An alarming high prevalence of resistance-associated mutations to macrolides and fluoroquinolones in Mycoplasma genitalium in Belgium: results from samples collected between 2015 and 2018.

OBJECTIVES: The number of reported cases of multiresistant Mycoplasma genitalium (MG) is increasing globally. The aim of this study was to estimate the prevalence of macrolide and possible fluoroquinolone resistance-associated mutations (RAMs) of MG in Belgium. METHODS: The study was performed retrospectively on two sets of MG-positive samples collected in Belgium between 2015 and 2018. The first set of samples originated from routine surveillance activities and the second set came from a cohort of men who have sex with men (MSM) using pre-exposure prophylaxis to prevent HIV transmission. Detection of RAMs to macrolides and fluoroquinolones was performed on all samples using DNA sequencing of the 23S ribosomal RNA gene, the gyrA gene and the parC gene. RESULTS: Seventy-one per cent of the MG samples contained a mutation conferring resistance to macrolides or fluoroquinolones (ParC position 83/87). RAMs were more frequently found among men compared with women for fluoroquinolones (23.9% vs 9.1%) and macrolides (78.4% vs 27.3%). Almost 90% of the MG infections among MSM possessed a RAM to macrolides (88.4%). In addition, 18.0% of the samples harboured both macrolides and fluoroquinolone RAMs; 3.0% in women and 24.2% in MSM. Being MSM was associated with macrolide RAMs (OR 15.3), fluoroquinolone RAMs (OR 3.8) and having a possible multiresistant MG infection (OR 7.2). CONCLUSION: The study shows an alarmingly high prevalence of MG with RAMs to macrolides and fluoroquinolones in Belgium. These results highlight the need to improve antimicrobial stewardship in Belgium in order to avoid the emergence of untreatable MG.

Origin and evolution of chloroplast group I introns in lichen algae.

The history of group I introns is characterized by repeated horizontal transfers, even among phylogenetically distant species. The symbiogenetic thalli of lichens are good candidates for the horizontal transfer of genetic material among distantly related organisms, such as fungi and green algae. The main goal of this study was to determine whether there were different trends in intron distribution and properties among Chlorophyte algae based on their phylogenetic relationships and living conditions. Therefore, we investigated the occurrence, distribution and properties of group I introns within the chloroplast LSU rDNA in 87 Chlorophyte algae including lichen and free-living Trebouxiophyceae compared to free-living non-Trebouxiophyceae species. Overall, our findings showed that there was high diversity of group I introns and homing endonucleases (HEs) between Trebouxiophyceae and non-Trebouxiophyceae Chlorophyte algae, with divergence in their distribution patterns, frequencies and properties. However, the differences between lichen Trebouxiophyceae and free-living Trebouxiophyceae were smaller. An exception was the cL2449 intron, which was closely related to ω elements in yeasts. Such introns seem to occur more frequently in lichen Trebouxiophyceae compared to free-living Trebouxiophyceae. Our data suggest that lichenization and maintenance of lichen symbiosis for millions of years of evolution may have facilitated horizontal transfers of specific introns/HEs between symbionts. The data also suggest that sequencing of more chloroplast genes harboring group I introns in diverse algal groups may help us to understand the group I intron/HE transmission process within these organisms.

[Evaluation of novel nucleic acid detection kit for Mycoplasma pneumoniae].

For diagnosis of Mycoplasma pneumoniae infection, highly sensitive and rapid diagnosis is important. Because antibiotics are limited for the treatment of M. pneumoniae infection. In this study, we evaluated new rapid nucleic acid detection kit for M. pneumoniae. This kit does not require excessive pretreatment of specimens and molecular diagnosis of M. pneumoniae is possible within 40 min. Using 120 nasopharyngeal specimens, we compared this kit with a commercially available molecular diagnostic reagent (LAMP). 51 of 120 cases were M. pneumoniae positive, and the results of both assays were all consistent. In addition, sequencing of 23S rRNA gene was performed on 51 cases positive for M. pneumoniae. As a result, macrolide resistance mutation (2063A>G) was observed in 19 cases (37.3%). The gene mutations estimated by this kit coincided completely with the sequencing. In conclusion, new rapid nucleic acid detection kit could detect M. pneumoniae with the same sensitivity as other molecular diagnostics, in a simple process.

RNAModMapper: RNA Modification Mapping Software for Analysis of Liquid Chromatography Tandem Mass Spectrometry Data.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) has proven to be a powerful analytical tool for the characterization of modified ribonucleic acids (RNAs). The typical approach for analyzing modified nucleosides within RNA sequences by mass spectrometry involves ribonuclease digestion followed by LC-MS/MS analysis and data interpretation. Here we describe a new software tool, RNAModMapper (RAMM), to assist in the interpretation of LC-MS/MS data. RAMM is a stand-alone package that requires user-submitted DNA or RNA sequences to create a local database against which collision-induced dissociation (CID) data of modified oligonucleotides can be compared. RAMM can interpret MS/MS data containing modified nucleosides in two modes: fixed and variable. In addition, RAMM can also utilize interpreted MS/MS data for RNA modification mapping back against the input sequence(s). The applicability of RAMM was first tested using total tRNA isolated from Escherichia coli. It was then applied to map modifications found in 16S and 23S rRNA from Streptomyces griseus.

Enrichment of Helicobacter pylori mutant strains after eradication therapy analyzed by gastric wash-based quantitative pyrosequencing.

The eradication of Helicobacter pylori reduces the risk of gastric cancer. A clear understanding of the factors underlying mixed infection with multiple clarithromycin-susceptible and clarithromycin-resistant H. pylori strains is necessary to design more effective therapies against H. pylori. We aimed to assess how the abundance and prevalence of H. pylori strains vary after clarithromycin-based eradication therapy. Using gastric wash samples, which represent the entire stomach, we sequentially analyzed the abundance and prevalence of H. pylori DNA by 23S ribosomal RNA pyrosequencing before and 1, 2, and 3 years after eradication therapy. Low levels of H. pylori DNA were still detectable at the first-year follow-up in all samples with negative post-treatment urea breath test results. The abundance of H. pylori DNA decreased significantly until the 2-year follow-up, but it switched to an increase at the 3-year follow-up. Importantly, the ratio of the prevalence of mutant strains to the prevalence of wild-type strains had already increased at the first-year follow-up and continued to increase, suggesting the selection and growth of clarithromycin-resistant strains during the follow-up periods. Being sensitive and representative, our assay will be useful in effectively addressing gastric cancer development by enhancing the long-term success of intervention strategies and consecutive surveillance for H. pylori eradication.

Azithromycin-resistant Neisseria gonorrhoeae isolates in Guangzhou, China (2009-2013): coevolution with decreased susceptibilities to ceftriaxone and genetic characteristics.

BACKGROUND: The recent emergence of azithromycin-resistant (AZM-R) N. gonorrhoeae isolates that have coevolved decreased susceptibility to extended-spectrum cephalosporins has caused great concern. Here we investigated the prevalence of decreased susceptibility to ceftriaxone (CRO(D)) in AZM-R isolates and genetically characterized AZM-R isolates in Guangzhou, China from 2009 to 2013. METHODS: The minimum inhibitory concentration (MIC) of AZM and ceftriaxone was determined using an agar-dilution method. All AZM-R isolates were screened for mutations in 23S rRNA, mtrR and penA genes and genotyped using N. gonorrhoeae multi-antigen sequence typing (NG-MAST). RESULTS: Of the 485 identified N. gonorrhoeae isolates, 445 (91.8%) were isolated from male urethritis subjects, and 77 (15.9%) were AZM-R (MIC ≥ 1 mg/L), including 33 (6.8%) with AZM low-level resistant (AZM-LLR, MIC = 1 mg/L) and 44 (9.1%) with AZM middle-level resistant (AZM-MLR, MIC ≥ 2 mg/L). Significantly more CRO(D) (MIC ≥ 0.125 mg/L) showed in AZM-MLR isolates (43.2%, 19/44) as compared with that in AZM-LLR isolates (18.2%, 6/33) (p < 0.05). For the 23S rRNA, mtrR, penA or combined 23S rRNA/MtrR/penA mutations, no significant difference was found between AZM-LLR isolates and AZM-MLR isolates (P > 0.05); similar results were detected between combined AZM-LLR/CRO(D) isolates and combined AZM-MLR/CRO(D) isolates (P > 0.05). No mutation A2059G or AZM high-level resistant (AZM-HLR, MIC ≥ 256 mg/L) isolate was detected. Among 77 AZM-R isolates, 67 sequence types (STs) were identified by NG-MAST, of which 30 were novel. Most STs were represented by a single isolate. CONCLUSIONS: The AZM-R together CRO(D) isolates are now present in Guangzhou, China, which deserve continuous surveillance and the mechanism of concurrent resistance needs further study.

More complications occur in macrolide-resistant than in macrolide-sensitive Mycoplasma pneumoniae pneumonia.

We sought to understand the situation of macrolide-resistant genotypes of Mycoplasma pneumoniae, and analyze the relationship between macrolide-resistant genotypes and clinical manifestations of Mycoplasma pneumoniae pneumonia (MPP). Full-length sequencing of the 23S rRNA gene of M. pneumoniae was performed in 235 nasopharyngeal aspirates (NPAs) from children with MPP. We also retrospectively compared the clinical characteristics of macrolide-resistant (MR) M. pneumoniae infections and macrolide-sensitive (MS) M. pneumoniae infections. A total of 206 patients had point mutations in the M. pneumoniae 23S rRNA gene, and these patients are referred to as MR patients. The remaining 29 patients without point mutations are referred to as MS patients. Among 206 MR patients, 199 (96.6%) had A2063G mutations, 6 had A2063T mutations, and the remaining patients had an A2064G mutation. Among the clinical manifestations, we found that the median fever durations were 8 days (range, 0 to 42 days) and 6 days (0 to 14 days) (P < 0.01), the median hospitalization durations were 8 days (2 to 45 days) and 6 days (3 to 16 days) (P < 0.01), and the median fever durations after macrolide therapy were 5 days (0 to 42 days) and 3 days (0 to 10 days) (P < 0.01), respectively, in the MR and MS groups. We also found that the incidence of extrapulmonary complications in the MR group was significantly higher than that in the MS group (P < 0.05). Moreover, the radiological findings were more serious in the MR group than in the MS group (P < 0.05). The increasing prevalence of MR M. pneumoniae has become a significant clinical issue in the pediatric patients, which may lead to more extrapulmonary complications and severe clinical features and radiological manifestations.

The identification and characterization of non-coding and coding RNAs and their modified nucleosides by mass spectrometry.

The analysis of ribonucleic acids (RNA) by mass spectrometry has been a valuable analytical approach for more than 25 years. In fact, mass spectrometry has become a method of choice for the analysis of modified nucleosides from RNA isolated out of biological samples. This review summarizes recent progress that has been made in both nucleoside and oligonucleotide mass spectral analysis. Applications of mass spectrometry in the identification, characterization and quantification of modified nucleosides are discussed. At the oligonucleotide level, advances in modern mass spectrometry approaches combined with the standard RNA modification mapping protocol enable the characterization of RNAs of varying lengths ranging from low molecular weight short interfering RNAs (siRNAs) to the extremely large 23 S rRNAs. New variations and improvements to this protocol are reviewed, including top-down strategies, as these developments now enable qualitative and quantitative measurements of RNA modification patterns in a variety of biological systems.

Harvested white-tailed deer as sentinel hosts for early establishing Ixodes scapularis populations and risk from vector-borne zoonoses in southeastern Canada.

Due to recent establishment of the blacklegged tick, Ixodes scapularis Say, in southeastern Canada, tick-borne zoonoses (Lyme disease, human granulocytotropic anaplasmosis, and babesiosis) are of growing concern for public health. Using white-tailed deer (Odocoileus virginianus) culled in southwestern Quebec during 2007-2008, we investigated whether hunter-killed deer could act as sentinels for early establishing tick populations and for tick-borne pathogens. Accounting for environmental characteristics of culling sites, and age and sex of deer, we investigated whether their tick infestation levels could identify locations of known tick populations detected in active surveillance, presumed tick populations detected by passive surveillance, or both. We also used spatial cluster analyses to identify spatial patterns of tick infestation and occurrence of tick-borne zoonoses infection in ticks collected from the deer. Adult ticks were found on 15% of the 583 deer examined. Adult male deer had the greatest number (approximately 90%) of adult ticks. Overall, 3, 15, and 0% of the ticks collected were polymerase chain reaction (PCR)-positive for Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti, respectively. Our statistical analyses suggest that sex and age of deer, temperature, precipitation, and an index of tick dispersion by migratory birds were significantly associated with tick infestation levels. Cluster analysis identified significant clusters of deer carrying ticks PCR-positive for A. phagocytophilum, and for deer carrying two or more I. scapularis. Our study suggests that hunter-killed deer may be effective as sentinels for emerging areas of tick-borne anaplasmosis. They may have limited use as sentinels for early emerging I. scapularis tick populations and emerging Lyme disease risk.

The aquatic environment as a reservoir of Vibrio cholerae O1 in hydrographic basins of the state of Pernambuco, Brazil.

After the worldwide cholera epidemic in 1993, permanent environmental monitoring of hydrographic basins was established in Pernambuco, Brazil, where cholera is endemic. After a quiescent period, 4 rfbN (serogroup O1) positive water samples that were culture negative were detected by multiplex single-tube nested PCR (MSTNPCR); 2 of these were also ctxA (cholera toxin) positive. From May to June 2012, 30 V. cholerae O1 isolates were obtained by culturing samples. These isolates were analyzed for the presence of virulence genes by PCR, intergenic spacer region 16S-23S PCR (ISR-PCR), and pulsed field gel electrophoresis (PFGE). The isolates were positive for the rfbN gene and negative for the assessed pathogenic genes and were classified into 2 groups by ISR and the same profile by PFGE. Close genetic similarity was observed between them (2012) and environmental strains from 2004 to 2005, indicating the permanence of endemic V. cholerae O1 in the region.

Insights into the ribosome function from the structures of non-arrested ribosome-nascent chain complexes.

During protein synthesis, the growing polypeptide threads through the ribosomal exit tunnel and modulates ribosomal activity by itself or by sensing various small molecules, such as metabolites or antibiotics, appearing in the tunnel. While arrested ribosome-nascent chain complexes (RNCCs) have been extensively studied structurally, the lack of a simple procedure for the large-scale preparation of peptidyl-tRNAs, intermediates in polypeptide synthesis that carry the growing chain, means that little attention has been given to RNCCs representing functionally active states of the ribosome. Here we report the facile synthesis of stably linked peptidyl-tRNAs through a chemoenzymatic approach based on native chemical ligation and use them to determine several structures of RNCCs in the functional pre-attack state of the peptidyl transferase centre. These structures reveal that C-terminal parts of the growing peptides adopt the same uniform ß-strand conformation stabilized by an intricate network of hydrogen bonds with the universally conserved 23S rRNA nucleotides, and explain how the ribosome synthesizes growing peptides containing various sequences with comparable efficiencies.

Direct Nanopore Sequencing for the 17 RNA Modification Types in 36 Locations in the E. coli Ribosome Enables Monitoring of Stress-Dependent Changes.

The bacterium Escherichia coli possesses 16S and 23S rRNA strands that have 36 chemical modification sites with 17 different structures. Nanopore direct RNA sequencing using a protein nanopore sensor and helicase brake, which is also a sensor, was applied to the rRNAs. Nanopore current levels, base calling profile, and helicase dwell times for the modifications relative to unmodified synthetic rRNA controls found signatures for nearly all modifications. Signatures for clustered modifications were determined by selective sequencing of writer knockout E. coli and sequencing of synthetic RNAs utilizing some custom-synthesized nucleotide triphosphates for their preparation. The knowledge of each modification's signature, apart from 5-methylcytidine, was used to determine how metabolic and cold-shock stress impact rRNA modifications. Metabolic stress resulted in either no change or a decrease, and one site increased in modification occupancy, while cold-shock stress led to either no change or a decrease. The double modification m4Cm1402 resides in 16S rRNA, and it decreased with both stressors. Using the helicase dwell time, it was determined that the N4 methyl group is lost during both stressors, and the 2'-OMe group remained. In the ribosome, this modification stabilizes binding to the mRNA codon at the P-site resulting in increased translational fidelity that is lost during stress. The E. coli genome has seven rRNA operons (rrn), and the earlier studies aligned the nanopore reads to a single operon (rrnA). Here, the reads were aligned to all seven operons to identify operon-specific changes in the 11 pseudouridines. This study demonstrates that direct sequencing for >16 different RNA modifications in a strand is achievable.

Multidrug resistance in European Clostridium difficile clinical isolates.

OBJECTIVES: Multidrug resistance and antibiotic resistance mechanisms were investigated in 316 Clostridium difficile clinical isolates collected during the first European surveillance on C. difficile in 2005. METHODS: MICs of eight different antibiotics were determined using Etest. Reserpine- and carbonyl cyanide m-chlorophenylhydrazone-sensitive efflux was tested using the agar dilution method. Molecular analysis of the resistance mechanisms was performed using PCR assays, PCR mapping and sequencing. RESULTS: One hundred and forty-eight C. difficile strains were resistant to at least one antibiotic and 82 (55%) were multidrug resistant. In particular, 48% of these isolates were resistant to erythromycin, clindamycin, moxifloxacin and rifampicin. New genetic elements or determinants conferring resistance to erythromycin/clindamycin or tetracycline were identified. Even if most multiresistant strains carried an erm(B) gene, quite a few were erm(B) negative. In-depth analysis of the underlying mechanism in these isolates was carried out, including analysis of 23S rDNA and the ribosomal proteins L4 and L22. Interestingly, resistance to rifampicin was observed in multidrug-resistant strains in association with resistance to fluoroquinolones. Mutations in the rpo(B) and gyrA genes were identified as the cause of resistance to these antibiotics, respectively. CONCLUSIONS: Characterization of multidrug-resistant C. difficile clinical isolates shows that antibiotic resistance is changing, involving new determinants and mechanisms and providing this pathogen with potential advantages over the co-resident gut flora. The present paper provides, for the first time, a comprehensive picture of the different characteristics of multidrug-resistant C. difficile strains in Europe in 2005 and represents an important source of data for future comparative European studies.

Community-acquired macrolide-resistant Mycoplasma pneumoniae pneumonia in patients more than 18 years of age.

A macrolide-resistant Mycoplasma pneumoniae strain was isolated from two patients with community-acquired pneumonia. The pneumonia severity score of both patients was mild, and rapid clinical improvement was seen after administration of fluoroquinolone. Clinical features of macrolide-resistant M. pneumoniae pneumonia were identical to those of macrolide-sensitive M. pneumoniae pneumonia. An A-to-G transition at position 2063 and 2064, respectively, in domain V of the 23S rRNA gene was identified. The minimum inhibitory concentration of erythromycin of these isolates was greatly elevated. In Japan, macrolide-resistant M. pneumoniae infections are common in pediatric patients but not in adults. However, physicians should pay attention to macrolide-resistant M. pneumoniae not only in children but also in adults.

'Candidatus Mycoplasma haematohydrochoerus', a novel hemoplasma species in capybaras (Hydrochoerus hydrochaeris) from Brazil.

Three different species of hemoplasmas have been described in rodents, Mycoplasma coccoides, 'Candidatus Mycoplasma haemomuris' and 'Candidatus Mycoplasma haemosphiggurus'. Additionally, potentially novel hemoplasma species have been detected in wild rodents from Brazil, including capybaras (Hydrochoerus hydrochaeris). Capybaras are the largest rodent in the world and are well adapted to live within close proximity to humans, which increases the risk to spread of zoonotic pathogens. Herein, we investigate the occurrence and genetic diversity of hemoplasmas infecting free-ranging capybaras from southern Brazil. Blood samples and ticks from 17 capybaras were collected. Packed cell volume and total plasma protein were measured, DNA was extracted, and further screened by species-specific and pan-hemoplasma PCR assays targeting the 16S rRNA gene of hemoplasmas. Sixteen out of 17 (94.12%; 95% CI: 73.02-98.95%) were anemic. Only one young female was hypoproteinemic. All capybaras were infested by adults and nymphs of Amblyomma dubitatum ticks. Using the PCR assay targeting the 16S rRNA gene of M. coccoides, 13/17 (76.47%; 95% CI: 52.74-90.44%) capybaras were positive for hemoplasmas. When DNA samples were tested by the pan-hemoplasma PCR, 16/17 (94.12%; 95% CI: 73.02-98.95%) animals were positive. One out of 11 (9.09%) adult ticks salivary glands tested positive for hemoplasma by the pan-hemoplasma PCR assay. Sequencing and phylogenetic analysis of the 16S and 23S rRNA gene fragments confirmed that animals were infected by a novel hemotropic Mycoplasma sp. previously reported in capybaras from Brazil. Additionally, sequencing and phylogenetic analysis of the 23S rRNA gene from three hemoplasma-positive capybaras samples from a previous study performed in midwestern Brazil also confirm our findings. Based on phylogenetic and Neighbor-Net network analysis of the 16S rRNA and 23S rRNA genes, the name 'Candidatus Mycoplasma haematohydrochoerus' is proposed for this novel organism.

Molecular detection and characterization of Anaplasma platys and Ehrlichia canis in dogs from the Caribbean.

Anaplasma platys is a tick-transmitted rickettsial pathogen, which is known to be the etiologic agent for cyclic thrombocytopenia in its primary canine host. Infections with this pathogen are also reported in cats, cattle and people. Similarly, Ehrlichia canis is another tick-borne rickettsial pathogen responsible for canine monocytic ehrlichiosis and is also reported to cause infections in people. We describe infections in dogs with these two pathogens on the Caribbean island of Grenada, West Indies by detection using molecular methods. We utilized a 16S rRNA gene-based PCR assay to detect both Ehrlichia and Anaplasma species by screening 155 canine blood samples from asymptomatic dogs. We found 18.7 % of the dogs to be positive for A. platys and 16.8 % for E. canis. Samples that tested positive for A. platys were further assessed by sequence analysis targeting 16S rRNA, 23S rRNA, citrate synthase (gltA) and heat shock protein (groEL) genes. Phylogenetic analysis revealed high correlation of A. platys 16S rRNA and gltA gene sequences with the geographic origins, while 23S rRNA and groEL gene sequences clustered independent of the geographic origins. This study represents an important step in defining the widespread distribution of active rickettsial infections in Caribbean dogs with no apparent clinical signs, thus posing a high risk for canine health and to a lesser extent to humans, as most dogs in the Caribbean are free-roaming.

Mycobacterium tuberculosis infection of domesticated Asian elephants, Thailand.

Four Asian elephants were confirmed to be infected with Mycobacterium tuberculosis by bacterial culture, other diagnostic procedures, and sequencing of 16S-23S rDNA internal transcribed spacer region, 16S rRNA, and gyrase B gene sequences. Genotyping showed that the infectious agents originated from 4 sources in Thailand. To identify infections, a combination of diagnostic assays is essential.

Rapid differentiation of Francisella species and subspecies by fluorescent in situ hybridization targeting the 23S rRNA.

BACKGROUND: Francisella (F.) tularensis is the causative agent of tularemia. Due to its low infectious dose, ease of dissemination and high case fatality rate, F. tularensis was the subject in diverse biological weapons programs and is among the top six agents with high potential if misused in bioterrorism. Microbiological diagnosis is cumbersome and time-consuming. Methods for the direct detection of the pathogen (immunofluorescence, PCR) have been developed but are restricted to reference laboratories. RESULTS: The complete 23S rRNA genes of representative strains of F. philomiragia and all subspecies of F. tularensis were sequenced. Single nucleotide polymorphisms on species and subspecies level were confirmed by partial amplification and sequencing of 24 additional strains. Fluorescent In Situ Hybridization (FISH) assays were established using species- and subspecies-specific probes.Different FISH protocols allowed the positive identification of all 4 F. philomiragia strains, and more than 40 F. tularensis strains tested. By combination of different probes, it was possible to differentiate the F. tularensis subspecies holarctica, tularensis, mediasiatica and novicida. No cross reactivity with strains of 71 clinically relevant bacterial species was observed. FISH was also successfully applied to detect different F. tularensis strains in infected cells or tissue samples. In blood culture systems spiked with F. tularensis, bacterial cells of different subspecies could be separated within single samples. CONCLUSION: We could show that FISH targeting the 23S rRNA gene is a rapid and versatile method for the identification and differentiation of F. tularensis isolates from both laboratory cultures and clinical samples.

Molecular Detection of Spotted Fever Group Rickettsia (Rickettsiales: Rickettsiaceae) in Ticks of Iran.

Ticks are reservoir hosts of pathogenic Rickettsia in humans and domestic animals. Most pathogenic Rickettsia species belong to the spotted fever group (SFG). The present study aimed to determine the tick species infected with Rickettsia based on the genus-specific 23S ribosomal ribonucleic acid (rRNA), 16S rRNA, and citrate synthase (gltA) gene fragments. A total of 61 tick specimens were selected for molecular assay and 12 samples for sequencing. Phylogenetic analysis was conducted using neighbor-joining and Bayesian inference methods. Argas persicus, Haemaphysalis sulcata, Ha. inermis, and Hyalomma asiaticum were infected by spotted fever Rickettsia. The SFG is the main group of Rickettsia that can be detected in the three genera of ticks from Iran.

Use of hidden correlations in short oligonucleotide array data are insufficient for accurate quantification of nucleic acid targets in complex target mixtures.

Nonspecific target binding (i.e., cross-hybridization) is a major challenge for interpreting oligonucleotide microarray results because it is difficult to determine what portion of the signal is due to binding of complementary (specific) targets to a probe versus that due to binding of nonspecific targets. Solving this challenge would be a major accomplishment in microarray research potentially allowing quantification of targets in biological samples. Marcelino et al. recently described a new approach that reportedly solves this challenge by iteratively deconvoluting 'true' specific signal from raw signal, and quantifying ribosomal (rRNA) sequences in artificial and natural communities (i.e., "Accurately quantifying low-abundant targets amid similar sequences by revealing hidden correlations in oligonucleotide microarray data", Proc. Natl. Acad. Sci. 103, 13629-13634). We evaluated their approach using high-density oligonucleotide microarrays and Latin-square designed experiments consisting of 6 and 8 rRNA targets in 16 different artificial mixtures. Our results show that contrary to the claims in the article, the hidden correlations in the microarray data are insufficient for accurate quantification of nucleic acid targets in complex artificial target mixtures.