureus bacteria are recovered from polymicrobial respiratory and wound sites by use of chromogenic primary media than by use of conventional culture.

Previous studies have validated the properties and documented the utility of chromogenic agar for surveillance of methicillin-resistant Staphylococcus aureus (MRSA). In this study, we used one of the chromagars, MRSASelect (Bio-Rad), as one of the primary isolation media for selected wound and respiratory clinical specimens which, in our institution, were typically polymicrobial. We examined a total of 638 specimens; 142 (22%) MRSA isolates were recovered. Twenty-six of these isolates were recovered only on the MRSASelect plate, representing a 28% (15/54) increase for endotracheal aspirates/sputa and a 15% increase for superficial wounds/ulcers (11/73) compared to the results with conventional culture. One isolate (1 CFU) was recovered by conventional medium alone. MRSASelect has generally been used for surveillance cultures; however, we document that an additional 21% of MRSA isolates would have gone unreported in these selected clinical specimens using only standard culture media. For 40% (6/15) of inpatients, MRSA isolated from the MRSASelect plate was the sole indicator of MRSA. Although these isolates can represent either colonization or infection, they are a potential reservoir of infection and nosocomial transmission. Our data support the focused use of chromogenic selective media for the increased detection of MRSA in polymicrobial wound and respiratory specimens, which could have an impact on both clinical treatment and infection control.

Site and clinical significance of Alloscardovia omnicolens and Bifidobacterium species isolated in the clinical laboratory.

Most of the members of the genus Bifidobacterium, including the related organism Alloscardovia omnicolens, are inhabitants of the gastrointestinal tract and oral cavity of humans and animals and have been considered nonpathogenic for humans. However, the actual site of isolation and the clinical significance of A. omnicolens and of Bifidobacterium species are unclear. This may be due in part to the difficulties in distinguishing these organisms from other genera such as Actinomyces. To determine the potential disease-causing role of these organisms, we analyzed the clinical significance of 15 A. omnicolens and Bifidobacterium isolates identified by 16S rRNA gene sequencing from a clinical laboratory. All of the organisms in this study were isolated from sterile sites or in significant numbers by standard clinical microbiological culture methods. Our 15 clinical strains fit into only four species: A. omnicolens (five isolates), Bifidobacterium scardovii (four isolates), B. longum (two isolates), and B. breve (four isolates). All five A. omnicolens isolates, one of the B. breve isolates, and three of the four B. scardovii isolates were cultured from urine at 10(5) CFU/ml. One B. scardovii isolate was from a patient with a genitourinary tract wound infection, two B. longum isolates were from abdominal wounds, and three B. breve isolates were from blood cultures. This study enlarges the spectrum of diseases and clinical sources associated with A. omnicolens and Bifidobacterium species and addresses identification problems.

Thumb infection caused by Streptococcus pseudoporcinus.

Streptococcus pseudoporcinus, a recently described organism found in the genitourinary tract of women, was isolated from a thumb wound in a male patient subsequent to trauma. This case describes a rarely reported non-genitourinary tract clinical isolate of S. pseudoporcinus.

Oral abscess caused by Campylobacter rectus: case report and literature review.

Campylobacter rectus was isolated under routine anaerobic conditions (no additional hydrogen gas in the atmosphere) from an oral, nonperiodontal abscess from a patient with gastroesophageal adenocarcinoma. We report the first case of a palate abscess caused by C. rectus and review the literature and atmospheric requirements of this organism.

Analyses of ampC gene expression in Serratia marcescens reveal new regulatory properties.

Serratia marcescens encodes an inducible, chromosomal beta-lactamase, ampC. Studies addressing the regulation of inducible ampC genes have focused primarily on Enterobacter cloacae and Citrobacter freundii. The purpose of this study was to clone and sequence the ampC, ampR and intergenic region of S. marcescens and examine both inducible and basal level ampC expression. Sequence analysis of the S. marcescens ampC gene identified an extended 5' untranslated region (UTR) of 126 nucleotides, which formed a prominent stem-loop structure. Induction of ampC expression required AmpR, and the start of transcription was determined using primer extension analysis. In vivo half-life analysis revealed that the half-life of the S. marcescens ampC transcript was 7 min. Confirmation of the in vivo half-life and the role of the stem-loop structure in the 5' UTR was demonstrated by comparing transcript half-life and luciferase expression between a wild-type (WT) and a 5' UTR stem-loop deletion mutant. These data demonstrated that the stem-loop structure was involved in transcript stability. Taken together, these findings indicate that constitutive expression of S. marcescens ampC is regulated by both transcriptional initiation and post-transcriptional events.