Conjunctiva Swabs Research Articles

Ophthalmia " Adultorum" Secondary to Cytomegalovirus and Candida albicans Isolates from Right Eye Ulcer of an Alcoholic: The Benin City Experience

Background: Eye injuries are more common and have more severe effects when they occur in developing countries. The factors reponsible for these eye injuries can be linked to socioeconomic status, insufficient safety precautions, a lack of ideal treatment facilities, the use of conventional eye medication, and low levels of education. Ocular trauma has been identified in studies from Nigeria and other African countries as a major contributing factor to monocular blindness.
 In neonates, conjunctivitis within the first 28 days of life is termed “Ophthalmia neonatorium”. Conjunctivitis arising from either viral, bacteria or fungal causes in an Adult is what we termed as “Ophthalmia Adultorum".
 In our study the individual was a chronic alcoholic who was already immunosuppressed from his large alcohol intake. This study therefore is case report of a traumatic innoculation causing Ophthalmia Adultorum in a male alcoholic, a university of Benin Teaching Hospital experience.
 Methods: This is a case report.
 Results: A 57 year old man who was admitted on 25 September, 2023, as a case of Cytomegalovirus (CMV ) and Candida albicans associated Conjunctivitis ,after a traumatic sandball inoculation to his right eye . Conjunctiva swab for microscopy and culture was sent to the medical microbiology laboratory. Gram stain showed yeasts cells, while 10%giemsa staining of the eye swab showed ztanck cells. Culture yielded Candida albicans.
 He was placed on chloramphenicol eye drop, steroid eye drops, Acyclovir (patient could not afford gangcyclovir the specific antiviral drug due to high cost) and fluconazole eye drops. Patient was managed by the ophthalmologists and the Medical Microbiology team.
 Patient was discharged home on the 5th day and was asked to come for follow up after one week .On follow up visit , previous symptoms had resolved and right and left eyes were both normal on examination.
 Conclusion: We are unaware of any previous reports of Ophthalmia Adultorium involving Cytomegalovirus and Candida albicans in patients with sandball eye accidents. Patients with sandball injuries should be assessed quickly and antibiotic and antifungal therapy should be considered if cultures are positive.

Clinical and Pathological Findings in Fatal Cases of Severe Fever With Thrombocytopenia Syndrome With High Viremia in Cats

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging zoonotic disease caused by the SFTS virus (SFTSV). SFTSV causes severe symptoms both in humans and cats. In this study, we report the clinical and pathological findings of 4 fatal cases of cats with high SFTS viremia levels. These cats showed an acute onset of fever, leukopenia, thrombocytopenia, and increased serum amyloid A and pro-inflammatory cytokine levels. A high viral copy number was detected in the blood, oral swabs, rectal swabs, conjunctiva swabs, and urine. Histopathologically, necrotizing lymphadenitis, splenitis with lymphoblastoid cell proliferation, and hemophagocytosis were observed in all 4 cats. Immunohistochemistry revealed the presence of SFTSV antigen on lymphoblastoid B cells. SFTSV-RNA was detected in systemic tissues, including the brain. The present findings provide useful information for understanding the features of fatal SFTS in cats. To elucidate the mechanisms of severe progress of SFTS cats, as well as its role as a source of human infection, further research is needed.

Successful treatment of corneal perforation due to gonococcal keratoconjunctivitis with a combinations amniotic membrane transplantation and antibiotics

AbstractPurpose: To present a case of gonococcal keratoconjunctivitis with corneal perforation as a rare case and its management.Methods: Case was reported from an outpatient clinic patient at Ophthalmology Department of Dr. Saiful Anwar General Hospital. The diagnosis was based on history taking, ophthalmological examinations and microbiological examination result.Results: A 19‐year‐old man complained of red, swollen, and painful eyes with sticky and copious discharge. The patient experienced clinical deterioration despite antibiotic treatment received at the previous eye clinic. There was a history of promiscuity and genital discharge 2 weeks before the eye symptoms. On eye examination, the visual acuity of the right eye was 1/60, and the left eye was 6/24. Both eyes showed conjunctival hyperemia and edema with purulent discharge. There was corneal perforation on the right eye sized 2.4 mm × 1.8 mm. Corneal erosion were noted on the left eye. Gram examination from conjunctiva swab of both eyes revealed intracellular Gram‐negative diplococci. The patient was hospitalized and received systemic antibiotics intravenous ceftriaxone, single dose oral azithromycin and topical moxifloxacin 0.5%. Patient also underwent amniotic membrane transplantation (AMT) in right eye due to small perforation. Improvement was noted as no discharge, closure of perforation, visual acuity of the right eye became 6/48, and the left eye became 6/6 at 1‐month follow‐up.Conclusions: Systemic and topical antibiotics with AMT effectively manage gonococcal keratoconjunctivitis with small corneal perforation.

Development and validation of portable, field-deployable Ebola virus point-of-encounter diagnostic assay for wildlife surveillance

Early detection of Ebola virus spillover into wildlife is crucial for rapid response. We developed and validated a portable, cold-chain independent Ebola virus RT-qPCR assay.MethodsThe field syringe-based RNA extraction method was compared with a conventional laboratory-based spin-column RNA extraction method. Next, the qPCR efficiency and limit of detection of the assay was compared to standard laboratory-based reagents and equipment. The specificity of the assay was confirmed by testing against multiple Zaire Ebolavirus (EBOV) variants and other ebolavirus species. Lastly, swabs from an EBOV-infected non-human primate carcass, stored at environmental conditions mimicking central and west Africa, were analyzed to mimic in field conditions.ResultsThe syringe-based RNA extraction method performed comparably to a standard laboratory spin-column-based method. The developed assay was comparable in sensitivity and specificity to standard laboratory-based diagnostic assays. The assay specifically detected EBOV and not any of the other tested ebolavirus species, including Reston ebolavirus, Sudan ebolavirus, Bundibugyo ebolavirus, and Tai Forrest ebolavirus. Notably, the assays limit of detection for EBOV isolates were all below 4 genome copies/μL. The assay was able to detect EBOV in oral, nasal, thoracic cavity, and conjunctiva swabs obtained from an infected non-human primate.ConclusionWe developed a field-based Ebolavirus assay which is comparable in sensitivity and specificity to laboratory-based assays. Currently, the assay is being incorporated into wildlife carcass surveillance in the Republic of the Congo and is being adapted for other infectious disease agents.

The Presence of SARS-CoV-2 in Conjunctival Secretions of COVID-19 Patients

ABSTRACT Purpose: To evaluate the presence of SARS-CoV-2 in conjunctival secretions of COVID-19 patients. Material and Methods: In this retrospective study, the records were examined of patients who were treated in the hospital with the diagnosis of COVID-19 between March-May 2020 and were referred to the eye clinic due to ocular symptoms. Conjunctival swabs from both confirmed and suspected COVID-19 cases during hospitalization were analyzed. Results: A total of 35 patients (22 suspected, 13 laboratory-confirmed COVID-19) were referred to the eye clinic. Conjunctival swab samples from 3 patients yielded positive PCR results. These three patients were being treated in the intensive care unit, and all were suspected COVID-19 patients. Conclusion: SARS-CoV-2 may be detected in patients with suspected COVID-19. Even with conjunctivitis findings, SARS-CoV-2 may not be detected in most conjunctiva swab samples of COVID-19 patients.

Ocular microbiota and lens contamination following Mel4 peptide-coated antimicrobial contact lens (MACL) extended wear

PurposeThe purpose of this study was to investigate the effect of Mel4 antimicrobial peptide-coated contact lenses (MACL) on the microbiota of the conjunctiva and lenses during three months of extended wear. MethodsOne hundred and seventy-six participants were recruited into a randomised, contralateral, double masked, biweekly extended wear MACL and uncoated control lens trial. At the one month and 3-month visit, the conjunctival microbiota was sampled using sterile cotton swabs and contact lenses were collected aseptically. Standard microbiological procedures were employed for culture of the swabs and contact lenses and identification of the isolated microorganisms. ResultsGram-positive bacteria (predominantly coagulase-negative staphylococci) were the most frequently isolated microbes from both contact lenses and conjunctiva. There was no difference in the frequency of isolation of most bacteria or fungi from the conjunctival swabs of eyes wearing either MACL or control lenses. The only exception was a higher frequency of eyes harbouring Staphylococcus arlettae when wearing control lenses (5%) versus MACL (<1%) (p = 0.002). There was no significant difference in the frequency of microbes isolated from MACL or control contact lenses. There were also no differences between lens types in the frequency of isolation of >1 microbial type per sampling occasion for either conjunctiva swabs or contact lenses. ConclusionMACL wear did not change the conjunctival microbiota during extended wear, and the types of microbes isolated from MACL were similar to those isolated from control lenses.

ISOLATION AND MOLECULAR CHARACTERIZATION OF MYCOPLASMA SYNOVIAE FROM INFECTED CHICKENS WITH RESPIRATORY SIGNS.

The aim of this study was to investigate the presence of Mycoplasma synoviae in broiler and layer chickens infected with respiratory signs. A total of 80 samples were collected randomly from layer and broiler chickens with respiratory signs in Baghdad from the period between January to May 2017 from the trachea, Lung, Air sac, oviduct, tracheal swabs, conjunctiva swabs, choanal swabs, and nasal swabs and cultured in PPLO medium with supplements, then positive culture subjected to DNA extracted and Polymerase Chain Reaction assay (PCR) to detect Mycoplasma as a genus and Mycoplasma spp by using specific primers targeting16S rRNA gene. The results of culture revealed that the total rate of Mycoplasma isolates was19/80(23.75%). DNA was extracted from 19 positive isolates, all nineteen isolates were positive for Mycoplasma genus by conventional PCR assay, and a product of 270 bp was generated by amplification of the 16SrRNA gene, while a 210 bp region of 16S rRNA gene was amplified for the Mycoplasma synoviae in 19 isolates. The products of amplification of Mycoplasma synoviae16SrRNA gene was sent to MACROGEM (Korea) for sequencing, then submitted in the Gene bank database and have accession number:ID: MG846121.1. Sequencing alignment showed that local MS isolates had highly identical with standard references at gene bank, analysis the phylogenetic tree revealed the presence of 100% identity of the Iraqi isolate to the USA: West Virginia, United Kingdom, Australia, and Brazil, also had 99% identity to South Africa, China, Sweden, USA, and VitNamHatey. This result was concluded that circulation of the Mycoplasma synoviae among birds of the flock and caused respiratory signs in chickens.

Meta-genomics study among four contact lens wearers from India

Human conjunctival flora plays an important role in protecting the eye. Contact lens wearers are prone to have changes in conjunctiva flora. This study aims at assessing the variability of conjunctiva flora among contact lens wearers. We performed Ion Torrent based 16s metagenomic sequencing from 4 contact lens wearers conjunctiva swab samples. Proteobacteria found to be the most predominant phylum present in all samples. On genus level we have detected 250 bacterial strains and we found upto 455 species bacterial strains at class level, the sample was found to be most enriched with Alpha proteobacteria (52%), Gamma proteobacteria (18%) and Actinobacteria (9%) and Bacilli (6%). Order level, analysis showed presence of 52% of Alpha proteobacteria with Rhodobacterales (29%), Sphingomanadales (20%), and 1% Caulobacterales order followed by 18% of Gamma proteobacteria with Pseudomonadales (15%), Xanthomanadales (1%), 13% of Beta proteobacteria occupies Burkholderiales and 5% of Bacillales. Wearing contact lens may increase the risk of eye infections and also from the data we found that opportunistic infections may not only be caused by predominant species like cultivable Pseudomanas but also be due to uncultivable bacteria found in conjunctival swab samples the detection of which was made possible by next generation sequencing.

Returning ex-patriot Chinese to Guangdong, China, increase the risk for local transmission of Zika virus

Fast expansion and linkage to microcephaly and Guillain Barre syndrome have made Zika virus (ZIKV) track attention of global health authority concerns. The epidemiology, virological characteristics and genetic evolution of introduced ZIKV to Guangdong, China, were investigated. Analyses of the epidemiological characteristics and genetic diversity of ZIKV isolates were performed. A total of twenty-eight confirmed ZIKV infection cases were imported into China in 2016, of which 19 were imported into Guangdong, China from Venezuela (16), the Samoa Islands (1), Suriname (1) and Guatemala (1). Serial sampling studies of the cases indicated longer shedding times of ZIKV particles from urine and saliva samples than from serum and conjunctiva swab samples. Seven ZIKV strains were successfully isolated from serum, urine and conjunctiva swab samples using cell culture and neonatal mouse injection methods. Genomic analysis indicated that all viruses belonged to the Asian lineage but had different evolutionary transmission routes with different geographic origins. The molecular clock phylogenetic analysis of the ZIKV genomes indicated independent local transmission that appeared to have been previously established in Venezuela and Samoa. Additionally, we found 7 unique non-synonymous mutations in the genomes of ZIKV that were imported to China. The mutations may indicate that ZIKV has undergone independent evolutionary history not caused by sudden adaptation to Chinese hosts. The increasing number of ex-patriot Chinese returning from ZIKV hyper-endemic areas to Guangdong combined with the presence of a variety of Aedes species indicate the potential for autochthonous transmission of ZIKV in Guangdong.

Prevalence and viral load of oncogenic human papillomavirus (HPV) in pterygia in multi-ethnic patients in the Malay Peninsula.

The aim of the study was to determine the prevalence of human papillomavirus (HPV) in primary and recurrent pterygia samples collected from different ethnic groups in the equatorial Malay Peninsula. DNA was extracted from 45 specimens of freshly obtained primary and recurrent pterygia from patients and from 11 normal conjunctival swabs from volunteers with no ocular surface lesion as control. The presence of HPV DNA was detected by nested PCR. PCR-positive samples were subjected to DNA sequencing to determine the HPV genotypes. Real-time PCR with HPV16 and HPV18 type-specific TaqMan probes was employed to determine the viral DNA copy number. Of 45 pterygia samples with acceptable DNA quality, 29 (64.4%) were positive for HPV DNA, whereas all the normal conjunctiva swabs were HPV negative. Type 18 was the most prevalent (41.4% of positive samples) genotype followed by type 16 (27.6%). There was one case each of the less common HPV58 and HPV59. Seven of the samples harboured mixed infections of both HPV16 and HPV18. All the four known recurrent pterygia samples were HPV-positive, whereas the sole early-stage pterygium sample in the study was HPV-negative. There was no significant association between HPV-positive status with gender or age. A high proportion of patients from the Indian ethnic group (five of six) were HPV-positive, whereas the Malay patients were found to have higher HPV positivity than the Chinese. The viral load of HPV18 samples ranged between 2 × 10(2) and 3 × 10(4) copies per μg, whereas the viral load of HPV16 specimen was 4 × 10(1) to 10(2) copies per μg. This report describes for the first time the quantitative measurement of HPV viral DNA for pterygium samples. The high prevalence of oncogenic HPVs in our samples suggests a possible role for HPV in the pathogenesis of pterygia. Moreover, the relatively low HPV viral load is concordant with the premalignant nature of this ocular condition.

Difficulties in species identification within the genus Haemophilus - A pilot study addressing a significant problem for routine diagnostics.

Diagnostic misidentifications of commensalic Haemophilus haemolyticus as pathogenic Haemophilus influenzae are frequent. This pilot study evaluates whether isolations of H. haemolyticus are frequent enough in Germany to cause a relevant diagnostic problem, considering the fact that even H. influenzae is a mere colonizer in about 30% of isolations. In microbiological laboratories of two hospitals located in Northern and Southern Germany, the distribution of Haemophilus spp. was analyzed during a six-month-period. Site of infection, sex, and age of the patients was taken into consideration. A total of 77 Haemophilus spp. isolates was acquired and discriminated on species level, comprising: 48 H. influenzae, 25 Haemophilus parainfluenzae, 3 H. haemolyticus, and 1 Haemophilus parahaemolyticus. The proportion of H. haemolyticus was calculated to range between 1.2% and 16.2 % within the 95% confidence limits. Commensalic Haemophilus spp. were isolated from oropharynx-associated sites only. H. influenzae, in contrast, was detected in clinically relevant materials like lower respiratory materials and conjunctiva swabs. Altogether, there was a low proportion of clinical H. haemolyticus isolates. Accordingly, the problem of unnecessary antibiotic therapies due to misidentifications of H. haemolyticus as H. influenzae is quantitatively negligible compared with the risk of confusing H. influenzae colonizations with infections.

Development and evaluation of hexaplex PCR for rapid detection of methicillin, cadmium/zinc and antiseptic-resistant staphylococci, with simultaneous identification of PVL-positive and -negativeStaphylococcus aureusand coagulase negative staphylococci

We developed a multiplex PCR to detect the presence of methicillin- (mecA), cadmium/zinc-(czrC) and antiseptic-resistant (qacA/B) staphylococci and to identify Panton-Valentine leukocidin (PVL)-positive and -negative Staphylococcus aureus and coagulase-negative staphylococci (CoNS) from infected and healthy eyes. The assay was validated on 177 staphylococci comprising of 55 each of S.aureus and CoNS isolated from infected eyes and five S.aureus and 62 CoNS isolated from healthy eyes and nine direct ocular samples. Nine direct ocular samples for in situ testing consisted of corneal scrapings (4), conjunctiva swabs (2) and others (3). Multiplex PCR result was correlated with genotype data obtained with single PCR and dot-blot assay. The control strains that were positive in multiplex PCR for 16S rRNA, nuc, mecA, pvl, czrC and qacA/B genes were also positive in the dot-blot assay. The specificity of amplified genes obtained with reference strains was further confirmed by DNA sequencing. The single step-hexaplex PCR method can be used for rapid detection of mecA, nuc, pvl, czrC and qacA/B genes in staphylococci with simultaneous identification of PVL-positive and -negative S.aureus and CoNS from a variety of ocular samples.

Evaluation of cytologic findings in feline conjunctivitis

Cytologic examination of smears prepared from ocular swabs of conjunctiva from cats with conjunctivitis permits identification of the type of inflammation and possibly specific microorganisms. Results of studies of the diagnostic utility of cytology for detection of infectious causes of feline conjunctivitis have been inconsistent. The objectives of this study were to describe cytologic findings in cats with conjunctivitis and to compare those findings with results of PCR analysis for feline herpesvirus (FHV-1), Chlamydophila felis (C felis), and Mycoplasma felis (M felis). Conjunctival smears from 88 cats with conjunctivitis and 10 healthy control cats were stained with a Romanowsky stain and evaluated for the type of inflammation and evidence of an infectious agent. PCR analysis for FHV-1, C felis, and M felis was performed. Infectious agents identified by PCR analysis were FHV-1 in 9 cats (10%), C felis in 8 cats (9%), and M felis in 6 cats (7%). Inclusions interpreted as chlamydial inclusions were found in all cytologic smears from cats positive for C felis by PCR analysis and in 3 PCR-negative cats. Inclusions interpreted as Mycoplasma organisms were found in 3 of 6 cats that were PCR-positive for M felis and in 1 PCR-negative cat. FHV-1 inclusion bodies were not detected on cytologic examination. Cytologic examination can be diagnostic for C felis infection when many typical inclusions are present. Cytologic examination was unreliable in diagnosing M felis infection, and viral inclusions of FHV-1 were not found in specimens stained with Romanowsky stains.

Pasteurella canisIsolation following Penetrating Eye Injury: A Case Report

A 3-year-old boy presented with history of trauma to the left eye after he accidentally injured his eye with a broom stick made up from coconut skewers. There was history of cats as their pets but not dogs. Ocular examination revealed left superonasal conjunctival laceration and scleral perforation with prolapsed vitreous. Fundus examination showed minimal vitreous haemorrhage and flat retina. Conjunctiva swab at the wound site was sent for gram staining, culture, and sensitivity. He underwent scleral suturing, vitreous tap, and intravitreal injection of Ceftazidime and Amikacin. Vitreous tap was sent for gram stained, culture and sensitivity. Postoperatively, he was started empirically on IV Ciprofloxacin 160 mg BD, Guttae Ciprofloxacin, and Guttae Ceftazidime. Conjunctiva swab grew Pasteurella canis which was sensitive to all Beta lactams, Ciprofloxacin, Chloramphenicol, and Aminoglycoside. Post-operative was uneventful, absent signs of endophthalmitis or orbital cellulitis.

Detection of elephant endotheliotropic herpesvirus type 1 in asymptomatic elephants using TaqMan real-time PCR

This study assessed the feasibility of identifying asymptomatic viral shedders using a novel TaqMan real-time PCR on trunk washes and swabs from the conjunctiva, palate and vulva of elephants. Six...

Performance evaluation of detecting adenovirus by using rapid diagnostic kits among Japanese people

This study evaluated the diagnostic performance of detecting adenovirus by rapid diagnostic kits among Japanese people. A meta-analysis was conducted to pool the sensitivity, specificity, positive and negative likelihood ratios, and diagnostic odds ratio of immunochromatography methods and latex agglutination tests; enzyme-linked immunosorbent assay tests had already been evaluated in another meta-analysis, and no other diagnostic kits have been appropriately studied. Immunochromatography methods were shown to have sufficiently high diagnostic power, regardless of whether conjunctiva or throat swabs, or stool specimens were sampled, based upon the area under the curve (0.961-0.991); this was significantly higher than enzyme-linked immunosorbent assay tests when sampling conjunctiva swabs (P=0.00). Additionally, the relative diagnostic odds ratio showed a higher diagnostic power of immunochromatography methods than latex agglutination tests (P=0.01) by sampling stool specimens. Furthermore, immunochromatography methods had a nearly confirmative level of diagnostic power when sampling throat swabs and stool specimens (positive likelihood ratio=28.0 and 43.6, respectively, and negative likelihood ratio=0.111 and 0.121, respectively), with enzyme-linked immunosorbent assay tests not sharing this characteristic when sampling either conjunctiva or throat swabs, or stool specimens. However, even regarding immunochromatography methods, the level of pooled sensitivity (0.667) when sampling conjunctiva swabs was not acceptable. It is apparent that, therefore, among the existing types of rapid detection methods, immunochromatography methods are overall preferable for diagnosing adenoviral infections, even though they still cannot completely rule out infections by negative results, especially when sampling conjunctiva swabs.

Pharmacodynamics of a New Ophthalmic Mydriatic Insert in Healthy Volunteers: Potential Alternative as Drug Delivery System Prior to Cataract Surgery*

Cataract surgery requires a satisfactory degree of mydriasis throughout the entire operation. A phase I, open-labelled, randomised, cross-over trial was conducted in 18 healthy volunteers to compare mydriasis obtained with subsequent administration of phenylephrine 10% and tropicamide 0.5% eyedrops or a new insoluble-matrix retropalpebral ophthalmic insert containing 5.38 mg phenylephrine and 0.28 mg tropicamide. Phenylephrine serum concentrations were measured over 6 hr following each treatment administration. Secondary end-points included cardiovascular, general and local tolerance and quantification of bacterial colonisation of the conjunctiva and the cultured insert, respectively. When normalized to the pupil diameter after conventional treatment, the diameter achieved with the insert was 1.13 (95% confidence interval, 0.94-1.48, P=0.38). Moreover, standard eye drops provided faster effective mydriasis than the insert, starting 30 min. as compared to 90 min. upon treatment administration (P<0.01, repeated-measures ANOVA). Phenylephrine concentrations remained almost undetectable for both treatments and no change in heart rate or blood pressure were observed throughout the study. Only three superficial punctuate keratitis were diagnosed with the insert and two with the eye drops. No significant bacterial contamination of conjunctiva swab and cultured insert was observed. The new insoluble-matrix retropalpebral ophthalmic mydriatic insert produced similar but delayed effective and prolonged mydriasis as compared to the standard delivery system. In addition to its potential usefulness in patients undergoing cataract surgery, such new ophthalmic delivery system may be an advantage in children who need to undergo fundus photography due to the single administration and excellent tolerance as well.

Bacteria and nematodes in the conjunctiva of mule deer from Wyoming and Utah.

Swabs of conjunctiva were collected from 44 live and 226 hunter-harvested mule deer (Odocoileus hemionus) from Wyoming and Utah (USA). We identified 29 gram negative and 22 gram positive bacterial taxonomic categories, but many isolates from hunter-harvested animals were environmental contaminants. Staphylococcus spp. and Micrococcus spp. were the most common gram positive bacteria isolated, and Enterobacter spp., Escherichia coli, and Pseudomonas spp. were common gram negative bacteria isolated. Thelazia californiensis were found in 15% of hunter-harvested deer in Utah in 1994 and in 8% in 1995. Nematodes were found in 40% of live deer in 1995 and 66% in 1996. Three live animals showed clinical signs of infectious keratoconjunctivitis (IKC) in 1996, but pathogenic bacteria were not isolated from these individuals. Hemolytic, non-piliated Moraxella ovis was isolated from two clinically normal live deer in 1996 and isolates were similar to those cultured from IKC cases from Wyoming and Utah.