Leona W. Ayers

Antibacterial activities of ciprofloxacin, norfloxacin, oxolinic acid, cinoxacin, and nalidixic acid.

In vitro studies were performed comparing ciprofloxacin (Bay o 9867) and norfloxacin with three related organic acids. Ciprofloxacin was two to eight times more active than norfloxacin against 658 bacterial isolates representing 30 species. For all species tested, ciprofloxacin MICs for 90% inhibition were less than or equal to 2.0 micrograms ml. Additional tests with 5,994 isolates detected only 37 (0.6%) strains resistant to 2.0 micrograms of ciprofloxacin per ml and 106 (1.8%) resistant to 1.0 micrograms/ml. Only 6 (0.1%) of the 5,994 strains were resistant to 16 micrograms of norfloxacin per ml, and 129 (2.1%) were resistant to 4.0 micrograms/ml. The majority of resistant strains were streptococci or Pseudomonas spp. Resistance among the Enterobacteriaceae was extremely rare (i.e., greater than 99.8% susceptible to both drugs.

Merkel cell carcinoma subgroups by merkel cell polyomavirus DNA relative abundance and oncogene expression

Merkel cell polyomavirus (MCPyV) was recently discovered in Merkel cell carcinoma (MCC), a clinically and pathologically heterogeneous malignancy of dermal neuroendocrine cells. To investigate this heterogeneity, we developed a tissue microarray (TMA) to characterize immunohistochemical staining of candidate tumor cell proteins and a quantitative PCR assay to detect MCPyV and measure viral loads. MCPyV was detected in 19 of 23 (74%) primary MCC tumors, but 8 of these had less than 1 viral copy per 300 cells. Viral abundance of 0.06–1.2 viral copies/cell was directly related to presence of retinoblastoma gene product (pRb) and terminal deoxyribonucleotidyl transferase (TdT) by immunohistochemical staining (p ≤ 0.003). Higher viral abundance tumors tended to be associated with less p53 expression, younger age at diagnosis and longer survival (p ≤ 0.08). These data suggest that MCC may arise through different oncogenic pathways, including ones independent of pRb and MCPyV.

Bloodstream infections in a neonatal intensive-care unit: 12 years' experience with an antibiotic control program.

OBJECTIVE To assess the prevalence of gram-positive coccal (GPC), gram-negative bacillary (GNB), and fungal blood-stream infections (BSIs) during a 12-year period in which a consistent antibiotic treatment protocol was in place; to evaluate the efficacy of these antibiotic policies in relation to treatment, to the emergence of bacterial or fungal resistance, and to the occurrence of infection outbreaks or epidemics. STUDY DESIGN Case series. METHODS Demographic, clinical, and bacteriological information from 363 infants born during 1986 through 1991 and 1992 through 1997 who developed 433 blood-culture-proven BSIs was analyzed. Early-onset BSIs were defined as those infections discovered within 48 hours of birth, and late-onset BSIs as those that occurred thereafter. Suspected early-onset BSIs were treated with ampicillin and gentamicin, and suspected late-onset BSIs with vancomycin and gentamicin. Antibiotics were changed on the basis of organism antimicrobial susceptibility. RESULTS Early-onset BSIs were noted in 52 of 21,336 live births and 40 of 20,402 live births during 1986 through 1991 and 1992 through 1997, respectively. GPC (83% due to group B streptococcus [GBS]) accounted for approximately one half of early-onset BSI cases and GNB (68% Enterobacteriaceae) for the remainder. Early-onset GBS declined from 24 to 11 cases (P=.04) and late-onset BSI increased from 111 to 230 cases (P<.01) from the first to the last study period. Sixty-eight percent of late-onset BSIs were due to GPC (primarily coagulase-negative Staphylococcus), 18% to GNB, and 14% to fungus. Over the study period, Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and Pseudomonas aeruginosa isolated from the newborn intensivecare unit (unlike those strains from other hospital units) remained fully susceptible to ceftazidime and gentamicin. Although the hospitalwide prevalence of methicillin-resistant Staphylococcus aureus increased, all 17 newborn BSI cases were due to methicillin-sensitive strains. Prevalence of methicillin-resistant coagulase-negative Staphylococcus increased, although all strains remained vancomycin-susceptible, as did the 16 Enterococcus faecalis isolates. All fungi recovered (from 48 patients) were susceptible to amphotericin. CONCLUSION We observed a decrease in the prevalence of early-onset BSIs due to GBS and an increase in late-onset BSIs due to GPC, GNB, and fungi. The combination of ampicillin and gentamicin for suspected early-onset BSIs and vancomycin and gentamicin for late-onset BSIs has been successful for treatment of individual patients without the occurrence of infection outbreaks or the emergence of resistance. Controlled antibiotic programs and periodic evaluations based on individual unit and not on hospitalwide antibiograms are advisable.

Immunological detection of viral large T antigen identifies a subset of Merkel cell carcinoma tumors with higher viral abundance and better clinical outcome.

To the editor, MCPyV, a new human oncogenic polyoma virus, appears etiologic for the classic, rare, aggressive Merkel Cell Carcinomas (MCC) of the skin characterized by MCPyV DNA integration, high virus copy numbers,1 expression of the potentially transforming MCPyV large T antigen (LTA) evidenced by reactivity to monoclonal antibody CM2B4,2–4 signature mutations in LTA,5 expression of retinoblastoma protein3,6 and a vigorous MCPyV specific neutralizing antibody response.7,8 Prevalence of this MCC phenotype has been reported as upward of 77–84% of MCC tested.1,3,9,10 Thus 16–23% of MCC may have a different etiology. MCPyV detection in MCC tissue includes MCCs with high and low virus copy numbers, detection ranges from 24% to 100% (Table 1a).1–4,6,9–19 Differences in relative proportions of MCC with low and high viral copies may also be significantly influenced by geographic region (Table 1b).13 All MCC tumors in our study population6 with viral abundance of 0.06–1.2 DNA copies/cell conform to the reported MCC MCPyV positive phenotype with demonstrable MCPyV LTA (IHC CM2B4, kindly provided by Dr. Moore) and retinoblastoma protein. (Figure 1A) Our tumor study population additionally contained variant MCC with low viral DNA (.0005–.0035 copies/cell) or no detectable virus; these later two subgroups were negative for LTA (CM2B4) and retinoblastoma protein. These data are similar to the more recent data from Busam et al.4 who found 5/15 MCPyV positive MCC non-reactive to CM2B4 and are consistent with the recent comprehensive characterization of MCC cell lines.20 Figure 1 Figure 1A: Immunohistochemical staining of MCC was performed for the detection of expression of retinoblastoma protein (pRb) using the commercially available antibody from Novocastra (clone 13A10) and for the detection of the LTA encoded by MCPyV using ... Table 1 Classic MCC viral oncogenesis characterized by abundance of MCPyV is clinically relevant as suggested by Sihto et al,16 who demonstrated that viral abundance in MCC correlates with survival. Our result6 supports this conclusion. When our patient MCC population is segregated by high vs. low or no viral copies/cell, survival is best for those with high viral copies/cell and worse for MCC likely initiated through other modes of oncogenesis (Figure 1B). We differ from the report by Becker et al.3,10 where high viral copy numbers were present in most of their samples. Lack of CM2B4 reactivity in a tumor with high viral copy number has been described.2 Some CM2B4 negative MCC might be ascribed to variations in large T antigenic epitopes. However, high correlation among viral load, retinoblastoma protein and CM2B4 reactivity in our data suggests that viral low-abundance or negative MCC tumor cells may have no or insufficient LTA for detection with CM2B4. Several recent reports have demonstrated presence of MCPyV in normal skin and other tumors.6,11,14,21 Our results do not allow us to discriminate between the presence of MCPyV in MCC tumor cells versus the presence of MCPyV in surrounding non-tumor cells; this issue is germane to the relevance of MCPyV mediated tumorigenesis particularly in MCC with low viral abundance. One approach to address this would be to examine whether MCPyV genome is integrated in the host genome, a process that is likely to indicate a direct role for MCPyV mediated oncogenesis. The lack of fresh/frozen tumor samples however, makes such a study technically unfeasible. Integration of MCPyV genome is also often associated with the presence of truncating mutations in the LTA and while it is unknown whether mutations occur prior to integration, the presence of such mutations may provide an alternative surrogate for supporting a pathogenic role of MCPyV. However recent data21 suggest that identical truncating mutations are detected in both tumor and adjacent normal skin tissue, thus the pathogenic relevance of detecting truncating mutations warrant additional investigation. While technically feasible, the sensitivities for detecting mutations particularly from low abundance viral DNA pose several challenges and will still fail to directly address the issue whether the viral DNA is associated with the tumor cells or the non tumor cells in the sample. Nonetheless the rarity of cells in MCC with low amplification of virus would be inconsistent with the presence of the viral DNA prior to clonal expansion in these tumors. To appreciate the differences in oncogenesis and MCC survival, the MCC study population must be heterogeneous and contain tumors with high, low and no viral detection. While artifact related to specimen quality and differences in detection methods could add to this heterogeneity, tumor factors such as mutations, ultraviolet (UV) DNA damage and patient clinical parameters such as immunosuppression must contribute. Compared to specimens from North America, MCC tissues from Australia might be expected to be virus negative because high UV radiation exposure of melanin-deficient skin probably contributes to MCC development.13 Our data (Figure 1A) imply that MCPyV positive and negative tumors differ in oncoprotein expression and prognosis. This may be important in establishing clinical correlates that will be useful worldwide. Clarifying the role of MCPyV in MCC will require accurate quantification of viral abundance. Houben et al.3 contend that most MCCs carry at least one viral copy per cell and do not find MCC with low viral abundance. In contrast, we and others6,13,16 have found very low viral loads (.0001–.0035 copies/cell) in 19 of 174 MCC tumors tested (Table 1). Both we and Garneski et al.6,13 observe what appear to be discrete categories of high- and low-abundance MCC tumors. Estimation of viral abundance is susceptible to differences in tissue quality, proportion of tumor and non tumor cells in the sample, primers selected for PCR amplification, target cellular genomic sequences used as a reference, and other technical differences, but it appears that at least a proportion of MCCs carry viral DNA that equates to less than 1 in 300 cells infected with MCPyV. Notably, similar low abundance of viral association has been described in MCC cell lines, suggesting that mere viral association may not provide growth advantage, at least in vitro, over non viral infected MCC.20 Finally, there is the question of how expression of oncoproteins in MCC differs among the classic MCPyV DNA and variant phenotypes. We find heterogeneity in MCC as to abundance or absence of detectable MCPyV and the presence of oncoproteins as well as geographic, clinical, or patient factors. All may be critically important for prognosis and therapy. To this end, the development of a multi-investigator, multi-institutional consensus study using an international, geographically diverse MCC tumor population with a pre-determined common set of reagents and methods to estimate MCPyV abundance and oncoproteins in primary, recurrent and metastatic lesions, geographic distributions of MCC types and correlations with clinical features of MCC would be an important step.

Neonatal airway colonization with Gram-negative bacilli : association with severity of bronchopulmonary dysplasia

BACKGROUND Airway colonization with Gram-negative bacilli (GNB) and Gram-positive cocci (GPC) is common in mechanically ventilated neonates. Whether GNB are related to nosocomial bloodstream infection (BSI) and/or to the severity of bronchopulmonary dysplasia (BPD) is unknown. METHODS We prospectively examine this relationship using a cohort design. Data from 260 < or = 1250-g birth weight inborn infants (1991 to 1995) intubated > or = 2 weeks included 917 serial tracheal cultures and 583 blood cultures. The severity of BPD was assessed by duration of mechanical ventilation, oxygen dependency at 36 weeks of postconceptional age and the use of home oxygen supplementation. RESULTS After 2 weeks of ventilation, 80% of the infants were colonized with GPC (Staphylococus epidermidis and Staphylococcus haemolyticus in 90% of the cases). Superimposed on 36% of these infants was GNB airway colonization with Klebsiella pneumoniae (25%), Enterobacter cloacae (25%), Escherichia coli (25%), Pseudomonas aeruginosa (10%), Serratia marcescen (10%), Acinetobacter baumannii and Haemophilus influenzae (5%). Comparison between 174 GPC- and 86 GNB-colonized infants showed that demographics, birth weight, gestational age, perinatal risk factors and mortality were similar. Fifteen percent of GNB-colonized infants developed BSI caused by GNB and 14% developed BSI caused by GPC. No significant temporal relationship between airway colonization and BSI was noted. GNB infants were ventilated longer and required oxygen at 36 weeks of postconceptional age and home oxygen supplementation twice as often as infants colonized only with GPC. GNB colonization was a predictor of severe BPD after controlling for ventilation. Ureaplasma colonization occurred in 28% of GNB-colonized and 33% of noncolonized infants and was not a predictor of BPD severity. CONCLUSION GNB airway colonization creates a moderate risk for BSI. Antibiotic treatment does not regularly eradicate GNB. GNB airway colonization is associated with severe BPD, but further studies will be necessary before therapeutic efforts to eradicate GNB from the airways should be undertaken.

MicroRNA-31 predicts the presence of lymph node metastases and survival in patients with lung adenocarcinoma

Purpose: We conducted genome-wide miRNA-sequencing (miRNA-seq) in primary cancer tissue from patients of lung adenocarcinoma to identify markers for the presence of lymph node metastasis. Experimental Design: Markers for lymph node metastasis identified by sequencing were validated in a separate cohort using quantitative PCR. After additional validation in the The Cancer Genome Atlas (TCGA) dataset, functional characterization studies were conducted in vitro. Results: MiR-31 was upregulated in lung adenocarcinoma tissues from patients with lymph node metastases compared with those without lymph node metastases. We confirmed miR-31 to be upregulated in lymph node-positive patients in a separate patient cohort (P = 0.009, t test), and to be expressed at higher levels in adenocarcinoma tissue than in matched normal adjacent lung tissues (P < 0.0001, paired t test). MiR-31 was then validated as a marker for lymph node metastasis in an external validation cohort of 233 lung adenocarcinoma cases of the TCGA (P = 0.031, t test). In vitro functional assays showed that miR-31 increases cell migration, invasion, and proliferation in an ERK1/2 signaling-dependent manner. Notably, miR-31 was a significant predictor of survival in a multivariate cox regression model even when controlling for cancer staging. Exploratory in silico analysis showed that low expression of miR-31 is associated with excellent survival for T2N0 patients. Conclusions: We applied miRNA-seq to study microRNomes in lung adenocarcinoma tissue samples for the first time and potentially identified a miRNA predicting the presence of lymph node metastasis and survival outcomes in patients of lung adenocarcinoma. Clin Cancer Res; 19(19); 5423–33. ©2013 AACR.

Comparison of a closed (Trach Care MAC) with an open endotracheal suction system in small premature infants.

OBJECTIVE:To determine whether ventilated, low birth weight infants treated with closed versus open tracheal suction in a neonatal intensive care unit (NICU) differ as to airway bacterial colonization, nosocomial pneumonia, bloodstream infection (BSI), incidence and severity of bronchopulmonary dysplasia (BPD), neonatal mortality, frequency of suction, reintubation, and nurse preference.STUDY DESIGN:A total of 175 low birth weight infants (≤1250 gm) consecutively born (1997 to 1999), intubated, and ventilated in the delivery room were randomized on admission to the NICU to a closed (Trach Care MAC) or open suction group. Closed multi-use catheters were changed daily; open catheters were changed after every use. Two-pass endotracheal suctioning (both groups) was performed every 8 hours or as needed. Side-port connectors were not used; thus open suction required disconnection from ventilators. Tracheal aspirate cultures were obtained on admission and weekly thereafter. Nosocomial BSI (occurring after 48 hours of life) was documented by positive blood cultures. Radiographs taken before, during, and after tracheal aspirate cultures or BSIs were graded using a semiquantitative system for pneumonia and a modified score for BPD. Nurse preference regarding suction method was recorded.RESULTS:Of the original 175 patients, 10 (5 from each group) died and 32 others (16 from each group) were extubated at or before 7 days of life. The study population comprised 67 patients in the closed group and 66 in the open group who were ventilated longer than 1 week. Groups were not statistically different in terms of demographic and clinical characteristics, such as birth weight (837 vs 876 gm), ventilation (27 vs 26 days), and length of stay (49 vs 40 days). Airway colonization with Gram-positive cocci occurred in the majority of patients by 2 weeks of life, regardless of group. A total of 39% of infants in the closed group and 44% of infants in the open group became airway colonized with Gram-negative bacilli; differences were statistically significant. No Gram-negative bacilli species was more likely to be associated with either suction. Nosocomial pneumonia was diagnosed in five patients from each group. Nosocomial BSIs occurred in six closed suction infants and five open suction infants. A comparable number of infants in each group developed severe BPD and were discharged from the hospital on oxygen. A total of 28% of closed suction patients and 27% of open suction patients died. Infants in the closed versus open group were suctioned on average 4.4 and 4.1 times per day and were reintubated 9.7 and 8.6 times per 100 ventilator days, respectively. A total of 40 of 44 NICU nurses considered closed suction to be easier to use, less time-consuming, and better tolerated by the patient.CONCLUSIONS:Closed suction obviates the physiological disadvantage of ventilator disconnection without increasing the rate of bacterial airway colonization, frequency of endotracheal suction and reintubation, duration of mechanical ventilation, length of hospitalization, incidence of nosocomial pneumonia, nosocomial BSI, severity of BPD, and neonatal mortality. Although slightly more expensive, closed suction is perceived by nursing staff to be easier, less time-consuming, and better tolerated by small premature infants requiring mechanical ventilation for ≥1 week.

The prevalence, six-month persistence, and predictive values of laboratory indicators of bacterial vaginosis (nonspecific vaginitis) in asymptomatic women☆

The natural course of signs and laboratory test findings indicative of bacterial vaginosis was followed in an observational noninterventive 6-month longitudinal study of 270 asymptomatic women. Only the minority of positive Gardnerella vaginalis cultures (5 of 33), wet mount clue cells (5 of 14), sniff tests (3 of 11), Papanicolaou smear clue cells (0 of 5), and discharge consistent with bacterial vaginosis (11 of 49) persisted in the absence of therapy. While these four laboratory parameters as well as chromatographic succinate/lactate ratios (performed only on the final visit) were abnormal significantly more often in patients with abnormal discharge than in those with normal discharge (p = 0.006, p less than 0.0001, p less than 0.0001, p = 0.0003, and p = 0.002, respectively), all were insensitive predictors of abnormal discharge with sensitivities ranging between 10.6% and 20.2% and abnormal test predictive values between 30.6% and 65.2%. We conclude that G. vaginalis represents indigenous flora in some normal women and that therapy is unwarranted for the incidental finding of a positive laboratory indicator of bacterial vaginosis in the patient without symptoms.

Lymphomas in sub-Saharan Africa--what can we learn and how can we help in improving diagnosis, managing patients and fostering translational research?

Approximately 30 000 cases of non‐Hodgkin lymphoma (NHL) occur in the equatorial belt of Africa each year. Apart from the fact that Burkitt lymphoma (BL) is very common among children and adolescents in Africa and that an epidemic of human immunodeficiency virus (HIV) infection is currently ongoing in this part of the world, very little is known about lymphomas in Africa. This review provides information regarding the current infrastructure for diagnostics in sub‐Saharan Africa. The results on the diagnostic accuracy and on the distribution of different lymphoma subsets in sub‐Saharan Africa were based on a review undertaken by a team of lymphoma experts on 159 fine needle aspirate samples and 467 histological samples during their visit to selected sub‐Saharan African centres is presented. Among children (<18 years of age), BL accounted for 82% of all NHL, and among adults, diffuse large B‐cell lymphoma accounted for 55% of all NHLs. Among adults, various lymphomas other than BL, including T‐cell lymphomas, were encountered. The review also discusses the current strategies of the International Network of Cancer Treatment and Research on improving the diagnostic standards and management of lymphoma patients and in acquiring reliable clinical and pathology data in sub‐Saharan Africa for fostering high‐quality translational research.

Duration of empiric antibiotics for suspected early-onset sepsis in extremely low birth weight infants

OBJECTIVES To study multicenter antibiotic practices for suspected early-onset sepsis (EOS) with negative blood cultures (NegBCs) and to identify opportunities for reduction of antimicrobial exposure. DESIGN Retrospective study. SETTING Thirty academic hospitals (University HealthSystem Consortium) located in 24 states. METHODS Data were from a survey of 790 extremely low birth weight (ELBW) infants. Total antibiotic exposures (antibiotic-days per patient) were calculated. RESULTS On admission to the NICU, 94% of 790 ELBW infants had BCs performed and empiric antibiotics initiated. When PosBC and NegBC infants were compared, 47 patients with PosBCs were similar to 695 with NegBCs in birth weight, gestational age (GA), and mortality. Patients with suspected EOS but NegBCs given ampicillin/aminoglycosides were grouped by length of administration and GA. For GA of 26 weeks or younger, 170 infants given a short (< or = 3 days) and 157 given a long (> or = 7 days) course were similar regarding birth weight, mortality, antepartum history, and CRIB scores, but were different (P < .01) in number receiving a third antimicrobial (3% and 17%) and antibiotic-days (23 and 38). For GA of 27 weeks or older, 113 infants given a short and 77 given a long course differed (P < .01) in number receiving a third antimicrobial (2% and 23%) and antibiotic-days (19 and 30). CONCLUSIONS Most suspected EOS infants with NegBCs are given antibiotics, but no antepartum historical risk factors or neonatal clinical signs explained prolonged administration. Discontinuing empiric antibiotics when BCs are negative in asymptomatic ELBW infants can reduce antimicrobial exposure without compromising clinical outcome.