Strongyloides stercoralis hyperinfection presenting pneumatosis intestinalis and acute respiratory distress syndrome after treatment for COVID-19.

A 91-year-old man was admitted with vomiting and abdominal pain. He had had COVID-19 pneumonia a month before and the treatment had consisted of remdesivir, dexamethasone and baricitinib. CT scans showed pneumatosis intestinalis. His respiratory condition rapidly deteriorated and chest CT scans showed ground-glass opacity and Strongyloides stercoralis was identified in the sputum, making a diagnosis of hyperinfection syndrome associated acute respiratory distress syndrome. Treatment of ivermectin was not achieved in time and he died of multiple organ failure. S. stercoralis is a soil-transmitted helminth endemic to tropical and subtropical areas. Immunosuppressive conditions can cause hyperinfection syndrome and life-threatening conditions. Our case highlights the importance of assessing for untreated chronic strongyloidiasis in COVID-19 patients requiring steroid treatment in endemic areas.

Platelet reactivity in twin pregnancies.

BACKGROUND: Gestational thrombocytopenia is more likely to occur in twin than singleton pregnancies. However, it is unclear whether platelets are more reactive in twin than singleton pregnancies. METHODS: Changes in spontaneous platelet aggregation and concomitant fall in platelet count were examined over 90min after blood sampling in 171 and 52 citrated whole blood (CWB) samples from 59 and 17 women with singleton and twin pregnancies, respectively. Soluble P-selectin (sP-selectin) levels in the plasma were also determined. RESULTS: CWB 60min after blood sampling during 2nd trimester exhibited significantly larger numbers of platelet aggregates (1297±1600 vs. 497±432/µl, P=0.040) concomitant with significantly greater net decrease in platelet count (152±55 vs. 115±45×10(9)/µl, P=0.036) in twin than singleton pregnancies, respectively. This was followed by significantly lower 3rd trimester platelet count (181±43 vs. 229±62×10(9)/l, P=0.009) with significantly greater mean platelet volume (8.0±1.2 vs. 7.1±1.1fl, P=0.021) in twin than singleton pregnancies, respectively. The 3rd trimester sP-selectin per platelet was significantly higher in twin than singleton pregnancies. CONCLUSIONS: Platelets were more reactive in the 2nd trimester of twin than singleton pregnancies. This enhanced platelet reactivity may explain the decreased platelet count in the 3rd trimester of twin pregnancy.

Platelet aggregation in citrated whole blood of the first trimester of pregnancy.

BACKGROUND: It was recently suggested that platelet reactivity is reduced in early pregnancy. This study was performed to determine whether the citrated whole blood from 33 pregnant women in their first trimester showed spontaneous platelet aggregation and whether it differed in extent from that of 11 non-pregnant women. METHODS: The platelet count and number of platelet aggregates (PA) were serially determined in the same citrated whole blood specimens at 15, 30, 45, 60, 75, and 90min after blood sampling using a hematology analyzer. RESULTS: The number of PA increased significantly at 30min and thereafter in both groups, but was consistently lower for pregnant than non-pregnant women over the 90-min observation period. The platelet count decreased significantly in a time-dependent manner in both groups, but was significantly lower at 30 and 90min for non-pregnant than pregnant women. The number of PA showed a significant positive correlation with net decrease in platelet count for both pregnant and non-pregnant women. PA counts were also significantly positively correlated with the mean platelet volume. CONCLUSION: Platelet reactivity monitored by the increase in number of PA and the fall in platelet count was reduced in early pregnancy compared with non-pregnant healthy controls.

Diverse CD36 expression among Japanese population: defective CD36 mutations cause platelet and monocyte CD36 reductions in not only deficient but also normal phenotype subjects.

INTRODUCTION: CD36 is a multifunctional glycoprotein expressed on various human cells, including platelets and monocytes. Five CD36 gene mutations (C268T, 949insA, 329-339del, 1228-1239del and 629-631del/insAAAAC) are mainly responsible for CD36-deficient phenotypes in Japan. It has also been reported that platelet CD36 expression varies widely among normal phenotype individuals. Here, in order to obtain further insight into CD36 expression, we investigated the association between platelet and monocyte CD36 expression levels and defective mutations in the Japanese population. MATERIALS AND METHODS: Blood samples were collected from 135 healthy Japanese volunteers. CD36 expression levels on platelets and monocytes were quantitatively analyzed by flow cytometry. Real-time PCR, PCR-RFLP and allele-specific PCR were performed to detect mutant genotypes. RESULTS: In this population, we found 2 (1.5%) and 9 (6.7%) CD36-deficient subjects as type I and type II, respectively. Among normal phenotype subjects, CD36 expression levels ranged from 1,259 to 11,002 (4,487±2,017) molecules/platelet and from 211 to 5,150 (1,628±986) molecules/monocyte. Genotyping assay showed that heterozygotes with the defective mutations were present in normal (12.9%) and type II-deficient (66.7%) subjects, and that these heterozygous mutations led to decreases in CD36 surface expression on platelets and monocytes. CONCLUSIONS: Heterozygous CD36 mutations, previously known to lead to deficiency in this molecule, are one of the factors responsible for the diversity of CD36 surface expression levels on platelets and monocytes in normal phenotype subjects.

[Companion Diagnostics for Thrombotic Disease].

In recent years, thrombotic disease, including myocardial infarction and ischemic stroke, has rapidly increased in Japan. To treat and prevent thromboembolism, warfarin has been commonly prescribed for a long period as an oral anticoagulant. However, it is difficult to define an appropriate warfarin dose because of large inter-individual variability in dose requirements and the narrow therapeutic range. Recent pharmacogenomic (PGx) studies have shown that several single nucleotide polymorphisms (SNPs) in CYP2C9 (warfarin metabolic enzyme) and VKORC1 (warfarin target enzyme) are responsible for an individual's warfarin sensitivity. In order to realize personalized warfarin treatment, algorithms to estimate the required warfarin dose based on PGx are under consideration, including the cost-effectiveness. Recently, novel oral anticoagulants (NOACs; dabigatran, rivaroxaban, apixaban, and edoxaban) have become available as well as alternatives to warfarin treatment for the prevention of ischemic stroke in non-valvular atrial fibrillation. Although NOACs are prescribed at a fixed-dose without frequent monitoring of blood coagulability, it has been reported that there is inter-individual variability in the blood concentration of dabigatran caused by gene polymorphisms. Further studies are needed to perform more effective and safer anticoagulant therapy using NOACs. Progress in PGx studies and the realization of personalized anticoagulant therapy are expected in the future. [Review].

[Monitoring of Oral Thrombin Inhibitor].

Dabigatran etexilate is a prodrug that is converted into its active metabolite, dabigatran, by hydrolysis. Dabigatran is a selective thrombin inhibitor that has been approved for the prevention of stroke in patients with non-valvular atrial fibrillation in Japan. Laboratory monitoring is not needed, but an assessment of its anticoagulant effect in certain clinical settings, such as emergency surgery, suspected overdose, or the occurrence of bleeding, is desirable. We overview the special coagulation assays, such as Hemoclot Thrombin Inhibitor (HTI), the thrombin generation assay (TGA), ecarin clotting time (ECT), ecarin chromogenic assay (ECA), prothrombinase-induced clotting time (PiCT), and activated clotting time (ACT). We also examined the relationship between dabigatran levels as determined by HTI, and the activated partial thromboplastin time (APTT) and prothrombin time (PT). APTT and PT demonstrated a positive correlation with the dabigatran levels. APTT, PT, and the combination of APTT and PT may estimate the dabigatran levels in plasma.

Development of a highly sensitive three-dimensional gel electrophoresis method for characterization of monoclonal protein heterogeneity.

Three-dimensional gel electrophoresis (3-DE), which combines agarose gel electrophoresis and isoelectric focusing/SDS-PAGE, was developed to characterize monoclonal proteins (M-proteins). However, the original 3-DE method has not been optimized and its specificity has not been demonstrated. The main goal of this study was to optimize the 3-DE procedure and then compare it with 2-DE. We developed a highly sensitive 3-DE method in which M-proteins are extracted from a first-dimension agarose gel, by diffusing into 150 mM NaCl, and the recovery of M-proteins was 90.6%. To validate the utility of the highly sensitive 3-DE, we compared it with the original 3-DE method. We found that highly sensitive 3-DE provided for greater M-protein recovery and was more effective in terms of detecting spots on SDS-PAGE gels than the original 3-DE. Moreover, highly sensitive 3-DE separates residual normal IgG from M-proteins, which could not be done by 2-DE. Applying the highly sensitive 3-DE to clinical samples, we found that the characteristics of M-proteins vary tremendously between individuals. We believe that our highly sensitive 3-DE method described here will prove useful in further studies of the heterogeneity of M-proteins.

Novel Bernard-Soulier syndrome variants caused by compound heterozygous mutations (case I) or a cytoplasmic tail truncation (case II) of GPIbα.

A defective platelet glycoprotein (GP) Ib/IX/V complex [von Willebrand factor (VWF) receptor] results in Bernard-Soulier syndrome (BSS), which is characterized by macrothrombocytopenia and impaired ristocetin- and thrombin-induced platelet aggregation. We found 2 independent BSS-variant families: Case I [compound heterozygous mutations, p.Glu331X and a frame shift by a deletion at c.1444delA of GPIbα (GP1BA) terminating at a premature stop codon (p.Thr452ProfsX58)], and case II [homozygous nonsense mutation at c.1723C>T, p.Gln545X]. Case I platelets expressed no GPIbα, resulting in absence of ristocetin-induced platelet aggregation (RIPA) and 50% reduction in thrombin-induced aggregation with no shape change. The mother's platelets had 50% the expression level of A-type GPIbα (4-repeated VNTR: variable number of tandem repeats, p.[Thr145Met; Ser399_Pro411[4]]); the father's platelets had the same expression level of C-type GPIbα (2-repeated VNTR, p.Ser399_Pro411dup) as the mother's platelets. The mother's RIPA was significantly higher than the father's. Thrombin-induced aggregation was normal in both parents. Case II platelets expressed a GPIbα with an abnormal cytoplasmic tail, p.Gln545X-truncated GPIbα, which complexed with GPIX and GPV on the cell surface; its expression level of the complex was normal. Case II platelets had reversible RIPA, with no ATP release, and weak thrombin-induced aggregation without shape change. These results suggest that a signaling process through the GPIbα cytoplasmic tail required for full platelet activation is defective in BSS variant case II and a length polymorphism of GPIbα is associated with a modified level of RIPA heterozygous BSS case I.

BDNF, produced by a TPO-stimulated megakaryocytic cell line, regulates autocrine proliferation.

While human platelets release endogenous brain-derived neurotrophic factor (BDNF) upon activation, a previous report on MEG-01, a megakaryocytic cell line, found no trace of BDNF production, and the pathophysiological function of platelet BDNF has remained elusive. In the present study, we demonstrate that MEG-01 produces BDNF in the presence of TPO and that this serves to potentiate cell proliferation. Our in vitro findings suggest that BDNF regulates MEG-01 proliferation in an autocrine manner, and we suggest that BDNF may be a physiological autocrine regulator of megakaryocyte progenitors.

Impact of asthmatic control status on serum cystatin C concentrations.

BACKGROUND: To determine whether cystatin C accurately reflects renal function in asthma, we investigated serum cystatin C concentrations in a large number of asthmatic patients by adjusting for several confounding factors that might affect serum cystatin C concentrations. METHODS: A total of 126 asthmatic patients and 126 healthy volunteers, matched for age and gender, were studied. RESULTS: Serum cystatin C concentrations in symptomatic subjects with asthma were significantly higher than in healthy controls (p < 0.001) and asymptomatic subjects with asthma (p = 0.007), whereas no significant difference was observed between healthy controls and asymptomatic subjects. In asthmatic subjects, serum cystatin C concentrations were not influenced by inhaled corticosteroid (ICS). However, serum cystatin C concentrations were significantly higher in subjects who were regularly treated by oral corticosteroid (OCS) (p = 0.001). CONCLUSIONS: Serum cystatin C concentrations are elevated in asthmatic patients; particularly while symptomatic and/or taking OCS but not ICS. Serum cystatin C concentrations may not accurately reflect renal function in those patients.

Effects of molecular structural variants on serum Krebs von den Lungen-6 levels in sarcoidosis.

BACKGROUND: Serum Krebs von den Lungen-6 (KL-6), which is classified as human mucin-1 (MUC1), is used as a marker of sarcoidosis and other interstitial lung diseases. However, there remain some limitations due to a lack of information on the factors contributing to increased levels of serum KL-6. This study was designed to investigate the factors contributing to increased levels of serum KL-6 by molecular analysis. METHODS: Western blot analysis using anti-KL-6 antibody was performed simultaneously on the bronchoalveolar lavage fluid (BALF) and serum obtained from 128 subjects with sarcoidosis. RESULTS: KL-6/MUC1 in BALF showed three bands and five band patterns. These band patterns were associated with the MUC1 genotype and the KL-6 levels. KL-6/MUC1 band patterns in serum were dependent on molecular size class in BALF. Significantly increased levels of serum KL-6, serum/BALF KL-6 ratio and serum soluble interleukin 2 receptor were observed in the subjects with influx of high molecular size KL-6/MUC1 from the alveoli to blood circulation. The multivariate linear regression analysis involving potentially relevant variables such as age, gender, smoking status, lung parenchymal involvement based on radiographical stage and molecular size of KL-6/MUC1 in serum showed that the molecular size of KL-6/MUC1 in serum was significant independent determinant of serum KL-6 levels. CONCLUSIONS: The molecular structural variants of KL-6/MUC1 and its leakage behavior affect serum levels of KL-6 in sarcoidosis. This information may assist in the interpretation of serum KL-6 levels in sarcoidosis.

Total serum IgE levels and atopic status in patients with sarcoidosis.

To date, two studies have reported lower total serum immunoglobulin E (IgE) levels and lower prevalence of atopy in patients with sarcoidosis compared with healthy subjects. However, those reports did not consider age or gender differences between cases and controls. In addition, the association between total serum IgE levels and clinical manifestations of sarcoidosis has not been clarified. This study assessed total serum IgE levels and prevalence of atopy in patients with sarcoidosis after taking age and sex differences into account and evaluated associations between total serum IgE levels and clinical manifestations of sarcoidosis. Total serum IgE levels and prevalence of atopy on initial visits were compared between 189 patients with sarcoidosis and 378 age- and sex-matched controls. Associations between total serum IgE levels and involvement of each affected organ were evaluated. Changes in total serum IgE levels during the clinical course of sarcoidosis were also evaluated. Total serum IgE levels were significantly lower in patients with sarcoidosis than in controls, independent of atopic status (atopic subjects, p = 0.025; nonatopic subjects, p < 0.001). Total serum IgE levels did not differ according to the involvement of different organs. Total serum IgE levels decreased further, albeit only slightly, after disease remission (p < 0.001). Increased susceptibility to sarcoidosis may be attributable to several underlying genetic or environmental factors that result in lower total serum IgE levels.

Overweight causes left ventricular diastolic asynchrony and diastolic dysfunction: a study based on speckle tracking echocardiography in healthy subjects.

BACKGROUND: Left ventricular (LV) diastolic dysfunction is often observed in healthy subjects and can be a cause of heart failure with preserved ejection fraction (EF). We aimed to investigate the role of LV diastolic asynchrony as a cause of diastolic dysfunction in healthy subjects. METHODS: In 40 healthy subjects, two-dimensional speckle tracking imaging (2DSTI) was performed to measure the peak early diastolic longitudinal strain rates (Esr) of the apical, mid-ventricular, and basal segments of the septum and posterior wall. A mean value of the Esr of the 6 segments (mEsr) was calculated. The time from aortic valve closure to the Esr was measured for each segment, and the standard deviation (SDTEsr) was calculated. The peak global early diastolic strain rate (gEsr) was measured with a region of interest (ROI) on the whole LV myocardium. LV flow propagation velocity (FPV) was measured using conventional Doppler techniques. RESULTS: SDTEsr was not correlated with age, but was significantly correlated with body mass index (BMI) (r = 0.41, p < 0.01). Although no significant correlation was observed between mEsr and FPV, gEsr and SDTEsr significantly correlated with FPV (r = 0.41, p < 0.01; r = -0.54, p < 0.001). As a result of the multiple regression analysis, SDTEsr was the single determinant of FPV. CONCLUSIONS: Diastolic asynchrony, associated with overweight but not with aging, may contribute to diastolic dysfunction in healthy subjects.

Release reaction of brain-derived neurotrophic factor (BDNF) through PAR1 activation and its two distinct pools in human platelets.

Brain-derived neurotrophic factor (BDNF) is a cytokine that plays important roles in the survival, development, and plasticity of neurons. BDNF is also expressed in peripheral tissues and cells. In this article, we report the BDNF release reaction through thrombin stimulation and its localization in human platelets. Platelets from healthy volunteers were subjected to PAR1-AP or PAR4-AP stimulation. Release of BDNF was measured by ELISA. Localization of BDNF in resting and thrombin-activated platelets was examined by immunoelectron microscopy and sucrose gradient ultracentrifugation following western blotting. BDNF was released dose-dependently with PAR1-AP concentrations with drastic release at low PAR1-AP concentrations and gently release at high PAR1-AP concentrations. Maximum BDNF release was approximately 37% at 132 µM PAR1-AP. In contrast, 3.8% BDNF was released with 1.13 mM PAR4-AP stimulation. In immunoelectron microscopy and sucrose gradient ultracentrifugation analyses, BDNF was detected not only in α-granules but also cytoplasm in of the resting platelets, and it was distributed in the swollen open canalicular system fused to α-granules at 1 min and disappeared at 5 min after stimulation by thrombin. However, BDNF in cytoplasm remained throughout platelet activation. In conclusions, we demonstrate that BDNF is released from platelets through predominately PAR1 regulation. Furthermore, we identified two pools of BDNF in the α-granules and cytoplasm of human platelets, and only BDNF in α-granules is released through platelet activation.

Early diastolic mitral annular velocity at the interventricular septal annulus correctly reflects left ventricular longitudinal myocardial relaxation.

AIMS: Early diastolic mitral annular velocity (e') obtained by tissue Doppler imaging (TDI) is widely used to evaluate left ventricular (LV) diastolic function based on the assumption that it reflects myocardial relaxation in the long-axis direction. In this study, we aimed to determine whether or not e' truly reflects early diastolic longitudinal myocardial relaxation, and which is the most useful for evaluating LV diastolic function among e' measured at the interventricular-septal annulus (IS-e'), that measured at the lateral annulus (LW-e') or their mean value (M-e'). METHODS AND RESULTS: IS-e', LW-e', and M-e' were measured using colour TDI in 15 patients with hypertrophic cardiomyopathy, 13 patients with hypertension, and 19 control subjects. Using two-dimensional speckle-tracking imaging, early diastolic myocardial strain rates (SR(E)) were measured for the IS (IS-SR(E)), LW (LW-SR(E)), and entire LV myocardium (G-SR(E)). IS-e' was excellently correlated with IS-SR(E) (r = 0.90, P < 0.001); the correlation was better than that between LW-e' and LW-SR(E) (r = 0.75, P < 0.001). IS-e' and M-e' were well correlated with G-SR(E) (r = 0.88, P < 0.001 and r = 0.86, P< 0.001, respectively) and with LV early diastolic flow propagation velocity (FPV) (r = 0.77, P < 0.001 and r = 0.78, P < 0.001, respectively). The correlations of LW-e' to G-SR(E) (r = 0.80, P < 0.001) and FPV (r = 0.75, P < 0.001) did not reach this level. CONCLUSION: IS-e' well reflected LV longitudinal myocardial relaxation and LV diastolic function, and was found to be more useful in evaluating LV diastolic function than LW-e'.

Chlamydophila pneumoniae attachment and infection in low proteoglycan expressing human lymphoid Jurkat cells.

This study investigated the proteoglycan (PG)-dependent mechanism of Chlamydophila pneumoniae attachment to lymphocytic cells. Lymphoid Jurkat cells and epithelial HEp-2 cells were statically infected with C. pneumoniae (TW183). Transmission electron microscopy and assessment of inclusion-forming units indicated that the bacteria grew normally in Jurkat cells and were capable of producing secondary infection; however, they grew at a slower rate than in HEp-2 cells. RT-PCR analysis indicated that HEp-2 cells strongly expressed PG-core protein encoding genes, thereby sustaining glycosaminoglycans (GAGs), such as heparin, on the cellular surface. Similar gene expression levels were not observed in Jurkat cells, with the exception of glypican-1. Immunofluorescence analysis also supported strong heparin expression in HEp-2 cells and minimal expression in Jurkat cells, although heparan sulfate pretreatment significantly inhibited bacterial attachment to both cell types. Immunofluorescent co-staining with antibodies against chlamydial LPS and heparin did not identify bacterial and heparin co-localization on Jurkat cells. We also confirmed that when C. pneumoniae was statically infected to human CD4(+) peripheral blood lymphocytes known not expressing detectable level of heparin, the bacteria attached to and formed inclusion bodies in the cells. Thus, the attachment mechanism of C. pneumoniae to Jurkat cells with low PG expression is unique when compared with HEp-2 cells and potentially independent of GAGs such as heparin.

Ciliates promote the transfer of the gene encoding the extended-spectrum ß-lactamase CTX-M-27 between Escherichia coli strains.

OBJECTIVES: The mechanism by which Escherichia coli acquires multidrug resistance genes from other bacteria in the natural environment or livestock is still unclear. The ability of ciliates to promote the transfer of genes encoding extended-spectrum ß-lactamases (ESBLs) between the CTX-M-27 donor and clinically isolated recipient E. coli strains was investigated. METHODS: Equal amounts (∼10(9) cfu) of donor cefotaxime-resistant E. coli and recipient ciprofloxacin-resistant E. coli strains were mixed together in the presence or absence of 10(5) ciliates in Page's amoeba saline for 24 h, in the presence or absence of certain drugs (cytochalasin D, cycloheximide and latrunculin B). RESULTS: Gene transfer frequency in the presence of ciliates was estimated at ∼10(-6); in the absence of ciliates it was ∼10(-10). Protein synthesis (cycloheximide) or phagocytosis (cytochalasin D or latrunculin B) inhibitors significantly reduced the frequency of gene transfer. CONCLUSIONS: Ciliates promote the transfer of genes encoding ESBLs between E. coli strains, implying that the presence of ciliates may provide a significant impact on emerging multidrug-resistant bacteria.

Ciliates rapidly enhance the frequency of conjugation between Escherichia coli strains through bacterial accumulation in vesicles.

The mechanism underlying bacterial conjugation through protozoa was investigated. Kanamycin-resistant Escherichia coli SM10λ+ carrying pRT733 with TnphoA was used as donor bacteria and introduced by conjugation into ciprofloxacin-resistant E. coli clinical isolate recipient bacteria. Equal amounts of donor and recipient bacteria were mixed together in the presence or absence of protozoa (ciliates, free-living amoebae, myxamoebae) in Page's amoeba saline for 24 h. Transconjugants were selected with Luria broth agar containing kanamycin and ciprofloxacin. The frequency of conjugation was estimated as the number of transconjugants for each recipient. Conjugation frequency in the presence of ciliates was estimated to be approximately 10⁻6, but in the absence of ciliates, or in the presence of other protozoa, it was approximately 10⁻8. Conjugation also occurred in culture of ciliates at least 2 h after incubation. Successful conjugation was confirmed by the polymerase chain reaction. Addition of cycloheximide or latrunculin B resulted in suppression of conjugation. Heat killing the ciliates or bacteria had no effect on conjugation frequency. Co-localization of green fluorescent protein-expressing E. coli and PKH-67-vital-stained E. coli was observed in the same ciliate vesicles, suggesting that both donor and recipient bacteria had accumulated in the same vesicle. In this study, the conjugation frequency of bacteria was found to be significantly higher in vesicles purified from ciliates than those in culture suspension. We conclude that ciliates rapidly enhance the conjugation of E. coli strains through bacterial accumulation in vesicles.

Impact of free-living amoebae on presence of Parachlamydia acanthamoebae in the hospital environment and its survival in vitro without requirement for amoebae.

Parachlamydia acanthamoebae is an obligately intracellular bacterium that infects free-living amoebae and is a potential human pathogen in hospital-acquired pneumonia. We examined whether the presence of P. acanthamoebae is related to the presence of Acanthamoeba in an actual hospital environment and assessed the in vitro survival of P. acanthamoebae. Ninety smear samples were collected between November 2007 and March 2008 (trial 1, n = 52) and between October 2008 and February 2009 (trial 2, n = 38) from the floor (dry conditions, n = 56) and sink outlets (moist conditions, n = 34) of a hospital. The prevalences of P. acanthamoebae DNA in the first and second trials were 64.3% and 76%, respectively. The prevalences of Acanthamoeba DNA in the first and second trials were 48% and 63.1%, respectively. A statistical correlation between the prevalence of P. acanthamoebae and that of Acanthamoeba was found (trial 1, P = 0.011; trial 2, P = 0.022), and that correlation increased when samples from just the dry area (floor smear samples, P = 0.002) were analyzed but decreased when samples from a moist area were analyzed (P = 0.273). The in vitro experiment showed that, without Acanthamoeba, P. acanthamoebae could not survive in dry conditions for 3 days at 30 degrees C or 15 days at 15 degrees C. Thus, both organisms were coincidentally found in an actual hospital environment, with the presence of Acanthamoeba having a significant effect on the long-term survival of P. acanthamoebae, suggesting that this potential human pathogen could spread through a hospital environment via Acanthamoeba.