SARS-CoV-2: preliminary study of infected human nasopharyngeal tissue by high resolution microscopy.

BACKGROUND: The novel coronavirus SARS-CoV-2 is the etiological agent of COVID-19. This virus has become one of the most dangerous in recent times with a very high rate of transmission. At present, several publications show the typical crown-shape of the novel coronavirus grown in cell cultures. However, an integral ultramicroscopy study done directly from clinical specimens has not been published. METHODS: Nasopharyngeal swabs were collected from 12 Cuban individuals, six asymptomatic and RT-PCR negative (negative control) and six others from a COVID-19 symptomatic and RT-PCR positive for SARS CoV-2. Samples were treated with an aldehyde solution and processed by scanning electron microscopy (SEM), confocal microscopy (CM) and, atomic force microscopy. Improvement and segmentation of coronavirus images were performed by a novel mathematical image enhancement algorithm. RESULTS: The images of the negative control sample showed the characteristic healthy microvilli morphology at the apical region of the nasal epithelial cells. As expected, they do not display virus-like structures. The images of the positive sample showed characteristic coronavirus-like particles and evident destruction of microvilli. In some regions, virions budding through the cell membrane were observed. Microvilli destruction could explain the anosmia reported by some patients. Virus-particles emerging from the cell-surface with a variable size ranging from 80 to 400 nm were observed by SEM. Viral antigen was identified in the apical cells zone by CM. CONCLUSIONS: The integral microscopy study showed that SARS-CoV-2 has a similar image to SARS-CoV. The application of several high-resolution microscopy techniques to nasopharyngeal samples awaits future use.

Macrolide-resistant Mycoplasma genitalium infections in Cuban patients: an underestimated health problem.

BACKGROUND: The increasing prevalence of macrolide resistant Mycoplasma genitalium is a major concern worldwide. In Cuba, several cases of clinical treatment failure with 1 g single dose and extended azithromycin regimen have been detected and the aim of the present investigation was to retrospectively determine the prevalence of macrolide-resistance mediating mutations (MRMM) in M. genitalium-positive samples conserved at the Cuban National Reference Laboratory of Mycoplasma Research between 2009 and 2016. METHODS: A total of 280 positive DNA extracts were analysed by a 5' nuclease assay for detection of M. genitalium MRMM. Ten urogenital specimens from patients with azithromycin treatment failure and MRMM were inoculated in Vero cell to obtain the isolates for subsequent determination of antimicrobial susceptibility. RESULTS: The overall prevalence of MRMM was 32%. No MRMM was detected in samples collected between 2009 and 2013 but since 2014 a dramatic increase to 90% (95% CI, 76-96%) in 2016 was seen. Three new M. genitalium isolates were isolated in Vero cell cultures and confirmed phenotypic resistance to macrolides in a cell-culture assisted susceptibility test. Preliminary observations suggest that combination therapy with levofloxacin and doxycycline may represent an affordable option for treatment of macrolide resistant M. genitalium infections. CONCLUSIONS: This investigation showed the rapid emergence and high prevalence of MRMM in M. genitalium-infected patients in Cuba and confirmed the phenotypic resistance in isolates carrying MRMM. We suggest that Cuban guidelines for sexually transmitted infections are modified to include testing for M. genitalium and detection of MRMM in patients with failure of syndromic treatment, to ensure that in these cases, the treatment will be guided by etiologic diagnosis.

Mycoplasma hominis in Cuban Trichomonas vaginalis isolates: association with parasite genetic polymorphism.

Trichomonas vaginalis can be naturally infected with intracellular Mycoplasma hominis. This bacterial infection may have implications for trichomonal virulence and disease pathogenesis. The objective of the study was to report the presence of M. hominis in Cuban T. vaginalis isolates and to describe the association between the phenotype M. hominis infected with RAPD genetic polymorphism of T. vaginalis. The Random Amplified Polymorphic DNA (RAPD) technique was used to determine genetic differences among 40 isolates of T. vaginalis using a panel of 30 random primers and these genetic data were correlated with the infection of isolates with M. hominis. The trees drawn based on RAPD data showed no relations with metronidazole susceptibility and significantly association with the presence of M. hominis (P=0.043), which demonstrates the existence of concordance between the genetic relatedness and the presence of M. hominis in T. vaginalis isolates. This result could point to a predisposition of T. vaginalis for the bacterial enters and/or survival.

SARS-CoV-2: theoretical analysis of the proposed algorithms to the enhancement and segmentation of high-resolution microscopy images-Part II.

Today is a reality that the novel coronavirus SARS-Cov-2 has become a global pandemic. For this reason, the study of real microscopic images of this coronavirus is of great importance, as it allows us to carry out a more precise research on it. However, as we pointed out in a former paper as reported by Roberto Rodríguez (SARS-CoV-2: Enhancement and Segmentation of High-Resolution Microscopy Images. Part I", Sent to Signal, Image and Video Processing Video Processing, Springer, New York, 2020), many times these microscopic images present some blurring problems, which are always susceptible to be improved. The aim of this work is to carry out a theoretical analysis of the proposed algorithms to enhancement and segmentation of these microscopic images, which is important for the design and development of future algorithms before new epidemics.

Defining Fluoroquinolone Resistance-Mediating Mutations from Non-Resistance Polymorphisms in Mycoplasma hominis Topoisomerases.

Often dismissed as a commensal, Mycoplasma hominis is an increasingly prominent target of research due to its role in septic arthritis and organ transplant failure in immunosuppressed patients, particularly lung transplantation. As a mollicute, its highly reductive genome and structure render it refractile to most forms of treatment and growing levels of resistance to the few sources of treatment left, such as fluoroquinolones. We examined antimicrobial susceptibility (AST) to fluoroquinolones on 72 isolates and observed resistance in three (4.1%), with corresponding mutations in the quinolone resistance-determining region (QRDR) of S83L or E87G in gyrA and S81I or E85V in parC. However, there were high levels of polymorphism identified between all isolates outside of the QRDR, indicating caution for a genomics-led approach for resistance screening, particularly as we observed a further two quinolone-susceptible isolates solely containing gyrA mutation S83L. However, both isolates spontaneously developed a second spontaneous E85K parC mutation and resistance following prolonged incubation in 4 mg/L levofloxacin for an extra 24-48 h. Continued AST surveillance and investigation is required to understand how gyrA QRDR mutations predispose M. hominis to rapid spontaneous mutation and fluoroquinolone resistance, absent from other susceptible isolates. The unusually high prevalence of polymorphisms in M. hominis also warrants increased genomics' surveillance.

SARS-CoV-2: enhancement and segmentation of high-resolution microscopy images-Part I.

Possibly, and due to poor eating habits and unhealthy lifestyle, many viruses are transmitted to human people. Such is the case, of the novel coronavirus SARS-Cov-2, which has expanded of exponential way, practically, to whole world population. For this reason, the enhancement of real microscopic images of this coronavirus is of great importance. Of this way, one can highlight the S-spikes and visualizing those areas that show a high density, which are related to active zones of viral germination and major spread of the virus. The SARS-Cov-2 images were captured from nasopharyngeal samples of Cuban symptomatic individuals (RT-PCR positives for SARS-CoV-2) and processed via scanning electron microscopy. However, many times these microscopic images present some blurring problems, and the S-spikes do not look well defined. Therefore, the aim of this work is to propose new computational methods to carry out enhancement and segmentation of SARS-Cov-2 high-resolution microscopic images. The proposed strategy obtained very satisfactory results, and we validated its performance, together with specialist physicians, on a set of 1005 images. Due to the importance of the obtained results, this first work will be addressed to the application of the proposed algorithm. A second paper will deeply analyze the theory related to these algorithms.

First detection and characterization of macrolide-resistant Mycoplasma pneumoniae strains in Cuba.

OBJECTIVES: Mycoplasma pneumoniae is a common cause of community-acquired pneumonia in humans. Treatment of infections can be complicated by the occurrence of macrolide resistant strains. The study was conducted to evaluate the presence of resistant strains in Cuba and to determine the corresponding genotypes. METHODS: DNA of M. pneumoniae isolates and positive respiratory tract specimens collected in the years 2012 and 2017 were tested for resistance-associated mutations of 23S rRNA. In addition, strain types (P1 and MLVA) were determined. RESULTS: Macrolide resistance mutations were confirmed in 5 out of 27 strains (18.5%). Whereas both P1 subtypes 1 and 2 as well variants V2a and V2c were identified, only two MLVA types (4/5/7/2 and 3/5/6/2) could be found. CONCLUSIONS: During both sampling years, circulation of macrolide resistant strains was demonstrated. No association of resistance with a particular P1/MLVA type was found. Future longitudinal sampling to monitor prevalence of macrolide resistance of M. pneumoniae is recommended to verify the resistance pattern of this important pathogen of human respiratory tract infections.

Mycoplasma genitalium infections in Cuba: surveillance of urogenital syndromes, 2014-2015.

Mycoplasma genitalium is an emerging sexually transmitted pathogen implicated in urethritis in men and several inflammatory reproductive tract syndromes in women. The prevalence of M. genitalium infections in Cuban patients with urogenital syndromes is unknown. The aim of this study was to analyse the prevalence of M. genitalium infection in sexually-active Cuban men and women with urogenital syndromes as a part of aetiological surveillance of urogenital syndromes in Cuba. Samples from men and women with urogenital syndromes submitted to the Mycoplasma Reference Laboratory for mycoplasma diagnosis from 1 January 2014 to 1 June 2015 were analysed by polymerase chain reaction (PCR) for detection of M. genitalium. A total of 971 samples were received and processed. Of the patients tested, 5.7% (47/824) of women and 27.9% (41/147) of men were positive for M. genitalium. This paper presents the largest study of M. genitalium infections among Cuban patients with urogenital syndromes and is Cuba's first M. genitalium survey. We suggest that M. genitalium should be considered in the Cuban sexually transmitted infection management protocols as an important pathogen, particularly in men.

Antimicrobial Susceptibility Patterns of Recent Cuban Mycoplasma genitalium Isolates Determined by a Modified Cell-Culture-Based Method.

Isolation of Mycoplasma genitalium from clinical specimens remains difficult and few strains are available for antimicrobial susceptibility testing. We describe the antimicrobial susceptibility of M. genitalium strains grown in Vero cell culture with first- and second- line antibiotics, using a modified cell-culture-based method. Macrolide- and -fluoroquinolone resistance determinants were detected by sequencing of the 23S and parC genes, respectively. Seven strains were examined, including three new, genetically distinct M. genitalium strains isolated from endocervical and urethral swab specimens from Cuban patients together with four reference strains isolated from specimens collected from men in Denmark, Sweden and Australia. Azithromycin was the most active drug against two of the Cuban M. genitalium strains with MICs values of 0.008 mg/liter, however, one strain was macrolide resistant with an MIC of >8 mg/liter, and the A2059G resistant genotype. Ciprofloxacin was the least active antimicrobial drug and moxifloxacin was the most active fluoroquinolone against the new clinical strains, although an MIC of 1 mg/l was found for two strains. However, no relevant parC mutations were detected. MICs for tetracyclines were 0.5-4 mg/liter. Although the number of Cuban strains was low, the results suggest that a single-dose azithromycin treatment could be ineffective, and that a second-line treatment with moxifloxacin, should become an option in Cuba. To our knowledge, this is the first report of isolation and antibiotic susceptibility testing of M. genitalium strains from the Latin-American region, and the first detection of macrolide resistance in such strains.

Design, optimization and evaluation of a polymerase chain reaction for detection of borrelia spp.

BACKGROUND: Lyme borreliosis and relapsing fever are important zoonotic diseases worldwide and the improvement of diagnostic strategies is a prioritized task considering the morbidity of these diseases in some areas. PCR based methods appear to be of utmost importance because of the high sensitivity and specificity of these assays. OBJECTIVES: To obtain a molecular method based on PCR for the detection of the genus Borrelia infection in different specimens. RESULTS: Sets of reported primers were evaluated "in silico" and they did not fulfill the proposal parameters. On the other hand, the two new, designed sets of primers were theoretically efficient for Borrelia DNA amplification. PCR procedures with these primers were standardized with borrelial DNA and optimum annealing temperatures, primer concentrations and reaction cycle numbers were determined. The PCR analytical sensitivity was 10 genomes per reaction for each technique. Both PCR were highly specific to different Borrelia species DNA and to samples (sera, cerebrospinal liquids and hard ticks) infected artificially with a Borrelia strain, visualizing the amplification of the expected DNA fragment. No amplification was obtained when other microorganisms were used. 36 human clinical samples were negatives in a preliminary study. CONCLUSIONS: Both sets of primers with their respective PCR protocols showed similar results, which suggest that each one can be used indistinctly in detecting Borrelia spp., mainly in countries where the situation of these diseases are unknown.