Temporal and spatial discordance of programmed cell death-ligand 1 expression and lymphocyte tumor infiltration between paired primary lesions and brain metastases in lung cancer.

BACKGROUND: The dynamics of PD-L1 expression may limit its use as a tissue-based predictive biomarker. We sought to expand our understanding of the dynamics of PD-L1 expression and tumor-infiltrating lymphocytes (TILs) in patients with lung cancer-related brain metastases. EXPERIMENTAL DESIGN: Paired primary lung cancers and brain metastases were identified and assessed for PD-L1 and CD3 expression by immunohistochemistry. Lesions with 5% or greater PD-L1 expression were considered positive. Agreement statistics and the χ(2) or Fisher's exact test were used for analysis. RESULTS: We analyzed 146 paired lesions from 73 cases. There was disagreement of tumor cell PD-L1 expression in 10 cases (14%, κ = 0.71), and disagreement of TIL PD-L1 expression in 19 cases (26%, κ = 0.38). Most paired lesions with discordant tumor cell expression of PD-L1 were obtained 6 or more months apart. When specimens were categorized using a proposed tumor microenvironment categorization scheme based on PD-L1 expression and TILs, there were significant changes in the classifications because many of the brain metastases lacked either PD-L1 expression, tumor lymphocyte infiltration or both even when they were present in the primary lung cancer specimens (P = 0.009). CONCLUSIONS: We identified that there are significant differences between the tumor microenvironment of paired primary lung cancers and brain metastases. When physicians decide to treat patients with lung cancer with a PD-1 or PD-L1 inhibitor, they must do so in the context of the spatial and temporal heterogeneity of the tumor microenvironment.

Multicenter Evaluation of BD Max Enteric Parasite Real-Time PCR Assay for Detection of Giardia duodenalis, Cryptosporidium hominis, Cryptosporidium parvum, and Entamoeba histolytica.

Common causes of chronic diarrhea among travelers worldwide include protozoan parasites. The majority of parasitic infections are caused by Giardia duodenalis, Entamoeba histolytica, Cryptosporidium parvum, and Cryptosporidium hominis Similarly, these species cause the majority of parasitic diarrhea acquired in the United States. Detection of parasites by gold standard microscopic methods is time-consuming and requires considerable expertise; enzyme immunoassays and direct fluorescent-antibody (DFA) stains have lowered hands-on time for testing, but improvements in sensitivity and technical time may be possible with a PCR assay. We performed a clinical evaluation of a multiplex PCR panel, the enteric parasite panel (EPP), for the detection of these common parasites using the BD Max instrument, which performs automated extraction and amplification. A total of 2,495 compliant specimens were enrolled, including 2,104 (84%) specimens collected prospectively and 391 (16%) specimens collected retrospectively. Approximately equal numbers were received in 10% formalin (1,273 specimens) and unpreserved (1,222 specimens). The results from the EPP were compared to those from alternate PCR and bidirectional sequencing (APCR), as well as DFA (G. duodenalis and C. parvum or C. hominis) or trichrome stain (E. histolytica). The sensitivity and specificity for prospective and retrospective specimens combined were 98.2% and 99.5% for G. duodenalis, 95.5% and 99.6 for C. parvum or C. hominis, and 100% and 100% for E. histolytica, respectively. The performance of the FDA-approved BD Max EPP compared well to the reference methods and may be an appropriate substitute for microscopic examination or immunoassays.

Multicenter evaluation of the BD max enteric bacterial panel PCR assay for rapid detection of Salmonella spp., Shigella spp., Campylobacter spp. (C. jejuni and C. coli), and Shiga toxin 1 and 2 genes.

Diarrhea due to enteric bacterial pathogens causes significant morbidity and mortality in the United States and worldwide. However, bacterial pathogens may be infrequently identified. Currently, culture and enzyme immunoassays (EIAs) are the primary methods used by clinical laboratories to detect enteric bacterial pathogens. We conducted a multicenter evaluation of the BD Max enteric bacterial panel (EBP) PCR assay in comparison to culture for the detection of Salmonella spp., Shigella spp., Campylobacter jejuni, and Campylobacter coli and an EIA for Shiga toxins 1 and 2. A total of 4,242 preserved or unpreserved stool specimens, including 3,457 specimens collected prospectively and 785 frozen, retrospective samples, were evaluated. Compared to culture or EIA, the positive percent agreement (PPA) and negative percent agreement (NPA) values for the BD Max EBP assay for all specimens combined were as follows: 97.1% and 99.2% for Salmonella spp., 99.1% and 99.7% for Shigella spp., 97.2% and 98.4% for C. jejuni and C. coli, and 97.4% and 99.3% for Shiga toxins, respectively. Discrepant results for prospective samples were resolved with alternate PCR assays and bidirectional sequencing of amplicons. Following discrepant analysis, PPA and NPA values were as follows: 97.3% and 99.8% for Salmonella spp., 99.2% and 100% for Shigella spp., 97.5% and 99.0% for C. jejuni and C. coli, and 100% and 99.7% for Shiga toxins, respectively. No differences in detection were observed for samples preserved in Cary-Blair medium and unpreserved samples. In this large, multicenter study, the BD Max EBP assay showed superior sensitivity compared to conventional methods and excellent specificity for the detection of enteric bacterial pathogens in stool specimens.

Novel fastidious, partially acid-fast, anaerobic Gram-positive bacillus associated with abscess formation and recovered from multiple medical centers.

We report a novel anaerobe causing abscess in four patients at three hospitals. In the clinical specimen, bacilli were branching, Gram positive, and acid fast. The organism grew slowly and was not identified by 16S rRNA sequencing. Our findings support the description of a new genus and species of the suborder Corynebacterineae.

Effect of preoperative shampoos with chlorhexidine or iodophor on emergence of resident scalp flora in neurosurgery.

Wound contamination with endogenous bacterial scalp flora plays an important role in the pathogenesis of postoperative neurosurgical infections. To assess the effect of preoperative antiseptic shampoos on the emergence of resident scalp flora during surgery and subsequent wound contamination, we randomized 151 neurosurgical procedures into four study groups: group A-preoperative shampoos with chlorhexidine, surgical scalp preparation with chlorhexidine; group B-no shampoos, surgical preparation with chlorhexidine; group C-shampoos with iodophor, surgical preparation with iodophor; group D-no shampoos, surgical preparation with iodophor. Quantitative cultures of the scalp were obtained preoperatively and at the end of surgery, and qualitative wound cultures were taken prior to wound closure. Group A had the lowest concentration of bacteria on the scalp both preoperatively and postoperatively (median range = 30 [0-5.7 x 10(5)] and 0 [0-2.5 x 10(3)] respectively). Group A also had significantly fewer positive postoperative scalp cultures (29%) than groups B (51%), C (58%), and D (53%) (P<0.05), as well as fewer positive wound cultures (20% v 25%, 42%, and 30% respectively). A density of bacteria on the scalp of < 10(2)/4 cm(2) best predicted the presence of bacteria in the wound. Repeated preoperative shampoos with chlorhexidine reduce intraoperative emergence of resident skin flora and subsequent contamination of the wound.

Evaluation of Pyrosequencing technology for the identification of clinically relevant non-dematiaceous yeasts and related species.

Pyrosequencing was used to identify 133 isolates of clinically relevant non-dematiaceous yeasts. These included 97 ATCC strains (42 type strains), seven UAMH strains, and 29 clinical isolates. Isolates belonged to the following genera: Candida (18 species), Trichosporon (10), Cryptococcus (7), Malassezia (3), Rhodotorula (2), Geotrichum (1), Blastoschizomyces (1), and Kodamaea (1). Amplicons of a hyper-variable ITS region were obtained and analyzed using Pyrosequencing technology. The data were evaluated by a BLAST search against the GenBank database and correlated with data obtained by conventional cycle sequencing of the ITS1-5.8S-ITS2 region. Cycle sequencing identified 78.9% of the isolates to the species level. Pyrosequencing technology identified 69.1%. In 90.1% of all of the strains tested, the identification results of both sequencing methods were identical. Most Candida isolates can be identified to the species level by Pyrosequencing. Trichosporon species and some Cryptococcus species cannot be differentiated at the species level. Pyrosequencing can be used for the reliable identification of most commonly isolated non-dematiaceous yeasts, with a reduction of cost per identification compared to conventional sequencing.

Genotypic analysis of invasive Streptococcus pneumoniae from Mali, Africa, by semiautomated repetitive-element PCR and pulsed-field gel electrophoresis.

As part of a large, ongoing study of invasive infections in pediatric patients in Bamako, Mali, 106 cases of invasive pneumococcal disease were identified from June 2002 to July 2003 (J. D. Campbell et al., Pediatr. Infect. Dis. J. 23:642-649, 2004). Of the 12 serotypes present, the majority of isolates were not contained in PCV7 (the 7-valent pneumococcal conjugate vaccine), including 1 isolate that was serotype 1, 12 isolates that were serotype 2, 58 isolates that were serotype 5, 7 isolates that were serotype 7F, and 1 isolate that was serotype 12F. To determine whether clonal dissemination of the predominant serotypes had taken place, genotyping was performed on 100 S. pneumoniae isolates by using two methods: pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA, and the Bacterial Barcodes repetitive-element PCR (rep-PCR) method. Criteria for delineating rep-PCR genotypes were established such that isolates of different serotypes were generally not grouped together. The two methods were equally discriminatory within a given pneumococcal serotype. PFGE separated the isolates into 15 genotypes and 7 subtypes; rep-PCR separated isolates into 15 genotypes and 6 subtypes. Using either method, isolates within serotypes 2, 5, and 7 formed three large, separate clusters containing 1 genotype each. Both methods further distinguished related subtypes within serotypes 2 and 5. Interestingly, one of the PFGE subtypes of serotype 5 is indistinguishable from the Columbia(5)-19 clone circulating in Latin America since 1994. The data support that serotypes 2 and 5 were likely to be the result of dissemination of particular clones, some of which are responsible for invasive disease over a broad population range.

Methicillin-resistant Staphylococcus caprae in a neonatal intensive care unit.

Staphylococcus caprae, a hemolytic coagulase-negative staphylococcus that is infrequently associated with humans, was initially detected in specimens from six infants in our neonatal intensive care unit due to phenotypic characteristics common to methicillin-resistant Staphylococcus aureus. These isolates were subsequently identified as S. caprae by the Automated RiboPrinter microbial characterization system. This prompted an 8-month retrospective investigation in our neonatal intensive care unit. S. caprae was the cause of 6 of 18 episodes of coagulase-negative staphylococcal bacteremia, was the most common coagulase-negative staphylococcus recovered from the nares of 6 of 32 infants surveyed in a methicillin-resistant S. aureus surveillance program, and was isolated from 1 of 37 health care providers' hands. Of 13 neonatal intensive care unit isolates tested, all were methicillin resistant and positive for the mecA gene. All 21 isolates were found to be a single strain by Automated RiboPrinter and pulsed-field gel electrophoresis with ApaI or SmaI digestion; ApaI was more discriminating in analyzing epidemiologically unrelated strains than Automated RiboPrinter or electrophoresis with SmaI. These findings extend the importance of S. caprae, emphasize its similarities to methicillin-resistant S. aureus, and demonstrate its ability to persist in an intensive care unit setting.

Gas-liquid chromatographic determination of morphine, heroin, and cocaine.

Morphine, heroin, and cocaine are quantitatively determined with the same gas-liquid chromatographic system. The compounds are separated on a 6 ft X 2 mm id glass column packed with a 1:1 mixture of 5% SE-30 on 80--100 mesh Chromosorb W and 3% OV-17 on 80--100 mesh Varaport 30. The column is temperature-programmed. Flame ionization detector responses are measured with a computer-based data system. Heroin and cocaine are chromatographed directly; morphine is derivatized first. The procedure was evaluated with previously analyzed commercial and forensic samples. Accuracy and precision were 5 and 3%, respectively.

Vancomycin resistance, esp, and strain relatedness: a 1-year study of enterococcal bacteremia.

The prevalence of esp, a gene associated with infection-derived and outbreak strains, in enterococcal blood isolates from 2002 was determined. Fifty-five of 137 (40.1%) Enterococcus faecalis isolates, 30 of 58 (51.7%) E. faecium isolates, 1 of 1 E. raffinosus isolate, 0 of 4 E. gallinarum isolates, and 0 of 1 E. casseliflavus isolate were positive. esp wasn't associated with vancomycin resistance (VR) or clinical service. VR E. faecium isolates were less genetically diverse than vancomycin-susceptible strains. A large cluster of VR isolates, belonging to esp-positive E. faecium, was revealed. These data support the hypothesis that esp and VR may contribute to dissemination of particular clones.

Recurrent, disseminated Mycobacterium marinum infection caused by the same genotypically defined strain in an immunocompromised patient.

An 81-year-old male with myasthenia gravis developed a cutaneous infection with Mycobacterium marinum, which apparently resolved following local heat therapy. Five months later, the patient developed new skin lesions and pancytopenia. M. marinum was isolated from his bone marrow. Pulsed-field gel electrophoresis was performed to determine if the skin and bone marrow isolates were clonally related. Digestion of the genomic DNA with the restriction enzymes SpeI and AseI yielded indistinguishable banding patterns. An epidemiologically unrelated control strain showed significant banding differences. The results suggest that the patient's recurrent, disseminated infection was due to recrudescence of his initial infection rather than reinfection by another strain.

Effect of preoperative shampoos with chlorhexidine or iodophor on emergence of resident scalp flora in neurosurgery.

Wound contamination with endogenous bacterial scalp flora plays an important role in the pathogenesis of postoperative neurosurgical infections. To assess the effect of preoperative antiseptic shampoos on the emergence of resident scalp flora during surgery and subsequent wound contamination, we randomized 151 neurosurgical procedures into four study groups: group A--preoperative shampoos with chlorhexidine, surgical scalp preparation with chlorhexidine; group B--no shampoos, surgical preparation with chlorhexidine; group C--shampoos with iodophor, surgical preparation with iodophor; group D--no shampoos, surgical preparation with iodophor. Quantitative cultures of the scalp were obtained preoperatively and at the end of surgery, and qualitative wound cultures were taken prior to wound closure. Group A had the lowest concentration of bacteria on the scalp both preoperatively and postoperatively (median range = 30 [0-5.7 x 10(5)] and 0 [0-2.5 x 10(3)] respectively). Group A also had significantly fewer positive postoperative scalp cultures (29%) than groups B (51%), C (58%), and D (53%) (P less than 0.05), as well as fewer positive wound cultures (20% v 25%, 42%, and 30% respectively). A density of bacteria on the scalp of greater than 10(2)/4cm2 best predicted the presence of bacteria in the wound. Repeated preoperative shampoos with chlorhexidine reduce intraoperative emergence of resident skin flora and subsequent contamination of the wound.

An outbreak of vancomycin-dependent Enterococcus faecium in a bone marrow transplant unit.

Outbreaks of vancomycin-resistant enterococci (VRE) are well described. The presence of mutants of VRE, such as vancomycin-dependent enterococci (VDE), in individual patients has been documented, but their potential to spread nosocomially has not been known. We present the first cluster of patients who acquired VDE nosocomially. Five bone marrow transplantation patients were infected or colonized by a genotypically indistinguishable multiantibiotic-resistant strain of Enterococcus faecium. Vancomycin dependence in 3 of the 5 isolates was demonstrated. All cluster patients had received protracted prophylactic treatment with vancomycin (mean, 22.6 days), and specimens from >/=2 body sites were repeatedly culture-positive for the outbreak strain. The outbreak was controlled with aggressive infection control strategies, and prophylactic antibiotic policies were revised. Awareness of the potential for nosocomial spread of multiantibiotic-resistant VDE is vital for the care of immunocompromised patients, especially those receiving prophylactic antibiotics.

Surveillance strategies and impact of vancomycin-resistant enterococcal colonization and infection in critically ill patients.

OBJECTIVE: To determine the optimal site and frequency for vancomycin-resistant enterococci (VRE) surveillance to minimize the number of days of VRE colonization before identification and subsequent isolation. SUMMARY BACKGROUND DATA: The increasing prevalence of VRE and the limited therapeutic options for its treatment demand early identification of colonization to prevent transmission. METHODS: The authors conducted a 3-month prospective observational study in medical and surgical intensive care unit (ICU) patients with a stay of 3 days or more. Oropharyngeal and rectal swabs, tracheal and gastric aspirates, and urine specimens were cultured for VRE on admission to the ICU and twice weekly until discharge. RESULTS: Of 117 evaluable patients, 23 (20%) were colonized by VRE. Twelve patients (10%) had VRE infection. Of nine patients who developed infections after ICU admission, eight were colonized before infection. The rectum was the first site of colonization in 92% of patients, and positive rectal cultures preceded 89% of infections acquired in the ICU. This was supported by strain delineations using pulsed-field gel electrophoresis. Twice-weekly rectal surveillance alone identified 93% of the maximal estimated VRE-related patient-days; weekly or admission-only surveillance was less effective. As a test for future VRE infection, rectal surveillance culture twice weekly had a negative predictive value of 99%, a positive predictive value of 44%, and a relative risk for infection of 34. CONCLUSIONS: Twice-weekly rectal VRE surveillance of critically ill patients is an effective strategy for early identification of colonized patients at increased risk for VRE transmission, infection, and death.