The prevalence of histologic acute chorioamnionitis among HIV infected pregnant women in Uganda and its association with adverse birth outcomes.

BACKGROUND: Preterm birth (PTB) is a leading cause of neonatal mortality and longer-term morbidity. Acute chorioamnionitis (ACA) is a common cause of PTB, however, there are limited data on the prevalence of ACA and its association with PTB in resource limited settings. METHODS: Data and samples came from a clinical trial evaluating novel strategies for the prevention of malaria in HIV infected pregnant women in Uganda. Women were enrolled between 12-28 weeks of gestation and followed through delivery. For each placenta delivered, three placental tissue types (membrane roll, umbilical cord and chorionic plate/villous parenchyma) were collected. Slides were assessed for diagnosis of maternal and fetal ACA by microscopic evaluation of neutrophilic infiltration using a standardized grading scale. The primary outcomes were PTB (<37 weeks), low birth weight (LBW, <2500 grams), and small-for-gestational age (SGA, birth weight <10th percentile for age). Univariate and multivariate logistic regression were used to estimate associations between 1) maternal characteristics (age, education, wealth, gravidity, gestational age at enrollment, placental malaria, anti-malarial prophylaxis treatment regimen, HIV disease parameters) and ACA, and 2) associations between ACA and adverse birth outcomes. FINDINGS: A total of 193 placentas were included in the analysis. The prevalence of maternal and fetal ACA was 44.5% and 28.0%, respectively. HIV infected women between 28-43 years of age had a higher risk of maternal ACA compared to those between 17-21 years of age (50.9% vs. 19.1%; aOR = 4.00 (1.10-14.5), p = 0.04) and the diagnosis of severe maternal ACA was associated with a significantly higher risk of PTB (28.6% vs. 6.0%; aOR = 6.04 (1.87-19.5), p = 0.003), LBW (33.3% vs. 9.4%; aOR = 4.86 (1.65-14.3); p = 0.004), and SGA (28.6% vs. 10.1%; aOR = 3.70 (1.20-11.4), p = 0.02). No maternal characteristics were significantly associated with fetal ACA and the diagnosis of fetal ACA was not associated with adverse birth outcomes. CONCLUSIONS: Histological evidence of severe maternal ACA was associated with an increased risk of PTB, LBW, and SGA in HIV infected, pregnant Ugandan women.

Outbreak of Tattoo-associated Nontuberculous Mycobacterial Skin Infections.

BACKGROUND: On 29 April 2015, the Florida Department of Health in Miami-Dade County (DOH Miami-Dade) was notified by a local dermatologist of 3 patients with suspected nontuberculous mycobacterial (NTM) infection after receiving tattoos at a local tattoo studio. METHODS: DOH Miami-Dade conducted interviews and offered testing, described below, to tattoo studio clients reporting rashes. Culture of clinical isolates and identification were performed at the Florida Bureau of Public Health Laboratories. Characterization of NTM was performed by the Centers for Disease Control and Prevention and the US Food and Drug Administration (FDA), respectively. Whole-genome sequencing (WGS) and single-nucleotide polymorphism (SNP) analyses were used to construct a phylogeny among 21 Mycobacterium isolates at the FDA. RESULTS: Thirty-eight of 226 interviewed clients were identified as outbreak-associated cases. Multivariate logistic regression revealed that individuals who reported gray tattoo ink in their tattoos were 8.2 times as likely to report a rash (95% confidence interval, 3.1-22.1). Multiple NTM species were identified in clinical and environmental specimens. Phylogenetic results from environmental samples and skin biopsies indicated that 2 Mycobacterium fortuitum isolates (graywash ink and a skin biopsy) and 11 Mycobacterium abscessus isolates (5 from the implicated bottle of graywash tattoo ink, 2 from tap water, and 4 from skin biopsies) were indistinguishable. In addition, Mycobacterium chelonae was isolated from 5 unopened bottles of graywash ink provided by 2 other tattoo studios in Miami-Dade County. CONCLUSIONS: WGS and SNP analyses identified the tap water and the bottle of graywash tattoo ink as the sources of the NTM infections.

Heartland Virus and Hemophagocytic Lymphohistiocytosis in Immunocompromised Patient, Missouri, USA.

Heartland virus is a suspected tickborne pathogen in the United States. We describe a case of hemophagocytic lymphohistiocytosis, then death, in an immunosuppressed elderly man in Missouri, USA, who was infected with Heartland virus.

Ebola Virus Disease in Pregnancy: Clinical, Histopathologic, and Immunohistochemical Findings.

Here we describe clinicopathologic features of Ebola virus disease in pregnancy. One woman infected with Sudan virus in Gulu, Uganda, in 2000 had a stillbirth and survived, and another woman infected with Bundibugyo virus had a live birth with maternal and infant death in Isiro, the Democratic Republic of the Congo in 2012. Ebolavirus antigen was seen in the syncytiotrophoblast and placental maternal mononuclear cells by immunohistochemical analysis, and no antigen was seen in fetal placental stromal cells or fetal organs. In the Gulu case, ebolavirus antigen localized to malarial parasite pigment-laden macrophages. These data suggest that trophoblast infection may be a mechanism of transplacental ebolavirus transmission.

Vertebrate Host Susceptibility to Heartland Virus.

Heartland virus (HRTV) is a recently described phlebovirus initially isolated in 2009 from 2 humans who had leukopenia and thrombocytopenia. Serologic assessment of domestic and wild animal populations near the residence of 1 of these persons showed high exposure rates to raccoons, white-tailed deer, and horses. To our knowledge, no laboratory-based assessments of viremic potential of animals infected with HRTV have been performed. We experimentally inoculated several vertebrates (raccoons, goats, chickens, rabbits, hamsters, C57BL/6 mice, and interferon-α/ß/γ receptor-deficient [Ag129]) mice with this virus. All animals showed immune responses against HRTV after primary or secondary exposure. However, neutralizing antibody responses were limited. Only Ag129 mice showed detectable viremia and associated illness and death, which were dose dependent. Ag129 mice also showed development of mean peak viral antibody titers >8 log10 PFU/mL, hemorrhagic hepatic lesions, splenomegaly, and large amounts of HRTV antigen in mononuclear cells and hematopoietic cells in the spleen.

Reductions in malaria in pregnancy and adverse birth outcomes following indoor residual spraying of insecticide in Uganda.

BACKGROUND: Indoor residual spraying of insecticide (IRS) is a key intervention for reducing the burden of malaria in Africa. However, data on the impact of IRS on malaria in pregnancy and birth outcomes is limited. METHODS: An observational study was conducted within a trial of intermittent preventive therapy during pregnancy in Tororo, Uganda. Women were enrolled at 12-20 weeks of gestation between June and October 2014, provided with insecticide-treated bed nets, and followed through delivery. From December 2014 to February 2015, carbamate-containing IRS was implemented in Tororo district for the first time. Exact spray dates were collected for each household. The exposure of interest was the proportion of time during a woman's pregnancy under protection of IRS, with three categories of protection defined: no IRS protection, >0-20 % IRS protection, and 20-43 % IRS protection. Outcomes assessed included malaria incidence and parasite prevalence during pregnancy, placental malaria, low birth weight (LBW), pre-term delivery, and fetal/neonatal deaths. RESULTS: Of 289 women followed, 134 had no IRS protection during pregnancy, 90 had >0-20 % IRS protection, and 65 had >20-43 % protection. During pregnancy, malaria incidence (0.49 vs 0.10 episodes ppy, P = 0.02) and parasite prevalence (20.0 vs 8.9 %, P < 0.001) were both significantly lower after IRS. At the time of delivery, the prevalence of placental parasitaemia was significantly higher in women with no IRS protection (16.8 %) compared to women with 0-20 % (1.1 %, P = 0.001) or >20-43 % IRS protection (1.6 %, P = 0.006). Compared to women with no IRS protection, those with >20-43 % IRS protection had a lower risk of LBW (20.9 vs 3.1 %, P = 0.002), pre-term birth (17.2 vs 1.5 %, P = 0.006), and fetal/neonatal deaths (7.5 vs 0 %, P = 0.03). CONCLUSION: In this setting, IRS was temporally associated with lower malaria parasite prevalence during pregnancy and at delivery, and improved birth outcomes. IRS may represent an important tool for combating malaria in pregnancy and for improving birth outcomes in malaria-endemic settings. Trial Registration Current Controlled Trials Identifier NCT02163447.

Centrofacial Balamuthiasis: case report of a rare cutaneous amebic infection.

Free-living amebae are ubiquitous in our environment, but rarely cause cutaneous infection. Balamuthia mandrillaris has a predilection for infecting skin of the central face. Infection may be restricted to the skin or associated with life-threatening central nervous system (CNS) involvement. We report a case of a 91-year-old woman, who presented with a non-healing red plaque over her right cheek. Several punch biopsies exhibited non-specific granulomatous inflammation without demonstrable fungi or mycobacteria in histochemical stains. She was treated empirically for granulomatous rosacea, but the lesion continued to progress. A larger incisional biopsy was performed in which amebae were observed in hematoxylin-eosin stained sections. These were retrospectively apparent in the prior punch biopsy specimens. Immunohistochemistry and polymerase chain reaction studies identified the organisms as Balamuthia mandrillaris. Cutaneous infection by B. mandrillaris is a rare condition that is sometimes complicated by life-threatening CNS involvement and which often evades timely diagnosis due to its rarity and nonspecific clinical manifestations. Moreover, these amebae are easily overlooked in histopathologic sections because of their small number and their resemblance to histiocytes. Dermatopathologists should be familiar with the histopathologic appearance of these organisms and include balamuthiasis and other amebic infections in the differential diagnosis of granulomatous dermatitis.

Abundance of megalin and Dab2 is reduced in syncytiotrophoblast during placental malaria, which may contribute to low birth weight.

Placental malaria caused by Plasmodium falciparum contributes to ~200,000 child deaths annually, mainly due to low birth weight (LBW). Parasitized erythrocyte sequestration and consequent inflammation in the placenta are common attributes of placental malaria. The precise molecular details of placental changes leading to LBW are still poorly understood. We hypothesized that placental malaria may disturb maternofetal exchange of vitamins, lipids, and hormones mediated by the multi-ligand (n ~ 50) scavenging/signaling receptor megalin, which is abundantly expressed in placenta but was not previously analyzed in pregnancy outcomes. We studied abundance of megalin and its intracellular adaptor protein Dab2 by immunofluorescence microscopy in placental biopsies from Ugandan women with (n = 8) and without (n = 20) active placental malaria. We found that: (a) abundances of both megalin (p = 0.01) and Dab2 (p = 0.006) were significantly reduced in brush border of syncytiotrophoblast of infected placentas; (b) amounts of megalin and Dab2 were strongly correlated (Spearman's r = 0.53, p = 0.003); (c) abundances of megalin and Dab2 (p = 0.046) were reduced in infected placentas from women with LBW deliveries. This study provides first evidence that placental malaria infection is associated with reduced abundance of megalin transport/signaling system and indicate that these changes may contribute to the pathology of LBW.

Clinical and Pathological Evaluation of Mycobacterium marinum Group Skin Infections Associated With Fish Markets in New York City.

BACKGROUND: From December 2013 through May 2014, physicians, dermatopathologists, and public health authorities collaborated to characterize an outbreak of Mycobacterium marinum and other nontuberculous mycobacterial skin and soft tissue infections (SSTIs) associated with handling fish in New York City's Chinatown. Clinicopathologic and laboratory investigations were performed on a series of patients. METHODS: Medical records were reviewed for 29 patients. Culture results were available for 27 patients and 24 biopsy specimens were evaluated by histopathology, immunohistochemistry (IHC) staining for acid-fast bacilli (AFB), and mycobacterial polymerase chain reaction (PCR) assays. RESULTS: All patients received antibiotics. The most commonly prescribed antibiotic regimen was clarithromycin and ethambutol. Of the 29 patients in this case series, 16 (55%) received surgical treatment involving incision and drainage, mass excision, and synovectomy. Of these, 7 (44%) had deep tissue involvement. All patients showed improvement. For those with culture results, 11 of 27 (41%) were positive for M. marinum; the remainder showed no growth. Poorly formed granulomas (96%), neutrophils (75%), and necrosis (79%) were found in 24 biopsies. Of 15 cases that were culture-negative and analyzed by other methods, 9 were PCR positive for M. marinum group species, 8 were IHC positive, and 3 were positive by AFB stains. CONCLUSIONS: A multidisciplinary approach was used to identify cases in an outbreak of M. marinum infections. The use of histopathology, culture, and IHC plus PCR from full thickness skin biopsy can lead to improved diagnosis of M. marinum SSTIs compared to relying solely on mycobacterial culture, the current gold standard.

Malignant Transformation of Hymenolepis nana in a Human Host.

Neoplasms occur naturally in invertebrates but are not known to develop in tapeworms. We observed nests of monomorphic, undifferentiated cells in samples from lymph-node and lung biopsies in a man infected with the human immunodeficiency virus (HIV). The morphologic features and invasive behavior of the cells were characteristic of cancer, but their small size suggested a nonhuman origin. A polymerase-chain-reaction (PCR) assay targeting eukaryotes identified Hymenolepis nana DNA. Although the cells were unrecognizable as tapeworm tissue, immunohistochemical staining and probe hybridization labeled the cells in situ. Comparative deep sequencing identified H. nana structural genomic variants that are compatible with mutations described in cancer. Invasion of human tissue by abnormal, proliferating, genetically altered tapeworm cells is a novel disease mechanism that links infection and cancer.

Novel poxvirus infection in 2 patients from the United States.

BACKGROUND: Some human poxvirus infections can be acquired through zoonotic transmission. We report a previously unknown poxvirus infection in 2 patients, 1 of whom was immunocompromised; both patients had known equine contact. METHODS: The patients were interviewed and clinical information was abstracted from the patients' medical files. Biopsies of the skin lesions were collected from both patients for histopathology, immunohistochemistry, and transmission electron microscopy analysis. Oral and skin swabs were collected from animals with frequent contact with the patients, and environmental sampling including rodent trapping was performed on the farm where the immunosuppressed patient was employed. "Pan-pox and high Guanine-cytosine" polymerase chain reaction assays were performed on patient, animal, and environmental isolates. Amplicon sequences of the viral DNA were used for agent identification and phylogenetic analysis. RESULTS: Specimens from both human cases revealed a novel poxvirus. The agent shares 88% similarity to viruses in the Parapoxvirus genus and 78% to those in the Molluscipoxvirus genus but is sufficiently divergent to resist classification as either. All animal and environmental specimens were negative for poxvirus and both patients had complete resolution of lesions. CONCLUSIONS: This report serves as a reminder that poxviruses should be considered in cutaneous human infections, especially in individuals with known barnyard exposures. The clinical course of the patients was similar to that of parapoxvirus infections, and the source of this virus is currently unknown but is presumed to be zoonotic. This report also demonstrates the importance of a comprehensive approach to diagnosis of human infections caused by previously unknown pathogens.

Tissue tropism, pathology and pathogenesis of enterovirus infection.

Enteroviruses are very common and cause infections with a diverse array of clinical features. Enteroviruses are most frequently considered by practising pathologists in cases of aseptic meningitis, encephalitis, myocarditis and disseminated infections in neonates and infants. Congenital infections have been reported and transplacental transmission is thought to occur. Although skin biopsies during hand, foot and mouth disease are infrequently obtained, characteristic dermatopathological findings can be seen. Enteroviruses have been implicated in lower respiratory tract infections. This review highlights histopathological features of enterovirus infection and discusses diagnostic modalities for formalin-fixed paraffin-embedded tissues and their associated pitfalls. Immunohistochemistry can detect enterovirus antigen within cells of affected tissues; however, assays can be non-specific and detect other viruses. Molecular methods are increasingly relied upon but, due to the high frequency of asymptomatic enteroviral infections, clinical-pathological correlation is needed to determine significance. Of note, diagnostic assays on central nervous system or cardiac tissues from immunocompetent patients with prolonged disease courses are most often negative. Histopathological, immunohistochemical and molecular studies performed on clinical specimens also provide insight into enteroviral tissue tropism and pathogenesis.

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.

Fatal hemophagocytic lymphohistiocytosis associated with locally acquired dengue virus infection - New Mexico and Texas, 2012.

Dengue is caused by infection with any of four mosquito-transmitted dengue viruses (DENV-1-4) and is characterized by fever, headache, myalgia, and leukopenia. Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal hyperinflammatory syndrome that can be familial or acquired, and is characterized by persistent fever, pancytopenia, hepatosplenomegaly, and increased serum ferritin. Acquired HLH is most frequently associated with Epstein Barr virus infection but also has been associated with dengue. This report describes a fatal case of acquired HLH that was apparently triggered by infection with DENV-3. The patient developed an acute febrile illness in August 2012 during a 1-month vacation in New Mexico. After returning to her home in Texas, she was initially diagnosed with West Nile virus (WNV) infection, developed pancytopenia, liver failure, and disseminated intravascular coagulopathy, and died. DENV-3 was detected in a premortem bone marrow biopsy in which erythrophagocytosis was evident. This case underscores the need for clinicians in the United States to be vigilant for dengue and request diagnostic testing for suspected cases, which should be reported to public health authorities.

Exserohilum infections associated with contaminated steroid injections: a clinicopathologic review of 40 cases.

September 2012 marked the beginning of the largest reported outbreak of infections associated with epidural and intra-articular injections. Contamination of methylprednisolone acetate with the black mold, Exserohilum rostratum, was the primary cause of the outbreak, with >13,000 persons exposed to the potentially contaminated drug, 741 confirmed drug-related infections, and 55 deaths. Fatal meningitis and localized epidural, paraspinal, and peripheral joint infections occurred. Tissues from 40 laboratory-confirmed cases representing these various clinical entities were evaluated by histopathological analysis, special stains, and IHC to characterize the pathological features and investigate the pathogenesis of infection, and to evaluate methods for detection of Exserohilum in formalin-fixed, paraffin-embedded (FFPE) tissues. Fatal cases had necrosuppurative to granulomatous meningitis and vasculitis, with thrombi and abundant angioinvasive fungi, with extensive involvement of the basilar arterial circulation of the brain. IHC was a highly sensitive method for detection of fungus in FFPE tissues, demonstrating both hyphal forms and granular fungal antigens, and PCR identified Exserohilum in FFPE and fresh tissues. Our findings suggest a pathogenesis for meningitis involving fungal penetration into the cerebrospinal fluid at the injection site, with transport through cerebrospinal fluid to the basal cisterns and subsequent invasion of the basilar arteries. Further studies are needed to characterize Exserohilum and investigate the potential effects of underlying host factors and steroid administration on the pathogenesis of infection.

Automated and unsupervised detection of malarial parasites in microscopic images.

BACKGROUND: Malaria is a serious infectious disease. According to the World Health Organization, it is responsible for nearly one million deaths each year. There are various techniques to diagnose malaria of which manual microscopy is considered to be the gold standard. However due to the number of steps required in manual assessment, this diagnostic method is time consuming (leading to late diagnosis) and prone to human error (leading to erroneous diagnosis), even in experienced hands. The focus of this study is to develop a robust, unsupervised and sensitive malaria screening technique with low material cost and one that has an advantage over other techniques in that it minimizes human reliance and is, therefore, more consistent in applying diagnostic criteria. METHOD: A method based on digital image processing of Giemsa-stained thin smear image is developed to facilitate the diagnostic process. The diagnosis procedure is divided into two parts; enumeration and identification. The image-based method presented here is designed to automate the process of enumeration and identification; with the main advantage being its ability to carry out the diagnosis in an unsupervised manner and yet have high sensitivity and thus reducing cases of false negatives. RESULTS: The image based method is tested over more than 500 images from two independent laboratories. The aim is to distinguish between positive and negative cases of malaria using thin smear blood slide images. Due to the unsupervised nature of method it requires minimal human intervention thus speeding up the whole process of diagnosis. Overall sensitivity to capture cases of malaria is 100% and specificity ranges from 50-88% for all species of malaria parasites. CONCLUSION: Image based screening method will speed up the whole process of diagnosis and is more advantageous over laboratory procedures that are prone to errors and where pathological expertise is minimal. Further this method provides a consistent and robust way of generating the parasite clearance curves.

Natural selection of FLT1 alleles and their association with malaria resistance in utero.

Placental malaria (PM) caused by Plasmodium falciparum contributes significantly to infant mortality in sub-Saharan Africa and is associated with pregnancy loss. We hypothesized that fetal genes that modify PM would be associated with fetal fitness. During PM, placental trophoblasts produce soluble fms-like tyrosine kinase 1 (sFlt1), also known as soluble VEGF receptor 1, an angiogenesis inhibitor associated with preeclampsia. Here we present a study examining the genotype of the fms-related tyrosine kinase 1 (FLT1) 3' UTR in Tanzanian mother-infant pairs. First-time mothers suffer the most PM, and newborn FLT1 genotype distribution differed by birth order, with newborns of first-time mothers outside of Hardy-Weinberg equilibrium (HWE) during peak PM season. Among first-time but not other mothers, maternal FLT1 genotype was associated with a history of prior pregnancy loss. During PM, newborn FLT1 genotype was associated with low birth weight and placental inflammatory gene expression. FLT1 genotype was also associated with Flt1 levels among study subjects and in vitro. Thus, FLT1 variants confer fetal fitness in utero and are associated with the maternal immune response during PM. This indicates that FLT1 is under natural selection in a malaria endemic area and that human exposure to malaria can influence the evolutionary genetics of the maternal-fetal relationship.

Genome-wide expression analysis of placental malaria reveals features of lymphoid neogenesis during chronic infection.

Chronic inflammation during placental malaria (PM) is most frequent in first time mothers and is associated with poor maternal and fetal outcomes. In the first genome-wide analysis of the local human response to sequestered malaria parasites, we identified genes associated with chronic PM and then localized the corresponding proteins and immune cell subsets in placental cryosections. B cell-related genes were among the most highly up-regulated transcripts in inflamed tissue. The B cell chemoattractant CXCL13 was up-regulated >1,000-fold, and B cell-activating factor was also detected. Both proteins were expressed by intervillous macrophages. Ig L and H chain transcription increased significantly, and heavy depositions of IgG3 and IgM were observed in intervillous spaces. The B cell phenotype was heterogeneous, including naive (CD27-negative), mature (CD138-positive), and cycling (Ki-67-positive) cells. B cells expressed T-bet but not Bcl-6, suggesting T cell-independent activation without germinal center formation. Genes for the Fc binding proteins FcgammaRIa, FcgammaRIIIa, and C1q were highly up-regulated, and the proteins localized to intervillous macrophages. Birth weight was inversely correlated with transcript levels of CXCL13, IgG H chain, and IgM H chain. The iron regulatory peptide hepcidin was also expressed but was not associated with maternal anemia. The results suggest that B cells and macrophages contribute to chronic PM in a process resembling lymphoid neogenesis. We propose a model where the production of Ig during chronic malaria may enhance inflammation by attracting and activating macrophages that, in turn, recruit B cells to further produce Ig in the intervillous spaces.

Hypertension and maternal-fetal conflict during placental malaria.

BACKGROUND: Malaria and hypertension are major causes of maternal mortality in tropical countries, especially during first pregnancies, but evidence for a relationship between these syndromes is contradictory. METHODS AND FINDINGS: In a cross-sectional survey of Tanzanian parturients, the rate of hypertension was similar in placental malaria (PM)-positive (11/85 = 13%) and PM-negative (73/602 = 12%) individuals. However, we found that PM was associated with hypertension in first-time mothers aged 18-20 y but not other mothers. Hypertension was also associated with histologic features of chronic malaria, which is common in first-time mothers. Levels of soluble vascular endothelial growth factor receptor 1 (sVEGFR1), a preeclampsia biomarker, were elevated in first-time mothers with either PM, hypertension, or both, but levels were not elevated in other mothers with these conditions. In first-time mothers with PM, the inflammatory mediator vascular endothelial growth factor (VEGF) was localized to maternal macrophages in the placenta, while sVEGFR1, its soluble inhibitor, was localized to the fetal trophoblast. CONCLUSIONS: The data suggest that maternal-fetal conflict involving the VEGF pathway occurs during PM, and that sVEGFR1 may be involved in the relationship between chronic PM and hypertension in first-time mothers. Because placental inflammation causes poor fetal outcomes, we hypothesize that fetal mechanisms that promote sVEGFR1 expression may be under selective pressure during first pregnancies in malaria-endemic areas.