Application of simplified MLST scheme for direct typing of clinical samples from human leptospirosis cases in a tertiary hospital in the Philippines

Despite the major threat of leptospirosis to public health in the Philippines, its epidemiologic data remain scarce. Multilocus sequence typing (MLST) is a method often used for identification of circulating Leptospira species and disease surveillance. Unfortunately, molecular typing of Leptospira isolates is not routinely done in most hospital settings. A simplified MLST scheme targeting three loci (adk, lipL41, mreA) was performed for rapid direct typing of Leptospira in clinical specimens. Blood samples from suspected or clinically diagnosed cases (n = 50) were initially screened via polymerase chain reaction (PCR) targeting 23S rRNA, 16S rRNA (rrs2), and lipL32 genes. From the nine positives, seven had interpretable data from MLST. Allelic profiles identified L. interrogans in all positive samples. Six were assigned to ST12 of serovar Manilae (serogroup Pyrogenes) while one sample cannot be clearly differentiated between two serovars/serogroups, Bataviae/Losbanos (serogroup Bataviae) or Australis (serogroup Australis), indicating possibility of a new ST. Phylogenetic analysis confirmed that the application of simplified MLST scheme produces consistent results with the seven-loci genetic profile of published Leptospira MLST schemes. Reduced scheme addressed the challenges often encountered in the amplification of full MLST genetic profile of Leptospira. The approach is a potential alternative to serological tests for rapid typing of clinical specimens and can also aid in investigations on disease epidemiology specifically to monitor occurrence, pathogen transmission, host specificity and susceptibility, and other factors that could lead to potential outbreaks.

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Detection of markers predictive of macrolide and fluoroquinolone resistance in Mycoplasma genitalium from patients attending sexual health services

Sexually Transmitted Infections ◽

10.1136/sextrans-2020-054897 ◽

2021 ◽

pp. sextrans-2020-054897

Author(s):

Michaela Joanne Day ◽

Michelle Jayne Cole ◽

Helen Fifer ◽

Neil Woodford ◽

Rachel Pitt

Keyword(s):

Clinical Significance ◽

Sequence Data ◽

Mycoplasma Genitalium ◽

Fluoroquinolone Resistance ◽

23S Rrna ◽

Reference Laboratory ◽

National Reference Laboratory ◽

Clinical Specimens ◽

National Reference ◽

Pcr Targeting

ObjectivesThis study sought to provide data on the prevalence of macrolide (23S rRNA) and fluoroquinolone (parC) resistance-associated mutations seen in Mycoplasma genitalium-positive specimens received in the UK national reference laboratory.MethodsIn total, 2580 clinical specimens from patients with suspected or confirmed M. genitalium infection were received at the national reference laboratory between September 2017 and November 2018. M. genitalium-positive clinical specimens were identified using a reverse transcription-PCR targeting two M. genitalium genes: MgPa and gap. Resistance-associated single nucleotide poylmorphisms were sought in all positive specimens by sequence analysis of the 23S rRNA and parC genes.ResultsEighteen per cent (458 of 2580) of clinical specimens were positive for M. genitalium and 389 had sequence data for both macrolide and fluoroquinolone resistance markers. Of these, 71% (275 of 389) had macrolide resistance-associated mutations, 8% (31 of 389) had fluoroquinolone resistance-associated mutations (S83I/R and D87Y/N) and 7% (26 of 389) had mutations associated with resistance to both antimicrobials. Only 28% (108 of 389) had no mutations associated with resistance to either class of antibiotic. Five specimens had mutations of unknown clinical significance in the parC gene (eg, G81C and S83N).ConclusionsMutations associated with resistance to macrolides were very frequent. By contrast, susceptibility to the second-line treatment, moxifloxacin (a fluoroquinolone), was estimated at 92% based on the absence of resistance-associated mutations. The few specimens with mutations of unknown clinical significance in the parC gene were excluded from the analysis and so the actual level of fluoroquinolone susceptibility may be slightly lower than that reported here. Surveillance of antimicrobial resistance in M. genitalium is imperative for this to remain a treatable infection.

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French Prospective Clinical Evaluation of the Aptima Mycoplasma genitalium CE-IVD Assay and Macrolide Resistance Detection Using Three Distinct Assays

Journal of Clinical Microbiology ◽

10.1128/jcm.00579-17 ◽

2017 ◽

Vol 55 (11) ◽

pp. 3194-3200 ◽

Cited By ~ 21

Author(s):

Chloé Le Roy ◽

Sabine Pereyre ◽

Nadège Hénin ◽

Cécile Bébéar

Keyword(s):

Clinical Evaluation ◽

Mycoplasma Genitalium ◽

Macrolide Resistance ◽

23S Rrna ◽

Clinical Samples ◽

Specific Method ◽

Rt Pcr ◽

Pcr Assay ◽

Content Type ◽

Clinical Specimens

ABSTRACTThe aim of this study was to evaluate the clinical performance of the AptimaMycoplasma genitaliumtranscription-mediated amplification (MG-TMA) CE-marked forin vitrodiagnosis (CE-IVD) assay for the detection ofMycoplasma genitaliumin male and female clinical samples in comparison with the in-house real-time PCR (in-house PCR) assay routinely used in our laboratory. A total of 1,431 clinical specimens obtained from 1,235 patients were prospectively collected at the Bacteriology Department of Bordeaux University Hospital (France). Additional research-use-only AptimaM. genitaliumtranscription-mediated amplification (TMA) assays, Alt1-TMA and Alt2-TMA, were performed on discordant specimens to determineM. genitaliuminfection status. All confirmedM. genitalium-positive specimens were tested for macrolide resistance using three assays: the in-house 23S rRNA FRET PCR assay, the SpeeDx ResistancePlus MG assay and the nested reverse transcription-PCR (RT-PCR) sequencing assay. The comparison of the MG-TMA assay with the in-house PCR results showed a moderate correlation (kappa value, 0.69). The MG-TMA assay had higher clinical sensitivity compared to that of the in-house PCR assay (100% versus 59.74%, respectively) and similar specificity (99.10% versus 100%, respectively) forM. genitaliumdetection. In this study, the prevalence ofM. genitaliuminfection was 5.90% (72/1,220 patients). The nested RT-PCR sequencing assay was the most sensitive but the most laborious assay for detecting macrolide-resistance-associated mutations. The prevalence of resistance was 8.33% (6/72). To our knowledge, this is the first clinical evaluation of the MG-TMA CE-IVD assay. The MG-TMA assay performed on the automated Panther system is a very sensitive and specific method for the detection ofM. genitaliumin clinical specimens.

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An extended MLST scheme for rapid direct typing of Leptospira from clinical samples

10.26226/morressier.56d5ba2dd462b80296c94fb3 ◽

2016 ◽

Author(s):

Sabrina Weiss

Keyword(s):

Clinical Samples ◽

Mlst Scheme

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Clinical guideline for diagnosis and management of melioidosis

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46652006000100001 ◽

2006 ◽

Vol 48 (1) ◽

pp. 1-4 ◽

Cited By ~ 29

Author(s):

Timothy J.J. Inglis ◽

Dionne B. Rolim ◽

Jorge L.N. Rodriguez

Keyword(s):

Oral Treatment ◽

Central Nervous System Infection ◽

Severe Infection ◽

Community Acquired Pneumonia ◽

Clinical Samples ◽

Clinical Microbiology Laboratory ◽

Serological Tests ◽

Wide Range ◽

Severe Community Acquired Pneumonia ◽

Diagnostic Microbiology

Melioidosis is an emerging infection in Brazil and neighbouring South American countries. The wide range of clinical presentations include severe community-acquired pneumonia, septicaemia, central nervous system infection and less severe soft tissue infection. Diagnosis depends heavily on the clinical microbiology laboratory for culture. Burkholderia pseudomallei, the bacterial cause of melioidosis, is easily cultured from blood, sputum and other clinical samples. However, B. pseudomallei can be difficult to identify reliably, and can be confused with closely related bacteria, some of which may be dismissed as insignificant culture contaminants. Serological tests can help to support a diagnosis of melioidosis, but by themselves do not provide a definitive diagnosis. The use of a laboratory discovery pathway can help reduce the risk of missing atypical B. pseudomallei isolates. Recommended antibiotic treatment for severe infection is either intravenous Ceftazidime or Meropenem for several weeks, followed by up to 20 weeks oral treatment with a combination of trimethoprim-sulphamethoxazole and doxycycline. Consistent use of diagnostic microbiology to confirm the diagnosis, and rigorous treatment of severe infection with the correct antibiotics in two stages; acute and eradication, will contribute to a reduction in mortality from melioidosis.

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Comparison of diagnostic clinical samples and environmental sampling for enterovirus and parechovirus surveillance in Scotland, 2010 to 2012

Eurosurveillance ◽

10.2807/1560-7917.es2014.19.15.20772 ◽

2014 ◽

Vol 19 (15) ◽

Cited By ~ 56

Author(s):

H Harvala ◽

J Calvert ◽

D Van Nguyen ◽

L Clasper ◽

N Gadsby ◽

...

Keyword(s):

Central Nervous System ◽

Cerebrospinal Fluid ◽

Rapid Identification ◽

Clinical Samples ◽

Environmental Sampling ◽

Clinical Specimens ◽

Young Infants ◽

Coxsackievirus A6 ◽

The Family ◽

Common Causes

Human enteroviruses (EV) and parechoviruses (HPeV) within the family Picornaviridae are the most common causes of viral central nervous system (CNS)-associated infections including meningitis and neonatal sepsis-like disease. The frequencies of EV and HPeV types identified in clinical specimens collected in Scotland over an eight-year period were compared to those identified in sewage surveillance established in Edinburgh. Of the 35 different EV types belonging to four EV species (A to D) and the four HPeV types detected in this study, HPeV3 was identified as the most prevalent picornavirus in cerebrospinal fluid samples, followed by species B EV. Interestingly, over half of EV and all HPeV CNS-associated infections were observed in young infants (younger than three months). Detection of species A EV including coxsackievirus A6 and EV71 in clinical samples and sewage indicates that these viruses are already widely circulating in Scotland. Furthermore, species C EV were frequently identified EV in sewage screening but they were not present in any of 606 EV-positive clinical samples studied, indicating their likely lower pathogenicity. Picornavirus surveillance is important not only for monitoring the changing epidemiology of these infections but also for the rapid identification of spread of emerging EV and/or HPeV types.

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First isolation of Mycobacterium ulcerans from Swabs and Fine-Needle-Aspiration Specimens in Togo.

10.21203/rs.2.17920/v1 ◽

2019 ◽

Author(s):

Tchalare Kondi Makagni ◽

Maman Issaka ◽

Piten Ebekalisai ◽

Disse Kodjo ◽

Essossimna A. Kadanga ◽

...

Keyword(s):

Fine Needle Aspiration ◽

Slow Growth ◽

Buruli Ulcer ◽

Needle Aspiration ◽

Mycobacterium Ulcerans ◽

Clinical Samples ◽

Direct Examination ◽

Fine Needle ◽

Pcr Targeting

Abstract Background Buruli ulcer is a skin disease caused by a mycobacterium called Mycobacterium ulcerans . It is prevalent in more than 33 countries on several continents but West Africa is the most affected. The isolation in culture of the bacteria is difficult because of its slow growth and the facilities required. In Togo, studies have been done on the risk factors for Mycobacterium ulcerans infection and the detection of cases by the Ziehl-Neelsen and PCR technique on clinical and environmental samples, but to date no data of isolates from clinical samples are available. The purpose of this study was to perform an in vitro culture of M. ulcerans from swab and fine needle aspiration samples through the confirmation stages of direct examination and IS2404 -PCR. Method A total of 70 clinical samples from Togo and 10 clinical isolates from Benin are analyzed by the three techniques indicated in the diagnosis, in particular the direct examination of acid-fast bacilli (AFB) using the Ziehl-Neelsen staining, the PCR targeting the IS2404 sequence, and the culture after transport of the samples in a transport medium made of Middlebrook 7H9 medium supplemented with a mixture of PANTA and OADC and decontamination by the modified Petroff method. Results The application of the three techniques of diagnosis for clinical samples yielded 44.28% of positivity rates on direct examination of AFB, 35.71% on culture and 77.14% on qPCR IS2404 with a significantly higher rate for qPCR (0.0001). All samples positive for Ziehl-Neelsen staining and culture were also positive for qPCR. Conclusion : Our results show that the culture, despite it difficulty and the slow growth of the bacteria, can be carried out with recommended tools of the mycobacteria culture and a good method of decontamination of the samples can improve the positivity rates. Its realization will allow the assessment of the in vitro sensitivity to the antibiotics used in the treatment and the discovery of new strains of Mycobacterium ulcerans .

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A broad-range survey of Borrelia burgdorferi sensu lato infection in small mammals in Yunnan Province, China

10.21203/rs.3.rs-124610/v1 ◽

2020 ◽

Author(s):

Zhihai He ◽

Baogui Jiang ◽

Zihou Gao ◽

Zongti Shao ◽

Yun Zhang ◽

...

Keyword(s):

Borrelia Burgdorferi ◽

Small Mammals ◽

Yunnan Province ◽

23S Rrna ◽

Borrelia Burgdorferi Sensu Lato ◽

Tropical Region ◽

Prevalence Rates ◽

High Genetic Diversity ◽

Landscape Types ◽

Pcr Targeting

Abstract Background: Lyme disease, caused by Borrelia burgdorferi sensu lato (BBSL), is commonly found in wild and domestic mammals worldwide. In China, human cases of B. burgdorferi infections have been identified across a wide distribution, but little direct surveillance of potential small mammal reservoirs has been performed in Yunnan Province, a tropical region in southwestern China. Here we report a thorough investigation of BBSL in small mammals collected from 2011 to 2016 from this region.Methods: Small mammals were captured using snap traps in 23 counties located in Yunnan Province. DNA was extracted from spleen tissue using DNA blood and tissue kits. A nested PCR targeting the 5S-23S rRNA intergenic spacer gene of BBSL was used for pathogen detection. Amplicons of 252bp expected sizes were sequenced directly and analyzed using BLAST algorithm. A phylogenetic tree was constructed using MEGA software and statistical analysis were conducted using SPSS version 17.0.Results: Overall, 3659 mammals belonging to 57 species were captured at 159 sample sites located in 23 counties in Yunnan Province. Borrelia burgdorferi s.l. was found in 146 mammals (3.99%), from 30 different species, 20 of which represent the first reported detection in that species. Sequence analysis revealed five genotypes of B. burgdorferi s.l., including B. afzelii, B. burgdorferi sensu stricto, B. japonica, B. garinii and B. valaisiana.Conclusions: Significant differences in prevalence rates of BBSL were observed at varying landscape types and altitudes. Small mammals in forested areas had higher prevalence rates than other landscape types, as did small mammals found at altitudes greater than 2500 meters. The five genotypes of BBSL detected, suggests high genetic diversity within this province.

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A comparative characterization of nasal and clinical isolates of Staphylococcus aureus from west of Iran

Iranian Journal of Microbiology ◽

10.18502/ijm.v13i6.8086 ◽

2021 ◽

Author(s):

Gholamreza Goudarzi ◽

Yaser Hasanvand ◽

Faranak Rezaei ◽

Somayeh Delfani

Keyword(s):

Staphylococcus Aureus ◽

Antibiotic Resistance ◽

Healthcare Workers ◽

Meca Gene ◽

Staphylococcal Enterotoxin ◽

Healthcare Personnel ◽

Clinical Samples ◽

Resistance Pattern ◽

Clinical Specimens ◽

Significant Difference

Background and Objectives: Recently, the rise of methicillin-resistant Staphylococcus aureus (MRSA) isolated from hos- pital healthcare workers (HCWs) and various infectious samples has become one of the main concerns in hospital settings. Therefore, epidemiological studies are necessary to monitor antibiotic resistance patterns in each region and to study the pathogenesis of this strain to control infections. Materials and Methods: In this cross-sectional study, a total of 100 S. aureus isolates, including 50 isolates obtained from the anterior nares of healthcare workers, as well as 50 other isolates cultured from the various clinical specimens from the referral hospitals in Khorramabad (West of Iran) were tested. All isolates were examined to determine antibiotic resistance pattern, and the presence of staphylococcal enterotoxin A (sea), staphylococcal enterotoxin B (seb) and mecA genes. Results: The mecA gene was found among 36% (18/50) of the clinical S. aureus isolates (CSIs) and 14% (7/50) of nasal S. aureus isolates (NSIs), with statistically significant difference (X2 = 6.53; p = 0.011). The difference between the frequency rate of sea gene among MRSA strains isolated from clinical specimens (46.6%, 7/15) was significant compared to strains isolated from nostrils (14.3%, 1/7) (X2 = 3.85; p = 0.049). Conclusion: The frequency of mecA, sea, and seb genes among the clinical samples was more than strains isolated from the nostrils of healthcare personnel.

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Sporosarcina newyorkensis sp. nov. from clinical specimens and raw cow’s milk

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.030080-0 ◽

2012 ◽

Vol 62 (2) ◽

pp. 322-329 ◽

Cited By ~ 16

Author(s):

William J. Wolfgang ◽

An Coorevits ◽

Jocelyn A. Cole ◽

Paul De Vos ◽

Michelle C. Dickinson ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

New York State ◽

23S Rrna ◽

Rrna Gene ◽

The Novel ◽

Gene Sequences ◽

16S Rrna Gene Sequences ◽

Clinical Specimens ◽

The 16S Rrna Gene

Twelve independent isolates of a Gram-positive, endospore-forming rod were recovered from clinical specimens in New York State, USA, and from raw milk in Flanders, Belgium. The 16S rRNA gene sequences for all isolates were identical. The closest species with a validly published name, based on 16S rRNA gene sequence, is Sporosarcina koreensis (97.13 % similarity). DNA–DNA hybridization studies demonstrate that the new isolates belong to a species distinct from their nearest phylogenetic neighbours. The partial sequences of the 23S rRNA gene for the novel strains and their nearest neighbours also provide support for the novel species designation. Maximum-likelihood phylogenetic analysis of the 16S rRNA gene sequences confirmed that the new isolates are in the genus Sporosarcina. The predominant menaquinone is MK-7, the peptidoglycan has the type A4α l-Lys–Gly–d-Glu, and the polar lipids consist of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The predominant fatty acids are iso-C14 : 0, iso-C15 : 0 and anteiso-C15 : 0. In addition, biochemical and morphological analyses support designation of the twelve isolates as representatives of a single new species within the genus Sporosarcina, for which the name Sporosarcina newyorkensis sp. nov. (type strain 6062T = DSM 23544T = CCUG 59649T = LMG 26022T) is proposed.

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