ABSTRACTCOVID-19, caused by SARS-CoV-2 virus infection, remains a public health concern in many countries. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid and cost-effective alternative test for COVID-19 diagnosis. In this study, we developed and evaluated a real-time RT-LAMP (rRT-LAMP) assay coupled with a melting curve analysis to detect SARS-CoV-2. The reaction was carried out in a real-time thermal cycler at 63 °C for 45 min to amplify the region of SARS-CoV-2 orf8; real-time monitoring of amplification was performed by fluorescence detection. The performance was assessed by comparing it to a real-time reverse transcription-polymerase reaction (rRT-PCR) as a reference. The rRT-LAMP could detect as few as 15 copies of SARS-CoV-2 RNA per reaction. Positive results appeared within 30 min, while the melting-temperature analysis could verify the amplification specificity. No positive results from non-SARS-CoV-2 templates and no mis-amplification were observed. The comparative analysis using 262 RNA extracted from nasopharyngeal swab samples revealed the overall accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive values (NPV) of the rRT-LAMP at 88.55% (95% CI: 77.52–100%), 84.13% (95% CI: 71.56–98.27%), 100% (95% CI: 78.38–100%), 100% (95% CI: 85.06–100%), and 70.87% (95% CI: 55.55–89.11%), respectively. The greatest sensitivity was as high as 98–100% for specimens with threshold rRT-PCR cycle (Ct) values of less than 30 cycles. Overall, this rRT-LAMP showed good performance for the rapid detection of SARS-CoV-2. It is proposed as a potential method for real-time amplification detection, offering increased laboratory capacity for SARS-CoV-2 testing in a cost-effective and timely manner.

ABSTRACTThe identification of Leishmania species is crucial for eco-epidemiological purposes and may be useful for clinical management. Notably, archived smear slides can be valuable in this scenario. ITS-1 PCR followed by sequencing was used to identify Leishmania species from archived lesion smears of patients with suspected cutaneous leishmaniasis in Santarem city, Para State, Brazil. A total of 44 microscopically positive lesion smears were analyzed, of which 34 yielded positive PCR results. Of these, 22 were subjected to Sanger sequencing and 15 were successfully sequenced, revealing five Leishmania species. This study demonstrates the applicability of molecular testing on archived samples. ITS-1 sequencing effectively differentiated between species, revealing significant diversity of Leishmania in the Brazilian Amazon.

ABSTRACT To assess SARS-CoV-2 reinfection incidence in the post-vaccination period, we carried out a prospective cohort study of blood donors from Amazonas and Sao Paulo States, Brazil. Anti-nucleocapsid immunoglobulin (IgG anti-N) tests carried out by blood centers in 2020 were used to identify previous SARS-CoV-2 infections in blood donors and divide them into two groups: prior infection (n=386) and no prior infection (n=111). From March 2021 to January 2022, donors were followed up for six months, during which IgG anti-N and real-time reverse transcription polymerase chain reaction tests were performed every two months to detect SARS-CoV-2 infections. Symptoms and vaccination status were also recorded. Most participants (93.6%) received at least one COVID-19 vaccine dose. Reinfection incidence in the prior infection group equaled 1.39 per 100 person-months (95% CI: 0.90–2.06), in comparison to 2.68 per 100 person-months (95% CI: 1.28–4.93) for new infections in those without prior infection. The incidence risk ratio showed no significant association (0.52, 95% CI: 0.25–1.13). However, prior infection significantly increased the probability of remaining uninfected (Log-rank: p=0.009). Most reinfections (84%) showed no symptoms and occurred post-vaccination during the Delta and Omicron waves. IgG anti-N seroprevalence decreased in the prior infection group (from 35.5% at baseline to 22.5% after six months, p=0.003). Despite no significant incidence risk ratio differences, donors with prior infection had lower infection rates and a higher likelihood of remaining uninfected. Persistent post-vaccination asymptomatic infections emphasize the need for ongoing prevention, genomic surveillance, and booster programs to address emerging variants and protect vulnerable populations.

ABSTRACTRhodococcus equi is an opportunistic soil-borne bacterium that is eliminated in feces of multi-host animals. An increase in multidrug-resistant R. equi isolates has been reported in humans and domestic animals, and it has been hypothesized that the treatment of R. equi in foals could increase the selective pressure on multidrug-resistant isolates and favor human infections by resistant isolates. We investigated the in vitro antimicrobial susceptibility/resistance of 41 R. equi strains from humans, which were isolated from patients with pulmonary signs, using 19 antimicrobials from 10 distinct classes, recommended exclusively to humans, recommended exclusively to domestic animals and used in both. All isolates were subjected to mass spectrometry and identified as R. equi. Among the antimicrobials used exclusively in humans, tigecycline and vancomycin showed 100% efficacy. Amikacin, amoxicillin/clavulanic acid, imipenem, levofloxacin, clarithromycin, rifampin, ciprofloxacin, and gentamicin, used in both humans and animals, revealed high efficacy (97–100%). Conversely, a higher frequency of isolates was resistant to penicillin (87.8%) and trimethoprim/sulfamethoxazole (43.9%), which are used in both humans and animals. Among the antimicrobials used only in animals, isolates were resistant to florfenicol (46.4%), ceftiofur (17.1%), and enrofloxacin (2.5%). Multidrug resistance was observed in 34% of isolates. The identification of drug-resistant R. equi isolated from humans used exclusively in animals is circumstantial evidence of the pathogen transmission from domestic animals to humans. This study contributes to the molecular identification of Rhodococcus species from humans and to the epidemiological vigilance of the multidrug-resistant isolates.

ABSTRACTMosquitoes (Diptera: Culicidae) are arthropods of medical importance because they can carry arboviruses. High-throughput sequencing (HTS) technology and metagenomic approaches conducted in mosquitoes have contributed to the discovery of many insect-specific viruses (ISVs), which have the potential to affect their vector competence. Mosquitoes were collected in urban parks in Sao Paulo city, Brazil and 20 pools with female mosquitoes were subjected to HTS by HiSeq 2500 sequencing system (Illumina). Long viral sequences (1,585-6,701 base pairs) were recovered from two pools of Aedes scapularis. BLASTx analyses revealed they had greater identity with segment L and S of Salarivirus and segment M of Furrundu phlebovirus, which encode, respectively, the RNA-dependent RNA polymerase (RdRd), the nucleocapsid protein, and a polyprotein. Phylogenetic tree of the segment L and S of the Phenuiviridae Family showed our sequences grouped with unverified sequences of Furrundu phlebovirus, an unclassified ISV that belongs to the Hareavirales Order and was first reported in mosquitoes in the Brazilian Pantanal, the largest natural tropical wetland worldwide. We report the second detection of Furrundu phlebovirus in mosquitoes collected in urban parks, showing it could be in mosquitoes from natural places and in green areas in urban cities. We conclude that Furrundu phlebovirus possibly occurs in Aedes scapularis in green areas, in Sao Paulo. Further studies should elucidate the role of this virus in the vector competence of Aedes scapularis and its interaction with different arboviruses.

ABSTRACT Mycobacterium marinum is a nontuberculous mycobacterium and an opportunistic pathogen which infects humans. Here, we report a case of an 87-year-old female who had extensive verrucous skin lesions on her upper limbs for 18 months. The patient was diagnosed with Mycobacterium marinum infection by pathology and quantitative polymerase chain reaction (qPCR) assay of skin tissue. After five months of oral rifampicin and clarithromycin, the skin lesions regressed completely.

ABSTRACTRickettsia species are Gram-negative, pleomorphic coccobacilli that are obligate intracellular pathogens transmitted by arthropod vectors such as ticks. Among them, Rickettsia conorii, the causative agent of Mediterranean spotted fever (MSF), is endemic in many Mediterranean countries, including Turkey. This case series describes three patients from Balıkesir, Turkey, who developed high-grade fever, generalized maculopapular rash involving the palms and feet soles, arthralgia, and necrotic eschars (tache noire) at the tick bite sites. All cases occurred during summer and had documented exposure to Rhipicephalus sanguineus. Laboratory evaluations ruled out other tick-borne diseases, while real-time PCR performed on skin biopsy samples confirmed Rickettsia spp. Subsequent DNA sequencing of the gltA and ompA gene regions enabled species identification. Additionally, serological tests showed a significant rise in IgM and IgG antibody titers reacting with Rickettsia conorii antigen by indirect immunofluorescence assay. All patients were treated with doxycycline and recovered without complications. This case series highlights the importance of considering rickettsial infections in the differential diagnosis of febrile patients with rash and recent tick exposure, especially in endemic regions during warm seasons.

Noroviruses are highly infectious, genetically diverse viruses. Global outbreaks occur frequently, making molecular surveillance important for infection monitoring. This cross-sectional descriptive study aimed to monitor cases of norovirus gastroenteritis in the Brazilian Amazon. Fecal samples were tested by immunoenzymatic assay, RT-PCR and genetic sequencing for the ORF1/ORF2 and protease regions. Bayesian inference with a molecular clock was employed to construct the phylogeny. The norovirus prevalence was 25.8%, with a higher positivity rate among children aged 0-24 months. Genogroup GII accounted for 98.1% of the sequenced samples, while GI accounted for 1.9% of them. The GII.P16/GII.4 genotype was the most prevalent, with an evolution rate of 2.87x10-3 and TMRCA estimated in 2012. This study demonstrates that norovirus is a primary causative agent of gastroenteritis and provides data on viral genetic diversity that may facilitate infection surveillance and vaccine development.

The worldwide monkeypox (mpox) outbreak in 2022 showed a high frequency of sexually transmitted infections (STI). A cross-sectional study was carried out using secondary data from the Brazilian official mpox surveillance systems. A total of 10,169 mpox cases were identified, with a median age of 32 years. Among them, 92.3% were male at birth and 57.5% were men who have sex with other men (MSM). Approximately 11% were diagnosed with STI, including 5.8% with syphilis and 2.5% with genital herpes. Individuals aged from 25 to 34 years, MSM, individuals with HIV-positive status, and those manifesting skin eruptions or penile edema were associated with STI. Laboratory investigation for mpox must be implemented as a priority in STI clinics (especially for MSM) to mitigate neglected cases, ensure appropriate treatments, and prevent misdiagnoses.