Demographic and Clinical Characteristics of Mpox Patients Attending an STD Clinic in Lisbon.

Mpox is a viral disease caused by the monkeypox virus, which marked the year of 2022 with a global outbreak. While previously considered to be a zoonosis of almost exclusive animal-to-human transmission, the current outbreak has been attributed to human-to-human transmission, particularly sexual transmission. As a new sexually transmissible disease, we studied the epidemiological and clinical features, as well as the concomitant occurrence of other sexually transmissible diseases, treatment approach, and outcome of our 291 patients, in the current outbreak. We found a total of 169 concomitant sexually transmissible infections of bacterial and viral origins, corresponding to 107 patients. Neisseria gonorrhoeae was the most common agent, particularly in the anal location. With this work, we emphasize the need for a thorough epidemiological and medical history, as well as a concomitant complete laboratorial screening for other STIs in patients with confirmed or suspected mpox.

Viral genetic clustering and transmission dynamics of the 2022 mpox outbreak in Portugal.

Pathogen genome sequencing during epidemics enhances our ability to identify and understand suspected clusters and investigate their relationships. Here, we combine genomic and epidemiological data of the 2022 mpox outbreak to better understand early viral spread, diversification and transmission dynamics. By sequencing 52% of the confirmed cases in Portugal, we identified the mpox virus sublineages with the highest impact on case numbers and fitted them into a global context, finding evidence that several international sublineages probably emerged or spread early in Portugal. We estimated a 62% infection reporting rate and that 1.3% of the population of men who have sex with men in Portugal were infected. We infer the critical role played by sexual networks and superspreader gatherings, such as sauna attendance, in the dissemination of mpox virus. Overall, our findings highlight genomic epidemiology as a tool for the real-time monitoring and control of mpox epidemics, and can guide future vaccine policy in a highly susceptible population.

Ongoing monkeypox virus outbreak, Portugal, 29 April to 23 May 2022.

Up to 27 May 2022, Portugal has detected 96 confirmed cases of monkeypox. We describe 27 confirmed cases (median age: 33 years (range: 22-51); all males), with an earliest symptom onset date of 29 April. Almost all cases (n = 25) live in the Lisbon and Tagus Valley health region. Most cases were neither part of identified transmission chains, nor linked to travel or had contact with symptomatic persons or with animals, suggesting the possible previously undetected spread of monkeypox.

Phylogenomic characterization and signs of microevolution in the 2022 multi-country outbreak of monkeypox virus.

The largest monkeypox virus (MPXV) outbreak described so far in non-endemic countries was identified in May 2022 (refs. 1-6). In this study, shotgun metagenomics allowed the rapid reconstruction and phylogenomic characterization of the first MPXV outbreak genome sequences, showing that this MPXV belongs to clade 3 and that the outbreak most likely has a single origin. Although 2022 MPXV (lineage B.1) clustered with 2018-2019 cases linked to an endemic country, it segregates in a divergent phylogenetic branch, likely reflecting continuous accelerated evolution. An in-depth mutational analysis suggests the action of host APOBEC3 in viral evolution as well as signs of potential MPXV human adaptation in ongoing microevolution. Our findings also indicate that genome sequencing may provide resolution to track the spread and transmission of this presumably slow-evolving double-stranded DNA virus.

The Population Structure of Borrelia lusitaniae Is Reflected by a Population Division of Its Ixodes Vector.

Populations of vector-borne pathogens are shaped by the distribution and movement of vector and reservoir hosts. To study what impact host and vector association have on tick-borne pathogens, we investigated the population structure of Borrelia lusitaniae using multilocus sequence typing (MLST). Novel sequences were acquired from questing ticks collected in multiple North African and European locations and were supplemented by publicly available sequences at the Borrelia Pubmlst database (accessed on 11 February 2020). Population structure of B. lusitaniae was inferred using clustering and network analyses. Maximum likelihood phylogenies for two molecular tick markers (the mitochondrial 16S rRNA locus and a nuclear locus, Tick-receptor of outer surface protein A, trospA) were used to confirm the morphological species identification of collected ticks. Our results confirmed that B. lusitaniae does indeed form two distinguishable populations: one containing mostly European samples and the other mostly Portuguese and North African samples. Of interest, Portuguese samples clustered largely based on being from north (European) or south (North African) of the river Targus. As two different Ixodes species (i.e., I. ricinus and I. inopinatus) may vector Borrelia in these regions, reference samples were included for I. inopinatus but did not form monophyletic clades in either tree, suggesting some misidentification. Even so, the trospA phylogeny showed a monophyletic clade containing tick samples from Northern Africa and Portugal south of the river Tagus suggesting a population division in Ixodes on this locus. The pattern mirrored the clustering of B. lusitaniae samples, suggesting a potential co-evolution between tick and Borrelia populations that deserve further investigation.

Borrelia lusitaniae Infection Mimicking Headache, Neurologic Deficits, and Cerebrospinal Fluid Lymphocytosis.

Headache with neurologic deficits and cerebrospinal fluid lymphocytosis (HaNDL) is a rare headache syndrome included in the Classification of Headache of the International Headache Society as a "headache attributed to non-infectious inflammatory intracranial disease." We report one 15-year-old patient with clinical history and cerebrospinal fluid findings compatible with the diagnosis of HaNDL in whom Borrelia lusitaniae was identified in cerebrospinal fluid by polymerase chain reaction.

Paralytic shellfish poisoning due to ingestion of contaminated mussels: A 2018 case report in Caparica (Portugal).

In Portugal, the potent paralytic shellfish toxins (PSTs) have appeared irregularly since the onset of a national monitoring program for marine biotoxins in 1986. In years where high contamination levels were attained in bivalves, sporadic cases of human poisonings have been recorded, as in 1994 and 2007. The reappearance of high contamination levels led to the appearance of new cases during the autumn of 2018. This study details toxin ingestion, symptomatology and toxin elimination and metabolization in the fluids of two patients, who ingested mussels from the Portuguese southwest coast and required hospitalization due to the severity of symptoms. Toxin elimination was confirmed by ELISA in plasma and urine samples. In mussel samples, the toxin profile obtained by HPLC-FLD displayed a wide diversity of toxins, typical of Gymnodinum catenatum ingestion. However, in the urine samples, the toxin profile was reduced to B1 and dcSTX. Abundant compounds in mussels having an O-sulphate at C11, such as C1+2 and dcGTX2+3, were absent in urine. In plasma, PSTs were not detected by HPLC-FLD. Calculated toxin ingestion, resulting from consumption of an estimated 200-g portion, was in the range of 104-120 µg STX eq./kg b. w.

Core genome phylogenetic analysis of the avian associated Borrelia turdi indicates a close relationship to Borrelia garinii.

Borrelia burgdorferi sensu lato comprises a species complex of tick-transmitted bacteria that includes the agents of human Lyme borreliosis. Borrelia turdi is a genospecies of this complex that exists in cryptic transmission cycles mainly between ornithophilic tick vectors and their avian hosts. The species has been originally discovered in avian transmission cycles in Asia but has increasingly been found in Europe. Next generation sequencing was used to sequence the genome of B. turdi isolates obtained from ticks feeding on birds in Portugal to better understand the evolution and phylogenetic relationship of this avian and ornithophilic tick-associated genospecies. Here we use draft genomes of these B. turdi isolates for comparative analysis and to determine the taxonomic position within the B. burgdorferi s.l. species complex. The main chromosomes showed a maximum similarity of 93% to other Borrelia species whilst most plasmids had lower similarities. All three isolates had nine or 10 plasmids and, interestingly, one plasmid with a novel partitioning protein; this plasmid was termed lp30. Phylogenetic analysis of multilocus sequence typing housekeeping genes and 113 single copy orthologous genes revealed that the isolates clustered according to their classification as B. turdi. In phylogenies generated from these 113 genes the isolates cluster together with other Eurasian genospecies and form a sister clade to the avian associated B. garinii and the rodent associated B. bavariensis. These findings show that Borrelia species maintained in cryptic ecological cycles need to be included to fully understand the complex ecology and evolutionary history of this bacterial species complex.

Characterization Through Multilocus Sequence Analysis of Borrelia turdi Isolates from Portugal.

Borrelia turdi is a spirochete from the Borrelia burgdorferi complex, first reported in Japan, that has been increasingly detected in Europe. This genospecies is mostly associated with avian hosts and their ornithophilic ticks such as Ixodes frontalis. In this study, we isolated B. turdi from five I. frontalis feeding on Turdus merula, Turdus philomelos, Parus major and Troglodytes troglodytes, and one Ixodes ricinus feeding on a T. merula in Portugal. These isolates were genetically characterised according to their 5S-23S rRNA intergenic spacer, 16S rRNA and through typing of seven housekeeping genes (multilocus sequence typing). Multilocus sequence analyses revealed that the strains isolated in our study, although belonging to B. turdi genospecies, are not identical to the B. turdi reference strain Ya501. Instead, our strains are separated into a clear defined group, suggesting that the European samples diverged genetically from the strain originally detected in Japan. Population analysis of 5S-23S rRNA sequences can further resolve subpopulations within B. turdi, but more samples from a large geographical scale and host range would be needed to assess potential phylogeographical patterns within this genospecies.

Ticks on passerines from the Archipelago of the Azores as hosts of borreliae and rickettsiae.

We examined the presence of borreliae and rickettsiae bacteria in ticks from wild passerine birds on three islands of the Archipelago of the Azores, the westernmost region of Palearctic. A total of 266 birds belonging to eight species from seven families were examined on São Miguel, Santa Maria and Graciosa islands in 2013. Ticks collected from these birds consisted of 55 Ixodes frontalis (22 larvae, 32 nymphs, 1 adult female) and 16 Haemaphysalis punctata nymphs. Turdus merula and Erithacus rubecula were the birds most infested with both tick species. Three T. merula in Santa Maria were infested with 4 I. frontalis infected with Borrelia turdi. No rickettsiae were found in the ticks. We report for the first time the presence of I. frontalis and B. turdi on the Azores islands and we showed that the spatial distribution reaches further west than previously thought.

The medical and veterinary role of Ornithodoros erraticus complex ticks (Acari: Ixodida) on the Iberian Peninsula.

Argasid ticks of the Ornithodoros erraticus complex are associated with traditional pig-farming practices on the Iberian Peninsula and are also found elsewhere in North Africa, West Africa, and western Asia. The ticks associated with pig farming on the Iberian Peninsula are the only biological vectors of African swine fever virus (ASFV) known to occur in Europe, and their ecology makes them an extremely effective reservoir of both ASFV and the Borrelia species which cause tick-borne relapsing fever (TBRF) in humans. The recent reappearance of ASFV in the European Union, coupled with evidence that Portuguese tick populations continue to harbor Borrelia despite a lack of confirmed human infections, suggest that these populations merit closer attention. In Portugal, a series of surveys over the last twenty-five years indicates that the number of farm sites with tick infestations has declined and suggest that populations are sensitive to changes in farm management, particularly the use of modern pig housing. Various technologies have been suggested for the control of farm-associated Ornithodoros ticks and related species but, in our opinion, farm management changes are still the most effective strategy for population control. Furthermore, we suggest that this species could probably be eradicated from Iberian pig farms.

Rickettsia lusitaniae sp. nov. isolated from the soft tick Ornithodoros erraticus (Acarina: Argasidae).

In this study a novel Rickettsia from the spotted fever group, isolated from Ornithodoros erraticus soft ticks collected from pigpens in the south of Portugal, is described. After initial screening revealed Rickettsia-positive ticks, isolation attempts were then performed. Successful isolates were achieved by shell-vial technique using Vero E6 cells at 28°C. Molecular characterization of the isolate was performed based on analysis of five rickettsial genes gltA, ompA, ompB, sca1 and htr with their subsequent concatenation along with other rickettsial species resulting in a clustering of the new isolate with Rickettsia felis and Rickettsia hoogstraalii. The degree of nucleotide sequence similarity with other rickettsiae fulfills the criteria for classification of our isolate as a novel species. The name Rickettsia lusitaniae sp. nov. (=CEVDI PoTiRo) is proposed for this new species found in O. erraticus.

Immune reactivity to dengue and Aedes albopictus mosquitoes in the population from Macao, China, before dengue occurrence.

A serological survey was conducted in Macao, China, in 753 individuals, with the objective of looking for antibodies to the mosquito, Aedes albopictus (Skuse 1894) (Diptera: Culicidae), and to dengue, before the occurrence of any autochthonous dengue cases. Blood samples were collected at several public health services, a questionnaire was answered, and enzyme-linked immunosorbant assay (ELISA) and Western blot techniques were performed with extracts of mosquito head and thorax (HT). Anti-Aedes albopictus IgG antibodies were present in titres 1:10(2)-1:10(3) in 9%, and in titres 1:10(4)-1:10(5) in 42% of the sera tested. This reactivity was more frequent (59%) in the population which had resided only in Macao in the 2 years previous to the survey, as opposed to those that had also resided in other areas (50%). From the 230 reactive sera selected for immunoblot, 48 (21%) reacted with a wide range of proteins from above 224 kDa to 21 kDa, with different patterns according to individual sera. Proteins in the intervals 35.3-28.7 kDa and 28.7-21.1 kDa were labelled by the greatest number of sera, 15 and 19 respectively. The presence of anti-Aedes albopictus antibodies presented a statistical relation to skin reaction to mosquito bites, but immunoblot patterns did not. Anti-dengue IgG antibodies were found in 48% of the subjects, with a higher proportion in people who had resided out of Macao, or who were nationals from dengue-endemic neighboring countries. Anti-dengue reactivity was in agreement with anti-mosquito reactivity in half of the population. It would be interesting to see if this proportion has changed since dengue became endemic in Macao in 2001.

Francisella-like endosymbiont in Dermacentor reticulatus collected in Portugal.

In Portugal, recent studies have confirmed the presence of Francisella tularensis in Dermacentor reticulatus. Bacterial endosymbionts with significant homology to F. tularensis have been described in several species of ticks. In this work we identified Francisella-like endosymbionts in D. reticulatus ticks (39%), confirming the presence of these bacteria in Portugal. This finding should be considered in future studies using molecular approaches to detect Francisella prevalence in ticks and environmental samples.

Coinfections of Rickettsia slovaca and Rickettsia helvetica with Borrelia lusitaniae in ticks collected in a Safari Park, Portugal.

Borrelia and Rickettsia bacteria are the most important tick-borne agents causing disease in Portugal. Identification and characterization of these circulating agents, mainly in recreational areas, is crucial for the development of preventive measures in response to the gradually increasing exposure of humans to tick vectors. A total of 677 questing ticks including Dermacentor marginatus, Rhipicephalus sanguineus, Ixodes ricinus, Hyalomma lusitanicum, H. marginatum, and Haemaphysalis punctata were collected in a Safari Park in Alentejo, Portugal, to investigate the prevalences of infection and characterize Borrelia and Rickettsia species. From a total of 371 ticks tested by PCR for Borrelia burgdorferi sensu lato (s.l.), of which 247 were tested for Rickettsia, an infection prevalence of 18.3% was found for B. lusitaniae and 55.1% for Rickettsia spp. Sequence analysis of positive amplicons identified the presence of B. lusitaniae (18.3%), R. monacensis strain IRS3 (51.7%), and R. helvetica (48.3%) in I. ricinus. R. slovaca (41.5%), R. raoultii (58.5%), and also B. lusitaniae (21%) were identified in D. marginatus ticks. One (5.9%) H. lusitanicum was infected with B. lusitaniae, and R. massiliae was found in one Rhipicephalus sanguineus. Coinfection was found in 7 (20%) I. ricinus and 34 (23.3%) D. marginatus ticks. We report, for the first time, simultaneous infection with R. helvetica and B. lusitaniae and also R. slovaca, the agent of TIBOLA/DEBONEL, with B. lusitaniae. Additionally, 6 isolates of B. lusitaniae were established, and isolates of Rickettsia were also obtained for the detected species using tick macerates cultured in mammalian and mosquito cell lines. This report describes the detection and isolation of tick-borne agents from a Portuguese Safari Park, highlighting the increased likelihood of infection with multiple agents to potential visitors or staff.

Molecular characterization of a new isolate of Borrelia lusitaniae derived from Apodemus sylvaticus in Portugal.

A total of 196 small mammals were collected in Portugal and tested for Borrelia burgdorferi sensu lato. Tissue samples were taken from each animal and cultured in Barbour-Stoenner-Kelly (BSK)-II medium. The single strain of spirochete isolated was confirmed as Borrelia lusitaniae by genetic analyses. This is the first report of B. lusitaniae isolated from Apodemus sylvaticus.

Detection of Borrelia lusitaniae, Rickettsia sp. IRS3, Rickettsia monacensis, and Anaplasma phagocytophilum in Ixodes ricinus collected in Madeira Island, Portugal.

A total of 300 Ixodes ricinus ticks were tested by polymerase chain reaction (PCR) for the presence of Borrelia spp., Rickettsia spp., and Anaplasma phagocytophilum. Sequence analysis demonstrated 8 (2.7%) ticks infected with B. lusitaniae, 60 (20%) with Rickettsia spp., and 1 (0.3%) with A. phagocytophilum. Seven (2.3%) ticks were coinfected with B. lusitaniae and Rickettsia spp., 2 (0.6%) with R. monacensis, and 5 (1.7%) with Rickettsia sp. IRS3. The results of this study suggest simultaneous transmission of multiple tick-borne agents on Madeira Island, Portugal.