Persistent long-term habitat use by Florida manatees at Fort Pierce, Florida from 1997 to 2020.

To survive cold winters, Florida manatees (Trichechus manatus latirostris) depend on artificial (i.e., power plants) and natural warm water sources such as springs and passive thermal basins. Passive thermal basins can provide critical habitat for manatees for short or extended periods of time. The Henry D. King Powerplant in Fort Pierce, Florida discharged warm water into Moore's Creek until it went offline in 1995. However, it is unknown to what degree manatees continue to occupy this area and how environmental factors influence their occurrence in the creek. To explore this, we examined the habitat use of Florida manatees in Moore's Creek after the shutdown from November 1997 to March 2020 from daily counts of manatees. In addition, we correlated local environmental data (ambient air, temperature, salinity) to assess if Moore's Creek had properties indicative of a passive thermal basin. Results indicated there was not an increase or decrease in habitat use over twenty years in the Creek. The consistent use of Moore's Creek over the study period suggests that this habitat possesses thermal and freshwater resources to support manatee occurrence long-term. These findings provide robust support for the importance of this habitat and passive thermal basins for Florida manatees.

Agonistic interactions initiated by adult bottlenose dolphins on Antillean manatee calves in the Caribbean Sea.

The dynamics and drivers of inter-species interactions in the wild are poorly understood, particularly those involving social animal species. Inter-species interactions between cetaceans and sirenians have rarely been documented and investigated. Here, we report 10 cases of interaction initiated by adult bottlenose dolphins (Tursiops truncatus) towards Antillean manatee (Trichechus manatus manatus). Interactions were documented through behavioral observations in the wild (n = 7) and from the examination of orphaned calves (i.e., tooth rake marks on their body; n = 4) that entered a rehabilitation facility, one individual both observed interacting with dolphins and found stranded with bite marks. Bottlenose dolphins were observed interacting with orphan manatee calves and with mother-calf pairs, exhibiting agonistic behavior (n = 2), affiliative or neutral behaviors (n = 1), but the behavioral contexts of these interactions remain unclear in most cases (n = 7). Information on stranded individuals was collected from four calves (of 13 examined calves) recovered in poor condition with bottlenose dolphin tooth rakes and bite wounds on their bodies, one of which died. Injury from bite wounds varied in extent and severity, ranging from superficial scratches leaving rake marks to deep lacerations. Our findings suggest the regular occurrence of agonistic behaviors initiated by adult bottlenose dolphins and directed toward manatee calves. However, the drivers of these interactions remain unknown and need to be further investigated.

Manatees display diel trends in acoustic activity at two microhabitats in Belize.

Many marine mammals exhibit diel trends in vocal production, which can provide information on habitat use and behavioral activity. In Belize, Antillean manatees (Trichechus manatus manatus) commonly inhabit small depressions in the substrate or deep-water coves known as "resting holes". Determining if manatees exhibit diel temporal trends in their call production rate and call types between microhabitats can provide insights into their diurnal and nocturnal activity patterns. Here, we investigate the diel vocalization patterns of wild Antillean manatees in two adjacent resting holes off of St. George's Caye, Belize. Recordings of manatees were made using a bottom-mounted hydrophone located near a reef barrier reef for nine days in July of 2017 and ten days in January of 2018. To explore if and how manatee acoustic activity differs between sites, we compared the number of calls per hour, the number of manatee positive hours, the number of tonal and atonal sounds, and the number of boats detected across sites. A total of 370 hours of acoustic recordings were analyzed resulting in the detection of 3,262 calls. There were no significant differences in the number of manatee calls produced per hour between sites. The average number of calls produced by manatees decreased over the course of several days. The proportion of tonal calls decreased with hours after sunset and increased in boat presence. These results suggest manatees in this region may exhibit different diel activity patterns which appear to be influenced by the characteristics of the environment. These findings can support ongoing conservation and management efforts to safeguard species in Belize.

A controlled evaluation of filter paper use during staining of sputum smears for tuberculosis microscopy.

Background: Some sputum smear microscopy protocols recommend placing filter paper over sputum smears during staining for Mycobacterium tuberculosis (TB) . We found no published evidence assessing whether this is beneficial. We aimed to evaluate the effect of filter paper on sputum smear microscopy results. Methods: Sputum samples were collected from 30 patients with confirmed pulmonary TB and 4 healthy control participants. From each sputum sample, six smears (204 smears in total) were prepared for staining with Ziehl-Neelsen (ZN), auramine or viability staining with fluorescein diacetate (FDA). Half of the slides subjected to each staining protocol were randomly selected to have Whatman grade 3 filter paper placed over the dried smears prior to stain application and removed prior to stain washing. The counts of acid-fast bacilli (AFB) and precipitates per 100 high-power microscopy fields of view, and the proportion of smear that appeared to have been washed away were recorded. Statistical analysis used a linear regression model adjusted by staining technique with a random effects term to correct for between-sample variability.   Results: The inclusion of filter paper in the staining protocol significantly decreased microscopy positivity independent of staining with ZN, auramine or FDA (p=0.01). Consistent with this finding, there were lower smear grades in slides stained using filter paper versus without (p=0.04), and filter paper use reduced AFB counts by 0.28 logarithms (95% confidence intervals, CI=0.018, 0.54, p=0.04) independent of staining technique. In all analyses, auramine was consistently more sensitive with higher AFB counts versus ZN (p=0.001), whereas FDA had lower sensitivity and lower AFB counts (p<0.0001). Filter paper use was not associated with the presence of any precipitate (p=0.5) or the probability of any smear washing away (p=0.6) during the staining process. Conclusions: Filter paper reduced the sensitivity of AFB microscopy and had no detectable beneficial effects so is not recommended.

Vocalizations of wild West Indian manatee vary across subspecies and geographic location.

Geographic variation in the vocal behavior of manatees has been reported but is largely unexplored. Vocalizations of wild West Indian manatees (Trichechus manatus) were recorded with hydrophones in Florida from Florida manatees (Trichechus manatus latirostris), and in Belize and Panama from Antillean manatees (Trichechus manatus manatus) to determine if calls varied between subspecies and geographic regions. Calls were visually classified into five categories: squeaks, high squeaks, squeals, squeak-squeals, and chirps. From these five categories, only three call types (squeaks, high squeaks and squeals) were observed in all three populations. Six parameters from the temporal and frequency domains were measured from the fundamental frequency of 2878 manatee vocalizations. A repeated measures PERMANOVA found significant differences for squeaks and high squeaks between each geographic location and for squeals between Belize and Florida. Almost all measured frequency and temporal parameters of manatee vocalizations differed between and within subspecies. Variables that may have influenced the variation observed may be related to sex, body size, habitat and/or other factors. Our findings provide critical information of manatee calls for wildlife monitoring and highlight the need for further study of the vocal behavior of manatees throughout their range.

Genetic confirmation of Octopus insularis (Leite and Haimovici, 2008) in South Florida, United States using physical features and de novo genome assembly.

The distribution of octopuses within the Octopus vulgaris species complex remains inadequately understood. Species determination can be complex and involves characterizing a specimen's physical features and comparing its genetic makeup to other populations. In this study, we present the first genetic confirmation of Octopus insularis (Leite and Haimovici, 2008) inhabiting the coastal waters of the Florida Keys, United States. We employed visual observations to identify species-specific body patterns of three wild-caught octopuses and used de novo genome assembly to confirm their species. All three specimens exhibited a red/white reticulated pattern on their ventral arm surface. Two specimens displayed body pattern components of deimatic display (white eye encircled by a light ring, with darkening around the eye). All visual observations were consistent with distinguishing features of O. insularis. We then compared mitochondrial subunits COI, COIII, and 16S in these specimens across all available annotated octopod sequences, including Sepia apama (Hotaling et al., 2021) as a control outgroup taxon. For species exhibiting intraspecific genomic variation, we included multiple sequences from geographically distinct populations. Laboratory specimens consistently clustered into a single taxonomic node with O. insularis. These findings confirm O. insularis presence in South Florida and suggest a more extensive northern distribution than previously assumed. Whole genome Illumina sequencing of multiple specimens enabled taxonomic identification with well-established DNA barcodes while also generating the first de novo full assembly of O. insularis. Furthermore, constructing and comparing phylogenetic trees for multiple conserved genes is essential for confirming the presence and delineation of cryptic species in the Caribbean.

Antillean manatee calves in captive rehabilitation change vocal behavior in anticipation of feeding.

Captive animals typically develop anticipatory behaviors, actions of increased frequency done in anticipation of an event such as feeding. Anticipatory behaviors can be an indicator of an animal's welfare. However, for rehabilitating animals that are expected to be reintroduced into the wild, these behaviors need to be extinguished to ensure successful release. Scheduled activities such as feeding occur daily and vocalizations could potentially be used to identify anticipatory behavior. Here, we tested the hypothesis that manatee calves modify their vocal production rate as a form of anticipatory behavior. Vocalizations of two Antillean manatee (Trichechus manatus manatus) calves were recorded for 10 min before, during, and after feeding sessions at Wildtracks, a manatee rehabilitation center in Belize. The number of calls were counted across recording sessions and three acoustic parameters were measured from calls including duration, frequency modulation, and center frequency. A repeated measures ANOVA comparing the number of calls across sessions indicated manatees produced significantly more calls before feeding sessions than during and after sessions. In addition, manatees increased the duration and lowered the frequency of calls before feeding sessions. This information can give further insight on ways to improve rehabilitation protocols and manage human interactions to increase the overall survival rate of rehabilitated manatees when released back into the wild.

Interchange of Southern Hemisphere humpback whales across the South Atlantic Ocean.

The cosmopolitan distribution of humpback whales (Megaptera novaeangliae) is largely driven by migrations between winter low-latitude breeding grounds and summer high-latitude feeding grounds. Southern Hemisphere humpback whales faced intensive exploitation during the whaling eras and recently show evidence of population recovery. Gene flow and shared song indicate overlap between the western (A) and eastern (B1, B2) Breeding Stocks in the South Atlantic and Indian Oceans (C1). Here, we investigated photo-identification evidence of population interchange using images of individuals photographed during boat-based tourism and research in Brazil and South Africa from 1989 to 2022. Fluke images were uploaded to Happywhale, a global digital database for marine mammal identification. Six whales were recaptured between countries from 2002 to 2021 with resighting intervals ranging from 0.76 to 12.92 years. Four whales originally photographed off Abrolhos Bank, Brazil were photographed off the Western Cape, South Africa (feeding grounds for B2). Two whales originally photographed off the Western Cape were photographed off Brazil, one traveling to the Eastern Cape in the Southwestern Indian Ocean (a migration corridor for C1) before migrating westward to Brazil. These findings photographically confirm interchange of humpback whales across the South Atlantic and Indian Oceans and the importance of international collaboration to understand population boundaries.

Manatee calf call contour and acoustic structure varies by species and body size.

Vocal activity and signal characteristics of mammals are driven by several factors that result in both stability and plasticity over multiple time scales. All three extant species of manatee communicate with several calls that are especially important for maintaining contact between cows and calves. Determining if calf calls differ across manatee species will provide insights into the evolution of species-specific acoustic communication traits. We investigated the interspecific differences in the vocalizations of calves of Amazonian manatees (Trichechus inunguis) and the two subspecies of the West Indian manatee (T. manatus). Vocalizations of individual calves were recorded in rehabilitation centers in Brazil, Puerto Rico, the United States, and Mexico. The acoustic structure of calls produced by manatee calves varied between species and with body size. Amazonian manatee calves produced shorter calls with multiple notes at higher frequency while West Indian calves produced modulated calls that were lower in frequency and longer in duration. Smaller West Indian calves produced frequency modulated, hill-shaped calls that flattened with an increase in body length. Our results provide evidence for divergence in the ontogeny of vocalizations across T. manatus and T. inunguis and suggest variation in body size contributed to the evolution of differences in the characteristics of their calls.

First characterization of vocalizations and passive acoustic monitoring of the vulnerable African manatee (Trichechus senegalensis).

Even among the understudied sirenians, African manatees (Trichechus senegalensis) are a poorly understood, elusive, and vulnerable species that is difficult to detect. We used passive acoustic monitoring in the first effort to acoustically detect African manatees and provide the first characterization of their vocalizations. Within two 3-day periods at Lake Ossa, Cameroon, at least 3367 individual African manatee vocalizations were detected such that most vocalizations were detected in the middle of the night and at dusk. Call characteristics such as fundamental frequency, duration, harmonics, subharmonics, and emphasized band were characterized for 289 high-quality tonal vocalizations with a minimum signal-to-noise ratio of 4.5 dB. African manatee vocalizations have a fundamental frequency of 4.65 ± 0.700 kHz (mean ± SD), duration of 0.181 ± 0.069 s, 97% contained harmonics, 21% contained subharmonics, and 27% had an emphasized band other than the fundamental frequency. Altogether, the structure of African manatee vocalizations is similar to other manatee species. We suggest utilizing passive acoustic monitoring to fill in the gaps in understanding the distribution and biology of African manatees.

Detection of drug resistant Mycobacterium tuberculosis by high-throughput sequencing of DNA isolated from acid fast bacilli smears.

BACKGROUND: Drug susceptibility testing for Mycobacterium tuberculosis (MTB) is difficult to perform in resource-limited settings where Acid Fast Bacilli (AFB) smears are commonly used for disease diagnosis and monitoring. We developed a simple method for extraction of MTB DNA from AFB smears for sequencing-based detection of mutations associated with resistance to all first and several second-line anti-tuberculosis drugs. METHODS: We isolated MTB DNA by boiling smear content in a Chelex solution, followed by column purification. We sequenced PCR-amplified segments of the rpoB, katG, embB, gyrA, gyrB, rpsL, and rrs genes, the inhA, eis, and pncA promoters and the entire pncA gene. RESULTS: We tested our assay on 1,208 clinically obtained AFB smears from Ghana (n = 379), Kenya (n = 517), Uganda (n = 262), and Zambia (n = 50). Coverage depth varied by target and slide smear grade, ranging from 300X to 12000X on average. Coverage of ≥20X was obtained for all targets in 870 (72%) slides overall. Mono-resistance (5.9%), multi-drug resistance (1.8%), and poly-resistance (2.4%) mutation profiles were detected in 10% of slides overall, and in over 32% of retreatment and follow-up cases. CONCLUSION: This rapid AFB smear DNA-based method for determining drug resistance may be useful for the diagnosis and surveillance of drug-resistant tuberculosis.

Tour boats affect the activity patterns of bottlenose dolphins (Tursiops truncatus) in Bocas del Toro, Panama.

Bottlenose dolphins (Tursiops truncatus) of the Bocas del Toro archipelago are targeted by the largest boat-based cetacean watching operation in Panama. Tourism is concentrated in Dolphin Bay, home to a population of resident dolphins. Previous studies have shown that tour boats elicit short-term changes in dolphin behavior and communication; however, the relationship of these responses to the local population's biology and ecology is unclear. Studying the effects of tour boats on dolphin activity patterns and behavior can provide information about the biological significance of these responses. Here, we investigated the effects of tour boat activity on bottlenose dolphin activity patterns in Bocas del Toro, Panama over 10 weeks in 2014. Markov chain models were used to assess the effect of tour boats on dolphin behavioral transition probabilities in both control and impact scenarios. Effect of tour boat interactions was quantified by comparing transition probabilities of control and impact chains. Data were also used to construct dolphin activity budgets. Markov chain analysis revealed that in the presence of tour boats, dolphins were less likely to stay socializing and were more likely to begin traveling, and less likely to begin foraging while traveling. Additionally, activity budgets for foraging decreased and traveling increased as an effect of tour boat presence. These behavioral responses are likely to have energetic costs for individuals which may ultimately result in population-level impacts. Boat operator compliance with Panamanian whale watching regulations is urgently needed to minimize potential long-term impacts on this small, genetically distinct population and to ensure the future viability of the local tourism industry.

The Antillean manatee produces broadband vocalizations with ultrasonic frequencies.

Antillean manatees produce vocalizations reported to be important for communication, but their vocal behavior throughout their geographic range is poorly understood. A SoundTrap recorder (sample rates: 288/576 kHz) was deployed in Belize to record vocalizations of wild manatees in a seagrass channel and of a young rehabilitated and released manatee in a shallow lagoon. Spectral analysis revealed broadband vocalizations with frequencies up to 150 kHz and a high proportion of calls with ultrasonic components. Ultrasonic frequency components appear prevalent in their vocal repertoire and may be important to manatee communication.

A household-level score to predict the risk of tuberculosis among contacts of patients with tuberculosis: a derivation and external validation prospective cohort study.

BACKGROUND: The epidemiological impact and cost-effectiveness of social protection and biomedical interventions for tuberculosis-affected households might be improved by risk stratification. We therefore derived and externally validated a household-level risk score to predict tuberculosis among contacts of patients with tuberculosis. METHODS: In this prospective cohort study, we recruited tuberculosis-affected households from 15 desert shanty towns in Ventanilla and 17 urban communities in Callao, Lima, Peru. Tuberculosis-affected households included index patients with a new diagnosis of tuberculosis and their contacts who reported being in the same house as the index patient for more than 6 h per week in the 2 weeks preceding index patient diagnosis. Tuberculosis-affected households were not included if the index patient had no eligible contacts or lived alone. We followed contacts until 2018 and defined household tuberculosis, the primary outcome, as any contact having any form of tuberculosis within 3 years. We used logistic regression to identify characteristics of index patients, contacts, and households that were predictive of household tuberculosis, and used these to derive and externally validate a household-level score. FINDINGS: Between Dec 12, 2007, and Dec 31, 2015, 16 505 contacts from 3 301 households in Ventanilla were included in a derivation cohort. During the 3-year follow-up, tuberculosis occurred in contacts of index patients in 430 (13%, 95% CI 12-14) households. Index patient predictors were pulmonary tuberculosis and sputum smear grade, age, and the maximum number of hours any contact had spent with the index patient while they had any cough. Household predictors were drug use, schooling of the female head of a household, and lower food spending. Contact predictors were if any of the contacts were children, number of lower-weight (body-mass index [BMI] <20·0 kg/m2) adult contacts, number of normal-weight (BMI 20·0-24·9 kg/m2) adult contacts, and number of past or present household members who previously had tuberculosis. In this derivation cohort, the score c statistic was 0·77 and the risk of household tuberculosis in the highest scoring quintile was 31% (95% CI 25-38; 65 of 211) versus 2% (95% CI 0-4; four of 231) in the lowest scoring quintile. We externally validated the risk score in a cohort of 4248 contacts from 924 households in Callao recruited between April 23, 2014, and Dec 31, 2015. During follow-up, tuberculosis occurred in contacts of index patients in 120 (13%, 95% CI 11-15) households. The score c statistic in this cohort was 0·75 and the risk of household tuberculosis in the highest scoring quintile was 28% (95% CI 21-36; 43 of 154) versus 1% (95% CI 0-5; two of 148) in the lowest scoring quintile. The highest-scoring third of households captured around 70% of all tuberculosis among contacts. A simplified risk score including only five variables performed similarly, with only a small reduction in performance. INTERPRETATION: This externally validated score will enable comprehensive biosocial, household-level interventions to be targeted to tuberculosis-affected households that are most likely to benefit. FUNDING: Wellcome Trust, Medical Research Council, Department of Health and Social Care, Department for International Development, Joint Global Health Trials consortium, Bill & Melinda Gates Foundation, Innovation for Health and Development.

Optimising fluorescein diacetate sputum smear microscopy for assessing patients with pulmonary tuberculosis.

BACKGROUND: Assessing Mycobacterium tuberculosis (TB) viability by fluorescein diacetate (FDA) microscopy can predict TB culture results, treatment response and infectiousness. However, diverse methods have been published. We aimed to optimise FDA microscopy, minimising sputum processing, biohazard and complexity for use in resource-constrained settings. METHODS AND RESULTS: Optimization: Patients with smear-positive pulmonary TB before treatment and healthy control participants provided sputa. These were divided into equal aliquots that were tested directly or after NaOH centrifuge-decontamination. Each aliquot was cultured and used to prepare slides (n = 80). FDA microscopy used: 1 or 3 drops of sputum; with/out acid-alcohol wash; with/out phenol sterilization; with 0/30/60 seconds KMnO4 quenching. Control samples all had negative culture and microscopy results. FDA microscopy had higher sensitivity when performed directly (without centrifuge-decontamination) on 1 drop of sputum (P<0.001), because 3 drops obscured microscopy. Acid-alcohol wash and KMnO4 quenching made bacilli easier to identity (P = 0.005). Phenol sterilization did not impair microscopy (P>0.1). Validation: The 2 protocols that performed best in the optimization experiments were reassessed operationally by comparing duplicate slides (n = 412) stained with KMnO4 quenching for 30 versus 60 seconds. FDA microscopy results were similar (P = 0.4) and highly reproducible, with 97% of counts agreeing within +/-1 logarithm. Storage: Smear microscopy slides and aliquots of the sputum from which they were made were stored for 4 weeks. Twice-weekly, paired slides (n = 80) were stained with freshly prepared versus stored FDA and read quantitatively. Storing sputum, microscopy slides or FDA solution at 4°C or room temperature had no effect on FDA microscopy results (all P>0.2). Cost: Material costs for each slide tested by FDA microscopy using reagents purchased locally were USD $0.05 and required the same equipment, time and skills as auramine acid-fast microscopy. CONCLUSIONS: We recommend a simple, bio-secure protocol for FDA microscopy that provides sensitive and repeatable results without requiring centrifugation.

Effect of Nasal Valve Shape on Downstream Volume, Airflow, and Pressure Drop: Importance of the Nasal Valve Revisited.

OBJECTIVES: The relative importance of the nasal valve relative to the remainder of the nasal airway remains unknown. The goal of this article was to objectively measure the shape of the nasal inlet and its effect on downstream airflow and nasal cavity volume using a physical model and a physiologic flow model. METHODS: A patient who had isolated nasal valve surgery and had pre- and postoperative computed tomography scans available for analysis was studied. Nasal inlet shape measurements, computational fluid dynamics, and nasal volume analysis were performed using the computed tomography data. In addition, a physical model was used to determine the effect of nasal obstruction on downstream soft tissue. RESULTS: The postoperative shape of the nasal inlet was improved in terms of length and degree of tortuosity. Whereas the operated-on region at the nasal inlet showed an only 25% increase in cross-sectional area postoperatively, downstream nonoperated sites in the nasal cavity revealed increases in area ranging from 33% to 51%. Computational fluid dynamics analysis showed that airway resistance decreased by 42%, and pressure drop was reduced by 43%. Intraluminal mucosal expansion was found with nasal obstruction in the physical model. CONCLUSION: By decreasing the degree of bending and length at the nasal valve, inspiratory downstream nonoperated sites of the nasal cavity showed improvement in volume and airflow, suggesting that the nasal valve could play an oversized role in modulating the aerodynamics of the airway. This was confirmed with the physical model of nasal obstruction on downstream mucosa.

Mobile phone interventions for tuberculosis should ensure access to mobile phones to enhance equity - a prospective, observational cohort study in Peruvian shantytowns.

OBJECTIVES: Mobile phone interventions have been advocated for tuberculosis care, but little is known about access of target populations to mobile phones. We studied mobile phone access among patients with tuberculosis, focusing on vulnerable patients and patients who later had adverse treatment outcomes. METHODS: In a prospective cohort study in Callao, Peru, we recruited and interviewed 2584 patients with tuberculosis between 2007 and 2013 and followed them until 2016 for adverse treatment outcomes using national treatment registers. Subsequently, we recruited a further 622 patients between 2016 and 2017. Data were analysed using logistic regression and by calculating relative risks (RR). RESULTS: Between 2007 and 2013, the proportion of the general population of Peru without mobile phone access averaged 7.8% but for patients with tuberculosis was 18% (P < 0.001). Patients without access were more likely to hold a lower socioeconomic position, suffer from food insecurity and be older than 50 years (all P < 0.01). Compared to patients with mobile phone access, patients without access at recruitment were more likely to subsequently have incomplete treatment (20% vs. 13%, RR = 1.5; P = 0.001) or an adverse treatment outcome (29% vs. 23% RR = 1.3; P = 0.006). Between 2016 and 2017, the proportion of patients without access dropped to 8.9% overall, but remained the same (18%) as in 2012 among the poorest third. CONCLUSION: Access to mobile phones among patients with tuberculosis is insufficient, and rarest in patients who are poorer and later have adverse treatment outcomes. Thus, mobile phone interventions to improve tuberculosis care may be least accessed by the priority populations for whom they are intended. Such interventions should ensure access to mobile phones to enhance equity.

Evaluation of the GeneXpert MTB/RIF in patients with presumptive tuberculous meningitis.

BACKGROUND: Meningitis caused by Mycobacterium tuberculosis is a major cause of morbidity and mortality worldwide. We evaluated the performance of cerebrospinal fluid (CSF) testing with the GeneXpert MTB/RIF assay versus traditional approaches for diagnosing tuberculosis meningitis (TBM). METHODS: Patients were adults (n = 37) presenting with suspected TBM to the Hospital Nacional Dos de Mayo, Lima, Peru, during 12 months until 1st January 2015. Each participant had a single CSF specimen that was divided into aliquots that were concurrently tested for M. tuberculosis using GeneXpert, Ziehl-Neelsen smear and culture on solid and liquid media. Drug susceptibility testing used Mycobacteria Growth Indicator Tube (MGIT 960) and the proportions method. RESULTS: 81% (30/37) of patients received a final clinical diagnosis of TBM, of whom 63% (19/30, 95% confidence intervals, CI: 44-80%) were HIV-positive. 22% (8/37, 95%CI: 9.8-38%), of patients had definite TBM. Because definite TBM was defined by positivity in any laboratory test, all laboratory tests had 100% specificity. Considering the 30 patients who had a clinical diagnosis of TBM: diagnostic sensitivity was 23% (7/30, 95%CI: 9.9-42%) for GeneXpert and was the same for all culture results combined; considerably greater than 7% (2/30, 95%CI: 0.82-22%) for microscopy; whereas all laboratory tests had poor negative predictive values (20-23%). Considering only the 8 patients with definite TBM: diagnostic sensitivity was 88% (7/8, 95%CI: 47-100%) for GeneXpert; 75% (6/8, 95%CI: 35-97%) for MGIT culture or LJ culture; 50% (4/8, 95%CI 16-84) for Ogawa culture and 25% (2/8, 95%CI: 3.2-65%) for microscopy. GeneXpert and microscopy provided same-day results, whereas culture took 20-56 days. GeneXpert provided same-day rifampicin-susceptibility results, whereas culture-based testing took 32-71 days. 38% (3/8, 95%CI: 8.5-76%) of patients with definite TBM with data had evidence of drug-resistant TB, but 73% (22/30) of all clinically diagnosed TBM (definite, probable, and possible TBM) had no drug-susceptibility results available. CONCLUSIONS: Compared with traditional culture-based methods of CSF testing, GeneXpert had similar yield and faster results for both the detection of M. tuberculosis and drug-susceptibility testing. Including use of the GeneXpert has the capacity to improve the diagnosis of TBM cases.

Comparison of Detection Limits of Fourth- and Fifth-Generation Combination HIV Antigen-Antibody, p24 Antigen, and Viral Load Assays on Diverse HIV Isolates.

Detection of acute HIV infection is critical for HIV public health and diagnostics. Clinical fourth-generation antigen (Ag)/antibody (Ab) combination (combo) and p24 Ag immunoassays have enhanced detection of acute infection compared to Ab-alone assays but require ongoing evaluation with currently circulating diverse subtypes. Genetically and geographically diverse HIV clinical isolates were used to assess clinical HIV diagnostic, blood screening, and next-generation assays. Three-hundred-member panels of 20 serially diluted well-characterized antibody-negative HIV isolates for which the researchers were blind to the results (blind panels) were distributed to manufacturers and end-user labs to assess the relative analytic sensitivity of currently approved and preapproved clinical HIV fourth-generation Ag/Ab combo or p24 Ag-alone immunoassays for the detection of diverse subtypes. The limits of detection (LODs) of virus were estimated for different subtypes relative to confirmed viral loads. Analysis of immunoassay sensitivity was benchmarked against confirmed viral load measurements on the blind panel. On the basis of the proportion of positive results on 300 observations, all Ag/Ab combo and standard sensitivity p24 Ag assays performed similarly and within half-log LODs, illustrating the similar breadth of reactivity and diagnostic utility. Ultrasensitive p24 Ag assays achieved dramatically increased sensitivities, while the rapid combo assays performed poorly. The similar performance of the different commercially available fourth-generation assays on diverse subtypes supports their use in broad geographic settings with locally circulating HIV clades and recombinant strains. Next-generation preclinical ultrasensitive p24 Ag assays achieved dramatically improved sensitivity, while rapid fourth-generation assays performed poorly for p24 Ag detection.

Lobomycosis-like disease in common bottlenose dolphins Tursiops truncatus from Belize and Mexico: bridging the gap between the Americas.

Lobomycosis and lobomycosis-like diseases (LLD) (also: paracoccidioidomycosis) are chronic cutaneous infections that affect Delphinidae in tropical and subtropical regions worldwide. In the Americas, these diseases have been relatively well-described, but gaps still exist in our understanding of their distribution across the continent. Here we report on LLD affecting inshore bottlenose dolphins Tursiops truncatus from the Caribbean waters of Belize and from the eastern tropical Pacific Ocean off the southwestern coast of Mexico. Photo-identification and catalog data gathered between 1992 and 2017 for 371 and 41 individuals, respectively from Belize and Mexico, were examined for the presence of LLD. In Belize, 5 free-ranging and 1 stranded dolphin were found positive in at least 3 communities with the highest prevalence in the south. In Guerrero, Mexico, 4 inshore bottlenose dolphins sighted in 2014-2017 were affected by LLD. These data highlight the need for histological and molecular studies to confirm the etiological agent. Additionally, we document a single case of LLD in an adult Atlantic spotted dolphin Stenella frontalis in southern Belize, the first report in this species. The role of environmental and anthropogenic factors in the occurrence, severity, and epidemiology of LLD in South and Central America requires further investigation.